Characterisation of Hardenbergia mosaic virus and development of ...

Characterisation of Hardenbergia mosaic virus and development of microarrays for detecting viruses in plants

This thesis is presented to Murdoch University for the degree of Doctor of Philosophy

June 2008

by

Craig Graham Webster B.Sc. Biotechnology and Chemistry Honours Molecular Biology

State Agricultural Biotechnology Centre School of Biological Sciences and Biotechnology Murdoch University Perth, Western Australia

DECLARATION

Declaration

I declare that this is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institute.

Craig Graham Webster

ii

ABSTRACT

Abstract A virus causing chlorosis and leaf distortion in the Western Australian endemic legume Hardenbergia comptoniana was detected by biological indexing to Chenopodium quinoa and Nicotiana benthamiana.

Enzyme linked immuno-sorbent assay (ELISA) using general

Potyvirus antiserum and amplification by reverse transcription polymerase chain reaction (RTPCR) with degenerate primers indicated that it was a species of Potyvirus. It was confirmed as an unknown member of the genus Potyvirus by comparing its coat protein sequence with those of other potyviruses. The name Hardenbergia mosaic virus (HarMV) is proposed for this new virus species. Isolates of HarMV were collected from 13 sites, covering much of the natural range of its host. An experimental host range was determined using nine virus isolates tested against plants from 11 species in three families. Its infectivity on three leguminous species important in agriculture (Lupinus angustifolius, L. luteus and Trifolium subterraneum) was established.

The nucleotide (nt) sequences of the coat proteins (CP) of 28 isolates determined there was 24.1- 27.6% diversity with the closest known relative, Passion fruit woodiness virus (PWV). Studies of the nucleotide sequences of the CP showed that there was considerable intra-species divergence (mean 13.5%, maximum 20.5%) despite its relatively small geographical distribution and single known natural host. The observed broad diversity strongly suggests long genetic isolation and that HarMV evolved in the region where it was collected. An examination of its phylogeny showed that 28 isolates clustered into eight clades with high bootstrap support (6.2-20.5% inter-clade diversity).

Isolates collected at locations distant to the Perth

metropolitan area (Margaret River and Seabird) diverged more from isolates collected in the metropolitan area (15.4-21.1% nucleotide sequence diversity). This virus represents the first endemic species to be characterised from Western Australia.

Differences in pathogenicity and symptoms induced on key host species were seen between isolates belonging to different phylogenetic clades.

Phylogenetic analysis confirmed the

inclusion of HarMV within the Bean common mosaic virus group of the potyviruses and also defined a previously unreported subgroup of six previously described Potyvirus species (Clitoria virus Y, Hibbertia virus Y, PWV, Siratro 1 virus Y, and Siratro 2 virus Y), from Australia, which is further evidence for a prolonged period of genetic isolation.

Both in relation to detection of strains of HarMV, and considering the broader issues of biosecurity and parallel detection of plant viruses, a microarray based detection system was established. To optimise conditions for the development of microarrays for virus detection iii

ABSTRACT

poly-L-lysine (PLL) coated microscope slides produced in the laboratory were compared to commercially produced PowerMatrix slides (Full Moon BioSystems). Variables tested for PLL slide production were: choice of printing buffer, probe concentration, method of immobilisation and slide blocking; and in particular the print buffer and immobilisation method had the greatest effect on the quality of PLL microarray slides. Slides printed on PLL surfaces in a high salt buffer (3x Saline sodium citrate) supplemented with 1.5M betaine and immobilised at 42oC overnight retained the highest amounts of probe DNA of the methods tested. Qualitative comparisons of the two showed more probe was retained on PowerMatrix slides which were also more reliable and consistent than the PLL slides.

Probes were designed for eight different virus species and six distinct strains of HarMV to test the potential to use microarrays to distinguish between them. Probes were designed to detect potyviruses at the genus, species and strain levels. Although there was evidence of non-specific hybridisation, the Potyvirus array was used to identify six strains of HarMV by hybridisation to species specific probes. Additionally the array was used to identify three other species of Potyvirus: Bean yellow mosaic virus, PWV and Passiflora foetida virus Y, following amplification with polyvalent PCR primers.

In further microarray tests, using labelled first strand cDNA of Potato virus X (PVX) and Potato virus Y (PVY) on an array, PVX was strongly detected in leaves known to be infected, but PVY was only weakly detected in infected leaves. Three methods of pre-amplification of virus nucleic acid before hybridisation to the array were investigated to improve the sensitivity of the assay. Two of the methods, Klenow amplification and randomly primed PCR, amplified the target virus; as confirmed by real time PCR. Of the methods tested only randomly primed PCR improved the sensitivity of the microarray. The best amplification method used genus-specific primers with adaptor sequences. This method when tested by real time PCR showed a 3.7Ct reduction for PVX and 16.8Ct for PVY. The microarray correctly identified both viruses.

In this work the first virus (HarMV) endemic to Western Australia was identified, and microarray methods were developed both to identify HarMV and other plant viruses of economic importance. The microarray approach, with further development, may be applicable as a means of identifying incursions of new viruses in a biosecurity situation.

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TABLE OF CONTENTS

Table of Contents

Declaration ................................................................................................................................... ii Abstract........................................................................................................................................ iii Table of Contents ..........................................................................................................................v Presentations and Publications ................................................................................................... x Publications ........................................................................................................................................x Oral Presentations..............................................................................................................................x Poster Presentations..........................................................................................................................xi

List of abbreviations................................................................................................................... xii Viruses and viroids .......................................................................................................................... xii Other abbreviations used in the text .................................................................................................xv Code for degenerate oligonucleotides .............................................................................................xvi

Acknowledgements................................................................................................................... xvii Chapter 1: Introduction and literature review ............................................................................ 1 1.1 Introduction to viruses ..................................................................................................... 1 1.2 Genomic structure of plant viruses ................................................................................. 2 1.2.1 Nucleic acid .............................................................................................................................. 2 1.2.2 Genome size.............................................................................................................................. 2 1.2.3 Genome organization................................................................................................................ 2 1.2.4 Satellite viruses, satellite RNA’s and viroids ............................................................................ 3 1.2.5 The Potyviridae......................................................................................................................... 3 The Potyvirus coat protein gene ................................................................................................... 5 Classification of Potyvirus species............................................................................................... 5 The BCMV group ........................................................................................................................ 7

1.3 Economic importance....................................................................................................... 9 1.4 Diagnostic methods......................................................................................................... 10 1.4.1 Visual detection and indicator hosts....................................................................................... 10 Electron microscopy .................................................................................................................. 10 1.4.2 Serological procedures........................................................................................................... 10 Enzyme-linked immunosorbent assay (ELISA) ...................................................................... 11 Tissue blot immunoassay (TBIA) ............................................................................................. 11 Lateral flow devices (LFDs) ...................................................................................................... 12 Quartz crystal microbalance (QCM) immunosensors ............................................................ 12 Nanowire field effect transistors............................................................................................... 13 1.4.3 Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) .................................................................................................................................................. 13 1.4.4 Nucleic acid procedures ......................................................................................................... 14 Dot Blots ..................................................................................................................................... 14 Reverse transcription-Polymerase Chain Reaction (RT-PCR) and PCR ............................. 14 Multiplex RT-PCR..................................................................................................................... 15 Nested PCR................................................................................................................................. 15 Polyvalent PCR .......................................................................................................................... 16 Immunocapture PCR (IC-PCR) ............................................................................................... 18 Fluorescence Real Time-PCR using Taqman technology ................................................... 18 Competitive fluorescence PCR (CF-PCR) ............................................................................... 19 Restriction fragment length polymorphism (RFLP)............................................................... 19 Rolling circle amplification (RCA) ........................................................................................... 19 Loop mediated isothermal amplification (LAMP).................................................................. 20 Macroarrays ............................................................................................................................... 21 1.4.5 Advantages and disadvantages of current methods................................................................ 21

1.5 Microarrays..................................................................................................................... 22 v

TABLE OF CONTENTS 1.5.1 Diagnostic applications of microarrays ................................................................................. 23 1.5.2 Probes for microarrays........................................................................................................... 27 Nucleic acid probes .................................................................................................................... 27 cDNA probes .............................................................................................................................. 27 Oligonucleotide probes .............................................................................................................. 28 Probe length................................................................................................................................ 28 Antibody/protein probes ........................................................................................................... 29 1.5.3 Slide surface and immobilisation methods.............................................................................. 30 Arraying of probes..................................................................................................................... 30 Slide surfaces .............................................................................................................................. 31 Covalent binding of probes ....................................................................................................... 31 1.5.4 Methods for labelling and hybridisation................................................................................. 32 Fluorescent labelling of DNA .................................................................................................... 32 Hybridisation and washing slides ............................................................................................. 33

1.6 Conclusions ..................................................................................................................... 34 1.7 Project Aims.................................................................................................................... 35 Chapter 2: General materials and methods .............................................................................. 36 2.1 Introduction .................................................................................................................... 36 2.2 Viruses and Inoculations................................................................................................ 36 2.3 RNA Extractions............................................................................................................. 36 2.3.1 Qiagen RNeasy Plant Mini Extractions.................................................................................. 36 2.3.2 Microarray RNA Extractions.................................................................................................. 37

2.4 Reverse Transcription.................................................................................................... 37 2.5 PCR Protocol................................................................................................................... 38 2.5.1 High fidelity PCR.................................................................................................................... 38 2.5.2 Lower-fidelity PCR ................................................................................................................. 39 2.5.3 Real time PCR ........................................................................................................................ 40

2.6 Agarose Gel Electrophoresis.......................................................................................... 40 2.7 Purification of DNA solutions........................................................................................ 41 2.7.1 Ethanol Precipitation.............................................................................................................. 41 2.7.2 Purification by QiaQuick PCR cleanup kit............................................................................. 41

2.8 Quantifying DNA............................................................................................................ 41 2.9 Cloning of PCR products ............................................................................................... 41 2.9.1 Ligation and Transformation using TOPO-Zero kit. .............................................................. 41 2.9.2 Ligation and Transformation using pGEM-T easy™ ............................................................. 42

2.10 Screening for recombinant plasmids .......................................................................... 42 2.11 Plasmid Extraction ....................................................................................................... 42 2.11.1 QIAprep Spin Miniprep Kit................................................................................................... 43 2.11.2: Sambrook Plasmid Extractions ........................................................................................... 43

2.12 Restriction Digest of Plasmid....................................................................................... 44 2.13 Storing clones as Glycerol Stocks................................................................................ 44 2.14 DNA Sequencing ........................................................................................................... 44 2.15 Coating Slides with Poly-L-lysine................................................................................ 45 2.16 Printing microarray slides ........................................................................................... 45 2.17 Quality control of printed slides.................................................................................. 46 2.18 Summary ....................................................................................................................... 46 Chapter 3: Bioinformatics for primer and probe design .......................................................... 47 3.1 Introduction .................................................................................................................... 47 vi

TABLE OF CONTENTS

3.2 Materials and methods ................................................................................................... 48 3.2.1 Sequence retrieval from online databases .............................................................................. 48 3.2.2 Sequence alignments............................................................................................................... 48 ClustalW alignments.................................................................................................................. 48 MEGA alignments ..................................................................................................................... 48 3.2.3 Comparisons of nucleotide sequences to international database ........................................... 48 3.2.4 Oligonucleotide design ........................................................................................................... 49 Primer design ............................................................................................................................. 49 Oligonucleotide probe design.................................................................................................... 50 3.2.5 Phylogenetic analysis of virus isolates ................................................................................... 52

3.3 Bioinformatics results..................................................................................................... 53 3.3.1 Sequences of legume specific PCR primers ............................................................................ 53 3.3.2 Design of oligonucleotide probes ........................................................................................... 53

3.4 Discussion ........................................................................................................................ 57 Chapter 4: Characterisation of an undescribed Potyvirus in the Australian native plant species Hardenbergia comptoniana .......................................................................................... 58 4.1 Introduction .................................................................................................................... 58 4.2 Method and materials .................................................................................................... 61 4.2.1 Plant Samples and Virus Isolates ........................................................................................... 61 4.2.2 Virus Inoculations................................................................................................................... 63 4.2.3 Enzyme Linked Immuno-Sorbent Assay (ELISA) .................................................................... 63 4.2.4 Incidence, host range and seed transmission studies.............................................................. 63 4.2.5 RT-PCR, Cloning and Sequencing.......................................................................................... 64 4.2.6 Phylogenetic Analysis of Isolates............................................................................................ 65

4.3 Results.............................................................................................................................. 67 4.3.1 Symptoms of infection ............................................................................................................. 67 4.3.2 Virus incidence ....................................................................................................................... 68 4.3.3 Alternative hosts of HarMV .................................................................................................... 68 4.3.4 Host range inoculations.......................................................................................................... 71 4.3.5 Aphid and seed transmission tests .......................................................................................... 71 4.3.6 Amplification of virus genome fragments ............................................................................... 72 Sequencing of coat protein genes and 3’ UTRs ....................................................................... 72 Amplification and sequencing of full length Nuclear Inclusion B gene................................. 73 4.3.7 Hardenbergia mosaic virus specific primers .......................................................................... 74 4.3.8 The CP genes – phylogenetic inferences ................................................................................ 76 4.3.9 Phylogenetic analysis of the 3’ UTR sequence ....................................................................... 85 4.3.10 Phylogeny of the HarMV NIb Gene ...................................................................................... 88 4.3.11 Evidence for recombination between HarMV isolates.......................................................... 91

4.4 Discussion ........................................................................................................................ 95 4.4.1 Phylogeny studies based on the CP sequence......................................................................... 95 Experimental host range and designation of virus strains ..................................................... 96 An Australian only subgroup to the BCMV group of Potyviruses ........................................ 97 4.4.2 Phylogeny of the 3’ untranslated region................................................................................. 98 4.4.3 Phylogeny of the NIb gene ...................................................................................................... 98 4.4.4 Determinants of virus symptoms and host range .................................................................... 99 4.4.5 Phylogeny of Potyviruses infecting Passiflora species ......................................................... 100 4.4.6 Recombination in Potyviruses .............................................................................................. 102 Recombination in MU-2A ....................................................................................................... 102 Recombination in other isolates.............................................................................................. 103 Detecting mixed infections ...................................................................................................... 104 Methods of detecting recombination ...................................................................................... 104 4.4.7 Surveys of Western Australia................................................................................................ 105 4.4.8 Surveys of Eastern Australia................................................................................................. 106 4.4.9 Full genome sequencing ....................................................................................................... 107 4.4.10 Conclusions ........................................................................................................................ 108

Chapter 5: Optimisation of microarray printing methods...................................................... 109 5.1 Introduction .................................................................................................................. 109 vii

TABLE OF CONTENTS 5.1.1 Creating arrays on PLL coated slides .................................................................................. 109 Printing buffer.......................................................................................................................... 109 DNA concentration .................................................................................................................. 110 Immobilisation of probes......................................................................................................... 110 Slide Blocking........................................................................................................................... 110 5.1.2 Pre-activated slide chemistries ............................................................................................. 111 5.1.3 Probe intensity and morphology........................................................................................... 111 5.1.4 Gene expression versus diagnostic arrays............................................................................ 112 5.1.5 Aims ...................................................................................................................................... 112

5.2 Methods and materials ................................................................................................. 113 5.2.1 Printing and immobilising PLL slides .................................................................................. 113 Immobilising probes on slides................................................................................................. 114 Blocking microarray slides...................................................................................................... 114 Slide scanning and data analysis............................................................................................. 114 5.2.2 Printing PowerMatrix slides................................................................................................. 115 5.2.3 Virus extraction, labelling and hybridisation ....................................................................... 116 Fluorescent labelling of PCR amplicons ................................................................................ 116 Purification and hybridisation................................................................................................ 116

5.3 Results............................................................................................................................ 117 5.3.1 Optimising PLL slides........................................................................................................... 117 Probe morphology.................................................................................................................... 117 DNA retention .......................................................................................................................... 117 5.3.2 Printing arrays on Power Matrix slides ............................................................................... 120 5.3.3 Detection and discrimination of Potyvirus species............................................................... 121 5.3.4 Probe design ......................................................................................................................... 123

5.4 Discussion ...................................................................................................................... 125 5.4.1 Arrays on PLL slides ............................................................................................................ 125 Printing buffer.......................................................................................................................... 125 Probe immobilisation............................................................................................................... 127 Probe concentration................................................................................................................. 127 5.4.2 Comparison of slide surfaces................................................................................................ 128 5.4.3 Virus identification by microarray........................................................................................ 129 Probe design ............................................................................................................................. 130 5.4.4 Conclusions .......................................................................................................................... 131

Chapter 6: Optimising methods of nucleic acid amplification for microarray-based virus detection.................................................................................................................................... 132 6.1 Introduction .................................................................................................................. 132 6.2 Materials and methods ................................................................................................. 135 6.2.1 Microarray slides.................................................................................................................. 135 6.2.2 Microarray hybridisation and scanning ............................................................................... 135 6.2.3 RNA extraction and precipitation ......................................................................................... 136 6.2.4 Gel Electrophoresis .............................................................................................................. 136 6.2.5 CyScribe Post-Labelling Kit (Indirect Labelling)................................................................. 136 6.2.6 Burton’s random amplification method ................................................................................ 137 6.2.7 Klenow based random amplification .................................................................................... 138 6.2.8 Genus-specific primer amplification..................................................................................... 139 6.2.9 Real Time PCR ..................................................................................................................... 140

6.3 Results............................................................................................................................ 142 6.3.1 Comparison of PVX and PVY virus titre............................................................................... 142 6.3.2 Burton’s amplification method ............................................................................................. 144 6.3.3 Amplification using Klenow labelling................................................................................... 147 6.3.4 Genus specific primer amplification..................................................................................... 148

6.4 Discussion ...................................................................................................................... 153 6.4.1 CyScript post labelling ......................................................................................................... 153 6.4.2 Burton amplification method ................................................................................................ 153 6.4.3 Klenow amplification............................................................................................................ 155 6.4.4 Genus specific primers ......................................................................................................... 155 6.4.5 A Multiplex polyvalent PCR ................................................................................................. 157

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TABLE OF CONTENTS 6.4.6 Other amplification approaches ........................................................................................... 158 6.4.7 Conclusions .......................................................................................................................... 159

Chapter 7: General discussion ................................................................................................ 162 7.1 Overview........................................................................................................................ 162 7.2 Characterisation of Hardenbergia mosaic virus........................................................ 162 7.3 Development of microarray based methods............................................................... 164 7.3.1 Cost effective methods for microarray slides........................................................................ 166 7.3.2 Strain specific microarrays................................................................................................... 166 7.3.3 Labelling methods for increased sensitivity.......................................................................... 167

7.4 Future work................................................................................................................... 170 7.5 Conclusions ................................................................................................................... 171 Appendix 1: .............................................................................................................................. 172 Appendix 2: .............................................................................................................................. 178 References ................................................................................................................................ 180

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PRESENTATIONS AND PUBLICATIONS

Presentations and Publications Some of the results presented in this thesis have been published and presented at scientific meetings.

Publications Chapter 1: Webster C.G., Wylie S.J. & Jones M.G.K (2004). “Diagnosis of plant virus pathogens.” Current Science 86: 1604-1607.

Chapter 4: Webster, C. G., Coutts, B. A., Jones, R. A. C., Jones, M. G. K. & Wylie, S. J. (2007). Virus impact at the interface of an ancient ecosystem and a recent agroecosystem: studies on three legume infecting potyviruses in the southwest Australian floristic region. Plant Pathology 56: 729-742.

Wylie, S. J., Webster, C. G., Coutts, B. A., Jones, M. G. K. & Jones, R. A. C. (2007). Hardenbergia mosaic virus, the first indigenous plant virus identified from the Southwest Australian Floristic Region. In ISHS International working group on legume and vegetable viruses: Annual newsletter 2006. Wageningen.

Oral Presentations Chapter 4: Australasian Plant Pathology Early Researchers Seminar Series, Perth, 19 October 2007. “An indigenous plant virus from the South West Australasian Floristic Region.”

Chapter 5: 6th Australasian Plant Virology Workshop (APVW), Gold Coast, Australia, 31 August – 2 September, 2004 “Towards highly parallel tests for plant virus diagnosis.”

Chapter 6: 7th Australian Plant Virology Workshop (APVW), Perth, Western Australia, 8-11 November, 2006. “Identification of unknown viral pathogens using microarrays.”

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PRESENTATIONS AND PUBLICATIONS

Poster Presentations Webster C. G., Coutts B. A., Jones R. A. C., Jones M. G. K., Wylie S. J. (2007) “The first indigenous plant virus identified from the South West Australian Floristic Region,” Proceedings of the CRC for National Plant Biosecurity Science Exchange, Melbourne, p26.

Webster C.G., Coutts B.A., Jones R.A.C., Jones M.G.K. & Wylie S.J. (2006). “The first indigenous plant virus identified from the South West Australian Floristic Region,” Proceedings of the 7th Australasian Plant Virology Workshop, Perth, p12.

Webster C.G., Wylie S.J., Chen G., Kenworthy W. & Jones M.G.K. (2005). “Towards highly parallel plant tests for plant virus diagnosis,” Proceedings of the AusBiotech 2005 National Biotechnology Conference, Perth, p20-State Winner of Student Excellence Award.

Webster C.G., Wylie S.J., Chen G., Kenworthy W. & Jones M.G.K. (2004). “Towards highly parallel plant tests for plant virus diagnosis,” Proceedings of the Combined ASBMB, ANZSCDB and ASPS Annual Meeting, Perth, p110.

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LIST OF ABBREVIATIONS

List of abbreviations

Viruses and viroids Virus and viroid species names are in italics if approved by the International Committee on Taxonomy of Viruses (ICTV) as listed in the 8th ICTV report (Fauquet et al., 2005) or ICTV database

(ICTVdB

-

The

Universal

Virus

Database,

version

4,

April

2006,

http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/). Unapproved or tentative names are given in Roman text.

APLV APMoV ApVY ApMV ArMV BanMMV BSV BaMMV BaYMV BCMNV BCMV BYMV BtMV BNYVV BBWV-2 BStMV CCFV CaLV CarVY CYBV CeMV CerMV ChiVMV CSMV CSVd CSFV ClVY CYVV ClYVV CSV CABMV CFMoMV CGMMV CMV CABYV CYMMV DsMV DCMV DiSMV DiVY EAPV EBV EVY FHV FMDV

Andean potato latent virus Andean potato mottle virus Apium virus Y Apple mosaic virus Arabis mosaic virus Banana mild mosaic virus Banana streak virus Barley mild mosaic virus Barley yellow mosaic virus Bean common mosaic necrosis virus Bean common mosaic virus Bean yellow mosaic virus Beet mosaic virus Beet necrotic yellow vein virus Broad bean wilt virus 2 Broome streak mosaic virus Cardamine chlorotic fleck virus Cardamine latent virus Carrot virus Y Cassia yellow blotch virus Celery mosaic virus Ceratobium mosaic virus Chilli veinal mottle virus Chloris striate mosaic virus Chrysanthemum stunt viroid Classical swine fever virus Clitoria virus Y Clitoria yellow vein virus Clover yellow vein virus Cocksfoot streak virus Cowpea aphid-borne mosaic virus Cucumber fruit mottle mosaic virus Cucumber green mottle mosaic virus Cucumber mosaic virus Cucurbit aphid-borne yellows virus Cymbidium mosaic virus Dasheen mosaic virus Dianella chlorotic mottle virus Digitara striate mosaic virus Diuris virus Y East Asian passiflora virus Epstein-Barr virus Eustrephus virus Y Florida hibiscus virus Foot-and-mouth disease virus

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LIST OF ABBREVIATIONS GFLV GFkV HarMV HBV HCV HiVY HAdV-A CV-A16 EV-71 HHV HIV HPV HPIV-3 HRSV HRV-A HRV-B HTLV INSV FLUVA JYMV JGMV KSHV KVY KYMV KGMMV LYSV LMV LMoV LASV MDMV MSSV NVMV OMV ONMV ORSV OYDV PLDMV PRSV PSMV PClV PCV PFVY PfMV PFRSV PaVY PCV PaMV PFMoV PFMTV PWV PEMV-1 PSbMV PMMoV PeMoV PStV PSV PepMV PepMoV PTV PlVY PPV PBRSV PLRV

Grapevine fanleaf virus Grapevine fleck virus Hardenbergia mosaic virus Hepatitis B virus Hepatitis C virus Hibbertia virus Y Human adenovirus-A Human coxsachievirus A-16 Human enterovirus 71 Human herpes virus Human immunodeficiency virus Human papillomavirus Human parainfluenza virus 3 Human respiratory syncytial virus Human rhinovirus A Human rhinovirus B Human T-cell lymphotrophic virus Impatiens necrotic spot virus Influenza A virus Japanese yam mosaic virus Johnsongrass mosaic virus Karposi's sarcoma-associated herpesvirus Kennedya virus Y Kennedya yellow mosaic virus Kyuri green mottle mosaic virus Leek yellow stripe virus Lettuce mosaic virus Lily mottle virus Lucerne Australian symptomless virus Maize dwarf mosaic virus Maize sterile stunt virus Nicotiana velutina mosaic virus Oat mosaic virus Oat necrotic mottle virus Odontoglossum ringspot virus Onion yellow dwarf virus Papaya leaf distortion mosaic virus Papaya ringspot virus Paspalum striate mosaic virus Passiflora chlorosis virus Passiflora crinkle virus Passiflora foetida virus Y Passiflora mosaic virus Passiflora ringspot virus Passiflora virus Y Passionfruit crinkle virus Passionfruit mosaic virus Passionfruit mottle virus Passion fruit mottle Thailand virus Passion fruit woodiness virus Pea enation mosaic virus-1 Pea seed-borne mosaic virus Pepper mild mottle virus Peanut mottle virus Peanut stripe virus Peanut stunt virus Pepino mosaic virus Pepper mottle virus Peru tomato mosaic virus Pleione virus Y Plum pox virus Potato black ringspot virus Potato leafroll virus

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LIST OF ABBREVIATIONS PMTV PSTVd PVA PVM PVS PVT PVV PVX PVY PYVV PDV PNRSV PtVY RhoVY RUBV RGMV SarVY ScaMV SARS-CoV S1VY S2VY SNMoV SrMV SAPV SMV SCMoV SCMV SCSMV SPFMV SPMMV TEV TMV TMSV TSV TVMV TYDV TAV TPMVd TSWV TYLCV TuMV VZV VTMoV WMV WSMV WYMV WSSV WPMV WVMV YMV ZGMMV ZYMV

Potato mop-top virus Potato spindle tuber viroid Potato virus A Potato virus M Potato virus S Potato virus T Potato virus V Potato virus X Potato virus Y Potato yellow vein virus Prune dwarf virus Prunus necrotic ringspot virus Pterostylis virus Y Rhopalanthe virus Y Rubella virus Ryegrass mosaic virus Sarcochilus virus Y Scallion mosaic virus Severe acute respiratory syndrome coronavirus Siratro 1 virus Y Siratro 2 virus Y Solanum nodiflorum mottle virus Sorghum mosaic virus South African passiflora virus Soybean mosaic virus Subterranean clover mottle virus Sugarcane mosaic virus Sugarcane streak mosaic virus Sweet potato feathery mottle virus Sweet potato mild mottle virus Tobacco etch virus Tobacco mosaic virus Tobacco mosaic satellite virus Tobacco streak virus Tobacco vein mottling virus Tobacco yellow dwarf virus Tomato aspermy virus Tomato planta macho viroid Tomato spotted wilt virus Tomato yellow leaf curl virus Turnip mosaic virus Varicella-zoster virus Velvet tobacco mottle virus Watermelon mosaic virus Wheat streak mosaic virus Wheat yellow mosaic virus White spot syndrome virus-1 Wild potato mosaic virus Wisteria vein mosaic virus Yam mosaic virus Zucchini green mottle mosaic virus Zucchini yellow mosaic virus

xiv

LIST OF ABBREVIATIONS

Other abbreviations used in the text o

C 3' 5' A. A. a. a. ACT ANGIS BHQ1 BLAST bp BSA CI cDNA CF-PCR COX CP CSL Ct CTAB C-terminus cv Da DAFWA DEPC DIG DMSO DNA dNTP ds DTT et al. EDTA ELISA EtOH FAM FMB fs cDNA h HCl HC-Pro IC-PCR ICTV ITS IVT JOE k kb KCl L LAMP LB medium LiCl LFD LNA µ µg µL µm M MAb

degrees Celsius hydroxyl-terminus of DNA molecule phosphate-terminus of DNA molecule amino acid amino allyl Australian Capital Territory Australian National Genome Information Service black hole quencher 1 basic local alignment search tool base pairs bovine serum albumin cytoplasmic inclusion complementary deoxyribonucleic acid competitive fluorescence PCR cytochrome oxidase coat (capsid) protein Central Science Laboratories, York, UK threshold cycle cetyl-trimethylammonium Bromide carboxy terminus cultivar Dalton Department of Agriculture and Food, Western Australia diethylpyrocarbonate digoxigenin dimethyl sulphoxide deoxyribose nucleic acid deoxyribose nuceltodie tri-phosphate (mix of A,C,G +T) double stranded dithiothreitol et alia, with others. ethylene diamine tetra-acetic acid enzyme-linked immunosorbent assay ethanol 5' carboxyfluorescein Full Moon Biosystems first strand complementary DNA hour hydrochloric acid helper component-protease immunocapture PCR International Committee on the Taxonomy of Viruses internal transcribed spaces in-vitro transcription 6-carboxy 4',5'-dichloro-2',7'-dimethoxy fluorescein label kilo kilobase potassium chloride litre loop-mediated isothermal amplification Luria-Bertani medium lithium chloride lateral flow device locked nucleic acid micro microgram microlitre micrometre molar monoclonal antibody

xv

LIST OF ABBREVIATIONS MALDI-TOF MEGA min mL mM mRNA MS n NaAc NaBH4 NaCl NaOH NaPO4 ng (ΝΗ4)2SO4 NIa NIb NSW nt oligo O/N pmol PAb PBS PCR pers. comm. Pfu PLL QCM Qld RDP RdRp RFLP RNA ROX rpm rPCR RT-PCR RTase, RT s ss SSC SDS SWAFR TAE buffer Taq TBIA TBE buffer TEM Tris UV vRNA WA WGA

matrix assisted laser desorption ionization time of flight molecular evolutionary genetics analysis minute mililitre millimolar messenger ribose nucleic acid mass spectrometry nano sodium acetate sodium borohydride sodium chloride sodium hydroxide sodium phosphate nanogram ammonium sulphate nuclear inclusion a nuclear inclusion b New South Wales nucleotide oligonucleotide overnight pico mole polyclonal antibody phosphate buffered saline polymerase chain reaction personal communication Pyrococcus furiosus DNA polymerase poly-L-lysine quartz crystal microbalance Queensland Recombination detection program RNA dependent RNA polymerase restriction fragment length polymorphism ribose nucleic acid 6-carboxy-X-rhodamine revolutions per minute random primed polymerase chain reaction reverse transcriptase polymerase chain reaction reverse transcriptase second single stranded sodium chloride/sodium citrate sodium dodecyl sulphate southwest Australian floristic region tris- acetic acid-EDTA electrophoresis buffer Thermus aquaticus DNA polymerase tissue blot immunoassay tris-boric acid-EDTA electrophoresis buffer transmission electron microscopy tris(hydroxymethyl)aminomethane ultra violet viral ribose nucleic acid Western Australia whole genome amplification

Code for degenerate oligonucleotides A C G I T U

Adenosine Cytosine Guanine Inosine Thymine Uracil

M R W S Y K

AC AG AT CG CT GT

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V H D B N

ACG ACT AGT CGT AGCT

ACKNOWLEDGEMENTS

Acknowledgements First I would like to sincerely thank my principle supervisor Dr. Steve Wylie for encouragement and furthering my interest in science and virology. I would like to thank you for always finding the time to give help or advice no matter how busy you were, for your patience and for making thorough corrections to this manuscript. All the support and guidance is much appreciated. Finally thanks for keeping me on track and motivated on the days when the end seemed such a long way off.

I would also like to thank Professor Mike Jones for giving me the opportunity to undertake a PhD and for his assistance and advice during the course of the project. Thank you also for the funding and for encouraging me to visit and work at CSL.

I also thank Dr. Roger Jones (DAWFA, Perth) for the useful advice on all aspects of research, particularly the advice and critical recommendations for manuscript preparation and on virus characterisation, and for providing samples and advice on virus collection. I would also like to thank Brenda Coutts (DAWFA) for carrying out the many of the host range inoculations of HarMV and preparation of some figures and Monica Kehoe (DAWFA) for the testing so many samples by ELISA during the project.

My sincere thanks go to Dr. Neil Boonham at CSL for giving me the opportunity to work with you and your group. The great advice and useful discussions on all aspects of developing microarrays are greatly appreciated. Thank you for sharing you knowledge and resources so freely. Thanks also for all the lifts to and from the gatehouse in the mornings. I would like to thank Jenny Tomlinson and Rachel Glover (CSL, UK) for the training in microarray methods and help with extractions, labelling and scanning.

I thank Violet Peeva (Lotteries State Microarray Facility, Perth) for protocols, information and advice on all the many different aspects of microarray research; Grace Chen for help with training and setting up of the microarray facilities both for printing and scanning of slides; Bill Kenworthy (Murdoch University) for sharing with me the basics of bioinformatics, for advice on how best to tackle phylogenetic reconstructions and on image adjustments and issues with scanning microarray slides.

The National Plant Biosecurity Cooperative Research Centre (NPBCRC) including Dr Kirsty Bayliss and Dr Simon McKirdy provided some financial assistance, training such as a writing

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ACKNOWLEDGEMENTS

workshop in Adelaide, and information-sharing opportunities, to help complete the thesis and funding for travel to CSL.

Barry Cayford deserves thanks for all the bizarre, and sometime scientific, conversations we’ve had over the last four years and for knowing when to say “stop complaining and just get on with it.” Past and present members of the Plant Biotechnology Research Group including John FosuNyarko, Kerry Ramsay, Linda Maccarone, Zhaohui Wang, Muhammad Saqib, and Sheila Mortimer-Jones for advice, protocols, support and friendship over the past four years. I also thank other friends and students from Murdoch including; Kylie Ireland, Kate Taylor and Monique Sakalidis for the lunches and coffee breaks.

Finally I would like to thank my family for their love, support and encouragement during this time. Without all your understanding and patience while I was completing this work I could not have achieved this.

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I: INTRODUCTION AND LITERATURE REVIEW

Chapter 1: Introduction and literature review

1.1 Introduction to viruses Viruses are obligate parasites that hijack host cellular machinery to replicate. "A virus is a set of one or more nucleic acid template molecules, normally encased in a protective coat or coats of protein or lipoprotein that is able to organize its own replication only within suitable host cells.

It can usually be horizontally transmitted between hosts.

Within such cells, virus

replication is (1) dependent on the host's protein synthesizing machinery, (2) organized from pools of the required materials rather than binary fission, (3) located at sites that are not separated from the host cell contents by a lipoprotein bilayer membrane, and (4) continually giving rise to variants through various kinds of change in the virus nucleic acid" (Hull 2002). Viruses lack the genetic information encoding the machinery essential to generate metabolic energy or for protein synthesis. As such, in older definitions of life, viruses are not alive. However, if the definition of an organism is broadened to “the unit element of a continuous lineage with an individual evolutionary history” (Luria et al., 1978) then viruses are alive. Viruses replicate and spread, often at considerable speed and their evolutionary history can be traced by comparing species. They can also be observed to change in response to environment, hosts etc.

Plant viruses are an important group of plant pathogens, which cause yield losses amongst economically important crops. Viruses use some of the cellular machinery of the host to replicate. Virus particles move between cells via plasmodesmata, then longer distances via the vasculature to cause infection throughout the host. In the process, cells are often weakened or die and this can result in plants with poor health and crops with lower yields and quality.

Plant viruses spread to other host plants via vectors, or by other methods such as transmission through seed, pollen, vegetative propagation and grafting. Important vectors include aphids, thrips, whitefly, nematodes and fungi, which are able to transmit virus particles between plants. The exact mechanism of transmission is complex and involves one or more virus proteins, receptors within the maxillae or gut of the vector and receptors within the host plant (Hull 2002).

The standard convention of italicising species names is followed through out this thesis; however where unapproved or tentative names are used they are given in Roman text. Approved species names can be found in the 8th report of the ICTV report (Fauquet et al., 2005) or ICTV online database (http://www.ncbi.nlm.hih.gov/ICTVdB/ICTVdB). 1


1.2 Genomic structure of plant viruses 1.2.1 Nucleic acid Viruses, like other living organisms, contain information encoded in nucleic acid sequences. The nucleic acid molecules may be either single-stranded (ss), double-stranded (ds), deoxyribose nucleic acid (DNA) or ribose nucleic acid (RNA), positive (+) sense or negative (-) sense. The sense of the molecule refers to whether a ribosome is able to act directly on the RNA molecule to transcribe protein (+) or whether the complimentary strand (-) must first be synthesised. In many plant virus species, the genome is typically single-stranded RNA of positive sense, encapsidated by a protein shell made of virus-encoded subunits. The particle may be rigid rod shaped such as with members of the genus Tobamovirus; flexuous rod shaped, such as with the Potyviridae and Potexviridae; or icosahedral/spherical shaped, such as the Bromoviridae. Other important families of plant viruses include the Geminiviridae which consist of ssDNA enclosed in an isometric protein shell. The genome size and type, along with particle morphology, host range and method of transmission are the main features used to classify plant viruses.

1.2.2 Genome size The genomes of plant viruses vary in size, but are usually smaller than those of animal viruses. The virus with the smallest known genome is that of Tobacco mosaic satellite virus (TMSV) which consists of a monocistronic ssRNA, coding for the coat protein, of about 1250 bp in length (Matthews 1991). The genome of the largest viruses found, the Phycodnaviridae, have genomes 180kbp to 560kbp in size with up to 375 genes coding for many different proteins (Van Etten et al., 2002). The genome may also consist of a single molecule of nucleic acid (monopartite) or several different segments (multipartite). Multipartite genomes usually require that all segments of the genome are present before the virus is said to be infectious (Walkey 1991). Where multipartite genomes occur the segments are usually packaged into individual particles.

1.2.3 Genome organization Virus genomes use the same codons as their host because the virus must use the cell’s ribosomal machinery to synthesize its proteins. Most plant virus genomes code for 4 to 10 proteins. The genome contains control and recognition sequences, which are usually in 5’ and 3’ non-coding regions, however they are sometimes found internally between open reading frames or in coding regions. The coding sequences are closely packed and coding regions may overlap. Leaky stop codons (e.g. as used by the genera Geminivirus and Sobemovirus) can also give rise to longer 2


read-through proteins where the ribosome continues to translate after the leaky stop codon giving longer versions of a protein. In some viruses, overlapping genes occur when they are located in different reading frames (e.g. as used be the genera Potexvirus and Sobemovirus) and coding regions are located on both the “+” and “-" strand.

1.2.4 Satellite viruses, satellite RNA’s and viroids Satellite viruses and satellite RNA’s are ssRNA molecules often found in purified virus preparations and can be considered as parasites of viruses. The host virus is often referred to as a helper virus. Satellite viruses encode their own coat protein, whereas satellite RNAs are packaged in a protein shell made from the coat protein of the helper virus. They are considered different from other viruses because they share no sequence homology with the helper virus. In contrast the sequence of a defective interfering RNA has homology with the virus sequence. Satellites are replicated in the cytoplasm of the host cell on their own RNA template and affect host symptoms ranging from attenuation to severe exacerbation of symptoms (Roossinck et al., 1992).

The replication of their RNA is dependent on a specific helper virus; however

replication of the satellite interferes to some degree with replication of the helper virus (Collmer & Howell 1992). For instance co-infection of Cucumber mosaic virus (CMV) infection in Nicotiana tabacum with CMV-Fsat shows low accumulation of CMV genomic RNA and attenuation of virus symptoms (Liao et al., 2007).

Viroids are separated from viruses and satellites because they replicate independently of any known associated plant virus and because they don’t encode for any proteins. There are structural similarities between some viroids (e.g. Tomato planta macho viroid, TPMVd) and transposons (Keifer et al., 1983).

They consist of a small unencapsidated circular RNA

molecule several hundred nucleotides long, which replicates via cellular components. There are no coding regions for polypeptides and they are transmitted mechanically between plants and cause several economically important diseases especially in citrus and potato (Hull 2002). For instance Potato spindle tuber viroid (PSTVd) which causes losses in Solanum tubersonum, S. melongena and Lycopersicon esculentum (Mackie et al., 2006). Complete sequences of 36 viroids are listed in the National Centre for Biotechnology Information (NCBI) database.

1.2.5 The Potyviridae The largest and one of the most economically important families of plant viruses is the Potyviridae, which consist of 111 species recognised by the ICTV and 86 possible species. This family is made up of six genera: Potyvirus, Rymovirus, Bymovirus, Ipomovirus, Macluravirus and Tritimovirus, of which five are monopartite, while those of the Bymovirus are bipartite. Assignment to a genus is based on nucleic acid homology, mode of transmission, aspects of 3


pathogenicity and cytopathology including inclusion body morphology and key host reaction differences (Fauquet et al., 2005). One of the main aims of this project is the characterisation of a newly found species of Potyvirus, so characteristics of viruses in this genus will be described in more detail.

Potyviruses have a single-stranded RNA molecule of positive-sense approximately 9.7kb in length,

although

this

varies

(http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/57010000.htm,

from accessed

9-12kb 22/5/07).

The

genome contains a single open reading frame that codes for a polyprotein, which is cleaved by virus-encoded proteases to give at least ten predicted mature proteins (Figure 1.1), although only eight have been isolated in vivo. This suggests that differential proteolytic cleavage plays a role in polyprotein processing (see Adams et al., (2005a) for a review of polyprotein cleavage in Potyvirus species). Viral RNA (vRNA) is bound covalently to a virus linked genome protein (VPg) at the 5' untranslated region (UTR) and contains a genome-encoded poly-adenylated tail downstream to the 3' UTR. Mature Potyvirus particles contain a single RNA molecule with approximately 2000 coat protein (CP) monomers to produce a particle of 720-850nm by 1112nm (Shukla et al., 1994) The genome also encodes a set of non-structural proteins including a RNA dependent RNA polymerase (Nuclear inclusion B, NIb gene) and suppressor of gene silencing (Helper component-protease, HC-Pro). For a more complete review of Potyvirus protein functions see Shukla et al., (1994) and Revers et al., (1999).

Figure 1.1: Genome organisation in the family Potyviridae. The upper diagram shows the ten predicted mature proteins produced by proteolytic cleavage of the polyprotein (arrows) in most members of the family. The two genomic RNAs of the Bymoviruses are shown below, aligned to show the homology between RNA1 and the 3’-section of the monopartite viruses (from Adams et al., 2005b).

4


Potyvirus species have been described in many plant species from around the (Shukla et al., 1994). In general, Potyviruses have narrow host ranges, not the broad host ranges of some viruses, such as Tomato spotted wilt virus (TSWV) and CMV, which infect over 1090 (Parrella et al., 2003) and 1000 (Roossinck et al., 2002 citing Edwardson & Christie 1991) species respectively. The exception to this is Turnip mosaic virus (TuMV) which has 318 recorded hosts in 43 dicot families (Walsh & Jenner 2002).

The Potyvirus coat protein gene The CP is responsible for encapsidating vRNA and plays an important role in short and long distance movement of virus particles (Shukla et al., 1994).

Its nucleic acid sequence is

commonly used to characterise Potyviruses (Adams et al., 2005b). The Potyvirus CP gene encodes 253-332 amino acids (Shukla et al., 1994) which are cleaved from the polyprotein at the 5' end from the NIb gene. The gene has the genome’s only translation stop codon, which is followed by the 3' UTR. Within the CP gene, there are three major domains: (i) a variable amino terminus (N-terminus), (ii) a conserved, trypsin-resistant ‘core’, and (iii) a variable carboxy terminus (C-terminus). Subunit folding leaves the N and C-terminals surface exposed where most virus-specific epitopes are located (Shukla et al., 1988b) (Figure 1.2).

The N-terminus of the coat protein is highly variable in length and sequence, and contains the amino acid (A.A.) triplet asp-ala-gly (the DAG or DAG-like motif), the presence and context of which allows aphid transmission (Lopez-Moya et al., 1999). A mutation in this motif can abolish aphid transmissibility (Wylie et al., 2002). The length of this region of the gene can vary considerably as seen in Shukla et al., (1988b) where N-terminus lengths varied from 30-67 amino acids, depending on the species.

The C-terminus is shorter in length (18-20A.A.)

(Shukla et al., 1988b) and no major conserved motifs have been identified.

The 'core' of the CP is resistant to trypsin digestion and constitutes the majority of the coat protein gene (216-218 A.A.). Conserved arginine residues may be involved in binding vRNA to CP subunits (Shukla et al., 1988b).

Classification of Potyvirus species Within the Potyvirus genus, the classification of newly described members has been challenging because of the large size of the group and significant variation seen in its members. Early efforts to classify them relied on detailed examination of host ranges, measurements of virus particles by electron microscopy, and antigenic properties such as serological differences.

5


Figure 1.2: Schematic drawing showing the linear CP subunit, the subunit folding pattern, and a cross section view through a Potato virus Y (PVY) particle. Modified from Shukla & Ward (1989).

However similar symptoms can be produced by distinct viruses and moderately different strains can produce diverse symptoms. Serology of Potyvirus species relies mainly on antibodies raised to epitopes located on the coat protein. The major immunological determinants are located in the surface exposed and highly variable N-terminal, while a core of conserved amino acids has produced broadly reactive and Potyvirus specific antibodies. Together these two regions are of most use, however several problems remain namely: (i) variable cross-reactivity, (ii) unexpected and inconsistent paired relationships and (iii) lack of cross-reaction between strains (Shukla et al., 1994).

Sequencing of virus nucleic acids has allowed more accurate classification of isolates into species based on nucleic acid homology and supported relationships deduced from serology and other methods. A study by Shukla & Ward, (1988) of coat protein sequences of 17 strains of eight Potyvirus species revealed a bi-modal distribution of sequence identities with species sharing 38 to 71% identity (mean 54%) and strains sharing 90-99% identity (mean 95%). Later, 6


this bi-modal distribution was confirmed in comparisons of 187 complete Potyvirus genomes (Adams et al., 2005b). In this study, pairings of more closely related species was seen, some of which represented incorrectly named species in the database. Potyvirus coat protein sequences are most commonly used in such comparative studies because of the relative ease of sequencing this region of the genome, and because of conserved motifs upstream (NIb gene) and downstream (polyA tail).

This has lead to the assigning of certain demarcation values below which a taxonomic species entity should exist. The 8th ICTV report (Fauquet et al., 2005) lists species demarcation values of 85% identity over full length sequences or 80% over complete CP sequences, however Adams et al., (2005b) suggests slightly less stringent values of ≥76% for full length CP sequences between species.

Analysis of sequence alignments (Figure 1.3), has shown significant groupings of more closely related species.

The three main groupings recognised in the genus Potyvirus are: (i) the

Sugarcane mosaic virus (SCMV) group infecting Gramineae (Shukla et al., 1992), (ii) the Potato virus Y (PVY) group infecting Solanaceae (Spetz et al., 2003; Spetz 2003) and (iii) the Bean common mosaic virus (BCMV) group mainly infecting Leguminosae (Ward & Shukla 1991).

Many Potyvirus species show lower levels of homology and do not fit into any

discernable groupings (Figure 1.3).

The BCMV group The virus identified in this thesis belongs to the BCMV group and this group will be looked at further. The group with its type member BCMV consists of mainly legume-infecting Potyvirus species. Membership of the group is based on sequence identity (Ward & Shukla 1991; Shukla et al., 1994; Berger et al., 1997; Adams et al., 2005b). Besides BCMV, members of the group for which full length sequences are available are: Zucchini yellow mosaic virus (ZYMV), Dasheen mosaic virus (DsMV), Soybean mosaic virus (SMV), Bean common mosaic necrosis virus (BCMNV) and Cowpea aphid-borne mosaic virus (CABMV) (Adams et al., 2005b). Shukla et al., (1994) also includes: Passion fruit woodiness virus (PWV), Peanut stripe virus (PStV) and South African Passiflora virus (SAPV) as members, although the latter two are now considered strains of BCMV and CABMV respectively (Fauquet et al., 2005).

7


SCMV Group

BCMV Group

PVY Group

Figure 1.3: Phylogenetic tree of the ORF nucleotide sequence (P3 to capsid protein) of members of the family Potyviridae with values at the forks indicating the number of times out of 100 trees that this grouping occurred after bootstrapping. Significant groupings of viruses are indicated. The scale bar shows the number of substitutions per base (Adams et al., 2005b). Virus Names: BaMMV, Barley mild mosaic virus; BaYMV, Barley yellow mosaic virus; BCMNV, Bean common mosaic necrosis virus; BCMV, Bean common mosaic virus; BYMV, Bean yellow mosaic virus; BtMV, Beet mosaic virus; BStMV, Broome streak mosaic virus; ChiVMV, Chilli veinal mottle virus; ClYVV, Clover yellow vein virus; CSV, Cocksfoot streak virus; CABMV, Cowpea aphid-borne mosaic virus; DsMV, Dasheen mosaic virus; JYMV, Japanese yam mosaic virus; JGMV, Johnsongrass mosaic virus; LYSV, Leek yellow stripe virus; LMoV, Lily mottle virus; LMV; Lettuce mosaic virus; MDMV, Maize dwarf mosaic virus; OMV, Oat mosaic virus; ONMV, Oat necrotic mottle virus; OYDV, Onion yellow dwarf virus; PLDMV, Papaya leaf distortion mosaic virus; PSRV, Papaya ringspot virus; PSbMV, Pea seed-borne mosaic virus; PeMoV, Peanut mottle virus; PepMoV, Pepper mottle virus; PTV, Peru tomato mosaic virus; PPV, Plum pox virus; PVA, Potato virus A; PVV, Potato virus V; PVY, Potato virus Y; RGMV, Ryegrass mosaic virus; ScaMV, Scallion mosaic virus; SrMV, Sorghum mosaic virus; SMV, Soybean mosaic virus; SCMV, Sugarcane mosaic virus; SPFMV, Sweet potato feathery mottle virus; SPMMV, Sweet potato mild mottle virus; TEV, Tobacco etch virus; TVMV, Tobacco vein mottling virus; TuMV, Turnip mosaic virus; WSMV, Wheat streak mosaic virus; WYMV, Wheat yellow mosaic virus; WPMV, Wild potato mosaic virus; YMV, Yam mosaic virus; ZYMV, Zucchini yellow mosaic virus.

8


1.3 Economic importance Viruses can infect most, if not all, economically important plants. A virus is economically important when the virus infection causes symptoms that reduce yield or quality of the crop. There are a number of symptoms of infection and these may include: localised lesions, stunting, mosaic patterns, yellowing disease, ring spots, necrosis, developmental abnormalities, wilting and reduced nodulation by Rhizobium (Matthews 1991). Importantly viruses do not always produce visible symptoms because leaves may escape infection because of age and/or position; while latent infections produce no outward signs of infection.

Crop production losses attributed to plant viruses can be enormous. There are few precise estimates of the losses that viruses have inflicted on crop plants but these losses can be as severe as total crop loss over wide areas. When such outbreaks occur the economic cost can run into millions of dollars. In the early 1990’s when Sweet potato feathery mottle virus (SPFMV) free sweet potato seed stock was introduced to 80% of the sweet potato growers of Shandong Province in China, the yields increased by 10t/ha or 30% with an increased value of $US145 million. It was estimated that sweet potato viruses cost the whole of China $US 1.5 billion per year (Fuglie et al., 1998). Further TSWV causes estimated annual global losses of US$ 1 billion per annum (Pappu 1997). Even where overall losses to not appear to be great, local areas may be severely affected when conditions are favourable to vectors. In Western Australia, Bean yellow mosaic virus (BYMV) is estimated to cause overall losses of 0.5 – 3.0% to the lupin crop annually, with a value of AUS$1 million to 6 million (R. Jones 2003 pers. comm.).

Apart from the losses inflicted by virus infection the preventative measures undertaken to control and limit virus outbreaks are often expensive. Costs include chemical sprays used to control insect vectors capable of spreading the virus between plants and across farms. Where a virus can spread via seeds testing is required to ensure seed stocks used to next season's crop are virus free. The costs of these tests can be expensive and often need to be repeated in successive seasons. The impact that plant viruses can have on the economy is large, so to limit the damage diagnostic methods that are quick, reliable and cheap are important so that plant virus spread can be minimised.

9


1.4 Diagnostic methods A wide variety of diagnostic methods have been developed to detect and diagnose viruses that infect plants. These range from the relatively simple methods of visual detection on indicator plants to more complex and costly methods of virus nucleic acid detection. Good diagnostic methods should be cheap, quick, accurate, sensitive and amenable to large sample numbers.

1.4.1 Visual detection and indicator hosts The infection of plants by viruses often produces visible symptoms, which can be used to detect and diagnose virus infection. Diagnosing plant virus disease using indicator plants involves infecting one or more species of indicator plant, for instance Nicotiana spp or Chenopodium quinoa. Indicator plants are those plants whose symptoms are known for many different virus species. They have been shown to have certain symptoms in response to a known species of virus. Nicotiana benthamiana is a particularly useful indicator species as it has been shown to be infected by 203 species of virus in 26 genera (Hovarth 1993). A diagnosis of the virus can be made by comparing the symptoms of the unknown virus in each indicator plant to the symptoms of known viruses in each of the indicator plants. The limitations of detection by symptomatology are that many different viruses can cause similar symptoms in a given indicator plant, and that different strains of a single virus may produce wide variation in the symptoms expressed.

Electron microscopy Transmission electron microscopy (TEM) has been used to visualise virions directly. The advantage of this method is that it is a generic method that can be used to detect many different viruses. Diagnosis to genus or family level can also often be made based on measurement of virus particles, most particularly with rod shaped virus particles, however diagnosis to species or strain requires further testing.

1.4.2 Serological procedures A number of serological methods for plant virus diagnostics have been developed which all rely on the ability of antibodies raised in animals to recognise antigenic determinants of plant viruses. Monoclonal antibodies (MAb) can also be developed if necessary but this is a complex and time consuming process. The highly specific nature of antibody-antigen binding makes these methods selective and coupled to colorimetric or chemiluminescent detection they can be very sensitive.

10


Enzyme-linked immunosorbent assay (ELISA) ELISA is not a new technique - it is widely used throughout the world because of its accuracy, simplicity and low cost. The technique utilizes the ability of antibodies to recognise proteins, usually the coat protein, of the virus of interest. Antibodies are fixed to the surface of a well within a microtiter plate, and a sap extract from the plant is added to the well. If the virus of interest is present it will bind to the antibodies fixed on the surface. A secondary antibody allows indirect detection of the virus because it has a reporter molecule attached to it, usually an enzyme that acts on a substrate that changes colour, (Clark & Adams 1977). ELISA can be quantitative as well as qualitative.

This method can be used to easily test multiple plants for a single virus using one well per plant sample, allowing rapid screening. Samples may also be grouped and tested with percentage incidence being estimated using the formula of Gibbs & Gower (1960). Group-specific antibodies are also useful as they can be used successfully to detect a number of species of virus. An important example of this is the Potyvirus specific antibody (Agdia Inc), which identifies the conserved core of the coat protein of many Potyvirus species (Shukla & Ward 1988).

The major constraint of ELISA is the requirement for polyclonal (PAb) or monoclonal antibody (MAb) sera specific for each virus of interest that do not cross-react with plant proteins. When novel or unexpected viruses are suspected in a sample, ELISA may not be suitable because of the requirement for appropriate antibodies.

A modification of ELISA named voltametric enzyme immunoassay, detects the change in electrical conductivity of the substrate, rather than a colour change, when acted upon by an enzyme attached to a secondary antibody. This method is claimed to be an order of magnitude more sensitive than ELISA and has been used to detect CMV (Sun et al., 2001). Other methods to improve sensitivity of enzyme immunoassays are given in Torrance (1998).

Tissue blot immunoassay (TBIA) Tissue blotting, like ELISA, utilizes antibodies raised against viruses. Sap from the plant tissue is expressed onto blotting paper, nitrocellulose or nylon membranes and the virus is detected by labelled antibodies, often using chemiluminescent detection. The procedure is less labour intensive than ELISA, rapid, sensitive, simple (no virus purification is required however samples must be homogenised to release virus particles from cells), inexpensive (minimal equipment is needed), suitable for surveys of 1000 to 2000 samples per day, and the samples can be taken in the field and processed some time later. Kits are available for a number of 11


viruses, notably from the International Centre for Agriculture Research in the Dry Areas, which offers kits for 19 viruses of legumes.

Lateral flow devices (LFDs) These small devices have been developed for plant pathogen detection (Danks & Barker 2000) and rely on a chromogenic change produced by antibody-antigen binding (Figure 1.4). Simple extractions and rapid results (as little as 2 minutes) make this suitable for field based testing (Mumford et al., 2006a) and LFD’s were recently evaluated for point of inspection testing of Phytophthora spp. (Lane et al., 2007). Tests are available for economically important plant viruses such as: Potato virus X, PVY, TSWV and Plum pox virus (PPV); and for bacterial and fungal pathogens such as the pathogens causing bacterial wilt, bacterial blight and Phytophthora spp from Agdia (www.agdia.com) and Pocket Diagnostics (www.pocketdiagnostics.com).

Figure 1.4: Outline of a Lateral Flow Device showing specific detection of Beet necrotic yellow vein virus (BNYVV). Figure taken from www.pocketdiagnostic.com

Quartz crystal microbalance (QCM) immunosensors In this novel technique for plant virus detection, a quartz crystal disk is coated with virusspecific antibodies. Voltage is applied across the disk, making the disk warp slightly because of the piezoelectric effect. Adsorption of virus particles to the crystal surface changes its resonance oscillation frequency in a concentration dependent manner. It is therefore qualitative and quantitative. The developers of the technique claim that it is as sensitive but more rapid than ELISA, and economical. In the first described use of QCM for plant viruses, as little as 1 ng of particles of Cymbidium mosaic virus (CYMMV) and Odontoglossum ringspot virus (ORSV) were detected in crude sap extracts (Eun et al., 2002). A more recent application can be found in Cooper & Singleton (2007), but there are no recent citations for detection of plant viruses.

12


Nanowire field effect transistors Nanowire field effect transistors are a novel diagnostic method. This has been used to detect groups of animal viruses, but not so far for plant virus detection. The method, described by Patolsky et al., (2004) uses nanowires conjugated with a MAb to detect an electrical conductance change resulting from the binding of a single virion. Its application was demonstrated in the parallel detection of Influenza A virus (FLUAV) and avian adenovirus group III. Figure 1.5 illustrates the principle of the technique.

Figure 1.5: Nanowire-based detection of single viruses. (Left) The schematic shows two nanowire devices, 1 and 2, where the nanowires are modified with different antibody receptors. Specific binding of a single virus to the receptors on nanowire 2 produces a conductance change (Right) characteristic of the surface charge of the virus only on nanowire 2. When the virus dissociates from the surface the conductance returns to the baseline value (Patolsky et al., 2004).

The authors speculate that larger nanowire arrays could be constructed (Jin et al., 2004), which could be used to screen for up to 100 viruses simultaneously (Patolsky et al., 2004). While PCR sensitivity is such that 3-11 virus particles can be detected (Pyra et al., 1994) the nanowire method is considerably quicker because no reaction and purification steps are required (Patolsky et al., 2004). A disadvantage may be that long flow times may be necessary to detect virus particles in very dilute sample volumes. Samples with higher virus concentrations would require shorter incubation times before virus-wire interactions are observed.

1.4.3 Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) MALDI-TOF MS relies on presuming the mass of ionized proteins from the time taken for them to reach the detector. The presence of proteins of certain masses, in this case the virus CP, can be determined and used to diagnose plant pathogens. For instance direct detection of virus CP 13


using MALDI-TOF MS was first used to detect Tobacco mosaic virus (TMV) in tobacco leaves where a peak corresponding to the known mass of the TMV CP was found (Thomas et al., 1998).

Later, two orchid infecting viruses were detected using MALDI-TOF and liquid

chromatography-MS (Tan et al., 2000). Their method was simple and amenable to processing multiple samples simultaneously.

The shortcoming of MALDI-TOF MS is that diagnosis is only possible when the mass of the coat protein is known (Padilya et al., 2006). Post transcriptional modifications may cause discrepancies between observed and theoretical masses (Srinivasan et al., 2002). Targets such as viroids which have no protein products, and low-abundance viruses, cannot be detected (Boonham et al., 2003).

1.4.4 Nucleic acid procedures Nucleic acid procedures detect plant vRNA or vDNA in a complex mixture of host and pathogen nucleic acids. These methods rely on the binding the binding of complementary DNA sequences in a double helix.

Dot Blots Positively charged membranes such as the Hybond-N+ membrane (Amersham Biosciences) can be used to immobilise pathogen nucleic acid. In this approach, termed a dot blot, nucleic acid from test plants is immobilised to the membrane and probed with a specific probe; usually amplified by PCR. The number of viruses tested for in each plant is limited, but many samples can be spotted in an array and this enables high throughput testing. This approach has been used to test orchid sap for CYMMV and ORSV (Hu & Wong 1998) and PSbMV in field pea (Ali et al., 1998) where picogram amounts of virus RNA were detected. That many samples can be tested is a clear advantage of this method, however if more than one virus is tested for the method cannot identify which of the viruses is present and further testing is required.

Reverse transcription-Polymerase Chain Reaction (RT-PCR) and PCR RT-PCR and PCR are widely used techniques for detection and identification of RNA and DNA plant viruses, respectively. These methods use short, specific oligonucleotide primers which bind to regions of complementarity. Thermostable DNA polymerases then synthesise a new complementary DNA strand from these primed sequences (Saiki et al., 1988). By cycling the temperature of the reaction completed strands can be disassociated, new primers annealed and extended. This cycling causes an exponential increase in the amount of template making it extremely sensitive. The procedures are also fairly inexpensive and require minimal skill to 14


carry out. In the case of RNA viruses, a DNA strand complementary (cDNA) to the virus is first made with reverse transcriptase (RT). For DNA viruses, this step is unnecessary.

PCR-based methods can be adapted to high-throughput applications (Walsh et al., 2001). PCR primers are designed to detect and distinguish closely related strains of a virus (Nemchinov & Hadidi 1998), and in addition, PCR amplicons can be sequenced to provide further data about strain types. The main drawback of the approach is that sequence information is required to design primers. With databases containing increasing numbers of virus sequences, this will be less of a problem over time. Careful primer design is crucial, whether the aim is to detect only a single strain, or all the members of a genus.

The sensitivity of PCR methods is the major advantage over serological methods such as ELISA. A RT-PCR assay of CMV in lupin grain was able to reliably detect one infected seed in one thousand healthy seeds (Wylie et al., 1993), and this test has been used in a conventional diagnostic service for WA farmers for 15 years. Detection of virus infections of Allium spp. also found a 102 to a 104 increase in sensitivity over an ELISA method (Dovas et al., 2001). High sensitivity can easily lead to false positive results from contamination, so adequate controls are essential.

There are a number of variations on the basic technique, designed to increase

sensitivity, alter specificity or allow automation of detection. The most common are listed below.

Multiplex RT-PCR Multiple species or strains are detected in a single reaction by combining oligonucleotide primers specific for different viruses. It is important that the amplicons are of different lengths and that there is no cross reactivity between them. This method was used to detect six citrus viroids and one virus (Ito et al., 2002) and three viruses of Gentian (Kuroda et al., 2002). In practice six to eight targets can be multiplexed (Ragozzino et al., 2004). Competing reactions and primer cross-reactivity have limited the development of assays for the simultaneous detections of more than eight targets. Alternatively, more PCR reactions can be combined after amplification, and multiplexed fragments detected in a capillary sequencer.

Nested PCR In this method, two PCRs are carried out sequentially; the first reaction increases the amount of template for the second. The method is particularly useful where the virus has very low titre or inhibitors of DNA polymerase are present in the plant extract. In the first PCR primers amplify a section of DNA and then an aliquot of the reaction is placed into a fresh tube for a second PCR.

In the second PCR primers that anneal within the first amplicon are used for 15


amplification. This at once increases the target molecule concentration and dilutes inhibitors. This method has been used successfully to detect members of Vitivirus and Foveavirus species in grapevines (Dovas & Katis 2003b).

Polyvalent PCR In this method the primers used are designed to hybridise to conserved regions of virus genomes and amplify a range of closely related species.

Amplification is then carried out and

amplification products be analysed on agarose gels to confirm amplification. Diagnosis to the species level can only be carried out using further sets of PCR primers, or cloning and sequencing of amplicons. Well known polyvalent primers are ‘Potyvirid’ primers of Gibbs & Mackenzie (1997) which are able to amplify a region of many, if not all the Potyviridae (Gibbs et al., 2003). Some other polyvalent primer sets available are listed in Table 1.1.

16


Table 1.1 Polyvalent primers sets available for plant viruses and viroids. Specificitya

Target regionb

Reference

Potyviridae family NIb or poly A Chen et al., 2001a Potyviridae family NIb or poly A Gibbs & Mackenzie, 1997 Potyvirus genus NIb or CP Hsu et al., 2005 Potyvirus genus NIb or CP Colinet et al., 1993 Potyvirus genus NIb or CP Langeveld et al., 1991 Potyvirus genus NIb or poly A Pappu et al., 1993 Potyvirus genus NIb or poly A van der Vlugt et al., 1999 Potyvirus genus HC-Pro Ha et al., 2007 Potyvirus genus CI Ha et al., 2007 Cucumovirus genus Upstream (intergeneic) or Choi et al., 1999 (CMV, PSV & TAV) downstream (3' UTR) of CP c CP + DNA B Chowda Reddy et al., 2005 Begomovirus genus Begomovirus genus Origin replication or AR1 gene Deng et al., 1994 Mastrevirus genus C2 Rybicki & Hughes 1990 Tobamovirus genus RdRp Dovas et al., 2004 Tobamovirus genus 3' NCR Letschert et al., 2002 (subgroup I) ORSV (Tobamovirus) & RdRp Seoh et al., 1998 CYMMV (Potexvirus) Potexvirus genus RdRp van der Vlught & Berendsen 2002 Closterovirus & HSP70 homolog Tian et al., 1996 Crinivirus genera Closteroviridae family HSP70 homolog Dovas & Katis, 2003a Vitivirus & Foveavirus RdRp Dovas & Katis, 2003b genera Vitivirus genus RdRp Saldarelli et al., 1998 Vitivirus & Trichovirus RdRp Saldarelli et al., 1998 genera Tricovirus, Capillovirus RdRp Foissac et al., 2005 & Foveavirus plus BanMMV Carmovirus, Dianthovirus RdRp Morozov et al., 1995 & Tombusvirus genera Tospovirus genus N gene or 3' NCR (S RNA) Okuda & Hanada, 2001 Luteovirus & Polerovirus N/A Robertson et al., 1991 genera Tymovirus and Marafivirus genera & RdRp Sabanadzovic et al., 2000 GFkV Badnavirus genus Reverse transcriptase Thompson et al., 1996 Ophiovirus genus RNA polymerase Vaira et al., 2003 Nepovirus genus (ArMV Movement protein gene Wetzel et al., 2002 & GFLV) Ilarvirus genus CP Candresse et al., 1998 (PNRSV & ApMV) Ilarvirus genus (PNRSV, 3' NCR or CP Saade et al., 2000 PDV & ApMV) Pospiviriod Central conserved region Bostan et al., 2004 Note: CI, Cytoplasmic inclusion; HC-Pro, Helper component-protease; CP, capsid protein; HSP70, heat shock protein 70kDa; 3' UTR, 3' untranslated region; NIb, Nuclear inclusion b; poly A, polyadenylated acid tail; S RNA, small RNA. Virus Abbreviations: ApMV, Apple mosaic virus; ArMV, Arabis mosaic virus; BanMMV, Banana mild mosaic virus; CMV, Cucumber mosaic virus; CYMMV, Cymbidium mosaic virus; GFkV, Grapevine fleck virus; GFLV, Grapevine fanleaf virus; ORSV, Odontoglossum ringspot virus; PDV, Prune dwarf virus; PNRSV, Prunus necrotic ringspot virus; PSV, Peanut stunt virus; TAV, Tomato aspermy virus; TSWV, Tomato spotted wilt virus. a The specificity is given as presented in the original publication. Since complete verification of the specificity was rarely performed, the specificity should be considered as only an indication. b An indication of the genome regions targeted by the polyvalent primers. Table is modified from James et al., 2006.

17


Immunocapture PCR (IC-PCR) IC-PCR combines capture of virus particles by antibodies with amplification by PCR. In this method, the virus is adsorbed by the antibody to a surface, then removed by heating in the presence of a non-ionic surfactant such as Triton X-100. The nucleic acids are then amplified by PCR. This method is especially useful in concentrating virus particles from plant species where virus titre is low, or where compounds that inhibit PCR are present, for example plum tree sap containing PPV (Wetzel et al., 1992) and Sugarcane streak mosaic virus (SCSMV) (Hema et al., 2003). A comparison of IC-PCR to other detection methods including TBIA, ELISA and Dot-blot immunoassay for the detection of Florida hibiscus virus (FHV) found it the most sensitive of the methods tested, able to detect as little as 500pg/mL of virus or 16 to 32 fold more, than DAS-ELISA (Kamenova & Adkins 2004). Sharman et al., (2000) also found IC-PCR up to 625 times more sensitive than ELISA for multiplexed detection of four viruses of banana. It has also been used to detect episomal Banana streak virus (BSV), parts of whose genome is present within the banana genome, and therefore increasing the chance of false positives from standard PCR tests (Harper et al., 1999).

Fluorescence Real Time-PCR using Taqman technology Two primers flank the sequence of interest and a third fluorescently labelled oligonucleotide anneals between them. As the flanking primers extend, the labelled primer is digested separating the dye and quencher molecules which leas to increased fluorescence (Figure 1.6). The advantages of this method are that no post-reaction processing is required to detect the reaction product (e.g. gel electrophoresis), and it is quantitative. Large scale implementation of this technology has been hampered by high costs but recent innovations have reduced the cost of both the required hardware (e.g. an ABI Prism 7900 Sequence Detection System) and the labelled primers.

Figure 1.6: TaqMan chemistry. Step 1: Intact probe anneals to a target sequence internal to the PCR primers; fluorescence emitted by the reporter is absorbed by the quencher. Step 2: during amplification, the probe is cleaved by the 5’-3’ nuclease activity of Taq, separating the dyes resulting in an increase in fluorescence (Mumford et al., 2006a).

18


A Taqman assay designed to detect PSTVd, a quarantine pathogen in Europe, was 1000 times more sensitive than a chemiluminescent dot-blot assay (Boonham et al., 2004) and as little as 10-20fg of PPV has been detected by real time PCR (Schneider et al., 2004). TaqMan real time PCR assays have also used to detect TSWV both in plant samples (Roberts et al., 2000) and its insect vector, Frankliniella occidentalis, (Boonham et al., 2002). Fluorescence PCR has been multiplexed to simultaneously detect two orchid viruses (Eun et al., 2000), and four potato viruses (Agindotan et al., 2007). Further examples of real time PCR systems, including field based methods can be found in Mumford et al., (2006b) and Tomlinson et al., (2005).

Many other real time PCR systems are available, e.g. Nucleic Acid Sequence Based Assay (Leone et al., 1997; Olmos et al., 2007) and molecular probes (Eun et al., 2000; Klerks et al., 2001; Goncalves et al., 2002) (see reviews by Bustin 2002; Mackay et al., 2002; James et al., 2006).

Competitive fluorescence PCR (CF-PCR) CF-PCR is a variation on the above technique. It is used for simultaneous differentiation between virus strains and multiple virus infections. Several primer sets, each labelled with a different fluorescent marker, are added to the reaction mixture. Virus strains are differentiated with primers that differ only at the 3’ end, complementary to a nucleotide position that is polymorphic between strains. Extension occurs only where the 3’ nucleotide is complementary. Only primers that generate amplicons fluoresce and the wavelength emitted identifies the primers that have been extended. Multiple strains of PVY were differentiated using this method (Walsh et al., 2001).

Restriction fragment length polymorphism (RFLP) RFLP is a method that can be used to differentiate isolates of viruses without the expense of cloning and sequencing. Viral nucleic acid is amplified by PCR and digested with restriction enzymes. RFLP relies on polymorphisms within restriction enzyme recognition sites which occur between virus species and strains. Digested PCR products are analysed by agarose electrophoresis. Different sized DNA fragments are produced and are compared to known samples to identify them. RFLP was used with RT-PCR to show that only CMV subgroup II was present in Western Australian lupin crops (Wylie et al., 1993).

Rolling circle amplification (RCA) The DNA polymerase of Bacillus subtilis bacteriophage Phi29 (Φ29) (Blanco et al., 1989) is used to amplify virus nucleic acids in a highly efficient amplification reaction (Dean et al., 19


2001). The method relies on the strand displacement activity of Φ29 polymerase which allows amplification to occur on newly synthesized template strands without having to cycle temperatures (isothermal amplification) (Figure 1.7). The authors also indicate the potential for nano- and Circovirus detection.

Figure 1.7: Scheme for multiply-primed rolling circle amplification. Oligonucleotide primers complementary to the amplification target circle are hybridized to the circle. The 3’ ends of the DNA strands are indicated by arrowheads to show the polarity of polymerization. Thickened lines indicate the location of the original primer sequences within the product strands. The addition of DNA polymerase and deoxynucleoside triphosphates (dNTPs) to the primed circle results in the extension of each primer, and displacement of each newly synthesized strand results from elongation of the primer behind it. Secondary priming events can occur subsequently on the displaced product strands of the initial rolling circle amplification step. It is possible that the last intermediate shown in the figure makes only a small contribution to the overall yield of amplified DNA (Dean et al., 2001).

The disadvantages of this method are that non-specific amplification products are produced in the reaction which requires further methods to identify infecting viruses to the species or strain level such as RFLP or sequencing. Secondly, long incubation times are required (18-20hrs) for amplification (Haible et al., 2006).

Loop mediated isothermal amplification (LAMP) The LAMP assay is another isothermal amplification assay which uses the high strand displacement activity of Bacillus stearothermophilus DNA polymerase and a set of four primers (Notomi et al., 2000). The primers are designed to produce stem-loop structures, which allow strand displacement and isothermal amplification by the B. stearothermophilus polymerase from a few copies to 109 copies in less than an hour (Notomi et al., 2000).

The isothermal

amplification also means that field testing applications are an option using this method.

This method has been applied to detect virus pathogens in infected plant material (Fukuta et al., 2003a; Fukuta et al., 2003b; Fukuta et al., 2004; Nie 2005) and vectors (Bemisia tabaci carrying Tomato yellow leaf curl virus (TYLCV)) (Fukuta et al., 2003b). This reaction is highly specific because four primers bind to six virus regions, and sensitive because of the highly efficient amplification.

The detection of amplification by pyrophosphate ion precipitation causing

visible turbidity means gel electrophoresis is unnecessary saving further time. IC-LAMP has 20


also been developed negating the need for extracting and purifying RNA (Fukuta et al., 2004). These advantages make LAMP a good method for virus diagnostics.

Macroarrays Positively charged membranes can also be used to immobilise cDNA or oligonucleotide probes. The typical size of spots of immobilised probes is much larger than that of microarrays (Section 1.5). An RNA extract from infected tissue is labelled and hybridised to the membrane to detect infecting viruses. An array of many virus specific probes can be developed which allows parallel detection. Arrays for Orthopoxvirus and Alphavirus detection (Fitzgibbon & Sagripanti 2006), plant fungal pathogens (Lievens 2006) and plant viruses (Agindotan & Parry 2007) have been developed using oligonucleotide probes. Hsu et al., (2005) also developed a cDNA array for 12 species of Potyvirus using degenerate primers for probe amplification and labelling. These methods allowed simple identification of multiple plant viruses in a single sample.

1.4.5 Advantages and disadvantages of current methods Of the methods described above, many are amenable to high-throughput processing of samples (ELISA, TBIA, PCR, dot blots and real time PCR) and can be used to process hundreds to thousands of samples per day. This allows rapid screening of many samples for the presence of a virus, but usually only one target virus at a time can be screened for. Novel or unexpected variants which are present will not normally be detected by these high-throughput methods.

Methods such as electron microscopy, multiplex or polyvalent PCR and macroarrays can be used to detect many more viruses in a single test, but all have their own drawbacks. Multiplex PCR is limited to detecting several targets in a single reaction, while electron microscopy and polyvalent PCR requires further tests to identify at the strain or species level. Macroarrays can be used to test for more viruses at a time but the large size of immobilised probes makes highly parallel detection difficult because larger sizes of membranes are needed with a requirement for pure labelled nucleic acid to ensure adequate hybridisation volumes.

Microarrays have been proposed as a 'multi-target' system capable of testing a full range of organisms in a generic format. This approach would streamline current diagnostic testing methods (Boonham et al., 2003) and improve detection of unknown or unexpected variants. As one of the major aims of the research in this thesis, the procedures and diagnostic applications of microarray methods are presented in detail below.

21


1.5 Microarrays Arrays, both microarrays and macroarrays, are a tool of choice for determining relative changes in expression levels of mRNAs. Microarrays were first used for determining changes in gene expression levels in Arabidopsis thaliana (Schena et al., 1995). Further developments have lead to arrays of the entire human transcriptome and other full genome arrays, allowing global gene expression studies. Full genome arrays are available from several vendors including: Affymetrix (ww.affymetrix.com) and Agilent (www.agilent.com). Microarrays have also been used for single nucleotide polymorphisms typing and host pathogen interactions (Kato-Maeda et al., 2001; Lareu et al., 2003).

Microarrays consist of probes fixed in an ordered array of discrete spots on a surface of glass, membrane or polymer (Hedge et al., 2000). Probes are arranged in high density with spot sizes typically smaller than 150 microns. They differ from macroarrays which have larger spot sizes (greater than 300 microns), which are printed on nylon membrane filters and typically have a smaller number of probes immobilised. A typical microarray slide can hold up to 30,000 spots, printed by high speed robotic workstations that deliver small volumes of probe solutions to chemically derivitised slides to produce a regular array of probes. Short length oligonucleotide probes can also be synthesised in-situ on the slide surface, most commonly by photolithography and combinatorial chemistry (www.affymetrix.com).

Hybridised to the probes is a fluorescently labelled solution of nucleic acids called the target solution, which in gene expression work is typically cDNA derived from total or purified messenger RNA. For diagnostic applications both DNA and RNA extracts may be labelled and hybridised. During reverse transcription fluorescently labelled nucleotides are incorporated in cDNA (direct labelling) or alternatively amino allyl (a.a.) labelled nucleotides can be incorporated during reverse transcription and fluorescent moieties coupled subsequently (indirect labelling). Unincorporated nucleotides, salts and enzyme are removed and the target solutions hybridised to the slide (typically overnight). See Figure 1.8 for an overview of the process.

22


RNA Extraction Cell Type A

RNA Extraction Cell Type B

Reverse Transcribe in presence

Cy5

Cy3

of Fluorescent Nucleotides

Hybridise to slide and wash to remove

Sli de

non-specific Binding.

00 00

Colour and intensity reveals level of gene expression

Slide scanned with laser

Sli

Sli

de

de

to reveal hybridisation

00

00

00

00

Figure 1.8: Overview of labelling, hybridisation and scanning process for microarray gene expression studies. RNA from two different sources is fluorescently labelled, most commonly during reverse transcription, and hybridised to a microarray slide. Following washing to remove non-specific hybridisation the slide is scanned. The intensity of fluorescence indicates the level of gene expression and this can be compared between the two samples.

Non-hybridised nucleic acids are removed from the slide surface by washing in a series of increasing stringency wash buffers, typically with decreasing concentrations of sodium chloride/sodium citrate (SSC) and sodium dodecyl sulphate (SDS). Slides are scanned to visualise hybridisation in a laser scanner at a wavelength appropriate for the fluorescent label. The intensity of fluorescence for each probe indicates the concentration of that particular nucleic acid species in the original sample. By comparing the level of expression in two tissue samples under different conditions relative changes in expression can be determined.

1.5.1 Diagnostic applications of microarrays The strength of microarrays for gene expression studies lies in their highly parallel nature, which allows an expression profile of thousands of genes to be made from a single sample (Schena et al., 1996). This advantage may be useful in cancer diagnostics where tumours that appear similar morphologically differ at the molecular level, which can impact on disease progression and prognosis. Gene expression microarrays have been proposed as a method for differentiating cancer types for example mesothelioma (Gordon et al., 2003) and breast cancer (Korkola et al., 2003; Yim et al., 2005; Glas et al., 2006). For a review of cancer diagnostics by microarrays see Schmidt & Begley (2003).

23


The highly parallel nature of microarrays is also applicable to diagnostic studies where a single sample can be tested for many pathogens at once, e.g. bacteria, fungi and viruses. Pathogen diagnostic microarrays ask whether a DNA sequence representative of a specific pathogen species is present or absent and quantification is unnecessary.

Microarrays have been used to diagnose fungal, bacterial and virus pathogens at the species and strain/pathotype level (Table 1.2). Diagnosis of plant virus pathogens has also been achieved by microarray (Table 1.2), but the majority report detection of human pathogens such as Escherichia coli, Bacillus anthracis, Human herpes virus spp. (HHV), Human papillomavirus virus (HPV) and Human immunodeficiency virus (HIV).

Microbial diagnostic arrays typically rely on multiplex or polyvalent PCR for amplification followed by hybridisation to an array to detect specific sequences. For reviews on microarrays in microbial diagnostics see Bodrossy & Sessitsch (2004); Call et al., (2003) and Ye et al., (2001).

In general for bacteria species-specific probes are chosen within the 16S rRNA

(ribosomal RNA) or Internal Transcribed Spacer (ITS) and a single set of primers are used to amplify this region e.g. Burton et al., (2005) and Fukushima et al., (2003). Detection of the nematode Meloidogyne chitwoodi (François et al., 2006) and phytoplasma spp. (Nicolaisen et al., 2007) was carried out in a similar way. Detection of plant viruses (e.g. Boonham et al., 2003) and animal viruses (e.g. Wang et al., 2002) has relied on alternative methods of labelling such as incorporation of labelled nucleotides during reverse transcription or random amplification. See Striebel et al., (2003) for a review of virus diagnostics by microarray.

The majority of published diagnostic microarrays (Table 1.2) have used DNA arrays (69 studies), and a minority have used protein/antibody arrays (three studies). One group report an array with both DNA and protein probes (Perrin et al., 2003). Probes for plant virus diagnostics have tended towards oligonucleotide probes because of the laborious preparation of PCR products as probes for cDNA arrays (Bystricka et al., 2005) and the improved selectivity of oligonucleotide probes to discriminate closely related isolates (Webster et al., 2004; Deyong et al., 2005). Arrays of plant viruses have also tended towards crop specific chips such as potato pathogen arrays (Boonham et al., 2003; Abdullahi et al., 2005; Bystricka et al., 2005) or a cucurbit virus array (Lee et al., 2003).

24


Table 1.2: Pathogens diagnosed by microarray-based methods. Species diagnosed A APLV, APMV, PBRSV, PSTVd, PVA, PVS, PVT, PVV, PVX, PVY, PYVV, Synchytrium endobiotichum, TSV 8 Phytophthora spp. Escherichia coli, Bacillus subtilis, B. thuringiensis FMDV E. coli PVA, PVSA, PVSO, PVX, PVY Chlamydia spp. & Chlamydophila spp. eight serotypes of human astroviruses

Microarray Probe

Reference

180 50mer oligonucleotide probes

Abdullahi et al., 2005

38 ITS1 probes 16S oligonucleotide probes 155 oligonucleotide probes 105 cDNA probes 5 cDNA probes 28 16S rRNA oligonucleotide probes 22 17mer oligonucleotide probes

B. anthracis, Enterobacteriacea, Mycobacterium, Streptococcus, Neisseria and Pseudomonas spp. PLRV, PMTV, PVA, PVS, PVX, PVY PLRV, PVA, PVMO, PVMI, PVSA, PVSO, PVX, PVYO, PVYNTN EV-71 & CV-A16

8 cDNA probes 29 40mer oligonucleotide probes 8 oligonucleotide probes

E. coli, Shigella spp. & Salmonella spp.

oligonucleotide probes

Human group A rotaviruses

50 oligonucleotide probes

E. coli, Staphylococcus aureas & Pseudomonas aeruginosa

120 cDNA probes

16S oligonucleotide probes

Influenza B Viruses

68,000 oligonucleotide probes 36 oligonucleotide probes

B. globigii & bacterial toxin proteins

4 capture antibodies

CSFV & Pestiviruses

8 oligonucleotide probes 5 24mer oligonucleotide probes

33 Staphylococcus spp.

CMV HHV-1, HHV-2, VZV (HHV-3), EBV (HHV4), Human cytomegalovirus (HHV-5) & HHV-6 Staphylococcus aureus Meloidogyne chitwoodi 14 Mycobacterium spp.

oligonucleotide probes 282 16S oligonucleotide probes oligonucleotide probes gyrB oligonucleotide probes

HPV spp. HIV-1, HTLV-I&II, HHV-6A&B, EBV (HHV4), KSHV (HHV-8), & HCV 19 HPV types 10 Mycobacterium spp. Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae, Aeromonas salmonicida, & Vibro parahaemolyticus


Anderson et al., 2006 Bavykin et al., 2001 Baxi et al., 2006 Bekal et al., 2003 Boonham et al., 2003 Borel et al., 2008 Brown et al., 2007 Burton et al., 2005 Bystricka et al., 2003 Bystricka et al., 2005 Chen et al., 2006 Chizhikov et al., 2001 Chizhikov et al., 2002 Cleven et al., 2006 Couzinet et al., 2005 Dankbar et al., 2007 Delehanty & Ligler, 2002 Deregt et al., 2006 Deyong et al., 2005 Foldes-Papp et al., 2004 Francois et al., 2003 Francois et al., 2006 Fukushima et al., 2003 Gharizadeh et al., 2003

264 cDNA probes

Ghedin et al., 2004

oligonucleotide probes oligonucleotide probes

Gheit et al., 2006 Gingeras et al., 1998


Gonzalez et al., 2004

Norovirus & Astrovirus spp.


Campylobacter jejuni & Campylocater coli WSSV typing of HPV lesions typing of HPV lesions Didymella bryoniae & Botrytis cinerea Salmonella enterica

5 oligonucleotide probes 3000 cDNA probes 22 oligonucleotide probes oligonucleotide probes 3 oligonucleotide probes 5 oligonucleotide probes

25

Jääskeläinen & Maunula, 2006 Keramas et al., 2003 Khadijah et al., 2003 Kim et al., 2003 Klaassen et al., 2004 Koch et al., 2005 Kostic et al., 2005


Table 1.2: Continued Species diagnosedA

Microarray Probe

Reference

10 genera of pathogenic bacteria

35 gyrB oligonucleotide probes

Kostic et al., 2007


Laassri et al., 2003


Lapa et al., 2002

9 cDNA probes

Lee et al., 2003

Variola, monkeypox, cowpox, vaccinia viruses and VZV Orthopox virus spp. CFMMV, CGMMV, CMV, KGMMV, PVX, TMV, PMMoV, ZGMMV, ZYMV

SARS-CoV

60 16S & 23S rRNA oligonucleotide probes 83 cDNA probes 27 cDNA probes 13 ITS oligonucleotide probes 100 ITS I or II oligonucleotide probes oligonucleotide probes cDNA probes 000’s oligonucleotide probes 18 oligonucleotide probes

8 Human adenovirus serotypes


Human group A rotavirus’s

33 oligonucleotide probes 132 16S oligonucleotide probes

19 Enterococcus spp. EBV (HHV-4) Human influenza virus A & B Fusarium oxysporum, Verticillum albo-atrum & Verticillum dahliae Fusarium oxysporum, Verticillium dahliae, Phytophthora nicotinae & Pythium ultimum Adenovirus & Influenza virus spp. virulence typing of E. coli typing Influenza A virus

41 spp. sulphate reducing prokaryotes typing influenza viruses


Toxoplasma gondii, RUBV, Human cytomegalovirus (HHV-5), and HHV-1 & -2

capture antibodies 21-33nt 16S oligonucleotide probes 57 ITS2 rRNA oligonucleotide probes

11 phytoplamsa disease groups 9 Fusarium spp.

6 Hantavirus spp. HIV-1, HBV, HCV six strains of PPV 30+ spp. human retrovirus Influenza virus spp. Staphylococcus aureus entertoxin genes genotyping VZV EV-71 Geobacter chapellei & Desulfovibrio desulfuricans Methylococcaceae spp. & Methylocystaceae spp. HHV -1, HHV-2, VZV (HHV-3), EBV (HHV4), HHV-5, and HHV-6. Influenza A virus Mycobacterium spp.& Staphylococcus spp. differentiation and antibiotic susceptibility testing 6 Listeria spp. E. coli

319 cDNA probes

Lehner et a., 2005 Li et al., 2006 Li et al., 2001 Lievens et al., 2003 Lievens et al., 2006 Lin et al., 2006 Ljperen et al., 2002 Lodes et al., 2006 Long et al., 2004 Lopez-Campos et al., 2007 Lovmar et al., 2003 Loy et al., 2002 Mehlmann et al., 2006 Mezzasoma et al., 2002 Nicolaisen & Bertaccini 2007 Nicolaisen et al., 2005

Nordstrom et al., 2004

oligonucleotide & antibody probes 19 70mer oligonucleotide probes 79 oligonucleotides 476 oligonucleotide probes 84 oligonucleotide probes 10 oligonucleotide probes 10 oligonucleotide probes 12 16S oligonucleotide probes

Sengupta et al., 2003


Stralis-Pavese et al., 2004


Striebel et al., 2004


Townsend et al., 2006

000’s oligonucleotide probes

Vernet et al., 2004

60 oligonucleotide probes 14 oligonucleotide probes

Volokhov et al., 2002 Vora et al., 2004

26

Perrin et al., 2003 Pasquini et al., 2007 Seifarth et al., 2003

Sergeev et al., 2004 Sergeev et al., 2006 Shih et al., 2003 Small et al., 2001


Table 1.2: Continued Species diagnosedA

Microarray Probe

Reference

respiratory tract viruses including: HRSV, HPIV-3, HAdV-12 (HadV-A), HRV-1b, -2, -21, -62 & -65 (HRV-A), & HRV -14 & -72 (HRV-B)

~1600 oligonucleotide probes

Wang et al., 2002

18 16S oligonucleotide Warsen et al., 2004 probes typing Group B Streptococcus agalacieae 35 oligonucleotide probes Wen et al., 2006 53660 oligonucleotide 11 prokaryotes, 2 eukaryotes and 5 virus spp. Wilson et al., 2002 probes HCV 4 HCV antigens Yuk et al., 2004 A: Virus names: APLV, Andean potato latent virus; APMoV, Andean potato mottle virus; CFMoMV, Cucumber fruit mottle mosaic virus; CGMMV, Cucumber green mottle mosaic virus; CMV, Cucumber mosaic virus; CSFV, Classical swine fever virus; CV-A16, Human coxsachievirus A16; EBV, EpsteinBar virus; EV-71, Human enterovirus 71; FMDV, Foot-and-mouth disease virus; HAdV-12, Human adenovirus-12; HAdV-A, Human adenovirus-A; HBV, Hepatitis B virus; HCV, Hepatitis C virus; HHV1-8, Human herpes virus1-8; HIV-1, Human immunodeficiency virus-1; HPIV-3, Human parainfluenza virus 3; HPV, Human papillomavirus; HRSV, Human respiratory syncytial virus; HRV-A, Human rhinovirus A; HRV-B, Human rhinovirus B; HTLV, Human T-lymphotrophic virus I&II; KGMMV, Kyuri green mottle mosaic virus; KSHV, Karposi's sarcoma-associated herpesvirus; PPV, Plum pox virus; PBRSV, Potato black ringspot virus; PLRV, Potato leaf roll virus; PMMoV, Pepper mild mottle virus; PMTV, Potato mop-top virus; PSTVd, Potato spindle tuber viroid; PVA, Potato virus A; PVM, Potato virus M; PVS, Potato virus S; PVT, Potato virus T; PVV, Potato virus V; PVX, Potato virus X; PVY, Potato virus Y; PYVV, Potato yellow vein virus; RUBV, Rubella virus; SARS-CoV, Severe acute respiratory syndrome coronavirus; TMV, Tobacco mosaic virus; TSV, Tobacco streak virus; VZV, Varicella-zoster virus; WSSV-1, White spot syndrome virus-1; ZGMMV, Zucchini green mottle mosaic virus; ZYMV, Zucchini yellow mosaic virus. 15 bacterial fish pathogens

1.5.2 Probes for microarrays Design of probes is of crucial importance in constructing high quality arrays. Probe design determines the amount of information that an array provides and for diagnostic applications the probes should be specific and sensitive for the target of interest. Nucleic acid probes were used first to create microarrays, and have been used predominantly since (Schena et al., 1996): protein (antibody) probes have been reported for the detection and quantification of specific proteins (Haab et al., 2001).

Nucleic acid probes Two types of DNA probes, cDNAs (PCR products) and oligonucleotides are used to construct arrays. Both types rely on the negatively charged phosphate backbone to enable attachment of DNA probes to derivitised surfaces. cDNA probes cDNA probes are relatively long (0.25-1.2kbp) denatured PCR amplicons derived from the target of interest. PCR amplicons are denatured and both strands are fixed to the slide, following purification to remove non-specific amplicons, un-incorporated nucleotides, enzymes and salts. 27


It is important to determine the sequence of the amplicon to ensure that it is not an unintended amplification product of PCR.

Originally cDNA probes were used because researchers had access to expressed sequence tag libraries from previous experiments; however there are a number of drawbacks to this approach over synthesis of oligonucleotide probes. These include the time consuming task of amplifying and purifying PCR products for each probe. A method used by Hedge et al., (2000) found only an 87.5% success rate for single band amplification for a 30,000 clone library. Sequencing may also be needed to verify clone identity (Taylor et al., 2003) further increasing costs and time taken. Difficulty in designing probes for differentiating strains of a virus (Webster 2003), and access to virus nucleic acid needed in order to amplify the probe are also disadvantages. Access to target nucleic acid for amplification may be problematic, e.g. for quarantine pathogens or those not available locally.

Oligonucleotide probes Oligonucleotide (oligo) probes are single-stranded DNA fragments of 20-70 nucleotides. Unlike cDNA probes, only one strand is present so, in the case of ss virus genomes, it is important that the probe corresponds to the coding strand of RNA viruses so that it hybridises to the labelled anticoding cDNA strand of the virus. It is not generally necessary to purify the probe, particularly where high performance liquid chromatography has been carried out after synthesis to remove partial-length oligonucleotides. Strain or pathotype specificity is easier to achieve with the relatively short oligo probes than the longer cDNA probes and, because probes can be designed from sequences available in international sequence databases, access to the target organism is not required for probe synthesis (Webster et al., 2004).

Probes can also be made from the nucleotide analogue locked nucleic acid (LNA) which show improved thermal stability and base stacking of complementary sequences (You et al., 2006). LNA monomers contain a modified ribose moiety which is locked into a state better suited for hybridisation experiments. Better sensitivity, better signal to noise ratios and discrimination of single nucleotide differences is reported for oligonucleotide probes of LNA (Castoldi et al., 2006), however their use has not so far been demonstrated for virus diagnostics.

Probe length The sensitivity of long (70mer) oligonucleotide probes has been shown to be higher, especially for low abundance transcripts (Ramdas et al., 2004). Tested on low abundance transcripts 70mer probes were 120 fold more sensitive compared to short (30mer) probes. No difference in the binding affinity of probes to the slides was observed between long and short probes. 28


Concentration was also found to have an effect on sensitivity, but this was not as significant as probe length (~ 5 fold increases in signal intensity, Ramdas et al., 2004).

With international databases, access to powerful bioinformatics software is vital to design microarray probes. The use of sequence alignment programs to identify conserved regions within virus genomes is important because single nucleotide differences dramatically reduce hybridisation efficiency especially in short length oligonucleotide probes (Lievens et al., 2006). Also of importance is knowledge of the genomic strategy of each target species, whether it is single-stranded or double-stranded, RNA or DNA, positive or negative sense because the single-stranded probe and fluorescent target species must be complementary.

To date pathogen diagnostic microarrays have used one or more specific probes for each pathogen of interest. Probes designed to be group or genus-specific have not been demonstrated for plant virus arrays; however Gibbs et al., (2005) suggested first to distinguish large sets of sequences by using a smaller number of shared sub-sequences. Using this approach they were able to effectively distinguish 201 Lepidoptera sequences using sixteen 18nt oligomers. Such a strategy may also be applicable to plant viruses (Gibbs et al., 2006) and be useful in microarray based work.

Antibody/protein probes Protein/antibody probes can be bound to derivitised slide supports and hybridised with biological extracts to test interactions. Proteins are much more structurally diverse than DNA and are folded into complex tertiary structures which may be important for their function. Thus providing a non-denaturing environment for protein immobilisation is a key goal (Angenendt et al., 2002). Few protein arrays have been demonstrated (Haab et al., 2001; Wiese et al., 2001). Protein arrays are therefore similar to ELISA, but multiple antibody-antigen interactions can be detected in a single experiment, unlike traditional ELISA which is limited to testing one interaction at a time.

Protein arrays are printed onto: (i) gel coated surfaces such as

polyacrylamide or agarose, or (ii) chemically derivitised glass or plastic surfaces coated with PLL. For an evaluation of slide surface chemistry for protein arrays see Angenendt et al., (2002). Fluorescently-labelled proteins are then hybridised to the array and following washing to remove non-specific binding and scanned in a similar way to DNA microarrays. See Espina et al., (2003) and Angenendt (2005) for reviews of protein microarrays; and Wacker et al., (2004) for a comparison of antibody immobilisation methods.

29


1.5.3 Slide surface and immobilisation methods Microarray slides use chemically activated surfaces to bind capture probes covalently following printing, and to prevent probe loss during hybridisation and washing steps. Glass slides are often used as a support for arrays because of their low auto-fluorescence (Zammatteo et al., 2000). A wide variety of surface modifications are available for covalent attachment of nucleic acid probes including; PLL, amine, epoxy and aldehyde derivitised slides. Specific methods are required to attach probes to the surface including chemical modification of the oligonucleotide probes. Novel surfaces like sugar coated slides (Willats et al., 2002), used for studying glycan interactions, are not widely applicable to diagnostic arrays.

Arraying of probes Probes must be positioned in a regular array to enable identification of specific probes following hybridisation. Arrays of DNA, protein or antibody probes can be made using robotic work stations for printing probes. Contact printing uses small amounts of probe solution transferred by surface tension to create arrays, while inkjet and piezoelectric printers are also available. See Venkatasubbarao (2004) for a review. Probes are first resuspended in a printing buffer, such as SSC or dimethyl sulphoxide, and small volumes (nano- to picoliters) transferred to the slide. Electrostatic attractions between DNA probes and slide surfaces must occur rapidly during the short period of buffer evaporation. Additives like betaine are commonly added to print buffers to improve spot morphology (Diehl et al., 2001). The concentration of the probe and choice of print buffer can have an effect on the signal intensity of the slides (Diehl et al., 2001; Hessner et al., 2003a; Hessner et al., 2004b; Wrobel et al., 2003).

Alternatively arrays of oligonucleotide probes can also be synthesised in-situ such as Affymetrix arrays (www.affymetrix.com) which contain hundreds of thousands of short oligonucleotide probes in a small (~2cm2) area. These arrays come synthesised from the vendor and no printing or immobilization is required. A commercially available test for genotyping single

nucleotide

polymorphisms

in

the

cytochrome

P450

system

is

available

(http://www.roche.com/med_backgr-ampli.htm, accessed 05-06-07) however no such arrays are available for plant virus diagnosis and detection. Therefore this type of array will not be considered further in this review.

Appropriate control probes, both positive and negative, must be included in microarrays. Positive controls used include probes to ubiquitously expressed genes such as Cytochrome oxidase or rRNA genes and are useful in normalising for differences in labelling efficiency between reactions. They also serve to orientate scanned images of slides and provide beacons for identifying probes. A commonly used negative control is a mix of water and print buffer to 30


check for carry over of probes on microarray pins. Genes, or sequences, which are not expected to be present in an RNA source, are used to check the stringency of washing where any signal on the array is due to non-specific binding. For diagnostic applications these controls are also important to verify all steps have worked as expected.

Slide surfaces A wide variety of slide surfaces have been developed to bind DNA probes in microarrays. Amino groups on slide surfaces are an effective way of binding probes which use the positive charge of amino groups at neutral pH (Venkatasubbarao 2004) binding to the negative charge of the phosphate backbone of DNA. Slides can also be coated with PLL which contains amino groups. These slides are most effective at binding long length oligonucleotide or cDNA probes (Venkatasubbarao 2004).

Other slide chemistries include epoxy (Lamture et al., 1994) and aldehyde (Schena et al., 1996) surface groups which bind DNA modified to include an amino crosslinker group (Zammatteo et al., 2000; Venkatasubbarao 2004). The crosslinker is synthesised with the probe and reacts with the slide surface to produce covalent bonds, for instance amino-modified oligonucleotides react with aldehyde and epoxy derivitised surfaces (Venkatasubbarao 2004). Terminal modification of microarray probes allows the probe sequence to extend above the slide surface and prevent steric hindrance during hybridisation and a spacer such as dT10 is also included in the probe sequence for the same reason (Shchepinov et al., 1997). These modifications help to relieve steric hindrance of the probe close to the slide surface but also increase the cost of oligonucleotide probe synthesis, which contributes a significant part of the array cost.

Covalent binding of probes The ionic bonding of probes to slide surfaces can be strengthened by Ultra Violet (UV) irradiation or thermal baking to produce covalent bonds. This is particularly important in amino modified slides. Slide surfaces, such as epoxy or aldehyde slides bind probes covalently by the amino crosslinker so this step is unnecessary and possibly deleterious. UV crosslinking induces free radical formation in pyrimidine residues of DNA (Church & Gilbert 1984) which creates covalent bonds between probe and slide. Thermal baking of slides can also be used, for instance 80oC for 1-2 hours, or longer overnight at 42oC. Optimisation of immobilisation is important as different slide chemistries, and unexpectedly, slides from different vendors have been shown to have a marked difference in optimal UV energy for immobilisation (Wang et al., 2003).

31


1.5.4 Methods for labelling and hybridisation The detection of hybridisation depends on nucleic acids being fluorescently labelled and binding to the immobilised probes of complementary sequence. A laser scanner is used to detect hybridisation.

The amount of fluorescence is linearly related to the amount of

complementary target.

Fluorescent labelling of DNA DNA is labelled through the attachment of fluorescent moieties which allow detection and quantification of hybridisation. The most commonly used fluorophores are the Cy series of fluorophores (Amersham Biosciences) including the Cy3 and Cy5 dyes. Also routinely used are the Alexa fluorescent dyes (Invitrogen). Each fluorophore is excited by a UV light source but emits visible light at a longer wavelength which allows the signal from each fluorophore to be quantified independently by the scanner. Comparisons of the dyes have shown higher resultant array intensity with CyDye fluorophores however these show lower photostablity (Ballard et al., 2007).

Fluorophores incorporated into nucleic acid either directly or indirectly. Direct incorporation of fluorophores involves using a fluorophore which has been conjugated to nucleotides. DNA is labelled by the DNA polymerase, including the labelled nucleotides, during DNA strand extension. This can be done during reverse transcription, PCR or other amplification methods. Methods for direct labelling of nucleic acids include those described by Hedge et al., 2000 and Bittner (2003).

Indirect labelling uses nucleotides modified with an amino-allyl group which is incorporated during PCR or reverse transcription in the same way as for direct labelling. In a separate coupling reaction an active N-hydroxyl succinimide form (e.g. NHS Cy3) of the fluorophore is conjugated to the free amino groups introduced into the DNA (DeRisi, 2003a). The benefits of indirect labelling are more efficient incorporation (compared to direct labelling) of the relatively small amino allyl group and reduced cost of the nucleotides, however the extra reaction time and purification steps required make it relatively time consuming. A biotin labelling method, used most commonly with Affymetrix arrays, is another type of indirect labelling utilizing biotin labelled nucleotides and an anti-biotin antibody which is conjugated to a suitable fluorophore for detection of hybridisation. For an example of this method see Tom et al., (2003).

A proprietary technology from Genishpere (3-DNA www.genisphere.com) uses a dendrimer based labelling method for low RNA input microarray technology. A random or poly dT primer 32


with a 5' adaptor sequence is used for reverse transcription. This is hybridised to the 3-DNA label (Figure 1.9). This is built up in four layers from simple oligonucleotide primers (which are cross-linked between layers to stabilize the interactions of the complementary arm sequences) and labelled with a fluorophore such as Cy3. A

B

C

D

Figure 1.9: Structure of 3DNA dendrimer oligonucleotides showing the building up of successive layers to form the final structure. A: Two oligomers bind to form double-stranded "waist" structure bordered by four single-stranded arms. B-D: Step wise assembly of monomers into final structure (D). Single stranded-arms bind to further oligomers through complementary sequences. Free arms in the final structure (D) bind the fluorescent label and the adaptor sequence used in reverse transcription. Figure modified from www.genishpere.com.

First strand cDNA is able to bind to probes on the array, and to the fluorescent 3-DNA by the adaptor sequence incorporated during reverse transcription. A single 3-DNA molecule contains an average of 200 fluorophores making this a highly sensitive method for labelling (www.genisphere.com).

Hybridisation and washing slides Following labelling and purification of unincorporated nucleotides the target solution is hybridised to the microarray slide. A buffer is used to promote hybridisation of labelled target to the probes and typically contains SDS and SSC and blocking agents such as tRNA, Cot-1 DNA and bovine serum albumin (BSA) (Ideker et al., 2003). Hybridisation is carried out at a constant temperature for a period of 1-16 hours. To prevent drying, the hybridisation is carried out in a sealed environment, typically under microscope slide covers, however many methods for hybridisation are available (Hedge et al., 2000; Ideker et al., 2003).

Non-specific hybridisation is prevented by washing slides in a series of increasing stringency wash buffers. These typically progress from high amounts of SSC/SDS (low stringency) to low amounts (high stringency). Slides are dried and scanned. For protocols on array hybridising and washing see Hedge et al., (2000) and Ideker et al., (2003).

33


1.6 Conclusions Available methods for plant virus diagnosis are of limited value when unknown or unexpected viruses are present because only one or a few viruses can be assayed at once. Parallel detection methods are particularly important in screening incoming plant material and preventing entry of quarantine pathogens. High-throughput assays, such as PCR and ELISA, while useful for screening samples in large numbers, are impractical for diagnosis of unknown or unexpected viruses/strains, which may not be detected.

Current polyvalent methods using PCR with

degenerate primers do not reliably detect all intended members and they require further tests for species or strain designation which limits their usefulness.

In contrast, microarrays can be used for highly parallel virus diagnostics. They can be used for assaying for hundreds of virus species on a single slide and diagnosing known and unknown viruses/strains to the genus, species and strain levels. Currently only small proof of concept microarrays have been reported for plant virus diagnostics (e.g. Boonham et al., 2003), however more elaborate studies have been undertaken for human pathogens (Wilson et al., 2002) indicating their potential.

Microarrays should be optimised for choice of probe and immobilisation method, and methods for labelling and hybridisation. These choices can have a large impact on the quality of arrays and the resulting fluorescent signal intensity. In this project, some of these parameters are optimised to increase the sensitivity of microarrays for plant virus diagnosis.

34


1.7 Project Aims There were two aims of the research undertaken in this thesis: these were to characterise a newly-discovered Potyvirus, and to develop a microarray based method to detect plant virus pathogens. To achieve these aims, the following steps were taken: •

Characterisation of a novel plant virus pathogen, Hardenbergia mosaic virus by serological and nucleic acid methods with determination of the phylogeny and probable origins of this species.

•

Development of methods to attach oligonucleotide probes to derivitised glass slides using robotic printing technology.

Printing variables included print buffers and

immobilisation methods. •

Demonstration of the use of a microarray based method to detect Hardenbergia mosaic virus and other plant virus pathogens and differentiation of virus strains.

•

Testing methods of fluorescent labelling to enable sensitive detection of plant virus pathogens, and comparing real time PCR with labelling methods to estimate amplification efficiency.

35

II: GENERAL MATERIALS AND METHODS

Chapter 2: General Materials and Methods

2.1 Introduction The aim of this project was to develop a microarray based diagnostic tool to detect plant viruses and to use this technology to evaluate detection of a novel Potyvirus called HarMV. The first step was to take leaf material infected with virus and extract RNA. This was then used to synthesise cDNA with reverse transcriptase. PCR was used to amplify different regions of the virus for microarray labelling and hybridisation, fragment cloning and direct sequencing.

Other general methods which were used included; gel electrophoresis, plasmid, nucleic acid purification, microarray slide fabrication and hybridisation.

2.2 Viruses and Inoculations Virus isolates were obtained from infected plants and transferred to appropriate indicator species, which were grown in an insect proofed, temperature controlled glasshouse. For sap inoculations young infected leaf material was ground and mixed with diatomaceous earth ‘celite’ and virus inoculation buffer (0.05M potassium phosphate buffer, pH 7.2). Young leaves of test plants were manually inoculated and left overnight in a humid environment to recover. Samples were preserved in a Maxi dry lo freeze dryer for up to 48 hours, or until samples were completely dry.

2.3 RNA Extractions Virus RNA was extracted from infected plant material using a RNeasy Plant Mini kit (Qiagen). For microarray experiments where large amounts of RNA were required this was modified to include a cetyl-trimethylammonium bromide (CTAB) extraction followed by a phenolchloroform precipitation.

2.3.1 Qiagen RNeasy Plant Mini Extractions Virus-RNA was extracted from infected plant material using a RNeasy Plant Mini (#74904, Qiagen) extraction kit as follows: • • • •

Weigh amount of tissue to be extracted (less than 100mg). Grind into a powder in liquid nitrogen. Decant powder plus liquid nitrogen into a RNase-free liquid nitrogen cooled 1.5mL microcentrifuge tube. Allow liquid nitrogen to evaporate but not tissue to thaw. 36


• • • • • • •

Add 450µL of buffer RLT and vortex vigorously. Pipette lysate onto QIAshredder spin column and centrifuge for 2min @ 20,000xg. Transfer supernatant flow-through to a new microcentrifuge tube. Add 225µL of 100% ethanol to cleared lysate and mix immediately by pipetting. Apply sample (650µL) to RNeasy mini column in 2mL collection tube. Centrifuge for 15s @ 20,000xg. Discard Flow through. Add 700µL Buffer RW1 to RNeasy column, centrifuge 15s @ 20,000xg to wash flow through. Discard Collection Tube. Transfer RNeasy column into a new collection tube, pipet 500µL buffer RPE onto RNeasy column and centrifuge 2min @ 20,000xg. Add 50µL of RNase free water and elute by centrifuging at 20,000xg for 15s. RNA used immediately or stored at -80oC.

• • •

2.3.2 Microarray RNA Extractions A modified CTAB extraction method was used for purifying the large amounts of total RNA required for microarray experiments. This method was carried out as follows: • • • • • • • • • • • • • •

Grind 150mg of infected leaf tissue in liquid nitrogen using a roller. This is done twice for each test plant. Add 1.5mL of CTAB grinding buffer (2% CTAB, 100mM Tris-HCl (pH 8.0), 20mM EDTA, 1.4M NaCl, 1.0% sodium sulphite, 2.0% PVP-40) and mix thoroughly using roller. Pour 1mL into a 2mL tube and incubate sap at 65oC for 10-15 min. After incubation add 1mL of choloform:isoamylalcohol. (24:1) and mix to emulsion by inverting the tube Centrifuge as 11,000 rpm for 10 min at room temperature Remove 800µL of the aqueous layer taking care not to disturb the interphase. Add an equal volume of 4M LiCl, mix well and incubate overnight at 4oC. Centrifuge at max speed for 25 min to pellet the RNA. Pour off the supernatant and resuspend each pellet in 50µL nuclease free water. For each sample pool together the two preps. Add 350µL buffer RLT and 250µL EtOH to the sample Apply sample to an RNeasy column (Qiagen) and spin at 10,000 rpm for 15s. Discard flow through and wash the column with 500µL of buffer RPE and spin at 10,000 rpm for 15s. Discard flow through and repeat the wash step Transfer the column to a RNase free collection tube, add 50µL of nuclease free water and spin at 10,000 rpm for 15s to elute, repeat elution step. Use RNA immediately or store at -80oC.

2.4 Reverse Transcription Reverse Transcription of total RNA was carried out using the ThermoScript Reverse Transcriptase-PCR kit (#122336-014, Invitrogen) as follows: •

In a 0.2mL tube combine: Component

Amount

Primer (T7T24) RNA total DEPC-treated H2O Total

1µL (10pmol/µL) 1µL (not quantified) 8µL 10

37


• • •

• •

Denature RNA and Primer by incubating at 65oC for 5min and place on ice Vortex 5x cDNA synthesis buffer prior to use Prepare master mix as follows: Component

Amount

5x cDNA synthesis buffer 0.1 M dithiothreitol (DTT) RNase OUT (400U/µL) DEPC-treated H2O 10mM dNTP mix Thermoscript RT (15U/µL) Total

4µL 1µL 1µL 1µL 2µL 1µL 10µL

Pipette 10µL Master Mix per reaction Incubate in thermocycler

The reaction mixture was incubated at the following temperatures and times.

• •

25oC

50oC

85oC

10min

50min

5min

Add 1µL of RNase H and incubate for 20min @ 37oC Store at -20oC or use for PCR immediately

2.5 PCR Protocol PCR was carried out using either a ‘high fidelity’ DNA polymerase (Pyrococcus furiosus, Pfu) or a 'lower fidelity' Thermus aquaticus (Taq) polymerase. High-fidelity PCR was used for cloning and direct sequencing of virus amplicons, while lower-fidelity PCR was used for screening samples for the presence or absence of particular gene products. PCR reactions were carried out on Applied Biosystems GenAmp PCR systems 2400, or Perkin Elmer-Applied Biosystems GenAmp PCR systems 9700 thermocyclers.

2.5.1 High fidelity PCR High-fidelity PCR was carried out using Pfu DNA polymerase (Promega Corp. Wisconsin USA, #M7741) using recommended conditions. Total reaction volumes were typically 25 to 50µL and contained: Component

Stock Concentration

Final Concentration

10x PCR buffer with MgSO4 20mM MgSO4 2mM dNTP mix 10mM 200µM upstream primer 10pmol/µL 1pmol/µL downstream primer 10pmol/µL 1pmol/µL DNA template† Pfu DNA polymerase 2-3u/µL 1.25u/50µL to final volume of 25 to 50µL Nuclease free H2O † Typically 1µL of first strand cDNA was used as template for PCR.

38


The thermocycling conditions for high fidelity PCR were as follows: Step

Temperature

Initial Denaturation Denaturation Annealing* Extension** Final Extension

o

Time

Number of Cycles

94 C 5min 1 94oC 10s 50-55oC 30s 30 72oC 2min 5min 1 72oC hold 1 4oC *The annealing temperature for reactions was dependant on the melting temperature (Tm) of the primer pair being used (Table 2.1) **The extension time for a PCR was 2min for every 1kb to be amplified

For the sequences of primers and the annealing temperatures used in PCR amplification see Chapter 3.

2.5.2 Lower-fidelity PCR Lower-fidelity PCR for diagnostic PCRs was carried out using the Taq DNA polymerase (Fisher Biotech, Australia, #TAQ-1) and the 5x polymerisation buffer containing dNTPs (Fisher Biotech, Australia, #PB-1) using recommended conditions. Typical reaction volumes were 10 to 25µL with the following component:

Component

Stock Concentration

Final Concentration

5x Buffer 0.2mM dNTPs 400µM dNTPs MgCl2 25mM 2.5mM Taq Polymerase 5u/µL 2.5u/20µL upstream primer 10pmol/µL 1pmol/µL downstream primer 10pmol/µL 1pmol/µL DNA template† to final volume of 10 to 25µL Nuclease free H2O †Typically 0.5 to 1µL of first strand cDNA was used as template for PCR.

The thermocycling conditions for diagnostic PCRs were as follows: Step

Temperature

Time

Number of Cycles

o

Initial Denaturation 94 C 5min 1 Denaturation 94oC 10s 50-55oC 30s 30 Annealing* ** 72oC 1min Extension 5min 1 Final Extension 72oC hold 1 Hold 4oC *The annealing temperature for reactions was dependant on the Tm of the primer pair being used (Table 2.1) **The extension time for a PCR was 1min for every 1kb to be amplified

For the sequences of the primers and the annealing temperatures used in PCR amplification see Chapter 3.

39


2.5.3 Real time PCR Real time PCR Assays for PVX, PVY and cytochrome oxidase (COX) were carried out using protocols from the Central Science Laboratories (CSL, York, UK) as provided by Dr Neil Boonham. The primer and probes sequences used are included in Table 2.1.

Table 2.1: Real time PCR probe and primer sequences Sequence 5’ → 3’

Name

ACACAGGCCACAGGGTCAA PVX F1 GGATCCACCAAATCAACTACCAC PVX F2 GGGATGGTGAACAGTCCTGAAG PVX R1 GGTATGGTGAATAGCCCTGAATTG PVX R2 ACTGCAGGCGCAACTCCTGC (FAM) PVX Probe* GGGCTTATGGTTTGGTGCA PVY F1 CCGTCATAACCCAAACTCCG PVY R1 TGAAAATGGAACCTCGCCAAATGTCA (FAM) PVY Probe* CGTCGCATTCCAGATTATCCA COX F1 CAACTACGGATATATAAGRRCCRRAACTG COX F2 AGGGCATTCCATCCAGCGTAAGCA (JOE/BHQ1) COX Probe* *Probes were labelled with either FAM or JOE/BHQ1 as indicated

Reactions were 25µL in volume with the final concentrations of reagents as follows: 1x PCR buffer, 5.5mM MgCl2, 0.2mM dNTP’s, 7.5µM forward primer, 7.5µM reverse primer, 5µM probe, 0.5µM ROX and 0.125µL of Taq DNA polymerase (Fermentas). Reactions were made up to 24µL with molecular biology grade water and 1µL of template DNA added. For reverse transcription real time PCR the only change was the inclusion of 0.125µL of a 1:50 dilution of MMLV reverse transcriptase. Cycling conditions were 95oC for 10 min, followed by 40 cycles of (95oC/15s, 60oC/60s). For reverse transcription real time PCR this included a 30 min step at 48oC. Reactions were run on an ABI 7900 sequence detection system using the ABI PRISM 7000 Sequence Detection System software.

2.6 Agarose Gel Electrophoresis PCR amplicons were visualised on 1.0% DNA grade agarose (Progen, Australia) gels by electrophoresis. The gels were run with 7µL of PCR product and 1µL of glycerol loading buffer. Tris-acetate acid-EDTA electophoresis buffer was used for gel and run buffer (50x TAE: 2M Tris-Acetate; 0.05M EDTA; pH 8.0 Sambrook et al. 1989). Five microliters (250ng) of 100bp ladder molecular weight marker (either #G8291, Promega; or #81-100, Fisher Biotech) was used as a size standard. The gel was run on Bio-Rad Mini-Sub Cells™ or BioRad Wide Mini-Sub Cells™ at 90 to 110V until products were separated. Gels were stained in a 1µg/mL EtBr solution for 15 min. The gel was then visualised on a transilluminator.

40


2.7 Purification of DNA solutions 2.7.1 Ethanol Precipitation DNA was purified and concentrated by ethanol precipitation. This was done as follows: • • • • • • • • •

Add 0.1 volume of 3M NaAc (pH= 5.2) to PCR. Add 2 volumes (110µL) of ice cold ethanol then vortex. Allow to sit at –20oC for 15-30 min to precipitate DNA Recover DNA by centrifugation for 10 min at max speed at 4oC. Remove supernatant Add 80µL 70% ethanol Centrifuge at max speed for 2min at 4oC, remove supernatant and repeat 70% ethanol wash Dry pellet at room temperature for 5 min Resuspend in ddH2O

2.7.2 Purification by QiaQuick PCR cleanup kit Samples of DNA were also purified to remove primers and unincorporated nucleotides using the QiaQuick PCR cleanup kit (#28104, Qiagen) as follows: • • • • • • •

Make up volume to be purified to 100µL with molecular grade H2O and add 500µL Buffer PB. Apply to QiaQuick column and spin at 13,000 rpm 30s, discard flow-through Add 700µL Buffer PE, spin at 13,000 rpm for 30s, discard flow through Spin at 13,000 rpm for further 1min to dry tube Transfer column to fresh collection tube Add 60µL elution buffer (or appropriate solution for elution) and leave 1 min. Spin at 13,000 rpm for 30s. Repeat elution with more buffer if needed.

2.8 Quantifying DNA Samples of DNA were quantified using a Nanodrop UV-Vis spectrophotometer (Nanodrop Technologies) following manufacturers protocols.

2.9 Cloning of PCR products To aid in sequencing PCR amplicons were ligated and cloned using appropriate vectors. Three cloning systems were used: the TOPO-Zero Blunt Kit for sequencing (#K2800-02, Invitrogen), the TOPO-Zero TA Kit for sequencing (#K4500-01, Invitrogen) and the pGEM-T easy cloning kit (#A1380, Promega).

2.9.1 Ligation and Transformation using TOPO-Zero kit. Both the blunt ended and TA TOPO kits were used for cloning PCR amplicons depending on whether the DNA polymerase used created “blunt” ends (Pfu polymerase) or “sticky” ends (Taq 41


polymerase). The protocols used were based on manufacturer’s recommendations. Following ligation, plasmids were transformed into Top10 competent cells (#C4040-10, Invitrogen). Ligation and Transformation were carried out as below: • • • • • • • • •

Combine: 0.5 to 4.0µL (depending on intensity of PCR band), 1µL Salt solution, 1µL TOPO vector (Blunt or TA depending on PCR product) and add sterile water to a total volume of 6µL. Mix gently and incubate for 5 min at room temperature. Thaw 1 vial of cells per ligation reaction. Add 2µL of TOPO cloning reaction to cells and mix gently Incubate on ice for 30 min. Heat shock cells at 42oC for 30s without shaking Immediately transfer cells to ice. Add 250µL SOC medium and shake for 1h at 37oC. Spread 50-100µL from each transformation onto pre-warmed LB plates with 100mg/mL Ampicillin and incubate overnight at 370C.

2.9.2 Ligation and Transformation using pGEM-T easy™ Amplicons from PCR were ligated into a plasmid and transformed into E coli after quantification. This was carried out as follows: • • • • • • •

1µL of pGEM-T easy™ vector, 1µL T4 DNA ligase, 5µL 2x rapid ligation buffer and 3µL of purified PCR product added and incubated for 1-2 hr at room temperature or in a 14oC water bath overnight. Add 100µL competent E. coli cells suspension and 5µL ligated plasmid mixed and chill for 30 min Transfer to water at exactly 42oC for exactly 45 s. Transfer to ice bath for 1-2 min Add 800µL LB media and incubate with shaking at 37oC for 45 min. Plate 200µL onto LB agar plates containing 100mg/mL ampicillin Incubate at 37oC for 12-16 hr.

2.10 Screening for recombinant plasmids The agar plates were incubated overnight then colonies were screened for recombinant plasmids using PCR the following day. • • •

Cells were picked directly from individual colonies and introduced to 10µL PCR reagent mixtures. PCR reagent mixtures contained 1x PCR buffer, 5mM MgCl2, 0.1pmol M13 F and M13 R primer and 0.25µL Taq polymerase (#TAQ-1, Fisher Biotech). A 5µL aliquot of each PCR was run on a 1% agarose gel at 100V for 60min and stained in 1µg/mL EtBr.

2.11 Plasmid Extraction Colonies that contained a fragment of the expected size were then inoculated into 5mL of LB media with 100mg/mL ampicillin and incubated at 37oC overnight. The plasmid was then

42


purified from the culture using either the QIAprep Spin Miniprep Kit (Qiagen, Catalogue #27104) or the method of Sambrook & Russell (2001).

2.11.1 QIAprep Spin Miniprep Kit Plasmid was extracted from 1.5mL of overnight culture as follows. Cells were centrifuged at 14,000 rpm for 2min and supernatant removed. • • • • • • • • •

Resuspend cells in 250µL Buffer P1 Add 250µL of Buffer P2 and mix by inverting six times Add 350µL Buffer N3 and mix by inverting six times Centrifuge for 10 min at 14,000 rpm Apply supernatant to QIAprep spin column and centrifuge for 1min, discard flow through Wash by adding 500µL Buffer PB and centrifuge for 1min, discard flow through. Wash QIAprep column by adding 750µL Buffer PE and centrifuging for 1min. Discard flow-through and centrifuge for additional 1min to remove residual wash buffer. Elute DNA by adding 50µL of Buffer EB and leave to stand for 1 min. Centrifuge for 1min.

2.11.2: Sambrook Plasmid Extractions Plasmid was extracted from overnight bacterial cultures using the method of Sambrook and Russel (2001). This method yielded more plasmid DNA, however it was of lower purity than QIAquick extractions, and the extraction took longer to carry out. This method was done as follows: • • • • • • • • • • • •

1.5mL of culture centrifuged at 14,000 rpm for 2min, supernatant removed. Resuspend bacterial pellet in 100µL ice-cold Alkaline lysis solution I (50mM glucose, 25mM Tris-Cl pH 8.0, 10mM EDTA pH 8.0) with vigorous vortexing Add 200µL of freshly prepared Alkaline lysis solution II (0.2N NaOH, 1% SDS) and invert 6 times to mix Add 150µL of ice-cold alkaline solution III (3M K+, 5M Ac-) and invert to mix. Store on ice for 5min. Centrifuge at 14,000 rpm for 5min and transfer supernatant to a new tube Mix and add an equal volume of phenol:chloroform, mix the organic and aqueous phases by vortexing and then centrifuge at 14,000 rpm for 2min. Transfer the aqueous layer to a new tube and ethanol precipitate by adding 2 volumes of 100% ethanol. Allow to stand at room temperature for 2min Precipitate nucleic acids by centrifugation at 14,00 rpm for 5min. Remove supernatant and allow drying at room temp. Add 1mL of 70% ethanol and invert several times. Centrifuge at 14,000 rpm for 2min and remove supernatant. Store open at room temperature to evaporate any remaining ethanol Resuspend in 50µL TE (pH 8.0) containing 20µg/mL DNase-free RNase A (Roche). Vortex for a few s and store DNA at -20oC.

43


2.12 Restriction Digest of Plasmid Purified plasmid DNA was analysed by restriction digestion as follows: • • •

5µL of extracted plasmid mixed with 1µL EcoR1 H (Promega) (activity = 12u/µL), 2µL EcoR1 H Buffer (10x), and 12 µL H2O. Incubated at 37oC for 1.5 hr Fragments analysed on a 1% Agarose gel and stained with EtBr

2.13 Storing clones as Glycerol Stocks Colonies that were had an insert of the right size and sequence were stored as glycerol stocks. This was done as follows: • • • •

0.5mL of fresh liquid culture was added to 0.5mL of 30% glycerol solution to create 15% glycerol solution. Solution was left at room temperature for a 30min to allow glycerol to be taken up by cells. Cells then dropped in liquid nitrogen to cool rapidly. Cells then stored at –80oC.

2.14 DNA Sequencing Virus DNA sequences were determined from either cloned fragments or by direct sequencing of PCR amplicons. Prior to sequencing plasmids were extracted using the QiaPrep Spin Mini Kit or the Sambrook & Russel (2001) method as described above. PCR products were purified using an ethanol precipitation as described above. The purified solutions were quantified using a Nanodrop Spectrophotometer.

For sequencing reactions 300-400ng of plasmid DNA (20-40ng PCR product) was combined with 4µL Big Dye version 3.1 (Applied Biosystems) dye terminator mix and 3.2pmoles of primer. Reactions were made up to 10µL with molecular biology grade water. Sequencing reactions were thermocycled at 96oC for 2 min, followed by 25 cycles of 96oC for 10s, 50oC for 5s, 60oC for 4min. Reactions were held at 14oC.

Sequencing reactions were cleaned using a modified ethanol procedure as described below: • • • • • • •

Combine 1µL 3M NaAc (pH 5.2), 1µL of 125mM Na2EDTA and 25µL 100% ethanol was added to the reaction mixed. Precipitate DNA at room temperature for 20 min. Centrifuge at 14,000 rpm for 30 min Discard supernatant and rinse with 125µL 70% ethanol Centrifuge at 14,000 rpm for 5 min Remove supernatant and leave on bench top for 5min to evaporate ethanol. Reaction placed at -20oC for sequencing.

44


The primers used were M13F and M13R universal primers (Promega Corp.) for plasmid sequencing, or primers used in the PCR reaction for direct sequencing. DNA templates were sequenced in both directions in all cases. Where appropriate sequencing reactions used diluted dye terminator mixes. Reactions were diluted to 1/4 or 1/8 reactions from the 1/2 reaction described above. The amount of plasmid or PCR product template used in each case was the same; however the amount of dye terminator was reduced. For 1/4 reactions only 2µL of Big Dye v3.1 was added with 1µL of 5x sequencing buffer (#4336697, Applied Biosystems). For 1/8 reactions only 1µL of Big Dye v3.1 was added with 1.5µL of 5x sequencing buffer.

2.15 Coating Slides with Poly-L-lysine Microarray slides were created by in-house coating of microscope slides with PLL.

The

protocol for coating slides was adapted from DeRisi (2003b).

Cleaning Microscope Slides: • • • •

100g NaOH in 400mL ddH2O Add 600mL 97% EtOH Incubate slides in cleaning solution for 2 hr Wash in ddH2O to remove cleaning solution (4 times for 2 min with stirring).

Once cleaned microscope slides were coated in PLL: • • • • •

100mL poly-L-lysine (#P8920, Sigma) + 100mL tissue culture PBS + 800mL ddH2O. Leave slides covered by PBS/poly-L-lysine for 1 hr Rinse once for 2 min in ddH2O with mixing Centrifuge dry for 5 min at 1200 rpm. Dry at 55oC for 10 min

Cleaned and coated microarray slides were then stored in a dark and dust free environment for two to four weeks to ‘age’ the slides. Following aging the slides were printed.

2.16 Printing microarray slides Microarray slides were printed with a Gene Tac 3000 Robotic Printer using a 4 x 12 horizontal printing tool with a single transfer per spot. Slides were printed from a 384 well microtiter plate. When frozen the 384 well plates were thawed, votexed to mix and spun down (5 min @ 1500 rpm) to collect the probes at the bottom of the wells. The sterilisation procedure was 3 s in a sonic bath, 3 s in 50% ethanol, 3 s in 70% ethanol, 3 s in the heater and 3 s air drying. Printed slides were stored sealed in alfoil wrapped containers within a low humidity (desiccator) environment unless otherwise stated.

45


In house created microarray slides were printed using the Buffer Comparison Plate (29/11/04) in a variety of printing buffers (50% DMSO, 3x SSC, 3x SSC with 1.5M Betaine, 150mM NaPO4, 150mM NaPO4 with 1.5M betaine, and 2x Array It).

Following printing slides were

immobilised by one of a number of methods. Briefly Slides were either UV cross-linked (BioRad™ GS Gene Linker) at 70mJ, 150mJ, 250mJ, 400mJ or incubated at 80oC for 2 h or 42oC overnight over saturated NaCl. Following immobilisation the slides were washed in 2x SSC, 0.1% SDS for 40 s, 0.1% SDS for 20 s and then dipped in ddH2O. Slides were then dried with a stream of compressed nitrogen and stored in a dry, dust free environment.

2.17 Quality control of printed slides A quality control slide for each batch of slides was produced using a modified version of the Quality Control of Spotting – Quick Hybridisation Protocol supplied by the Lotteries State Microarray

Facility

(LSMAF).

Cyanine

3

labelled

random

nonamers

(Proligo,

http://www.proligo.com) were hybridised to the slide for 20 min before washing to remove unbound oligonucleotides. Slides were then scanned on a GeneTac UC4 Laser Scanner.

The protocol for hybridisation for random nonamers hybridisation is as follows:

Prepare the following hybridisation mixture: Reagent

Stock Solution

Final Concentration

Volume for 80µL mix

Cy3 random oligo SDS† SSC Tris HCl pH 7.5 Water

100µM 20% 20X 1M

7.5µM 0.2% 4X 50mM

3µL 0.8µL 16µL 4µL 56.4µL

†SDS added last as this may precipitate out of solution if not enough water is present • • • • • • • •

Heat mixture at 90oC for 5 min in a heating block Centrifuge at 14,000 rpm for 2 min Apply lifterslip (rough side down) to microarray and pipette 80µL of hybridisation solution underneath Allow to hybridise at room temperature for 20 min. Cover with foil to protect from direct light. Wash slide in 2x SSC, 0.2% SDS for 40 s. Wash slide in 0.05x SSC for 20 s. Immediately spin dry at 1200 rpm for 7 min Scan on Cy3 channel.

2.18 Summary These general materials and methods were used successfully to characterise the novel virus HarMV and in the printing and testing of microarray slides. In the following chapters more details of the specific experiments leading to these goals are described.

46

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Chapter 3: Bioinformatics for primer and probe design

3.1 Introduction Bioinformatics was used to design primers and probes to detect plant viruses on DNA microarrays. DNA microarrays rely on the complementation between a single-stranded DNA probe immobilised on a slide surface and a fluorescently-labelled target. Careful design of the probe is essential to ensure that the microarray works. For example, where a probe is designed to detect all strains of a virus species, a region of the virus genome that is conserved amongst all known strains should be chosen. On the other hand, where detection and discrimination of closely related strains is required the probe should be designed within a region of the virus that is specific for the strain.

The bioinformatic processes used in this study are combined in this chapter. The resulting primers and probes were used to generate results on phylogeny of a new virus in Hardenbergia comptoniana (Chapter 4) and for constructing and testing microarray slides (Chapter 5).

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3.2 Materials and methods 3.2.1 Sequence retrieval from online databases Sequences were retrieved from international sequence databases such as GenBank using the Entrez-Nucleotide

search

tool

available

online

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide). Sequences were downloaded into either a *.fasta file format, or into an online program database and file storage program (Biomanager) available through the Australian National Genome Information Service (ANGIS) at http://www.angis.org.au/.

3.2.2 Sequence alignments Alignments of nucleotide sequences were carried out to identify conserved regions for primer design. Alignments of virus isolate sequences were also carried out in Chapter 4.

ClustalW alignments Multiple sequence alignments were carried out using the ClustalW (accurate) program (Thompson et al., 1994, Version 1.9) available from Biomanager (http://www.angis.org.au). The default parameters used which were: IUB DNA weight matrix, Gap opening and extension penalties of 10 and 0.05 respectively and Gap Separation distance of 8. End gap separation penalties were included.

MEGA alignments The

Molecular

Evolutionary

Genetics

Analysis

(MEGA)

program

(Version

3.1,

www.megasoftware.net) also used ClustalW for sequence alignments. Nucleotide sequence alignments were created with default parameters which were: Gap Opening Penalty of 15, and a Gap Extension Penalty of 6.66 for both pairwise and multiple sequence parameters. Amino acid alignments were also aligned with ClustalW with Pairwise Alignment parameters of: Gap Opening Penalty of 10, and Gap Extension Penalty of 0.1. Multiple Alignment parameters were: Gap Opening Penalty of 10 and Gap Extension Penalty of 0.2. Alignments were checked and adjusted manually.

3.2.3 Comparisons of nucleotide sequences to international database Nucleotide sequences were compared against those held in international databases using the BLASTN algorithm available on the National Centre for Bioinformatics Information (NCBI) 48

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website (http://www.ncbi.nlm.nih.gov/BLAST) using default options. Sequences were checked against the Nucleotide collection (nr/nt) database. Sequences from coding regions were also checked against the protein and translated protein databases using the BLASTX algorithm available at the same website using default parameters.

3.2.4 Oligonucleotide design Short length (18-24 mers) oligonucleotides were designed for use in PCR amplifications and longer length (40-50 mers) oligonucleotides were used as capture probes in microarray experiments. The sequences of probes designed by others and used during this project are provided in Table 3.1. The materials and methods used to design both types of probes are included.

Table 3.1: Primers designed by others and used in this project Primer Name

Sequence (5’ → 3’)A

Tm

Reference

Actin F Actin R BCMV12F BCMV12R

ACCTGATGAAGATCCTCAC ATCCTCCAATCCAGACAC AGCAACAGTGTCTGAATCTTC CCCYTGTATTGTGCTCCAAC

49 48 60 55

M13F

CGCCAGGGTTTTCCCAGTCACGAC

78

M13R

TCACACAGGAAACAGCTATGAC

60

Potex1RCB

GTTTCCCAGTCACGATCTCAGTRTTDGCRTCRAARGT

75.6

Potex 5B

GTTTCCCAGTCACGATCCAYCARCARGCMAARGAYGA

79.6

Poty1 Poty2B Poty3 Random A Random B

GTTTCCCAGTCACGATC(T17)VC GTTTCCCAGTCACGATCGGBAAYAAYAHYHHDCARCC GGBAAYAAYAHYHHDCARCC GTTTCCCAGTCACGATCNNNNNNNNN GTTTCCCAGTCACGATC

72.4 76.6 52.3 66.7 56.0

T7

TAATACGACTCACTATAGGG

56

S. Wylie 2004 pers. comm. S. Wylie 2004 pers. comm. Cayford (2005) Cayford (2005) Zero Blunt TOPO PCR Kit for sequencingD Zero Blunt TOPO PCR Kit for sequencingD van der Vlugt & Berendsen (2002) van der Vlugt & Berendsen (2002) Gibbs & Mackenzie (1997) Gibbs & Mackenzie (1997) Gibbs & Mackenzie (1997) Bohlander et al., 1992 Bohlander et al., 1992 Zero Blunt TOPO PCR Kit for sequencingD

B

CCCTATAGTGAGTCGTATTA 56 This thesisC TTTTTTTTTTTTTTTTTTTTTTTT A: Abbreviations of extended IUPAC codes included in Lists of Abbreviations, B: Sequences in italics are adaptor sequences used for amplifications not included in the original reference C: A T24 primer was modified to include an anti T7 primer sequence. D: Primers were from the Zero Blunt TOPO PCR kit for sequencing available at www.invitrogen.com T7T24

Primer design Short length oligonucleotides were designed for use in PCR amplification in conserved regions of virus genomes. Primers were chosen to conform, as closely as possible, to the following general characteristics: 18-24 nucleotides in length, the calculated melting temperature of the two primers was similar, the G+C content was 50% or more, there were no runs of one nucleotide (e.g. GGGG), the two primers were not self-complementary (primer dimer) and 49

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could not bind to each other (heterodimer), and the primers did not bind elsewhere in the sequence of interest. The sequences of each primer were analysed by the BLASTN program against the GenBank database to check for complementarity with other known sequences to check for primer specificity. (www.geneworks.com.au)

as

Primers were synthesised by Geneworks, Australia Sequencing/PCR

grade

(desalted)

oligonucleotides

and

resuspended to 100µM in ddH2O. Primers were diluted to 10pmol/µL for use.

Oligonucleotide probe design Sequences were accessed through Biomanager (Australian National Genome Information Service). Alignments were done on EClustalW Multiple Sequence alignment probes version 1.9 using standard parameters. RNA viruses can be quite variable within a species so alignments of sequences from several isolates were carried out to identify conserved regions suitable for probe design. Probes approximately 50 nucleotides in length were designed in virus (+ve) sense. The sequences were examined for 2o structures with OligoAnalyzer (version 3.0) using a

hybridisation

temperature

of

45oC

(available

at

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx).

Microarray probes for BYMV and CMV were designed as part of a BSc (Honours) project and were used on PLL slides (Webster 2003). The probe sequences are listed in Table 3.2.

Virus speciesA

Table 3.2: Oligonucleotide probes and the probe sequences. Microarray probe Oligonucleotide probe sequence

Cucumovirus B

CucumoOligo1

TATCAAGAGCGTACGGTTCAACCCCTGCCTCCCCTGT

BYMV

BYMVOligo1

CTTAATGTGTTCACTTCTATACCACAAAACTTTGAGG

CMVOligo1

CTCATTCGACATTGTATTTGGTTCCCCTCTATTAAGGAC

CMVOligo2

GGTGCTGAAGATTACCTTGAAAAATCTGATGATGAGCTCC

CMVOligo3

CTTGTTTCGCGCATTCAAATTCGAGTTAATCCTTTGC

C

CMV

A: Virus names: Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), B: Probes designed to detect many members of a virus genus. C: Three probes were designed to detect different genomic segments of CMV (RNA1, 2 & 3 respectively).

A microarray to detect PVX, PVY and other viruses was also used during this project. The sequences of probes used are provided in Table 3.3. These were kindly supplied by Dr Neil Boonham of CSL, UK and were used in a collaborative project on testing methods of amplification for microarrays (Chapter 6).

50

NumberA

TargetB

Sequence (5’→ 3’)

Table 3.3: Names and sequences of oligonucleotide probes provided by Dr N. Boonham (CSL) Name

LengthC

TTGTACACACCGCCCGTCGCTCCTACCGATTGAATGATCCGGTGAAATGT 1and23 18SrRNA 18s rRNA gene 50 GAAACCTGGAGGAAGTCCGACGAGATCGCGGTTGGGGCTTAGGAC 2 CSVd 97 CSVd 45 AAGCTTCCTTTGGCTACTACCCGGTGGAAACAACTGAAGCTTCAA 3 CSVd 242 CSVd 45 AACTCGTGGTTCCTGTGGTTCACACCTGACCTCCTGAGCAGAAAA 4 PSTVd 9 PSTVd 45 CTTCGGCTACTACCCGGTGGAAACAACTGAAGCTCCCGAGAACCG 5 PSTVd 250 PSTVd 45 AGGGAAAGTTTGCACTGTGCTGAAAAGCAAAGCATTTGAAATGACTGAAG 6 TSWV 04#1 TSWV NC 50 GACAGGATTGGAGCCACTGACATGACCTTCAGAAGGCTTGATAGCTTGAT 7 TSWV 04#2 TSWV NC 50 CTACCCCTTCCAACCCTGCACTATCACGCCGAGCCCTCGCTGCTCAGTTT 8 PepMV 03#1 PepMV CP 50 CACAAGGTTTCAAGCCTGAGCACAAATTCGCAGCATTTGACTTCTTCAAT 9 PVX 4#1 PVX CP 50 TCCTGCCAACTCAGGACTGTTCACAATACCGGATGGAGACTTCTTTAGCA 10 PVX C#26 PVX CP 50 ACTGCTGCCTTTGTGAAGATTACGAAGGCCAGGGCACAATCCAACGACTT 11 PVX C#27 PVX CP 50 GATAGATACGGGTCCCTATTCCAATGGCATCAGTAGAGCTAGACTGGCAG 12 PVX 11#1 PVX CP 50 AACAAGTTGAATACCCGTTGAAACCAATCGTTGAAAATGCAAAGCCAACC 13 PVY 55#1 PVY CP 50 TCATACTGTGCCACGAATTAAAGCTATCACGTCCAAAATGAGAATGCCCA 14 PVY 57#1 PVY CP 50 GGAAGCGCACATTCAAATGAAGGCCGCAGCATTGAAATCAGCCCAACCTC 15 PVY 59#1 PVY CP 50 GTGCCACGAATTAAGGCTATCACGTCCAAAATGAGAATGCCCACAAGCAA 16 PVY 58#1 PVY CP 50 CCAGTTACAACACGAGCGTCGTACAGATTATGGAAAAAAATTATCTAAA 17 PVY 97#1 PVY 38kDa 50 AGGTTGATGGCAGAACAATGAAGCACGGTTGCCTAGAAATTGTCACAAAA 18 PVY 99#1 PVY CI 50 CTGAACTAACACACCAAATCAGGAGATTCTACTCATGGTTATTGCAACAG 19 PVY 103#1 PVY NIb 50 AGAGATATTCAAGAGAGATTTAGTGAAGTGCGAAAGAAAATGGTTGAGAA 20 PVY 101#1 PVY VPg 50 ACATCTGGAACGCATACAGTGCCGAGAATAAAAGCTATCACGTCAAAAAT 21 PVY 54#1 PVY CP 50 CGTTGAAACTTAAAGACAGCACTCCCAAAGGGTTATTCAAAACAACAAAG 22 PVY 102#1 PVY NIb 50 Virus and viroid acronyms: CSVd, Chrysanthemum stunt viroid; PepMV, Pepino mosaic virus; PSTVd, Potato tuber spindle viroid; PVX, Potato virus X; PVY, Potato virus Y; TSWV, Tomato spotted wilt virus. A: refers to location of the probe on the microarray in Figure 6.1. Probes were printed in three replicate patches across the array to provide replicate hybridisation intensity signal. B: approximate location of genomic region to which probe hybridises; 18S rRNA; 18S ribosomal RNA subunit gene; 38kDA, 38 kilodaltaon protein; CI, cytoplasmic inclusion gene; CP, capsid protein gene; NC, Nucleocapsid protein; NIb, Nuclear inclusion b gene; VPg, virus RNA linked genome protein. C: length of oligonucleotide probes in nucleotides

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3.2.5 Phylogenetic analysis of virus isolates Alignments of sequences were done in ClustalW within MEGA (Version 3.1) as described above. Phylogenetic trees were constructed from the alignments using four models: Neighbour joining, minimum evolutions, maximum parsimony and UPGMA. All were carried out using default parameters and showed congruent trees. The bootstrapped Neighbour joined trees were included in this thesis and were constructed from alignments of both deduced amino acids and nucleotide sequences.

The Kimura 2-parameter model was used with 1000 re-samplings of

alignments. A matrix of percent differences between genes at the nucleotide and amino acid levels was constructed using OLD DISTANCES (GCG Accelrys).

Recombination analysis was carried out using the Recombination Detection Program (RDP) Version 3.1 (Martin et al., 2005b). Nucleotide sequences were first aligned in MEGA 3.1 using default parameters. Sequences were checked for recombination using nine methods: RDP (Martin & Rybicki 2000), GENECONV (Padidam et al., 1999), Bootscan (Martin et al., 2005a), MaxChi (Maynard Smith 1992), Chimaera (Posada & Crandall 2001), SiScan (Gibbs et al., 2000b), PhylPro (Weiller 1998), LARD (Holmes et al., 1999) and 3Seq (Boni et al., 2007). Default parameters were used for all algorithms. A recombinant event was only considered when two of the nine methods detected the event with a P-value of less than 1x10-5. Events with probabilities below this level were excluded from further analysis.

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3.3 Bioinformatics results The results presented in this chapter are the sequences of PCR primers and oligonucleotide capture probes designed during the project. These primers were used to amplify and sequence virus isolates (Chapter 4) and for microarray fabrication and testing (Chapter 5).

3.3.1 Sequences of legume specific PCR primers Primers to amplify legume-infecting Potyviruses were designed based on an alignment of Potyviruses known to infect legumes, with the hope that motifs common to all of them would be revealed. Seven full length Potyvirus genome sequences were aligned [(GenBank accession numbers: AY112735 (BCMV), AY575773 (Blackeye cowpea mosaic virus), U47033 (BYMV), AF348210 (CABMV), AB011819 (Clover yellow vein virus, ClYVV), AJ252242 (PSbMV) and AB100442 (SMV)] as described above and conserved regions were found by eye. These were used to design primers, which were then compared to sequences on GenBank to confirm the specificity and check for any cross reactivity.

Four primers were designed: LegPotyF,

LegPotyR, LegForNIA and LegRevUTR (Table 3.4).

Table 3.4: Names and sequences of legume-specific Potyvirus primers Name

Genomic locationA

Sequence (5’ → 3’)B

TmC

GCWKCHATGATYGARGCHTGGG LegPotyF NIb 57 AYYTGYTYMTCHCCATCCATC LegPotyR CP 52 GGBACWAAYTTCCARGARAARAG LegFor NIa NIa-Pro 53 GGCRTTGCAAYGGTTCTCCC LegRevUTR 3’ UTR 56 A: NIb, Nuclear inclusion B; CP, coat protein; NIa-Pro, Nuclear inclusion A protease domain; 3’ UTR, 3’ untranslated region. B: Abbreviations of extended IUPAC codes included in Lists of Abbreviations. C: The melting temperature for double stranded DNA, where the lowest temperature of a pair of primers was used as the annealing temperature in PCR reactions (Chapter 2).

3.3.2 Design of oligonucleotide probes Within the GenBank sequence database ~4900 Potyvirus sequences are available, with ~2700 being partial or complete coat protein sequences. The bias towards coat protein sequences is largely due to the availability of degenerate primers that anneal to conserved motifs at the 3’ end of the NIb and the polyadenylated tail at the 3’ end of the genome (as listed in Table 1.1).

Other oligonucleotide probes were designed for microarray work during this project from nucleotide sequence alignments. Microarray probes were needed to differentiate clades of HarMV which were characterised in this project (Chapter 4). The alignment of 30 HarMV sequences showed a good region for microarray design, which requires both clear differences between the clades and conserved sequences within each clade (Table 3.5 and 3.6). From this 53

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region microarray probes for distinguishing between clades of the virus were designed as shown in Table 3.7.

Table 3.5: Differences in Hardenbergia microarray probe sequences indicating strain specificity of probes for virus diagnostics. Probe Name

Sequence (5’ → 3’)B

GGCAAAGNAAGNNCNTCTGCAGCAAAAGACAAAGATGTGGATGCTGGCTCG ConsensusA GGCAAAGAAAGCACTTCTGCTGGAAAAGACAAAGATGTGGATGCTGGTTCG HarMV-I GGAAAGGAGGGCACCTCTACAACAAAGGATAAGGATGTGGACGCTGGCTCA HarMV-II GGCAAAGGGAGCACCTCCGCAGCAAAAGATAAGGATGTAGATGCTGGCTCG HarMV-V GGCAAAGCTAGTGCCTCTGTAGAAAAAGACAAAGATGTAGACGCTGGCACG HarMV-VI GATAAAGTAGGTGTATCAGCTGCAAGAGACAAAGATGTTGATGCTGGGTCG HarMV-VII AGCAAAGCAAGTGCCTCTACAGTAAAAGACAAAGACGTGGATGCTGGTTCA HarMV-VIII A: The consensus sequence showing bases in the majority of probe sequences. B: Nucleotide positions differing from the consensus are highlighted.

Probes for other species of Potyvirus were designed including PWV and Passiflora foetida virus Y (PFVY) to make a microarray chip capable of detecting and identifying multiple Potyviruses to the species level.

54

Table 3.6: Alignment of HarMV sequences showing conservation within clades. Clades are listed from I to VIII

HarMV-I

Probe NameB

AGGAAAAAGGTTGATGGAGGCAACAAAGATAAAGGCAAAGAAAGCACTTCTGCTGGAAAAGACAAAGATGTGGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTTCAGAAGATAAC AGGAAAAAGGCTGATGGAGGCAACAAAGATAAAGGCAAAGAAAGCACTTCTGCTGGAAAAGACAAAGATGTGGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTTCAGAAGATAAC AGGAAAAAGGCTGATGGAGGCAACAAAGATAAAGGCAAAGAAAGCACTTCTGCTGGAAAAGACAAAGATGTGGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTTCAGAAGATAAC AGGAAAAAGGCTGATGGAGGCAACAAAGATAAAGGCAAAGAAAGCACTTCTGCTGGAAAAGACAAAGATGTGGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTTCAGAAGATAAC

HarMV-II

Sequence (5’ to 3’)A

AAGAAGACAGCTGATGGAGATGACAGAGACAAAGGAAAGGAGGGCACCTCTACAACAAAGGATAAGGATGTGGACGCTGGCTCACAAGGAAAAGTCGTGCCACGTCTTCAGAAAATAAC AAGAAGACAGCTGGTGGAGGTGACAAAGACAAAGGAAAGGAGGGCACCTCTACAACAAAGGATAAGGATGTGGACGCTGGCTCACAAGGGAAAGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAGCTGGTGGAGGTGACAGAGACAAAGGAAAGGAGGGCACCTCTACAACAAAGGACAAGGATGTGGACGCTGGCTCACAAGGGAAAGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAGCTGGTGGAGGTGACAGAGACAAAGAAAAGGAGGGCACCTCTACAACAAAGGATAAGGATGTGGACGCTGGCTCACAAGGGAAAGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAGTTAGTGGAGGTGATAAGGACAAAGGCAAGGGAAGCGCTTCTACAGCAAAGGATAAAGATGTAGATGCTGGCTCACAAGGGAATGTTGTGCCGCGTCTTCAAAAGATAAC

HarMV-VIII

HarMV-VII

HarMV-VI

HarMV-V

AAAAAGACAACTGATGGAGGCAACAAAGACAAGAGCAAAGCAAGTGCCTCTACAGTAAAAGACAAAGACGTGGATGCTGGTTCACAAGGGAAAGTTGTGCCACGTCTTCAAAAGATAAC AAAAAGACAACTGATGGAGGCAACAAAGACAGGAGCAAAGCAAGTGCCTCTACAGTAAAAGACAAAGACGTGGATGCTGGTTCACAAGGGAAAGTTGTGCCACGTCTTCAAAAGATAAC AAAAAGACAACTGATGGAGGCAACAAAGATAAGAACAAAGCAAGTGCCTCAACAGTTAAAGACAAGGATGTTGACGCTGGTTCACAAGGGAAAGTTGTACCGCGTCTTCAAAAGATAAC AAAAAGACAACTGATGGAGGCAACAAAGATAAGAACAAAGCAAGTGCCTCAACAGTTAAAGACAAGGATGTTGACGTTGGTTCACAAGGGAAAGTTGTACCGCGTCTTCAAAAGATAAC

AAGAAAGCAACTGATGGAGGCAACAAAGACAAAGGCAAAGCTAGTGCCTCTGTAGAAAAAGACAAAGATGTAGACGCTGGCACGCAGGGAAAGGTTGTGCCACGTCTCCAAAAGATCAC AAGAAAGCAACTGATGGAGGCAACAAAGACAAAGGCAAAGCTAGTGCCTCTGTAGAAAAAGACAAAGATGTAGACGCTGGCACGCAGGGAAAGGTTGTGCCACGTCTCCAAAAAATCAC AAGAAAGCAACTGATGGAGGCAACAAAGACAAAGGCAAAGCTAGTGCCTCTGTAGAAAAAGACAAAGATGTAGACGCTGGCACGCAGGGAAAGGTTGTGCCACGTCTCCAAAAGATCAC AAGAAAGCAACTGATGGAGGCAACAAAGACAAAGGCAAAGCTAGTGCCTCTGTAGAAAAAGACAAAGATGTAGACGCTGGCACGCAGGGAAAGGTTGTGCCACGTCTCCAAAAGATCAC AAGAAAACAACTGATGGTGGAAATAAAGGTAAAGATAAAGTAGGTGTATCAGCTGCAAGAGACAAAGATGTTGATGCTGGGTCGCAGGGAAAGGTTGTGCCGCGGCTTCAGAAAATCAC AAGAAAACAACTGATGGTGGAAATAAAGGTAAAGATAAAGTAGGTGTATCAGCTGCAAGAGACAAAGATGTTGATGCTGGGTCGCAGGGAAAGGTTGTGCCGCGGCTTCAGAAAATCAC

AAGAGGACAACTGGTGGGGGTGACAAAGACAAAGGCAAAGGGAGCACCTCCGCAGCAAAAGATAAGGATGTAGATGCTGGCTCGCAAGGGAAGGTCGTGCCGCGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGACAAAGACAAAGGCAAAGGGAGCACCTCCGCAGCAAAAGATAAGGATGTAGATGCCGGCTCGCAAGGGAAGGTCGTGCCGCGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGACAAAGACAAAGGCAAAGGGAGCACCTCCACAGCAAAAGATAAGGATGTAGATGCTGGCTCGCAAGGGAAGGTCGTGCCGCGTCTCCAAAAAATAAC AAGAAGACAACTGGTGGGGGTGACAAAGACAAAGGCAAAGGGAGCACCTCCACAGCAAAAGATAAGGATGTAGATGCTGGCTCGCAAGGGAAGGTCGTGCCGCGTCTCCAAAAAATAAC AAGAAGACAACTGGTGGGGGTGATAAAGACAAAGGCAAAGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGCTGGCTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGATAAAGACAAAGGCAAAGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGATAAAGACAAAGGCAAAGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGATAAAGACAAAGGCAAAGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGATAAAGACAAAGGCAAGGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAACTGGTGGGGGTGATAAAGACAAAGGCAAGGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGTTGGTTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC AAGAAGACAACCGGTGGGGGTGATAAAGATAAAGGCAAAGGAAGCACCTCTGCAACAAAAGACAAGGATGTAGACGCTGGTTCGCAAGGGAAGGTCGTGCCACGTCTCCAGAAAATAAC

Isolate Name Bun-9 MU-1C Med-7 Wel-1 He-2 KiP-1 WHP-2 He-1 Cgt-1 MU-3A BB-6 WHP-1 Can1-1 MU-2A Med-1 BB-1 BB-2 MU-1A Can1-2 Co-1 Sb-3 Sb-15 Sb-5 Sb-6 Sb19-1 Sb19-4 MR-13 MR-21 MR-30 MR-3

A: Sequences of microarray probes are underlined with any positions which differ from the consensus sequence highlighted. B: Virus sequences were organised into six major groups (named I to VIII) based on phylogenetic trees.

NumberA General Poty Probe PWV (Gld-1) PFVY HarMV-all HarMV-I (MU-1C) HarMV-II (He-2) HarMV-V (MU-1A) HarMV-VI (Sb-15) HarMV-VII (Sb-19) HarMV-VIII (MR-13) BCMV (R) BYMV (MI) SMV TuMV ZYMV 18S rRNAD

NameB CP CP CP CP CP CP CP CP CP CP CP 3’ UTR CP CP CP 18s rRNA gene

Genomic Location

GGAAAGGAAAGCGCCAGTGGTGAAAAGGACAAGGACGTCAATGCAGGATCA GGGCAGGAAGGAACTAGCGGGACAAAGGACAAGGACAAAGATATTAATGCCGG GCAGCTGAAGCGTAYATHGARATGAGGTGYTCAACYGGACCRTACATGCCT GGCAAAGAAAGCACTTCTGCTGGAAAAGACAAAGATGTGGATGCTGGTTCG GGAAAGGAGGGCACCTCTACAACAAAGGATAAGGATGTGGACGCTGGCTCA GGCAAGGGAAGCACCTCTGCAGCAAAAGACAAGGATGTAGATGCTGGTTCG GGCAAAGCTAGTGCCTCTGTAGAAAAAGACAAAGATGTAGACGCTGGCACG GATAAAGTAGGTGTATCAGCTGCAAGAGACAAAGATGTTGATGCTGGGTCG AGCAAAGCAAGTGCCTCTACAGTAAAAGACAAAGACGTGGATGCTGGTTCA CTTCACCGGATGTAAATGGAACATGGGTGATGATGGACGGAGACGAGCAA GGGCAGAGTATCAGGCAAATAGTGCCAGACAGAGATGTGAATGCAGGAACT GATGCTAATGGCGTGTGGGTGATGATGGATGGAGAGGAACAGATTG CGGCGACGATCAGGTGGAATTCCCGATCAAACCGCTCATTGACCACGCC GTCATGATGGACGGAAATGAGCAGGTTGAGTATCCTTTGAAACCAATAGTTG TTGTACACACCGCCCGTCGCTCCTACCGATTGAATGATCCGGTGAAATGT

NH2-ATGGTITGGTGYATHGAIAATGGC

Probe Sequence (5’ → 3’)

23 51 53 51 51 51 51 51 51 51 50 51 46 49 52 50

Length

Table 3.7: Microarray probe sequences designed in this project

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

A: Refers to location of the probe on the microarray in Figure 5.2. Probes were printed in four replicate patches across the array to provide replicate hybridisation intensity signal. An 18S rRNA control was also included (Table 3.3). B: Name of probes used on microarray with the names in brackets representing the strain of the virus most closely related to the probe. Virus acronyms: BCMV, Bean common mosaic virus; BCMNV, Bean common mosaic necrosis virus; BYMV, Bean yellow mosaic virus; CarVY, Carrot virus Y; CeMV, Celery mosaic virus; HarMV, Hardenbergia mosaic virus; PFVY, Passiflora foetida virus Y: PWV, Passion fruit woodiness virus; SMV, Soybean mosaic virus; TuMV, Turnip mosaic virus; ZYMV, Zucchini yellow mosaic virus. C: The General Poty Probe was modified to include a 5’ amino linker group to improve attachment to the slide surface D: The 18S rRNA probe was provided by Dr. Neil Boonham of CSL (Section 3.2.4).

III: BIOINFORMATICS

3.4 Discussion Choice of oligonucleotide probe sequences is crucial in determining the specificity of any microarray diagnostic test. The bias towards coat protein sequences available combined with the high conservation amongst strains made this the best region to design probes to. There was enough diversity between the nucleotide sequences of isolates to distinguish them from other members of the species. These probes targeted to the coat protein showed conservations amongst strains and diversity between strains/virus species. The large number of Potyvirus isolates sequenced, high error rate of RNA genomes and degenerate nature of the genetic code made identifying a region conserved amongst all Potyvirus species impossible, however by incorporating nucleotide analogues, such as deoxyinosine, at degenerate positions a universal Potyvirus probe could be designed. In the following chapters these primers will be shown to amplify and can be used to identify virus isolates. DNA arrays were created with oligonucleotide probes for plant virus detection and diagnosis.

57

IV: CHARACTERISATION OF HARDENBERGIA MOSAIC VIRUS

Chapter 4: Characterisation of an undescribed Potyvirus in the Australian native plant species Hardenbergia comptoniana

4.1 Introduction Plant virology in Australia has focussed mainly on the study of viruses of concern to economically-important plants. These viruses have often been introduced to Australia in infected propagules since European settlement in 1788 (1829 in Western Australia). Within the endemic plant species in Australia many native viruses have been identified. For instance collections of early European explorers show symptoms typical of Kennedya yellow mosaic virus (KYMV, Tymovirus) on Kennedia rubicunda from as early as 1802-5 in eastern Australia and 1839 in Western Australia (Gibbs & Guy 1979). In some cases it is difficult to determine the geographical and host plant origin of a virus. One notable case is Subterranean clover mottle virus (SCMoV), which has been identified only in Australia, but occurs only on introduced pasture legumes. Its geographic and original host, if not pasture legumes, remains unknown (R. Jones 2006 pers. comm.). More than 28 viruses have only been identified in Australia, however many of them are poorly characterised (R. Jones 2007 pers. comm.).

Many viruses believed to have an Australian origin belong to the Potyviridae. A survey of native orchids in eastern Australia revealed a number of previously undescribed Potyviruses including: Clitoria virus Y (ClVY), Diuris virus Y (DiVY), Eustrephus virus Y (EVY), Pleione virus Y (PlVY), Pterostylis virus Y (PtVY), Rhopalanthe virus Y (RhoVY) and Sarcochilus virus Y (SarVY) (Gibbs et al., 2000a). Other Australian Potyviruses include Apium virus Y (ApVY) (Moran et al., 2002), Ceratobium mosaic virus (CerMV) (Mackenzie et al.,1998), Dianella chlorotic mottle virus (DCMV) (unpublished), Hibbertia virus Y (HiVY) (KiratiyaAngul & Gibbs, 1992), Kennedya virus Y (KVY) (Dale et al.,1975), Passiflora virus Y (Parry et al., 2004), PFVY (unpublished), PWV (Taylor & Kimble 1964), Siratro 1 virus Y (S1VY) (unpublished and Siratro 2 virus Y (S2VY) (unpublished).

Viruses from Australia, within other families are: Cassia yellow blotch virus (CYBV, Bromovirus) (Dale et al., 1984); (Cardamine chlorotic fleck virus (CCFV, Carmovirus) (Skotnicki et al., 1992); Cardamine latent virus (CaLV, Carlavirus) (Guy & Gibbs 1985); Chloris striate mosaic virus (CSMV, Mastrevirus) (Francki et al., 1979); Digitara striate mosaic virus (DiSMV, tentative Mastrevirus) (Greber 1989); KYMV (Ding et al., 1990); Lucerne Australian symptomless virus (LASV, tentative Sadwavirus) (Remah et al., 1986); Maize sterile 58


stunt virus (MSSV, tentative Rhabdoviridae) (Greber 1982); Paspalum striate mosaic virus (PSMV, tentative Mastrevirus) (Greber 1989); Nicotiana velutina mosaic virus (NVMV, unassigned) (Randles et al., 1976); Solanum nodiflorum mottle virus (SNMoV, Sobemovirus) (Greber 1981); SCMoV (Sobemovirus) (Francki et al., 1983); Tobacco yellow dwarf virus (TYDV, Mastrevirus) (Helson 1950) and Velvet tobacco mottle virus (VTMoV, Sobemovirus) (Randles 1981).

The genus Hardenbergia contains two species, H. violacae and H. comptoniana (Figure 4.1). Both

are

perennial

climbers

of

coastal

regions

of

southern

Australia

(http://florabase.calm.wa.gov.au). H. violacae is endemic to the coastline of New South Wales, Victoria, South Australia, Queensland and Tasmania, and H. comptoniana is endemic to the southwest Australian floristic region (SWAFR) of Western Australia. The two species can be most easily distinguished by the number of leaflets present. H. violacae has one leaflet, H. comptoniana has three to five.

Figure 4.1: Typical morphology of Hardenbergia comptoniana and H. violacae, which occurs as a perennial climbing or twining shrub across Australia. A: Trifoliate leaves of H. comptoniana. B: Pentafoliate leaves of H. comptoniana. C: Monofoliate leaves of H. violacae. D: Form of H. comptoniana climbing over bottlebrush (Callistemon sp.). E: Form of H. comptoniana as a twining shrub. F: Close up of purple pea shaped flowers of H. comptoniana.

Surveys of native flora, including H. comptoniana, have found natural infections of TSWV in 19 species (Latham & Jones 1997) and BYMV in four species (McKirdy et al., 1994). However, neither virus was found in H. comptoniana. In another study, natural infection of H. comptoniana and H. violacae by TSWV and Impatiens necrotic spot virus (INSV) was reported 59


in North America (Sclar & Anisko 2001). There are no other reports of virus infection in H. comptoniana or H. violacae.

An aim of this research was to characterise a virus found infecting Hardenbergia plants of both species in the south-west coastal region of Western Australia, and one of H. comptoniana in Canberra, Australian Capital Territory. Another aim was to identify and characterise isolates of a virus observed in three Passiflora species from Kununurra, Carnarvon and Perth, WA.

60


4.2 Method and materials

4.2.1 Plant Samples and Virus Isolates Leaf samples were collected from symptomatic and asymptomatic H. comptoniana plants at 14 locations in the southwest of Western Australia (Conservation and Land Management Licence Number SW010462) (Figure 4.2 and Table 4.1). Symptomatic plants typically had mosaic patterns on leaves, and some with leaf distortion were also observed. Similarly, symptomatic leaves of P. edulis, P. caerulea and P. foetida plants were collected. Symptomatic leaves of a H. violacae plant purchased from a commercial plant nursery and a leaf sample of a symptomatic H. comptoniana plant was collected from a private garden in Canberra, ACT. Leaves from plants that appeared healthy were also collected. Leaves were freeze-dried and stored at 4oC.

Inset 1

Inset 2

Figure 4.2: Locations of sampled HarMV and PWV isolates. Sites sampled in Western Australia: map of the Australian continent showing the location of the southwest Australian floristic region (SWAFR): inset 1, boundaries of the SWAFR; inset 2, southern Perth region where most virus isolates were collected (site names in Table 4.1).

61

Site

Virus & Site NameA symptomsB

Predominant

symptomsC

% plants with

sequencedD

Virus isolates partially

Table 4.1: Study areas, sites, symptoms and isolates of HarMV and PWV isolates. Site type

HarMV 1 Seabird Coastal dune vegetation YS, YB, YR, M, LD 16 (167) Sb-3, Sb-5, Sb-6, Sb-15, Sb-19 2 Burns Beach Coastal dune vegetation M, LD ND (7) BB-1, BB-2, BB-6 3 Kings Park Nature reserve M, LD 20 (40) KiP-1 4 South Perth/Como Roadside vegetation M, LD ND (15) Co-1 5 State Herbarium Research facility (grounds) YB, M, LD 20 (50) He-1, He-2 6 Wireless Hill Park Nature reserve M, LD 73 (110) WHP-1, WHP-2 MU-1A, MU-1C, MU-2A, MU7 Murdoch University Research facility (campus) M, LD 28 (104) 3A 8 Cannington Plant Nusery M, LD ND (1) Cgt-1 9 Medina Research facility (verge) M, LD 52 (62) Med-1, Med-7 10 Wellard Roadside vegetation M, LD ND (1) Wel-1 11 Bunbury Roadside vegetation M, LD 20 (50) Bun-9 12 Margaret River Roadside vegetation M, LD 60 (62) MR-3, MR-13, MR-21, MR-30 13 Scullin, Canberra Garden M, LD ND (1) Can-1 PWV 4 South Perth Research facility (grounds) SM, LD 100 (40) Not sequenced 4 Como Garden MM ND (1) CoP-1 14 Kununurra Irrigation channel MM ND (20) Ku-1 15 Carnarvon Market garden M, LD ND (6) Car-1 16 Guildford Garden M, LD ND (5) Gld-1 A: Virus names: HarMV (Hardenbergia mosaic virus) = the name suggested for the Potyvirus isolated from Hardenbergia spp. and PWV (Passion fruit woodiness virus). PWV was isolated from Passiflora edulis (site 16), P. foetida (sites 14 and 15) and P. caerulea (site 4). B: Coded symptom descriptions: M, mosaic; SM, severe mosaic; LD, leaf distortion; YS, yellow spotting; YB, yellow blotchy mosaic; YR, yellow rings. C: Incidences based on numbers of plants with symptoms out of 40-167 inspected/site. ND, not determined (too few plants). Numbers in parentheses are total numbers of plants inspected for symptoms at each site. D: Isolates of HarMV used in host range inoculations shown in bold. Isolate Cgt-1 from site 8 was from H. violacae, but all others of HarMV were from H. comptoniana.


4.2.2 Virus inoculations Virus isolates of HarMV and PWV were inoculated on to indicator hosts and cultivated plants and grown in insect-proofed glasshouses by either sap or aphid inoculations.

For sap

inoculations, young infected leaf material was ground up and mixed with diatomaceous earth (celite) and chilled virus inoculation buffer (0.05M potassium phosphate buffer, pH 7.2). Young leaves of test plants were manually inoculated with this mixture and left in a humid environment overnight to recover. For aphid inoculations, non-winged Myzus persicae were (i) starved for 2 h and then given 10 min acquisition feeds on infected H. comptoniana or Nicotiana benthamiana leaves followed by 2 h inoculation feeds on N. benthamiana plants (5 aphids/plant), or (ii) starved for 12 h and then given 6 h acquisition feeds on infected leaves of N. benthamiana followed by 18 h inoculation feeds on H. comptoniana plants (10 aphids/plant). Cultures of HarMV and PWV isolates were maintained in N. benthamiana plants. These cultures were used for subsequent inoculations and to provide material for ELISA and RT-PCR.

4.2.3 Enzyme Linked Immuno-Sorbent Assay (ELISA) Leaf samples of plants were tested for the presence of HarMV and PWV using a Plate Trapped Antigen ELISA (PTA-ELISA) (Clark & Adams 1977) using a generic Potyvirus monoclonal antibody (Agdia Inc, Elkhart, IN, USA).

4.2.4 Incidence, host range and seed transmission studies Where sufficient plants were present in natural populations, the incidence of HarMV in a population was estimated by counting numbers of symptomatic and asymptomatic H. comptoniana plants.

Symptomatic and asymptomatic leaves were sampled from different

plants, and samples tested individually (symptomatic) or in groups of appropriate size (asymptomatic) as described previously (McKirdy et al., 1994). For test results from grouped samples, incidence was determined using the formula devised by Gibbs & Gower (1960) as shown below.

Percentage Incidence in asymptomatic plants =

1/groupC

1-

1-

+veA totalB

Where A = number of positive groups, B = total number of groups tested and C = the size of the group.

63


In host range studies, plants (two or more/species) were inoculated with infective N. benthamiana sap. Samples from inoculated and tip leaves were tested by ELISA for Potyvirus infection, three or 4-6 weeks after inoculation, respectively. Symptoms that developed were recorded. In seed transmission studies, seeds from N. benthamiana plants infected with seven different HarMV isolates were germinated and the seedlings tested by ELISA in groups of ten. An insufficient number of H. comptoniana seeds were available to test whether HarMV is seed borne.

4.2.5 RT-PCR, Cloning and Sequencing Total RNA was extracted from infected leaf material using the RNeasy Plant Mini kit (Qiagen, #74904) and the protocol provided by the manufacturer was followed. Leaf material used for HarMV came from infected H. comptoniana leaves or cultures maintained in N. benthamiana. For PWV leaf material came from infected Passiflora spp. leaves or cultures maintained in N. benthamiana. Virus cDNA was synthesised using the ThermoScript™ Reverse Transcription system (Invitrogen, #122336-014) following manufacturers instructions as outlined in Chapter 2. The primer used was a modified polyT primer (T7T24) with an antisense T7 primer adaptor sequence at the 5’ end (Table 3.1).

For amplification and sequencing of Potyviruses in legumes, degenerate Potyvirus-specific primers (LegPotyF and LegPotyR, sequences Table 3.4) were designed from alignments of fulllength sequences of legume-infecting Potyviruses as described in Chapter 3.

Polymerase chain reactions (PCR) were carried out to amplify the coat protein and 3’ UTR of the Potyvirus using 1st strand cDNA as template and supplied reaction buffer and LegPotyF and T7 primers, Pfu DNA polymerase (1.25units, Promega). A temperature regime of 94oC/5min, 10x (94oC/20s, 60oC-1oC/cycle/30s, 72oC/3min), 30x (94oC/20s, 50oC/30s, 72oC/3min), 72oC/7min, hold @ 4oC was followed. Further full length CP were amplified using virus specific primers (HarMV-CP F&R) and Pfu DNA polymerase as described above. Actin gene primers (sequences in Table 3.1) were used in amplification reactions to confirm the presence of cDNA.

The entire CP and 3’ UTR of five isolates of HarMV were cloned into the ZeroTOPO Vector (Invitrogen) (Chapter 2). A reaction mix consisting of 1µL of vector, 2µL PCR product and 3µL of H2O was incubated 10 min. Competent Top-10 Escherichia coli cells (Invitrogen) were transformed with the plasmid by manufacturer’s recommendations (Chapter 2).

A 50µL

aliquot of cells was plated out on LB Agarose plates with 100mg/ml Ampicillin and incubated overnight at 37oC. Individual colonies were screened by PCR for an insert using the following 64


reaction mixture: 1µM each of M13F and M13R primers (sequences in Table 3.1), 1x PCR buffer, 2.5mM MgCl2 and 1U Taq DNA polymerase (Fisher Biotech). o

o

o

Thermocycling

o

conditions were as follows: 94 C/5min, 30x (94 C/20s, 55 C/30s, 72 C/2min), 72oC/7min, Hold at 4oC. After thermocycling, a 10µL aliquot of the mixture was run on a 1% agarose gel to determine if a fragment of DNA was cloned.

Primers specific for HarMV CP and 3’UTR regions (HarMV CP-F & HarMV CP-R, sequences were designed according to sequence data obtained using primers LegPotyF and LegPotyR. These primers were used to amplify the CP of further HarMV isolates. Colonies containing cloned amplicons from isolates MU-1C, MU-2A, MU-3A, Sb-3 and Sb-19 were sequenced using M13F and M13R primers. Amplicons from other isolates of HarMV and PWV were sequenced in both directions.

4.2.6 Phylogenetic Analysis of Isolates Chromatograms of sequences were checked manually, and where necessary, annotated using Finch V3.1 and compared against the GenBank databases using BLAST. Bootstrapped trees of nucleotide and deduced amino acid sequences were made using MEGA and BioManager as described in Chapter 3. Full-length gene sequences were obtained from raw sequence data by removing primer sequences and other external sequences. Deduced amino acid sequences were generated from nucleotide sequences using translated BLAST.

Related sequences for

comparisons were downloaded from GenBank (NCBI) (Table 4.2).

Recombination analysis was done using the Recombinant Detection Program version 3.1 (Martin et al., 2005b) as described in Chapter 3 using default parameters. Sequences for all 28 HarMV isolates and 4 PWV isolates which were sequenced in this study were used. Also included was all available sequence data for the Potyvirus isolates included in Table 4.2. The only exception to this was that where full length sequences were available (e.g. BCMV #NC003397) these were trimmed to only include the 3’ terminal 3kbp which was sequenced for 4 isolates of HarMV (MU-1C, MU-2A, MU-3A, Sb-19). All sequences were aligned using CLUSTAL W in MEGA v3.1 as described in Chapter 3.

65


Table 4.2: Isolate names and database accession numbers of sequences used in phylogenetic trees. Virus Name*

Strain/Isolate

Location**

GenBank Accession

Reference

ApVY BCMV BCMNV

Cm R NL3

Australia: NSW China: Zhejiang USA: Michigan

AY049716 NC_003397 NC_004047

BYMV

MI-NAT

Australia: WA

AF434661

CarVY CerMV ClVY CABMV DsMV DiVY EAPV EVY

1 Z M13 AO -

Australia: Vic Australia Australia: Qld Zimbabwe China: Zhejiang Australia Japan: Kagoshima Australia

AF203537 AF022443 AF228515 AF348210 AJ298033 AF203527 AB246773 DQ098904

HiVY

Kiola

Australia: NSW

AF228516

PaVY PClV PFVY

RD-1711 #299 #386 Culnes Newrybar UniSyd K† TB† S† M† Pangda12 Pangda15 Ts Pterostylis G7 WMV-Fr Beijing TW-TN3

Indonesia: Papua USA: Florida Australia Australia: Qld Australia: Qld Australia: NSW Australia: NSW Australia: NSW Australia Australia Australia Australia Thailand Thailand Australia Australia Australia Australia Australia Australia USA France China Taiwan

AY461661 DQ860147 DQ112219 AJ430527 AY461662 U67149 U67151 U67150 1906186A P32576 P32575 P32574 AM409187 AM409188 AY968604 AF185958 AF185964 AF185956 AF185957 DQ098900 DQ098901 AY216010 NC_006262 AY656816 AF127929

Moran et al.,2002 Zheng et al., 2002 Fang et al., 1995 Jones 1992; Wylie et al., 2002 Moran et al.,2002 Mackenzie et al.,1998 unpublished Mlotshwa et al., 2002 Chen et al., 2001b Gibbs et al., 2000a Iwai et al. ,2006a unpublished Kiratiya-Angul & Gibbs 1992; Gibbs & Mackenzie 1997 Parry et al., 2004 Baker & Jones 2007 unpublished unpublished Parry et al., 2004 Sokhandan et al., 1997 Sokhandan et al., 1997 Sokhandan et al., 1997 Gough & Shukla 1992 Shukla et al., 1988a Shukla et al., 1988a Shukla et al., 1988a unpublished unpublished unpublished Gibbs et al., 2000a Gibbs et al., 2000a Gibbs et al., 2000a Gibbs et al., 2000a unpublished unpublished Hajimorad et al., 2003 Desbiez & Lecoq 2004 unpublished Lin et al., 2001

PWV

PStV PlVY PtVY RhoVY SarVY S1VY S2VY SMV WMV WVMV ZYMV

*ApVY, Apium virus Y; BCMV, Bean common mosaic virus; BCMNV, Bean common mosaic necrosis virus, BYMV, Bean yellow mosaic virus; CarVY, Carrot virus Y ; CABMV, Cowpea aphid-borne mosaic virus; CerMV, Ceratobium mosaic virus; ClVY, Clitoria virus Y; DsMV, Dasheen mosaic virus; DiVY, Diuris virus Y; EAPV, East Asian passiflora virus; EVY, Eustrephus virus Y; HiVY, Hibbertia virus Y; PWV, Passion fruit woodiness virus; PaVY, Passiflora virus Y, PClV, Passiflora chlorosis virus, PFVY, Passiflora foetida virus Y; PlVY, Pleione virus Y; PStV, Peanut stripe virus; PtVY, Pterostylis virus Y; RhoVY, Rhopalanthe virus Y; SarVY, Sarcochilus virus Y ; S1VY, Siratro 1 virus Y; S2VY, Siratro 2 virus Y ; SMV, Soybean mosaic virus; WMV, Watermelon mosaic virus; WVMV, Wisteria vein mosaic virus; ZYMV, Zucchini yellow mosaic virus. **ACT, Australian Capital Territory; NSW, New South Wales; Qld, Queensland; WA, Western Australia. † Sequences correspond to protein sequences (Shukla et al., 1998a; Gough & Shukla 1992) and as such, no nucleotide sequences were available.

66


4.3 Results

4.3.1 Symptoms of infection From April to November (autumn to spring) virus symptoms were often apparent in newly emerged leaves of H. comptoniana in the study areas. Where sufficient numbers of plants were available the incidence of symptomatic plants and typical virus symptoms were collected from 11 sites for HarMV and four sites for PWV. Typical virus symptoms included mosaic and leaf deformation (Figure 4.3B&C) on recently emerged leaves. Another symptom seen at the Seabird and State Herbarium locations consisted of bright yellow spots, rings and mosaic (Figure 4.3E). Symptoms of PWV included chlorotic spots, mosaic, leaf distortion and reduced plant growth (Figure 4.3F).

Figure 4.3: Typical virus symptoms of Hardenbergia mosaic virus (HarMV) and Passion fruit woodiness virus (PWV) showing symptoms in infected host plants. A: Uninfected H. comptoniana leaf (narrow-leafed form), B: Infected H. comptoniana with mild mosaic symptoms, C: H. comptoniana with severe mosaic and leaf distortion, D: Asymptomatic H. comptoniana (broad-leafed form), E: Infected leaf of H. comptoniana from Seabird showing yellow mosaic symptoms, F: PWV on P. caerulea with mottling.

In Hardenbergia plants infected with HarMV, often only some shoots were symptomatic while the rest were asymptomatic. The presence of both HarMV and PWV in symptomatic shoots was confirmed by testing leaf samples by ELISA with generic Potyvirus antibodies or by RTPCR using degenerate Potyvirus primers.

Virus presence within some, but not all,

asymptomatic shoots was confirmed by ELISA, using a number of asymptomatic shoots from a 67


positive sample. The results showed all symptomatic shoots were positive, however only some asymptomatic shoots were positive.

4.3.2 Virus Incidence A total of 678 wild plants were surveyed by ELISA for the presence of a Potyvirus. The percentage infection in asymptomatic plants was estimated (Table 4.3) and ranged from 0% (Bunbury), to 11.7% (Seabird) with a mean of 6.15%. The total percentage incidence ranged from 13.5% (Piney Lakes) to 72.6% (Wireless Hill Park), and the mean was 31.7%. Both the percentage of symptomatic and asymptomatic plants varied widely at the different locations surveyed. The total percentage of infection showed a close correlation with the percentage of symptomatic plants.

Table 4.3: Incidence of Hardenbergia mosaic virus in H. comptoniana plants at eight sites Location

% of Plants

% of Plants A

AsymptomaticB

Symptomatic

Total % Infected

Piney Lakes 9.5% (8/84) 4% (3/910 Bulks) 13.5% Murdoch University 28.4% (31/104) 9.3% (5/810 Bulks) 37.7% 72.6% Wireless Hill Park 63.6% (70/110) 9.0% (3/85 Bulks) 20.1% DAWA/Herb 17.5% (7/40) 2.6% (1/85 Bulks) 27.9% Seabird 16.2% (27/167) 11.7% (13/285 Bulks) 52.5% Medina 43.5% (27/62) 9.0% (3/95 Bulks) 20.4% Bunbury 20.4% (10/49) 0% (0/55 Bulks) 60.1% Margaret River 56.5% (35/62) 3.6% (1/65 Bulks) 31.7% (215/678) 6.15% 38.1% Mean A: values in brackets represent number of symptomatic plants out of total number of plants surveyed. B: values in brackets represent the number of bulk samples which were positive by ELISA, over the total number tested. The superscript represents the number of individual plants in each bulk sample. Percentages determined by formula of Gibbs & Gower (1960) see above.

4.3.3 Alternative hosts of HarMV The host range of HarMV was determined by inoculating nine isolates onto 19 species of plants. Of these, nine plant species in four families became infected (Table 4.4). Plants were tested for the presence of the virus in inoculated leaves and tip leaves to determine local lesion and systemic host species. Typical virus symptoms of HarMV isolates on indicator and crop species are included in Figure 4.4

68


Figure 4.4: HarMV symptoms on alternative host species. A: Mosaic symptoms on N. benthamiana, B: Necrotic spots on Chenopodium quinoa C: Necrotic spots surrounded by red rings on C. amaranticolor, D: Apical meristem death in Lupinus angustifolius.

69

Table 4.4: Plant responses to inoculation with nine isolates of the newly described Potyvirus from Hardenbergia spp.

Species Isolate and cladeA I I II IV V V V VI VII Symptom Group MU-1C Wel-1 He-2 MU-2A BB-6 Co-1 MU-3A Sb-3 Sb-19 Amaranthaceae Gomphrena globosa RSB RS RS RS RS RS RS RS Chenopodiaceae Chenopodium NS SI CS CS CS NS CS SI NS amaranticolor C. quinoa SI SI CS CS SI CS CS SI Fabaceae M, LD, M, LC, M, B, Lupinus angustifolius LC, B, S M, B, S M, B, S B, LC, S M, B, S LC, S B, S LD, S M, LD, M, LD, M, LD, L. cosentinii M, L, C M, LD, D LD, S M, S M, LD LD, S S, D S, D S, D M, LD, L. luteus M, LD, S M, LD M, LD, S SI M, LD, S M, B, LD M, LD, S S L. mutabilis MM MM MM MM SI Pisum sativum M M SI SI Solanaceae Nicotiana M, LD M, LD M M M, LD M, LD M M, LD M, LD benthamiana A: Leaves were inoculated with infective N. benthamiana sap. Samples from inoculated and tip leaves of all plants were tested for a Potyvirus infection by ELISA after 2-3 and 4-6 weeks, respectively. B: Coded symptom descriptions: B, bunching of young leaves; CS chlorotic spots in inoculated leaves; D, plant death; LC, leaf downcurling; LD, leaf distortion; M, mosaic; MM, mild mosaic; NS, necrotic spots in inoculated leaves; SI, symptomless infection in inoculated leaves only; S, stunting; RS, red spots in inoculated leaves; -, no virus detected by ELISA in inoculated leaves or tip leaf samples.


4.3.4 Host range inoculations Host range experiments showed that N. benthamiana was infected systemically by all nine isolates of HarMV tested, causing symptoms of mosaic and leaf distortion. Apart from isolate Sb-3, all HarMV isolates infected only inoculated leaves of members of the Amaranthaceae (Gomphrena globosa), and Chenopodiaceae (Chenopodium amaranticolor and C. quinoa). Systemic infection was induced by nearly all isolates (except Sb-3) in Lupinus angustifolius, L. cosentinii and L. luteus), causing severe mosaic symptoms, leaf distortion and plant stunting associated with minimal seed production (Figure 4.3 & 4.4).

Three further plant species were hosts for HarMV, however each species was infected by one isolate. Isolate MU-1C infected the legume species Medicago truncatula and Vigna unguiculate causing vein clearing on Medicago truncatula and necrotic local lesions on inoculated leaves in V. unguiculate. Trifolium subterraneum was infected by isolate He-2 causing vein clearing. Isolate Sb-3 was less virulent than the others only infecting N. benthamiana and L. cosentinii symptomatically and C. amaranticolor, L. mutabilis and Pisum sativum asymptomatically. Other plant species tested that did not support infection by any virus isolate included: Brassica rapa, Capsicum annuum, Vicia faba, Lycopersicon esculentum, N. glutinosa, N. tabacum, Apium graveolens, Daucus carota, and Passiflora edulis.

The HarMV isolates could be separated into six groups on the basis of host range (Table 4.4). The largest of these is Group V which contains three isolates. The isolates do not group according to sampling location. For example, Murdoch University isolates separate into three groups (Groups I, IV & V). Isolates from close to the metropolitan area: MU-1C, MU-2A, MU3A, Co-1, He-2 and Wel-1 have a host range with clear differences to those from Groups VI and VII (Sb-3 and Sb-19).

4.3.5 Aphid and seed transmission tests Inoculation using the aphid Myzus persicae with brief virus acquisition and inoculation times transmitted the virus successfully from symptomatic H. comptoniana leaves (isolate Co-1) to plants of N. benthamiana. Symptoms of mosaic or leaf distortion appeared within 2 to 3 weeks and a Potyvirus was detected using a generic Potyvirus antiserum in an ELISA on tip leaf samples from the inoculated plants.

Koch’s postulates were satisfied under glasshouse

conditions when healthy H. comptoniana plants inoculated with isolate MU-2A using aphids, developed the characteristic mosaic and leaf distortion symptoms in young leaves and a Potyvirus was detected in tip leaf samples by ELISA. Similar aphid inoculations transmitted isolates MU-2A and Co-1 from infected to healthy N. benthamiana plants. 71


No seed transmission of the virus was observed. When seed harvested from infected N. benthamiana plants was sown and leaves tested by ELISA, no virus was detected.

4.3.6 Amplification of virus genome fragments The full CP gene and 3’ UTR, as well as ~400bp of the NIb gene was amplified for virus isolates MU-1C, MU-2A, MU-3A, Sb-3 and Sb-19. Amplification products of PWV were obtained for isolates Car-1, CoP-1, Gld-1 (Figure 4.5).

The amplicon produced was

approximately 1.3kbp in size, which was of the expected size.

These fragments were

successfully cloned into TOPO-Zero.

A:

1

2

3

4

5

6

B:

7

8

9

10

11

1300bp 1000bp

Figure 4.5: Amplicons of the CP and 3’ UTR regions of five HarMV and four PWV isolates showing bands of expected size (1.2kbp). A: Amplicons of HarMV; Lane 1, 5µL of 100bp molecular weight marker (Fisher Biotech); Lane 2, MU-1C; Lane 3-MU-2A; Lane 4-MU3A; Lane 5-Sb-3 and Lane 6-Sb-19. B: Amplicons of PWV; Lane 7, 5µL of 100bp molecular weight marker (Promega); Lane 8, Car-1; Lane 9 CoP-1; Lane 10, Gld-1 and Lane 11, Ku-1.

Sequencing of coat protein genes and 3’ UTRs The coat protein genes of five isolates (MU-1C, MU-2A, MU-3A, Sb-3 and Sb-19) were determined by sequencing cloned PCR products. A BLAST search of the complete coat protein nucleotide sequence (819bp) of isolate MU-1C indicated it was from an undescribed Potyvirus species; the closest sequence was PWV-299 with 79% identity (E-value of 1e-180) over 743 of the 819 nucleotides searched. Other closely related viruses from the BLAST search were ClVY, S2VY, S1VY, HiVY & BCMV (BLAST results can be seen in Appendix 1). The name Hardenbergia mosaic virus (HarMV) is proposed for this virus.

The CP gene of HarMV isolates ranged from 819 to 834 nucleotides (271-277 amino acids) with a mode of 825 nucleotides (275 amino acids). The CP has a variable N-terminal of ~60 nucleotides. A DAG motif, like those involved in aphid transmission (Shukla et al., 1994), was 72


present twice in all isolates tested at amino residues 8-10 and 48-50. The remainder of the CP was more conserved amongst the isolates sequenced.

The CP genes of four isolates of the Potyvirus from P. caercula, P. edulis and P. foetida were sequenced. These had previously been determined by ELISA to be infected with a Potyvirus using the same generic Potyvirus antiserum described above. The CP gene sequence of the four isolates was 837 nucleotides (284 amino acids) and contained a variable N-terminal and single DAG motif at amino acids 27-29. Comparing the sequences to those on GenBank showed they to belonged to the species Passion fruit woodiness virus (PWV) and were most similar to isolate #299 from Queensland and more distantly related to the isolates from NSW.

Amplification and sequencing of full length Nuclear Inclusion B gene Two sets of primer pairs, used to amplify genome fragments from the genomes of BYMV and BCMV, were used with a HarMV (isolate MU-1C) cDNA template in an attempt to amplify other parts of the HarMV genome. These primer sets were designed by S. Wylie (2006, pers. comm.) and Cayford (2005). The sequences of the primers are shown in Table 3.1. The results of the PCR indicated that only BCMV primer pair 12 (partial NIa/NIb) allowed amplification of a HarMV genomic fragment (Figure 4.6). Four more isolates of the virus from Hardenbergia (MU-2A, MU-3A, Sb-3 & Sb-19) were amplified using BCMV primer pair 12 as shown in Figure 4.7. Amplicons were cloned and the sequence of the 750bp fragment showed homology to the NIa/NIb region of other Potyviruses, particularly BCMV group Potyviruses (Appendix 1), for all five isolates. No clones of the 850bp band were identified during colony screening.

M 1

2

3 4

5

6 7 8 M 9 10 11 12 13 14 15 16 M 17 18 19 20 21 22 23 24

750bp

Figure 4.6: Amplification of HarMV MU-1C with BCMV and BYMV sets of full length primers. M: 100bp molecular weight marker (Fisher Biotech), 1-8: BYMV primer pairs 9-16: BCMV primer pairs 1 F&R to 8 F&R, 17-24: BCMV primer pairs 9 F&R to 16 F&R.

73


M

1

2

3

4

5

1000bp

850bp 750bp

Figure 4.7: Amplification of a partial NIa/NIb gene of HarMV isolates using BCMV 12 F&R. Lane M, 5µL of 100bp Molecular weight marker (Fisher Biotech); Lane 1, MU-1C amplicon; Lane 2, MU-2A amplicon; Lane 3, MU-3A amplicon; Lane 4, Sb-3 amplicon; Lane 5, Sb-19 amplicon.

4.3.7 Hardenbergia mosaic virus specific primers Hardenbergia mosaic virus specific primers were designed to amplify the CP and NIb gene and where used to sequence further isolates. The sequences of the primers are shown in Table 4.5. Using these primers the CP of a further 23 HarMV isolates (to give 28 in total) were sequenced; for two isolates two clones were sequenced, giving in total 30 sequences (BB-1, BB-2, BB-6, Bun-9, Can-1, Cgt-1, Co-1, He-1, He-2, KiP-1, Med-1, Med-7, MR-3, MR-13, MR-21, MR-30, MU-1A, Sb-5, Sb-6, Sb-15, Wel-1, WHP-1, WHP-2). At least one virus isolate from each location surveyed was sequenced, as well as at least one isolate from each different symptom group. An amplicon of approximately 1kbp was seen for 28 of the 48 isolates tested. This amplicon consisted of the full coat protein gene with a 3’ part of the NIb and 5’ part of the 3’ UTR (Figure 4.8). These were sequenced in both directions and deposited in GenBank with accession numbers as listed in Table 4.6. The full length NIb gene of four isolates was also amplified.

Table 4.5: Names and sequences of HarMV specific primers Name

Sequence (5’ → 3’)A

TmB

CYCCTTACATTGCTGAATCAGC HarMV CP-F 54 GACTACGAGCCAATAACTGTG HarMV CP-R 52 CYCCMTAYATTGCWGAGTCAGC HarMV CP-2F 56 GACKGSGAGCCAATAACTCTG HarMV CP-2R 55 GCAACAGTGTCTGAATCTTC HarMV NIb-F 50 TTYTGRAGACGYGGCAC HarMV NIb-R 48 YTCTCTYAAYCTBAARGCAGC HarMV NIb2F 52 TGCGWVCCAGCRTCHACATC HarMV NIb2R 55 A: Abbreviations of extended IUPAC codes included in Lists of Abbreviations. B: The melting temperature for double stranded DNA, where the lowest temperature of a pair of primers was used as the annealing temperature in PCR reactions (Chapter 2).

74


A: 1 2

3 4

5 6

7

8

M 9 10 11 12 13 14 15 16 M 17 18 19 20 21 22 23 24

1000bp

B: 25 26 27 28 29 30 31 32 M 33 34 35 36 37 38 39 40 M 41 42 43 44 45 46 47 48

1000bp

Figure 4.8: Amplicons of full CPs of 48 isolates of HarMV. M: 5µL of 100bp molecular weight marker (Fisher Biotech). Lanes 1-48 Amplicons of; MU-1A, MU-1B, MU-2B, MU-2C, MU-4A, MU-5, He-1, Cgt-1, WHP-1, WHP-2, Sb-5, Sb-6, Sb-7, Sb-9, Sb-11, Sb-15, Med-1, Med-2, Med-4, Med-5, Med-6, Med-7, Med-8, Med-10, Med-11, PL-2, BB-1, BB-2, BB-5, BB-6, Bun-1, Bun-2, Bun-9, MR-3, MR-6, MR-11, MR-13, MR-21, MR-30, MR-31, Co-1, Wel-1, Can-1, He-2, MR-6rpt, MR-11(rpt), Can-1(rpt), Co-1(rpt).

Table 4.6: Results of molecular diagnostic tests on HarMV and PWV isolates and accession numbers. PCR test - primer pairs HarMV CP GenBank Accession Leg-PotyB HarMV Isolates Sb-3 + + + DQ898204 Sb-5 + + + DQ898205 Sb-6 + DQ898214 Sb-15 + + + DQ898213 + + + DQ898202, DQ898203 Sb-19C BB-1 NT + + DQ898189 BB-2 NT + + DQ898190 BB-6 NT + + DQ898188 KiP-1 NT NT + DQ898210 Co-1 + + + DQ898192 He-1 + + + DQ898209 He-2 + + + DQ898193 Cgt-1 + + + EF375606 WHP-1 + + + DQ898206 WHP-2 + + + DQ898207 MU-1A + + + DQ898194 MU-1C + + + DQ898195 MU-2A + + + DQ898196 MU-3A + + + DQ898197 Med-1 + + + DQ898200 Med-7 + + + DQ898201 Wel-1 + NT + DQ898208 Bun-9 + + DQ898191 MR-3 + + DQ898211 MR13 + + + DQ898198 MR21 + + + DQ898199 MR-30 + + + DQ898212 + + + EF375607, EF375608 Can-1C PWV Isolates Ku-1 NT + DQ898217 Car-1 NT + NT DQ898216 Gld-1 NT + DQ898215 CoP-1 NT + NT DQ898218 A: Tested by ELISA using generic Potyvirus monoclonal Ab (Agdia), B: Using LegPoty F/R or LegPoty F/T7 primers (Sequences Table 3.4), C: Two clones of each isolate sequenced, NT: not tested Virus isolate name

ELISAA test

75


From this sequence the HarMV-specific primer, HarMV-NIb-F, was designed as described in Chapter 3. A NIb reverse primer, HarMV-NIb-R was designed from the full coat protein sequences. These primers were used to amplify the full NIb gene of five HarMV isolates, however only four isolates were successfully amplified (MU-1C, MU-2A, MU-3A & Sb-19 (Figure 4.9). The 5’ portion of Sb-3 was not amplified (Lane 10; Figure 4.9). Generation of a single continuos sequence of the whole NIb showed the length of the four isolates sequenced to be 1551 nucleotides (517 amino acids).

1 2

3

4

5

6 M 7

8

9 10 11 12

2100bp 1300bp

Figure 4.9: 5’ and 3’ NIb gene of HarMV Lane M: 100bp Molecular Weight Marker (Promega); Lane 16: HarMV NIb F and HarMV NIb2R on MU-1C, MU-2A, MU3A, Sb-3, Sb-19 and He-2, Lane 7-12: HarMV NIb2F and HarMV NIb R on MU-1C, MU-2A, MU3A, Sb-3, Sb-19 and He-2.

4.3.8 The CP genes – Phylogenetic Inferences The CP gene sequences of virus isolates were compared with each other and with other virus species to determine the phylogeography of the species. Nucleotide (Figure 4.10) and deduced amino acid (Figure 4.11) sequences were aligned and compared. PWV-299 from Queensland was included as an out-group in both trees because it showed the least genetic distance to the HarMV isolates. The percentage difference between isolates was determined at the nucleotide and amino acid levels. Table 4.7 shows the averages of nucleotide and amino acid differences between groups. The actual figures of identity between individual isolates are in Appendix 2.

76

IV: CHARACTERISATION OF HARDENBERGIA MOSAIC VIRUS Bun-9

32 100

MU-1C Med-7

I

53 Wel-1

WHP-2

27

He-2

45

99

100

II

KiP-1

62

He-1 Cgt-1

III

MU-2A 66

MU-3A

54

IV

BB-6

100 88

WHP-1 99

41 99

99

Can1-1

MU-1A Can1-2

V

Co-1

100

63 Med-1

BB-1

96

96 BB-2 53

Sb-3 Sb-15

VI

100 Sb-5 86 Sb-6 100

Sb19-1 Sb19-4

100

76

VII

MR-13 MR-21

100

MR-3 100

VIII

MR-30 PWV-299

0.02 Figure 4.10: A bootstrapped neighbour joined phylogenetic tree of 30 full-length HarMV CP gene nucleotide sequences created using MEGA v3.1. PWV-299 was included as an out-group. Values at the forks represent the number of times out of 1000 the groupings occurred (shown as a percentage), thereby providing a confidence value of relatedness. The scale bar represents the number of substitutions per base. Roman numerals represent names of the clades.

77

IV: CHARACTERISATION OF HARDENBERGIA MOSAIC VIRUS Bun-9 100

MU-1C

I

Med-7

70

71 Wel-1

WHP-2

97

49

He-2

98 43

II

He-1 KiP-1 Cgt-1

80

MU-2A 47

57

MU-3A

54

BB-6

III IV

WHP-1

65 62 68

Can1-1 Can1-2

57

V

Med-1

74

MU-1A BB-1

63 BB-2

Co-1 Sb-3 100 Sb-6

VI

Sb-5 Sb-15 45

68 MR-13

MR-21 100

VIII

MR-3

66 MR-30

Sb19-1 100

VII

Sb19-4 PWV-299

0.02 Figure 4.11: A bootstrapped neighbour joined phylogenetic tree of 30 full-length HarMV CP deduced amino acid sequences created using MEGA v3.1. PWV-299 was included as an out-group. Values at the forks represent the number of times out of 1000 the groupings occurred (shown as a percentage), thereby providing a confidence value of relatedness. The scale bar represents the number of substitutions per amino acid. Roman numerals represent names of the clades.

78


Table 4.7: Average percentage differences of HarMV isolates using PWV-299 as an out-group. Nucleotide values are given in bold and amino acid values in italics.

I

I 0.80 0.51

II

III

IV

V

VI

VII

VIII

PWV

6.23

8.33

5.13

6.04

8.33

10.81

9.89

16.03

I

6.20

5.59

5.15

10.13

11.36

9.90

17.71

II

12.30 8.64

1.76 1.76 10.49 10.93

10.99

6.23 -

10.95 9.16

13.32 10.81

11.40 9.98

18.25 16.12

III IV

V

12.87

11.32

11.24

6.24

5.07 1.83 1.17 2.51

9.27

10.99

9.50

16.41

V

VI

16.64

16.12

15.97

15.84

16.47

0.00 0.34

10.77

7.75

15.33

VI

VII

17.76

20.42

19.59

17.83

19.46

18.49

0.36 0.24

10.82

15.82

VII

II

13.89

III IV

0.42 16.70 VIII 3.65 PWV 25.27 PWV 25.55 24.45 23.93 24.75 24.79 24.97 26.96 I II III IV V VI VII VIII PWV Clades III, IV and PWV only contained the one isolate and therefore within clade figures are not given. VIII

17.55

17.83

18.37

18.47

18.51

16.67

17.36

The phylogenetic trees of all 30 HarMV sequences grouped into eight distinct clades numbered I to VIII, with bootstrap percentages of at least 99 for each cluster (Figure 4.10). Within-clade variation was 0-3.6% nucleotide differences (0-1.8% amino acid differences) (Table 4.7) and between-clade variation was 6.2-20.4% nucleotide differences (1.8-13.3% amino acid differences). The mean difference between isolates was 13.2% (7.0% amino acids). Isolates MU-2A and Cgt-1 which did not group closely to any other isolates showed a minimum of 8.4210.1% difference, respectively, over the full length CP gene at the nucleotide level and were considered as separate clades (III and IV) during further analysis.

The bootstrap values

separating them from other clades (62 and 88, respectively) and relatively deep branch lengths support this conclusion (Figure 4.10).

Clades contained from one to nine isolates. The Seabird isolates of HarMV from the northernmost collection site were the most divergent from the other HarMV isolates. The Sb-19 sequences (cluster VII) were the most divergent with up to 20.4% divergence at the nucleotide level. The remaining four Seabird isolates clustered together (cluster VI) and also diverged considerably (16.6%) from all other isolates. Cluster VIII containing the four Margaret River isolates showed an average percentage nucleotide difference to cluster VII (isolate Sb-19) of 17.4%, while cluster VI (the four other Seabird isolates) showed 16.7% nucleotide divergence to cluster VII. Clusters VII and VI were 18.5% different from each other at the nucleotide level. The metropolitan isolates (clusters I to V) showed up to 16.6% difference at the nucleotide level to each other.

When deduced amino acid sequences were used to compare isolates, they grouped into the same eight clusters (Figure 4.11). The only difference in this analysis was the position of clade VII 79


in which the branch length was longer compared to the other clades in Figure 4.10. When compared to HarMV sequences, the sequence of PWV (isolate 299 from Queensland) was at least 23.9% different at the nucleotide level and 15.3% at the amino acid level.

The CP sequences of all HarMV and PWV isolates sequenced in this study were aligned with published Potyvirus sequences to determine relatedness (Table 4.2). Potyviruses were included if BLAST alignments indicated they were related. Alignments of both nucleotides and amino acids were represented as phylogenetic trees to show the relationship of HarMV and PWV to other Potyviruses (Figures 4.12, 4.13). The alignments showed all HarMV sequences clustered together in a single group with a high level of confidence (bootstrap value of 99%, Figure 4.12). HarMV is most closely related to five Potyvirus species described only from Australia: ClVY, HiVY, PWV, S1VY and S2VY. The HarMV isolates were 23.9% to 28.1% different to these viruses at the nucleotide level and 17.3% to 19.8% different at the amino acid level. HarMV was 27.8% to 34.2% different at the nucleotide level and 15.5% to 29.4% different at the amino acid level to other non-Australian members of the BCMV group of the Potyviruses (Table 4.8). BYMV which was included as an out-group was 40.3% and 43.2% different from HarMV at the nucleotide and amino acid levels respectively. Percentage differences of the BCMV group Potyviruses are given in Table 4.8. The percentage differences at the nucleotide and amino acid levels of all the Australian-only subgroup isolates are given in Appendix 2.

The four PWV isolates from WA showed closest identity with PWV isolate 299 from Queensland (2.23 to 6.45% identity, Appendix 2) forming a single clade (bootstrap support 100%) and were more distinct from the three New South Wales isolates (23.1 to 25.0% identity, Appendix 2), which formed a distinct group together. Two isolates of PWV recently reported from Thailand were only distantly related to other PWV sequences with 34.2 and 35.1% nucleotide difference from Australian PWV clades (27.6 & 27.9% amino acid difference, Table 4.8) and, therefore, are not considered to be members of the species for this study. This analysis shows with a high confidence value (bootstrap value of 98%, Figure 4.12) that both PWV and HarMV are part of the mainly legume-infecting BCMV group of Potyviruses (Adams et al., 2005b; Ward et al., 1991) which includes BCMV, BCMNV, CABMV DsMV, SMV and ZYMV.

HarMV together with the two clades of PWV, ClVY, HiVY, S1VY and S2VY formed an exclusively Australian-only subgroup within the BCMV subgroup. The two strains (clades above) of PWV were up to 24% different at nucleotide level and 12.7% at the amino acid level over the complete CP.

80


Figure 4.12: A bootstrapped neighbour joined tree of 66 full length Potyvirus CP gene nucleotide sequences created using MEGA v3.1 from an alignment on Clustal W. A, Australian-only subgroup of the BCMV group. Clades of the Potyvirus isolates from Hardenbergia spp. are numbered I to VIII; B, Remainder of the BCMV group containing members of the genus Potyvirus known only from Australia and non-Australian members; C, non-BCMV group members of the genus Potyvirus known only from Australia (one isolate/virus) and BYMV included as an out-group. Values at the forks represent the percentage of times out of 1000 the grouping occurred (shown only if greater than 75%). The scale bar represents the number of substitutions per nucleotide. Virus accession numbers are included in Table 4.6. The names of the clades of HarMV and PWV are included.

81

Figure 4.13: A bootstrapped neighbour joined tree of 70 full length Potyvirus deduced CP amino acid sequences created using MEGA v3.1. A, ‘Australian only subgroup of the BCMV group, clades of the Potyvirus isolates from Hardenbergia spp. are numbered I to VIII, additional PWV isolates TB, S, M & K are labelled; B, remainder of the BCMV group containing members of the genus Potyvirus known only from Australia and others; C, non-BCMV group members of the genus Potyvirus known only from Australia, and BYMV included as an out-group. Values at the forks represent the percentage of times out of 1000 the grouping occurred. The scale bar represents the number of substitutions per nucleotide. Virus accession numbers are included in Table 4.6.


82

Table 4.8: Percentage differences of BCMV Group Potyviruses. Nucleotide differences in bold and amino acid differences in italics.

26.1

25.2

22.7

20.7

21.1

19.7

28.8

24.1

24.8

23.6

42.1

42.1

41.0

42.4

S1VY

HiVY

S2VY

PWV-Qld & WA CliVY

HarMV

28.6

29.4

23.0

23.0

43.2 27.6

29.9

24.8

25.9

23.2 24.9

28.0

27.7

25.7

PWV-NSW

19.8 24.3

27.0

29.7

29.5

42.6

41.8

21.7

23.1

26.6

25.2

30.8

25.3

26.5

25.9

17.7

25.9

25.9

27.7

17.9

20.7

27.3

24.0 18.8

23.4

25.5

23.2

25.1

29.4

23.0 22.6

17.6

25.9

26.2

26.1

23.7

23.1 22.6 23.3

18.0

30.7

28.7

22.3

24.5 24.2 25.2 24.4

27.9

28.2

22.7

21.8 24.1 23.8 24.4

29.3

27.6

15.5

20.8 21.7 22.3 23.4

23.6

24.0

23.1

20.2 23.0 22.3 27.0

23.2

25.2

22.9

12.8 21.9 21.9 23.8

16.5

18.4

PStV

BCMV

24.2

14.5 17.8 21.5 21.9

24.6

24.1

42.1

41.8

24.1

15.6 18.7 16.2 21.2

23.7

25.2

22.8

22.4

22.2

19.4 17.9 16.1

22.7

22.3

17.6

17.6

19.7

16.1

18.6 -

15.1

24.6

25.1

22.3

22.3

18.6

15.4 13.5 -

21.9

22.4

21.7

23.2

18.3

22.8 22.6

22.2

27.5

28.2

18.9

4.84 5.03 22.2 20.2 24.5

26.6

22.2

22.2

26.4

25.6

19.7

26.8 23.9 23.7

27.7

20.4

25.1

23.7

19.0

27.5 23.5

23.9

24.4

22.9

22.6

19.8

PWV-Qld & WA CliVY 28.1

24.0

21.2

21.5

17.3

S2VY 26.8 24.0

15.7

16.1

HarMV

HiVY

23.9

20.7

19.6

8.01 12.9

S1VY 25.6

20.0

19.6

CerMV

28.9

30.4

30.7

33.0

33.2 30.7

31.7

30.8 30.7

32.3

29.6 28.6

32.5

29.0 28.0

33.9

29.5 31.0

31.2

33.2

-

23.5

-

22.0

20.2

24.4

21.9

20.4

24.4

21.9

21.1

17.6

18.8

18.0

25.3

22.6

22.6

25.3

26.6

25.5

26.0

25.8

24.6

28.2

29.1

26.7

26.7

26.2

28.5

24.2

25.4

21.4

24.2

26.2

23.8

22.2

19.5

19.9

25.3

24.4

23.5

44.0

42.5

42.1

PFVY

ZYMV

CerMV

EVY

SarVY

31.1

41.9

43.0

34.3

24.1

23.0

32.0

22.2

22.2

32.7

26.3

24.8

30.9

24.8

25.6

33.9

26.7

25.9

29.9

29.3

29.6

33.3

25.6

23.7

32.3

27.4

28.2

31.3

23.7

24.8

PFVY

21.1

21.1 33.1

33.3

-

30.4

32.0

10.0

33.5

33.6

-

30.7

30.4

20.0

30.5

PClV

BCMNV

31.4

43.2

40.2

EAPV

CABMV

31.5

20.4

17.2

42.1

43.2

30.8

17.6

18.4

23.1

22.9

31.1

23.0

17.2

19.5

20.7

32.6

24.5

22.2

23.4

21.8

32.8

28.5

23.4

24.6

25.8

32.8

25.6

23.0

31.0

27.6

30.9

20.4

19.9

27.3

32.5

17.9

14.9

-

24.0

31.1

-

32.2

-

14.2

30.9

32.7

-

30.7

32.2

25.0

26.6

32.4

29.3

32.6

28.9

EVY

35.6

SarVY

31.6

33.1 35.8

32.7

33.2

PWV-Thai

34.1 31.1

47.1

DiVY

DsMV

31.6

32.4

27.8

42.9

34.6 35.8

24.9

28.8

32.2

32.6

28.6

23.4

32.6 34.1

31.1

25.9

33.0

32.3

33.0

30.0

31.6 32.2

-

29.1

33.6

28.6

26.7 2.93 1.46 36.7

27.6

32.0

35.4

36.0

28.0

31.8

30.2

37.3

29.6

32.2

30.2

35.8

28.4

35.2

33.3

30.0

32.1

33.1

34.8

35.1

31.6

35.1

36.0

36.2

33.6

36.2

35.5

34.7

30.3

37.2

31.0

36.2

34.2

32.5

32.5

36.9

41.8

42.5

33.0

33.8

31.3

35.4

17.1

24.3

33.2

32.9

35.1

33.3

12.3

21.1

29.6

33.2 32.9

37.9

-

24.6

31.6

34.0

36.1 37.4

-

28.9

34.6 32.7 37.5

30.3

33.0

32.6

33.3 36.9

35.6

38.4

29.8

32.4 34.8 36.2

34.4

36.5

31.5

EAPV

CABMV

34.2 36.2

30.1

33.3

30.0

PWV-Thai 33.0

30.6

35.8

PClV

DsMV

28.8

33.3

SMV

WMV

WVMV

29.5

SMV

31.6

WMV

33.5

39.9

BYMV

35.4

7.7

-

41.8

32.4

-

37.4

-

39.7

31.6

21.3

37.3

16.0

33.7

30.4

40.1

24.3

36.6

31.9

42.4

32.4

34.3

31.4

40.5

36.8

31.1

28.6

42.9

35.0

30.0

29.3

41.5

32.4

29.2

29.6

40.3

31.6

31.0

30.9

43.2

30.9

29.3

31.9

40.5

31.0

31.2

31.9

43.1

34.6

31.1

30.1

41.5

33.5

33.1

33.0

41.3

33.7

31.9

29.8

42.4

37.8

32.7

25.9

42.3

32.7

33.2

26.8

39.2

31.3

34.1

28.8

38.5

31.2

29.6

28.6

39.3

33.2

34.1

31.7

39.9

32.7

29.9 29.4

42.0

35.6

33.2

29.8

39.2

32.2

31.2 29.2

39.9

32.3

33.4

29.2

39.6

32.4

29.0

SMV

40.3

30.8

32.8

WMV

DiVY

BYMV

WVMV

BCMNV

ZYMV

23.9

PWV-NSW

22.0

22.0

BYMV

22.2

WMV

22.6

SMV

20.9

WVMV

20.2

DiVY

-

DsMV

6.6

PWV-Thai

-

EAPV

13.2

CABMV

31.8

PClV

15.3 2.85 2.82 30.3

BCMNV

29.8

EVY

30.1

SarVY

29.4

PFVY

30.9

ZYMV

27.3

CerMV

29.9

PStV

28.2

BCMV

30.3

PWV-NSW

30.1

S1VY

30.0

HiVY

27.8

S2VY

28.6

CliVY

PStV

PWV-Qld &WA

BCMV

HarMV HarMV

PWV-Qld &WA CliVY

S2VY

HiVY

S1VY

PWV-NSW

BCMV

PStV

CerMV

ZYMV

PFVY

SarVY

EVY

BCMNV

PClV

CABMV

EAPV

PWV-Thail

DsMV

DiVY

WVMV

BYMV

Percentage differences of the BCMV group Potyvirus CP’s as measured by Old Distances (GCG). Isolates were first aligned with ClustalW in MEGA v3.1. Accession numbers of isolates used are included in Table 4.2 & 4.6.


It is interesting that the virus described as PWV falls into two separate groups (excluding the Thailand isolates and other incorrectly labelled isolates on GenBank now considered to be EAPV and CABMV) based on geographical locations, and there is good bootstrap support for this (100%). In fact, ClVY, HiVY, S1VY and S2VY fall between the two Australian PWV groups, indicating that the current classification of the NSW and Queensland PWV isolates as members of one species needs reconsideration.

The two groups of PWV showed 23.9% nucleotide differences and 12.8% amino acids to each other (Table 4.8). The PWV-NSW isolates share more homology at the nucleotide level with HiVY and S1VY than with the clade containing the Queensland and WA PWV isolates. Similarly the Queensland and WA PWV isolates share more homology with ClVY and S2VY than with PWV-NSW again at the nucleotide level. This is not seen at the amino acid level where the isolates only differ by 12.8%.

Some other Australian Potyviruses, including CerMV, EVY, PFVY and SarVY, closely aligned with ZYMV within the BCMV group. Diuris virus Y also grouped with the BCMV group even though it showed low identity with the other BCMV Potyvirus sequences (30.0% to 38.4% nucleotide differences, Table 4.8). Several other Australian Potyviruses did not group within the BCMV group, and these included the apiaceous Potyviruses ApVY and CarVY (Moran et al., 2002) and several orchid-infecting Potyviruses (PlVY, PtVY and RhoVY).

Four other passionfruit-infecting Potyviruses: CABMV, EAPV, PClV and PFVY, all clustered independent of each other, and separate from PWV. The Thai isolates of PWV showed most homology to EAPV, PClV and EVY (Table 4.8). A sixth species of passionfruit-infecting Potyvirus, PaVY, isolated from Queensland, was not included because only a partial CP sequence is available. A number of other Passiflora-infecting Potyviruses were not included because no sequences are available. These include: Passiflora crinkle virus (PCV, Chang et al., 1996), Passionfruit mosaic virus (PaMV, Martini 1962), Passionfruit mottle virus (PFMoV, Chang 1992) and Passiflora ringspot virus (PFRSV, De Wijs 1974).

Deduced amino acid sequences derived from the nucleotide sequences grouped the viruses in the same way as the nucleotide sequences, providing confirmation of the relationships determined by nucleotide alignments (Figure 4.13). The BCMV group was present with high bootstrap support (99%) and the Australian Potyvirus sub-group again formed with a high degree of confidence (bootstrap 77%) indicating that these viruses share a more recent ancestor. The existence of two sub-clades within the sub-group was suggested, one with HarMV from the west of Australia and the other with the species found mainly in the east of Australia (the two PWV groups, S1VY, S2VY, ClVY and HiVY). 84


Included as amino acid-only sequences are four additional isolates of PWV; TB, S, M & K (Shukla et al, 1988a, Gough & Shukla 1992) which, when aligned with deduced amino acid sequences, clustered with the other PWV isolates, confirming their identities. Isolates TB, S and M clustered with the Qld & WA strain of PWV, while isolate K clustered with the NSW strain of PWV (Figure 4.13).

4.3.9 Phylogenetic analysis of the 3’ UTR sequence The 3’ UTRs of five isolates of HarMV and three isolates of PWV was sequenced (Figure 4.5) and aligned with the corresponding sequences of 29 other Potyviruses and isolates from GenBank (Accession numbers given in Table 4.2). These were aligned and represented as a neighbour joined tree generated using MEGA (Figure 4.14). The length of the 3’ UTR was 251-255 nucleotides. The length of the polyA tail was not determined and was trimmed from all sequences used. The percentage differences are shown in the Table 4.9.

The five HarMV isolates formed a single clade with a bootstrap value of 68%. The greatest percentage difference between two HarMV isolates was 21.1%; between Sb-19 and the two isolates MU-1C and MU-2A. The most closely related virus, as determined by the 3’UTR, was PWV (Car-1) with 31.3% difference. The 3’UTRs of three PWV isolates (Ku-1, Car-1 and Gld1) were also included. These formed a monophyletic cluster with the two available PWV 3’UTR sequences from GenBank (#386 and #299). The Potyvirus spp. known only from Australia: ClVY, HiVY, S1VY and S2VY, all had relatively low percentage differences in the 3’ UTR compared to PWV and HarMV (27.0 to 38.2% nucleotide difference).

PaVY, another passionfruit-infecting virus, which was included, differs from PWV, EAPV, and CABMV (46.3%, 43.8% & 56.7% at the nucleotide level respectively Table 4.9), however groups closely to PFVY because it was only 13.5% different at the nucleotide level. This virus clusters most closely with other BCMV group Potyviruses such as BCMV, CerMV, WVMV, and EAPV. Other significant groupings included BCMV and PStV (bootstrap 100%) and SMV and WMV (bootstrap 84%).

85


Figure 4.14: Phylogenetic tree of 27 Potyvirus 3’ UTR nucleotide sequences created using MEGA v3.1. Values at the forks represent the percentage of times out of 1000 the grouping occurred after bootstrapping. The scale bar represents the number of substitutions per nucleotide. BYMV was included as the outgroup sequence. Virus accession numbers are included in Table 4.2 & 4.6.

86

HiVY S1VY PWV S2VY CliVY HarMV ZYMV BCMNV CABMV DsMV SMV WMV WVMV PFVY PaVY EAPV BCMV PStV BYMV HiVY

S1VY

31.0 35.4 35.4 38.2 51.6 43.3 54.1 53.6 40.0 40.0 37.6 49.0 46.8 37.6 40.8 42.0 56.9

PWV

2.12A 30.1 34.2 32.7 48.6 39.3 51.3 54.1 37.2 39.3 33.3 46.7 46.3 33.8 39.2 39.9 55.2

PWV S2VY

30.5 35.1 48.4 41.6 48.5 55.1 39.5 42.0 39.9 54.3 48.2 36.6 41.2 42.8 60.3

S2VY CliVY

37.4 50.2 47.3 52.8 57.6 36.2 40.3 37.5 48.1 45.7 37.5 36.2 39.5 59.8

CliVY HarMV

15.3A 44.7 42.9 48.6 57.6 41.5 41.3 37.6 49.7 48.7 37.0 45.3 45.5 58.5

HarMV ZYMV

55.9 49.8 58.7 47.0 46.5 49.8 66.8 52.6 47.4 52.1 53.5 73.6

ZYMV BCMNV

39.0 56.7 44.9 42.9 37.6 54.8 49.8 36.7 42.9 42.0 62.1

BCMNV CABMV

58.0 51.1 51.1 46.8 60.1 56.7 48.9 53.3 55.0 68.4

CABMV 53.8 53.4 51.8 61.1 57.8 57.0 57.0 55.8 67.2

DsMV SMV

17.1 28.2 43.8 41.4 32.8 37.1 37.9 61.5

SMV WMV

23.8 45.2 41.3 36.1 37.3 36.5 65.5

WMV

WVMV

47.1 43.9 30.2 32.2 33.7 63.2

WVMV

PFVY

13.5 45.2 39.4 38.9 56.3

PFVY

PaVY

43.8 37.9 39.3 57.5

PaVY

EAPV

35.9 37.7 60.9

EAPV

BCMV

9.8 60.9

BCMV

60.9

PStV

-

BYMV

Table 4.9: Percentage differences of isolates of aligned 3’ UTR’s.

23.6 27.0 34.8 36.5 35.9 51.2 42.5 53.7 54.9 35.6 36.1 33.5 52.4 45.1 35.2 40.8 39.5 62.1

S1VY

HiVY S1VY PWV S2VY CliVY HarMV ZYMV BCMNV CABMV DsMV SMV WMV WVMV PFVY PaVY EAPV BCMV PStV BYMV DsMV

PStV

BYMV

Percentage differences of 27 Potyvirus 3’ UTR nucleotide sequences as measured by Old Distances (GCG). Isolates were aligned with ClustalW in MEGA v3.1 A: Multiple isolate used (values represent the average of all isolates). Accession numbers of isolates used included in Table 4.2 & 4.6.

HiVY


4.3.10 Phylogeny of the HarMV NIb Gene The full length NIb gene sequences were aligned against 11 other full-length NIb sequences from other Potyviruses listed on GenBank (Table 4.2). Neighbour joined trees of nucleotide and amino acids sequences were constructed (Figure 4.15) and percentage differences of isolates (Table 4.10) showed the four HarMV sequences clustering together with high bootstrap values (100%). They clustered with isolates of WMV, SMV and WVMV (bootstrap value 86%).

The amount of diversity within the NIb of HarMV was from 8.06% to 22.5% at the nucleotide level and 3.09% to 9.09% at the amino acid level (Table 4.10). The closest virus to HarMV over the NIb was SMV with 28.3 to 29% nucleotide differences (16.1 to 17.2% amino acid differences) however full length NIb sequences of PWV were not available for comparisons. The BCMV12 F&R, HarMV NIb F&2R or HarMV NIb 2F&R primer pairs failed to amplify the NIb gene of the four PWV isolates tested (CoP-1, Gld-1, Car-1 & Ku-1).

88


Figure 4.15: Phylogenetic tree of 15 Potyvirus NIb nucleotide (top) and amino acid (bottom) sequences created using MEGA v3.1. Values at the forks represent the percentage of times out of 1000 the groupings occurred after bootstrapping. Scale bar represents the number of nucleotide substitutions per nucleotide. Virus accession numbers are included in Table 4.2 & 4.6.

89

-

6.2 8.63

8.23

12.5

11.8

11.6

11.9

10.4

11.0

15.4

15.7

16.2

17.2

15.4

16.7

16.7

18.2

14.7

17.0

16.1

19.1

16.7

16.8

16.7

17.8

16.7

18.8

17.2

17.4

18.4

17.1

19.0

17.6

19.7

18.0

17.9

18.6

17.3

DsMV

21.3

19.4

18.7

21.1

19.2

ZYMV

17.4

17.6

16.7

15.9

16.7

33.7

33.9

31.3

32.9

33.3

BCMV

EAPV

BCMNV

WVMV

WMV

SMV

Table 4.10: Percent difference matrix of NIb gene of HarMV and other selected Potyviruses (nucleotides in bold and amino acids in italics)

SMV 19.7 17.6

17.0

32.9

WMV 23.0 14.1

16.0

19.0

SMV

24.3 -

15.9

21.1

WVMV 26.4 14.9

18.8

WMV

27.0 14.3

21.3

25.6 -

21.3

BCMNV 28.2

21.3

PStV

WVMV

25.8

20.3

32.78

27.9 2.71

19.2

26.2 -

21.3

EAPV 28.4

18.2

BCMNV

29.7

21.1

27.0

21.1

29.4

21.5

28.6

20.7

BCMV

-

EAPV

29.4

14.6

27.9

Sb-19

27.1

32.7

30.2

18.2

28.9

22.1

PStV

22.0

BCMV

31.0

9.09

28.5

7.93

30.1

8.32

27.8

-

30.1

30.9

28.3

MU-1A

Sb-19A

33.5

PStV

32.1

19.6

31.6

23.5

29.1

22.8

29.7

8.12

29.5

7.74

30.3

-

28.3

22.5

MU-1AA

Sb-19

20.0

MU-2A 31.1

33.5

31.1

19.6

28.8

23.1

28.2

22.6

28.8

3.09

28.8

MU-1A

19.3

-

29.0

20.8

18.6

MU-2AA

31.3

MU-3A 31.8

33.9

28.9

19.4

28.4

MU-2A

31.1

23.3

29.5

31.2

22.6

28.8

31.7

-

28.5

30.9

8.06

MU-3AA

29.1

MU-3A

33.1

CABMV 31.0

33.3

33.5

28.6

33.3

19.2

27.5

32.8

20.4

30.2

33.0

-

29.3

32.9

31.4

CABMV

31.6

CABMV

31.1

DsMV 32.8

30.2

32.6

31.7

30.6

21.3

32.8

32.3

-

31.9

31.7

30.9

DsMV

33.0

ZYMV 32.7

32.6

32.4

31.3

33.5

31.7

BYMV

30.5

-

BYMV

Percentage differences of 15 Potyvirus NIb genes as measured by Old Distances (GCG). Isolates were first aligned with ClustalW in MEGA v3.1. Accession numbers of isolates used included in Table 4.2 & 4.6. A: Isolates of HarMV that were sequenced

32.8

39.4

BYMV

ZYMV

40.3

ZYMV 38.0

DsMV 39.6

CABMV

39.5

MU-3A

39.0

MU-2A

39.7

MU-1A

40.2

Sb-19

39.4

PStV

40.8

BCMV

38.9

EAPV

37.9

BCMNV

39.3

WVMV

39.8

WMV

BYMV

SMV


4.3.11 Evidence for recombination between HarMV isolates Incongruence was seen between the phylogenetic trees of isolates MU-1C and MU-2A (Figures 4.10, 4.14 & 4.15). The 3’ UTRs of both isolates were identical, but the partial NIa (NIa-Pro), the NIb and the CP genes of the isolates diverge enough to be placed in two different clades which indicates a recombination event. An alignment of a 3kbp contig of the 3’ end of the genome of isolates MU-1C, MU-2A, MU-3A and Sb-19 showed a change in pairwise identity consistent with recombination (Table 4.11). The alignment of nucleotide sequences showed that isolates MU-1C and MU-2A have undergone a recombination event within the CP region around nucleotide position 603. A second potential recombination event was seen between isolates MU-2A and MU-3A where the NIa-Pro and 3’ UTR show high nucleotide diversity (~15%, Table 4.11) while the NIb and CP show lower diversity (6-8%, Table 4.11).

Table 4.11: Nucleotide pairwise comparisons of four HarMV isolates in each genomic region. Isolates compared

NIa-ProA

NIb

CP

3’ UTR

entire region

1-366nt

367-1917nt

1918-2742nt

2743-2997nt

2995ntB

MU-1C to MU-2A MU1C to MU-3A MU1C to Sb-19 MU-2A to MU-3A MU-2A to Sb-19 MU-3A to Sb-19 Average

22.40 18.63 8.42 0.0 14.73 22.68 19.34 12.45 15.14 17.51 24.04 22.50 17.46 20.72 21.16 15.57 8.06 6.72 15.14 9.21 13.66 19.99 17.83 20.72 18.68 15.57 20.76 19.52 19.29 19.66 18.99 18.21 13.73 15.17 16.83 Figures represent the percentage diversity between sequences A: only partial sequences included which represent 366nt of the 3’ end of the NIa gene B: full length sequenced for MU-2A is only 2989nt because of a 6nt deletion.

A systematic check for recombination in the BCMV group Potyvirus sequences used in the thesis was conducted using the RDP program (Beta version 1.5; Martin et al., 2005b). Ninety four potential recombination events were detected but excluded from further analysis because of a high probability of them occurring by chance. Six potential events were detected which had a low probability of arising by chance (Table 4.12).

The P-value for each event differed

considerably between the different methods and the PhylPro method did not detect recombination between any of the sequences examined.

A plot of pairwise identity as

determined by the RDP method is included in Figure 4.16 for each significant event; however similar results were obtained for each method used where a P-value is included in Table 4.12 (data not shown).

91

Table 4.12: The locations and average probability (P) values of the six recombination events in Potyvirus sequences which were examined by nine recombination

MaxChi

Bootscan

GENECONV

RPD

Genomic region B

Position (bp) A

2.378x10-1

1.071 x10-1

3.371 x10-9

3.224 x10-6

4.712x10-11

NIb

Med-1 & Unknown 478-1776

A

9.148 x10-13

4.978 x10-2

6.909 x10-7

-

-

7.789x10-2

NIb to 3’ UTR

MU-1C & MU3A 1123-2997

B

-

4.725 x10-6

1.130 x10-6

1.578 x10-12

-

1.398 x10-4

1.474 x10-3

NIa to NIb

12-779

2.889 x10-43

-

6.992 x10-26

9.702 x10-16

1.123 x10-15

5.203 x10-37

3.923 x10-34

3.913 x10-31

CP to 3’ UTR

MU-2A & MU-1C 2402-3117

D

4.536 x10-9

4.553x10-32

-

6.579 x10-22

1.591 x10-1

7.590 x10-6

1.070 x10-3

2.505 x10-7

1.207 x10-10

3’ UTR

Sb-3 & EAPV 2865-3117

E

9.776 x10-17

4.120x10-21

-

1.697 x10-15

-

3.920 x10-10

3.657 x10-8

3.477 x10-8

9.659 x10-8

3’ UTR

Sb-3 & MU-3A 2865-3012

F

detecting methods.

Chimaera 1.807 x10-20

-

1.244 x10-26

7.756 x10-26

C

SiScan -D

7.719 x10-7

1.190 x10-3

Event

PhylPro 9.223x10-13

1.853 x10-6

MU-2A & Sb-19

LARD

5.318 x10-12

Sequences

3Seq

A: Start and end point of recombinant event in base pairs from the 5’ end of the alignment including spaces. B: Region of genome in which recombination occurred. NIa: Nuclear inclusion A; NIb: Nuclear inclusion B; CP: Coat protein; 3’ UTR: three prime untranslated region. C: P-values represent the probability that the recombinant region occurred by chance. Events were only included when they were detected by two or more methods with a P-value of less than 1x10-5. D: Methods for which a P-value is not included did not detect the event.

Average P-value C

Figure 4.16: Pairwise identities of six recombinant events detected by the Recombination Detection Program (RDP: Martin et al., 2005b) using the RDP method. A: Event A between Med-1 and an unknown isolate; B: Event B between MU-1C and MU-3A; C: Event C between MU-2A and Sb-19; D: Event D between MU-1C and MU-2A; E: Event E between Sb-3 and EAPV; F: Event F between Sb-3 and MU-3A. Blue shaded areas were not available for comparisons because of partial sequence information, and red areas represent probable recombinant areas as determined by the RDP method (Martin et al., 2005b).


The event between MU-1C and MU-2A, which was suspected from pairwise comparisons (Table 4.11), was confirmed by RDP (Table 4.16) as event D by eight of the nine methods. The location of the recombination event also matches the expected regions from Table 4.11 and nucleotide alignments. The P-values for the various methods ranged from 1.1x10-15 to 2.9x10-43 which provides good evidence that the event is real and did not occur by chance. The second suspected event (event B) between MU-2A and MU-3A was not detected by any of the RDP methods and therefore is unlikely to be a represent a real recombination event.

Five other events were detected using multiple methods in the RDP program in HarMV isolates. Three of these events were between two HarMV isolates (Event B, C and F: Table 4.12), one was between two virus species (Event D: Table 4.12) and one was between HarMV and an unknown virus (Event A: Table 4.12). While there was less agreement between the results for these methods, and some estimated a high probability of occurring by chance, all events were detected by at least two methods with a low P-value.

94


4.4 Discussion In this study, the biological and genetic properties of a newly-found Potyvirus are described. This virus causes a widespread mosaic disease in the native legume H. comptoniana and the name Hardenbergia mosaic virus (HarMV) was proposed. The experimental host range was narrow for nine isolates of the virus tested, but isolate-specific differences were observed. As expected, the aphid species M. persicae was found to transmit the virus non-persistently. A previously described Potyvirus of Australian origin, PWV, was found in three species of introduced Passiflora, and partially characterised. Partial genome nucleotide sequences of 28 isolates of HarMV and four isolates of PWV from WA were obtained and used to elucidate the phylogenetic origin of the two species.

4.4.1 Phylogeny studies based on the CP sequence The Potyvirus identified in Hardenbergia spp. showed a minimum of 24.3% nucleotide difference (15.8% amino acids) over the full-length coat protein gene from it closest known relative, PWV isolate #299 from Queensland. This level of coat protein diversity is higher than species demarcation values of 23-24% nucleotide diversity which have been suggested for the Potyviruses (Adams et al., 2005b, Fauquet et al., 2003, Ward et al., 2005). The phylogenetic trees of coat protein sequences (Figures 4.10 & 4.11) showed all isolates of the virus from Hardenbergia spp. cluster together with high bootstrap support. From this information the name Hardenbergia mosaic virus (HarMV) is proposed.

Neighbour joined trees showed that the 28 isolates of HarMV grouped into 8 distinct clusters which were present in the nucleotide (Figure 4.10) and deduced amino acid trees (Figure 4.11). These eight clades show high genetic diversity, particularly isolates Sb-15 and Sb-19 (Clade VI and VII) from Seabird which both gave bright yellow blotch and ring symptoms on H. comptoniana (Figure 4.3). This symptom was not seen elsewhere except for isolate He-1, however because this was collected close to the state herbarium where samples have been collected from a wide area, its origin is uncertain. It is not known whether the yellow symptoms reflect infection by the virus, genotype differences in the host, environmental conditions, or a combination of reasons. Clade VIII was also divergent and contains all four isolates from Margaret River which show up to 19.8% nucleotide and 11.7% amino acid diversity to all other isolates (Appendix 2). The nucleotide and amino acid diversity (up to 21.1% and 12.4% respectively) between the Seabird and Margaret River isolates, and between them and all other isolates, do not warrant classification as distinct species, instead they clearly belong to divergent strains of HarMV.

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Very little information is available on the diversity of the Potyviruses in undomesticated hosts. Several studies have looked at wild Potyviruses in domesticated plants that have an intrinsically low genetic diversity, for example Spetz (2003), however this is not the case with H. comptoniana.

What occurs for HarMV infecting H. comptoniana is a remarkably large

diversity within a small geographical region (13.2% nucleotide diversity), as opposed to the diversity seen worldwide in other Potyviruses eg: 11.52% nucleotide differences for 27 isolates of Yam mosaic virus (YMV), BYMV: 9.23% and Plum pox virus (PPV): 8.72% (Bousalem et al., 2000). Furthermore the Seabird population, which is an undisturbed population of H. comptoniana, contains isolates of HarMV which show up to ~18.5% divergence at the nucleotide level. Until host diversity can be determined quantitatively, I can only speculate that virus diversity is a function of this.

Although differences in morphological features (e.g. leaf size and shape, See Figure 4.3) between locations were obvious, H. comptoniana populations are classified as one species based on classical taxonomy. The morphological differences seen in H. comptoniana, which is an undomesticated plant, suggest genetic differences, but molecular taxonomy has not been applied. Differentiation of natural virus populations according to host plant species, as for KYMV, can be evidence of host-associated selection (Skotnicki 1996). Is the large diversity seen within HarMV typical of a virus population in a natural host, and how can this diversity be explained? Evolutionary theory predicts that genetic drift, founding effects and selection, such as adaptation to host genotype, can reduce genetic diversity and increase diversity between populations (Garcia-Arenal et al., 2001). It is unclear which is responsible for the diversity seen in HarMV.

Genomic divergence is thought to be roughly proportional to the evolutionary distance from a common ancestor (Van Regenmortel 2000), and a high degree of sequence diversity over a small geographic range is typical for a virus species that co-evolved with indigenous wild plants over a long period of time (Jones 1981; Spetz et al., 2003). These lines of evidence indicate strongly a proximity to the point of origin of the species and suggest that HarMV co-evolved within indigenous plants in the SWAFR, and was not introduced by Europeans during the relatively short period of cultivation since 1829.

Experimental host range and designation of virus strains The isolates of HarMV did not group into clades based only on sampling locations. For instance, isolates from Murdoch fell into three clades (Clade I, IV & V), while all isolates from Margaret River grouped together in Clade VIII. The clustering of isolates did however show a stronger correlation with the experimental host range (Table 4.4). In particular isolate Sb-3 96


(Clade VI) was unable to infect Gomphrena globosa and Chenopodium quinoa which were susceptible to all other isolates. Pisum sativum could also be used to distinguish isolates of Clade I, II and VI to which it was susceptible. Lupin species were useful in differentiating isolates such as L. angustifolius and L. luteus which were resistant to Sb-3 (VI). No host range was completed for isolates from Clade III (Cgt-1) or Clade VIII (MR-3) and this should be carried out for completeness.

Viruses are designated strain status when they have been characterised at the nucleotide and biological levels. The eight clades of HarMV (Figure 4.10) show greater than 10% nucleotide differences within the CP (except Clade IV which shows 5.4 to 7.6% difference to Clade V). The eight clades of HarMV should be regarded as representing eight strains of the virus and it is proposed to name them Strain I through VIII as indicated in the phylogenetic trees (Figure 4.10 & 4.11). Classification of newly-found isolates into strains would require sequencing the CP or inoculations onto several indicator species.

An Australian only subgroup to the BCMV group of Potyviruses Alignment of sequences of HarMV to the BCMV group of Potyviruses as seen in the CP alignments shows that HarMV belongs to this group (B: Figure 4.12 & 4.13). A BCMV group of Potyviruses has previously been seen in nucleotide alignments (Ward et al., 1991, Mink et al., 1994, Shukla et al., 1994, Berger et al., 1997, Desiez & Lecoq 2004) with members typically infecting leguminous plants and a common ancestor suggested for PStV (BCMV), WMV, ZYMV & PWV (McKern et al., 1991). The members of this group can be expanded to include CerMV, PFVY, SarVY, EVY, PClV and DiVY; all of which have only been reported in Australia. Previously unreported is an Australian only subgroup; which includes HarMV, PWV, S1VY, S2VY, HiVY and ClVY; that had high bootstrap support (96% at the nucleotide level) confirming a strong relationship. HarMV differed from the other Potyviruses in the Australian sub-group with nucleotide differences ranging from 24.1 to 30.6% (14.9% to 22.1% amino acids). This grouping together of Potyviruses further confirms the Australian origin of HarMV and indicates these Australian native Potyviruses have been present in Australia for a long time, diverging from a common ancestor as suggested for the ‘PVY group’ (Spetz et al., 2003).

Furthermore the separation of HarMV from other Australian Potyvirus species (Figure 4.12 & 4.13) indicates a distinction between the Potyviruses of eastern Australia and Western Australia, possibly related to the increasing aridity of central Australia. This occurred approximately 3-5 million years ago (Hopper & Gioia 2004) and caused the biological isolation of eastern and Western Australia.

HarMV is most closely related to PWV, especially the isolate from 97


Queensland, but it also shows high homology to other Australian Potyviruses species (HiVY, S1VY, S2VY & ClVY). This finding indicates the SWAFR is the centre of origin for HarMV. Until a survey for HarMV in the Canberra region can be carried out, the presence of the virus in eastern Australia cannot be ruled out. The isolate found in Canberra, and the isolate found infecting H. violacae from eastern Australia fall into different clades, suggesting the possibility that HarMV may be present in states other than WA. It is also reasonable to propose that both isolates originated in WA and were spread by the movement of plant material for nurseries.

Other Potyvirus species only reported from Australia, and presumably indigenous to it, including CerMV, PFVY, EVY and SarVY were present in the BCMV group (Figure 4.12b & 4.13b). Only EVY and SarVY grouped together with 100% bootstrap support indicating a common ancestor. All other viruses were independent of each other.

4.4.2 Phylogeny of the 3’ untranslated region The phylogeny of the 3’ UTR sequences was very similar to the phylogeny of the CP sequences. The analysis of all Potyvirus 3’UTR sequences grouped HarMV as a single clade separate from all other viruses (Figure 4.14). As with the CP gene, it was closest to PWV, ClVY, S1VY, S2VY and HiVY Table 4.8. BYMV was also only distantly related. The main difference to the CP analysis was that a distinct Australian Potyvirus subgroup did not form with good bootstrap support (Figure 4.14).

The remainder of BCMV group viruses showed the expected

relationships i.e. a clade of SMV, WMV and WVMV and BCMV and PStV.

The 3’ UTR sequences were less phylogenetically useful than coat protein sequences for two reasons. First the bootstrap support for the 3’UTR phylogenetic trees was lower than for the coat protein gene and predicted amino acid trees because the 3’ UTR is more variable (up to 73.6%, Table 4.8). Second fewer 3’UTR sequences were available for comparison compared to the coat protein. This included five species of the BCMV group (EVY, SarVY, DiVY, PClV & CerMV) however for PaVY a 3’ UTR sequence is available but no coat protein sequence. The limited number of sequences and the high genetic diversity of 3’ UTR sequences made estimating geographical origin impossible, however as Adams et al., (2005b) found, 3’ UTR sequences tend to be conserved within species and these results agree with the establishment of HarMV as a separate species of Potyvirus.

4.4.3 Phylogeny of the NIb gene The NIb gene showed the same high diversity and relationship to BCMV group viruses as seen in the CP alignments. The genetic distance of HarMV to other Potyvirus species is higher than the suggested value of 23.4% (Adams et al., 2005b), but the most divergent HarMV strains 98


show less than 22.5% nucleotide diversity (9.09% amino acid diversity, Table 4.10). This supports including all strains of HarMV as a new species of Potyvirus. The phylogenetic trees of amino acid and nucleotide sequences further support this conclusion. Both (Figure 4.15) show a distinct cluster of HarMV isolates clearly separated from the other BCMV group viruses.

These conclusions were similar for all the genomic regions examined at both

nucleotide and amino acid levels which improves confidence in them.

The lack of sequence available for the NIb, particularly for other Australian Potyviruses such as ClVY, S1VY, S2VY and HiVY, which limited the amount of information gained. To try and correct this I attempted to amplify and sequence the full NIb of PWV which is not available. Both the BCMV primers and HarMV primers failed to produce a band and given the high diversity seen between HarMV isolates, this was not surprising. Other approaches to increase sequence information such as genome walking, could be used instead.

4.4.4 Determinants of virus symptoms and host range It is unlikely that the yellow symptoms seen in H. comptoniana are caused solely by differences in the virus; interactions of the virus and host are responsible for symptom development. The diversion of plant resources into the synthesis of virus nucleic acids and proteins, as well as disruption of normal cellular processes probably lead to the generation of plant symptoms (Revers et al., 1999). The HC-Pro as a suppressor of post transcriptional gene silencing may have an effect on both symptom expression and virus host range (Revers et al., 1999; Hull 2002).

H. comptoniana is a wild plant with a range of leaf morphologies (Figure 4.3),

presumably reflecting its genetic diversity. For example, at Seabird, leaves of H. comptoniana are large and broad, while in metropolitan locations some plants have leaves that are short and narrow.

It is, therefore, reasonable to speculate that the variety of symptoms to HarMV

infection is a function of the host as much as the virus. Environmental factors may also play a role as the climate ranges from a cool, inland, high rainfall area (Margaret River) to warmer and drier coastal sand dunes (Seabird) in the areas sampled. An investigation into the causes of symptom variety should look at the role of host diversity in symptoms as well as isolate diversity. Inoculations of virus isolates onto H. comptoniana plants sourced from distant locations, especially isolates causing yellowing from Seabird onto host plants from Margaret River and visa-versa, would indicate whether differences in the host or the virus cause the yellow symptoms seen.

There was no evidence that any other virus, in addition to HarMV, was present in symptomatic leaves of H. comptoniana. No unusual symptoms that might have indicated the presence of another virus developed on inoculation of sap from symptomatic leaf tissue to indicator hosts. 99


Also, extracts from symptomatic leaf samples were always positive when tested by ELISA and/or RT-PCR using generic Potyvirus antibodies and primers respectively. However, it is possible that other viruses co-infected the plants. Not all viruses induce symptoms on indicator plants or would be detected by the molecular and serological tests used. This could be further investigated by electron microscopy on sap preparations for virus particles, or using degenerate primers as given in Table 1.1.

That infection with HarMV causes severe symptoms in L. angustifolius, L. cosentinii and L. luteus is an important finding because southwest Australia supports the world’s largest lupin industry, exporting its grain to many other parts of the world. A severe disease that greatly diminished seed set would be a concern for the lupin industry. The symptoms caused by nonnecrotic strains of BYMV in L. angustifolius resemble those caused by HarMV (R. Jones 2006, pers. comm.) and past surveys for BYMV in wild populations and crops of lupins were mainly based on recording symptomatic plants (Cheng & Jones 1999). Inadvertently, such surveys may have attributed symptoms of HarMV infection to non-necrotic BYMV strains.

Further varieties of lupins should also be tested against more HarMV strains to determine the potential risk to agriculture. This is also true for the other locally important crop legumes in the region that became infected with some isolates of HarMV (P. sativum, field pea; M. polymorpha, burr medic; T. subterraneum, subterraneum clover).

This work would allow

variations in susceptibility and resistance to be identified as was done for BYMV in L. angustifolius (Cheng & Jones 2000). Infection of a host requires an interaction of both host and virus genes to allow replication of virus genes followed by cell-to-cell movement through the plasmodesmata, and finally longer distance movement through the vasculature. If at any level the interactions break down, then the host is resistant. Testing of further varieties would help to identify whether R genes for other viruses in legume hosts provide protection against HarMV.

4.4.5 Phylogeny of Potyviruses infecting Passiflora species A number of Potyviruses naturally infect Passiflora species including PWV, CABMV, PClV, PaVY, PFVY and EAPV. The species PWV, PaVY and PFVY have only been reported in or around Australia. PWV provides an example of a new encounter between an indigenous virus, that has apparently emerged from an unknown indigenous host, possibly Passiflora aurantia, to infect introduced Passiflora and legume species, both cultivated and wild. Its CP gene sequences show a similar high level of diversity to the HarMV CP gene, suggesting its origin too is within the Australian native vegetation (Jones 1981; Thresh 1982).

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The PWV sequences from Western Australia are closest in identity to the PWV isolates from Queensland. These isolates form a single clade which is distinct from the NSW strain of PWV and from other Australian Potyviruses, including HarMV. PWV isolate #386 probably belongs to the Qld & WA clade based on its 3’ UTR sequence however a full length CP sequence would confirm this. Two clades of PWV from Australia have been reported based on amino acid sequences (Gough & Shukla 1992) and the low level of identity between the two clades of PWV (Appendix 2) confirms the suggestions of Adams et al., (2005b) that they should be reclassified as distinct species. The name PWV should be retained for the clade containing the Queensland isolate and the four Western Australian isolates because its members were described first (Shukla et al., 1988a). The name Passiflora mosaic virus (PfMV) is suggested for the clade containing the three New South Wales isolates sequenced by Sokhandan et al., (1997).

The virus reported from Thailand (isolate Pandga 12 and 15) is incorrectly named PWV, presumably because it causes a woodiness disease on Passiflora species.

Passionfruit

woodiness disease is caused by several other viruses including CABMV and EAPV (Nascimento et al., 2006; Iwai et al., 2006b; Adams et al., 2005b) and Cucumber mosaic virus (CMV, Magee 1948; Taylor & Kimble 1964) and is of limited diagnostic value (Taylor & Greber 1973). To date only isolates from Australia causing Passionfruit woodiness disease have been attributed to PWV (Gough & Shukla 1992; Shukla et al., 1988a; Sokhandan et al., 1997). The low nucleotide and amino acid identity over full length coat protein sequences to both Australian clades of PWV (Table 4.8) confirm the misnomer. The Thai isolates were most closely related to EAPV and PClV where they showed 77-78% CP nucleotide identity, which is enough to consider it a new species. The name Passion fruit mosaic Thailand Virus (PFMTV) is proposed for this virus. The Thai isolates therefore represent a newly described Potyvirus causing disease in Passiflora (Table 4.8).

No complete Passiflora virus Y (PaVY) coat protein sequences were available for phylogenetic comparisons. However the 3’ UTR alignment shows that is it a different species of Potyvirus which is more closely related to SMV, WMV and WVMV than to the Australian Potyviruses (Figure 4.14).

This agrees with the finding of Parry et al., (2004), who found 3’ UTR

nucleotide differences of 59.1%, 62.2% and 46.9% to PWV, CerMV and ZYMV respectively. The alignment of the 3’ UTR (Figure 4.14) showed that PaVY and PFVY are closely related with only 13.5% nucleotide difference (Table 4.9).

This is well below the percentage

difference of at least 28.1% in the UTR found by Adams et al., (2005b) as the delineation point between Potyvirus species. Further sequencing of the PaVY CP needs to be carried out to establish the taxonomic status of this proposed species. If the CP sequences show the same low diversity as seen in the 3’ UTR, then PaVY and PFVY should be classified together as isolates of PFVY, as this species was described first. 101


No sequences are available for PCV, PaMV, PFMoV and PFRSV although host range differences have been reported between some of these viruses (Chang et al., 1992; Chang et al., 1996; De Wijs 1974).

Coat protein sequences, however, will provide more accurate

classification of these species and allow synonymous virus names to be identified and removed. Given the divergent strains of virus reported causing woodiness disease of passionfruit in Australia (Gough & Shukla 1992) such misnomers may be common and this will simplify the taxonomy of Potyviruses.

4.4.6 Recombination in Potyviruses Recombination has been reported in plant viruses, including within and between several species of Potyvirus, for example within species recombination - PPV, Cevera et al., (1993); PVY, BCMV, BYMV, ZYMV, Revers et al., (1996); YMV, Bousalem et al., (2000); TuMV, Ohshima et al., (2002), BYMV (Chare & Holmes 2006) and between species - Cucurbit aphidborne yellows virus (CABYV) & Pea enation mosaic virus -1 (PEMV-1): Gibbs & Cooper (1995); WMV & SMV: Desbiez & Lecoq (2004). Co-infection of two parental BCMV isolates resulting in daughter isolates showing recombination of phenotypic characteristics has also been reported, however subsequent nucleotide sequencing was not carried out (Silbernagel et al., 2001). Recombination is thought to play a role in the evolution of virus genomes by removing mutations, by combining different mutation free parts of the genome, and helping spread advantageous traits (Worobey & Holmes 1999). Isolates of HarMV and other BCMV group Potyvirus sequences were checked for recombination by pairwise comparisons and using the Recombination Detection Program (RDP: Martin et al., 2005b).

Recombination in MU-2A In HarMV a recombination event was seen when comparing isolates MU-1C and MU-2A in phylogenetic trees (Figure 4.10, 4.14 & 4.15). A sharp change in nucleotide diversity was seen in the CP in pairwise comparisons of HarMV isolates (Table 4.11). Within species of the Potyvirus there is up to 28% variation in the 3’ UTR, which is only slightly more than other genes such as the CP (up to 22.0%) and NIb (up to 23.4%) and is considerably lower than for the 5’ UTR (up to 40.3%) and P1 gene (up to 38.6%; Adams et al., 2005b).

The difference

seen in the 3’ NIa (22.4%), NIb (18.6%) and the 3’ end and core of the CP (11.4%) is normal for strains of HarMV; however the 100% identity from nucleotide 603 of the CP until the end of the 3’ UTR is unusual and indicates a recent recombination event. This recombinant event was detected by eight of the nine methods employed in the RDP with a low probability of it occurring by chance (Table 4.12).

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A change in pairwise identifies was also seen in different genomic regions between isolates MU-2A and MU-3A (Table 4.11), but this event was not detected by any method in the RDP program. The RDP method of Martin & Rybicki (2000) uses a sliding window of 30nt to compare triplets of sequences and looks for a change in the relative distance of the three sequences to each other which it identifies as a potential recombination event. However the pairwise comparisons in Table 4.11 examined relatedness in whole genome regions of hundreds of base pairs in length. Therefore the high percentage homology in the NIb and CP of MU-2A and MU-3A represents closely related sequences without the corresponding change in relatedness to a third sequence. It is therefore unlikely any recombination has occurred between these sequences.

A second recombination event was identified in MU-2A by the RDP program with Sb-19 located in the NIa and NIb gene (Table 4.12). This was detected with a low P-value by seven of the nine methods which indicate it is real and not an artefact, which could occur if only a single method is used. This event could not be identified in the pairwise comparisons in Table 411 because high diversity (13%) remained in the NIa gene following recombination, which could be due to mutations which can obscure older recombination events (Posada & Crandall 2001).

Isolate MU-2A appears to be a recombinant of HarMV isolates MU-1C and Sb-19 as seen in the results of the RDP program. Two likely events were identified (Table 4.12) which together make up ~52% of the length of the sequence analysed. Recombination has been suggested as a mechanism to allow expansion of a virus host range, for example in WMV, which is a recombinant of BCMV and SMV (Desiez & Lecoq 2004). This recombination may explain why isolate MU-2A does not group together with any other isolates studied (Figure 4.10). Although the CP and NIb proteins, which are the result of a recombination in MU-2A, have roles in virus replication and movement (Revers et al., 1999), there is no evidence of an increased host range or recombinant virus symptoms (Table 4.4).

Recombination in other isolates Recombination was suggested in other HarMV isolates including Med-1 (event A),

MU-1C

(event B), MU-3A (event C & F) and Sb-3 (event E & F). Of these events 1 and 6 are unlikely to represent real events because of the short region of sequence information available. In the case of the Med-1 isolate recombination was detected across the whole coat protein (Figure 4.16A). This is likely to be an artefact of the program and not a real recombination event because of the high diversity in the 3’ and 5’ ends of Potyvirus coat proteins. Sequencing 3’ UTR and NIb of Med-1 with subsequent recombinant analysis may confirm this. 103


Detecting mixed infections Co-infection of parental sequences is required for recombination to occur (Worobey & Holmes 1999). Three clades are present on the Murdoch campus so co-infection could occur, but the detection method was not designed to pick up co-infection. Direct sequencing may show coinfections as double peaks in chromatograms as long as both templates are amplified evenly, however sequencing of multiple clones could pick up sequences present at a low level. Divergent variants of isolates co-infecting a single host were seen in isolates Sb-19 (Sb19-1 & Sb19-4) and Can-1 (Can1-1 & Can1-2). Two variants of the CP of each isolate were sequenced which showed 0.36% and 2.55% nucleotide divergence, respectively (Appendix 2).

Recombination of viruses probably arises when the RNA dependent RNA polymerase switches templates during replication (Simon & Bujarski 1994).

To persist, the recombinant must

demonstrate a selective advantage over other non-recombinant virions. Recombination (and mutation) may occur quite frequently but the majority of recombinant sequences/mutants are removed by natural selection. Those that can be identified, such as the ones here, probably have beneficial combinations of advantages mutations, or have removed deleterious mutations promoting their fitness. For instance the recombination that produced WMV from SMV and BCMV gave rise to a virus with a broader host range than the parental isolate (Desbiez & Lecoq 2004). Successful recombinants probably arise very rarely because, over time, they would increase homogeneity, whereas considerable diversity was seen between isolates, even those taken from plants growing beside one another.

Methods of detecting recombination Recombination can be detected in sequences by several methods. Phylogenetic reconstructions of multiple regions of sequence regions can indicate recombination has occurred because of chances in the relative positions of sequences which is how a potential event was detected between MU-1C and MU-2A (Figure 4.10, 4.14 & 4.15). The limits of this approach are that multiple regions must be examined and compared, which as no automated methods are available, must be done by eye making it time consuming and inaccurate. Similarly pairwise comparisons of HarMV isolates (Table 4.11) indicated several events, some of which were false positives, and missed others.

Perhaps the easiest, and most accurate method, is the use of computer programs to examine multiple sequence alignments of which many are available. The program chosen (RDP: Martin et al., 2005b) combines nine methods in a single, easy-to-use Windows compatible program. Using this program six recombination events were detected by the various methods with varying 104


probabilities (Table 4.12). As no single method can be judged to detect recombination best under all conditions (Posada & Crandall 2001; Posada 2002) this gave reliable detection of recombination.

Comparisons of 14 methods using real data sets indicated that MaxChi and

RDP work well (Posada 2002) which is in agreement with simulated data using the same methods (Posada & Crandall 2001).

4.4.7 Surveys of Western Australia The SWAFR is a relatively wet, island-like refuge, isolated from other ecosystems by a buffer of arid lands and oceans. The drying of central Australia started in the Oligocene (25-45Mya) with the separation of Australia from Antarctica enclosing the SWAFR as early as 30Mya (Hopper & Gioia 2004 citing Frakes 1999). Currently 7400 indigenous higher plants are recognised and when the taxonomic survey is complete the region will have at least 8000 indigenous plant species (Hopper & Gioia 2004), with an estimated endemism rate of 53% (Beard et al., 2000). Although the SWAFR represents one of the worlds plant biodiversity hotspots, plant viruses in the indigenous flora of the region have been almost completely ignored, with HarMV being the first indigenous virus species described. It is expected that there are many more indigenous species of viruses within the flora of the SWAFR.

The PCR assay developed in this study using the HarMV CP F&R primers, combined with direct sequencing, was a quick and simple method for strain determination. The natural range of H. comptoniana extends further south to Albany and isolates from this region should be collected and sequenced. Due to the large amount of diversity seen at Seabird and Margaret River sequencing of more isolates from these regions should be carried out, but could not be done because of time constraints. The status of HarMV in the leguminous crops L. angustifolius, M. polymorpha and T. subterraneum should also be determined by collection and PCR based assays.

A survey of Australian native orchids for Potyvirus infection (Gibbs et al., 2000a) found four new species. Furthermore, a survey of ten other Australian wild plant species by KiratiyaAngul & Gibbs (1992) found serological evidence of Potyvirus infection in seven wild plant species which were subsequently sequenced by Gibbs & Mackenzie (1997) who identified the isolates as six new species of Potyvirus (CVY, EVY, HVY, KVY, GVY). Similar surveys in Western Australia should be undertaken, particularly as the region has a rich orchid flora, for example within the genus Caladenia, which includes the spider orchids, there are 162 described species. Within the family Fabaceae, 540 indigenous species are described (Hopper & Gioia 2004). It is reasonable to assume that this rich native flora harbours many new virus species.

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During the collection of H. comptoniana samples potential virus symptoms on other native plants were looked for, both as alternative hosts for HarMV and other virus species. The only other plant with virus symptoms noted were Kennedia prostrata, which were subsequently found by sequencing to be infected with BYMV (Webster et al., 2007). BYMV had previously been shown to infect several species of native legume in Western Australia including K. prostrata, K. coccinea, Hovea elliptica and H. pungens (McKirdy et al., 1994) in a limited survey. Clearly, more work should be done on the virus pathogens, both native and introduced, of the SWAFR.

Aphid transmissibility was confirmed using Myzus persicae to transmit the virus from symptomatic H. comptoniana to N. benthamiana. However, Myzus persicae is not native to the SWARF and natural vectors for HarMV remain unknown. An aphid species found feeding on H. comptoniana during the collection period could not be identified. It is highly probable that aphid species native to the SWAFR will be found to transmit HarMV and future surveys for native viruses should also aim to identify the natural vectors responsible for virus transmission. Natural mechanisms for virus transmission will also play an important role for the potential impact of HarMV on cultivated lupin species.

4.4.8 Surveys of Eastern Australia One isolate of HarMV was collected from an atypical host, H. violacae (Cgt-1), and one isolate was collected from beyond the borders of the SWAFR, Canberra (Can-1). Both species of Hardenbergia are often sold as ornamental plants, particularly in native gardens because of their striking purple flowers and movement of plant material is likely to have occurred.

The

occurrence of HarMV infecting the eastern Australian species H. violacae within the regions and H. comptoniana in a garden in eastern Australia do not necessarily alter the conclusion that HarMV evolved in the SWAFR.

HarMV isolate Can-1 was most closely related to isolates collected from around the Perth metro area of WA, and it grouped with them into Clade V ((Figure 4.10). Given only a single isolate from the eastern states location was studied, no conclusions could be made about the status of the virus in eastern Australia, however transport of an infected plant from WA to Canberra is the most likely explanation for isolate Can-1. Virus surveys of Hardenbergia plants in eastern Australia would need to be carried out to investigate this.

Isolate Cgt-1 was closely related to isolates in Clade II, but different enough to form its own clade. As a comprehensive survey was not undertaken the incidence of the virus in H. violacae cannot be stated with any certainty. Transplantation of an uninfected H. violacae plant imported 106


to Western Australia from eastern Australia, and its subsequent infection, could explain the isolate, however clearly a more detailed survey should be conducted. As isolate Cgt-1 was most similar to those collected from the Perth metropolitan area (KP, He, WHP) and because it does not form a clear out-group, its origin probably in WA and from the Perth area. Selection pressures faced by virions upon entering a new host can cause differentiation into new strains or divergent isolates (Spetz et al., 2003) and could explain the relative deviation of isolate Cgt-1.

4.4.9 Full genome sequencing Adams et al., (2005b) found whole genome comparisons to give the most accurate means of comparing Potyvirus species. If only a small region of the genome was available, the cytoplasmic inclusion (CI) gene was the region of choice for classification of Potyviruses because it most accurately represented the genome diversity seen over the length of the genome. Spetz et al., (2003) made similar conclusions when resolving the phylogeny of the PVY group viruses from Peru. In the genus Potyvirus the coat protein has been extensively sequenced, mainly because of the conserved regions of the GNNSGQ motif in the NIb gene, and the 3’ polyT region, which flanks the CP gene. Primers Potyvirid 1 and Potyvirid 2, which amplify this region (Gibbs & Mackenzie 1997), have allowed sequencing of the full CP gene of many Potyviruses. Such widely conserved motifs have not been found in other regions of the genome, including the CI gene.

Regions of the Potyvirus genome, such as the HC-Pro, CI gene and P3 protein have important functions in Potyvirus vector transmission, genome amplification and long distance movement via the plasmodesmata and the vasculature (Revers et al., 1999). The information gained from the sequencing of limited regions of HarMV has allowed comparisons to other Potyvirus species and has demonstrated its status as an independent species. Further sequencing of the genome may answer several important questions which have arisen from this research including: determinants of host range, further phylogenetic characterisation of the Australian-only subgroup and the role of recombination in Potyvirus speciation.

Further sequencing of the Australian-only subgroup of Potyviruses should be done to investigate their origins. To better characterise the relationship between HarMV and other Australian Potyviruses further sequence information is needed, particularly the CI gene of Australian Potyviruses including PWV, HiVY, ClVY, S1VY & S2VY. The CP and CI gene sequences of PaVY would also confirm whether this represents a new species of Potyvirus or should be renamed as PFVY. The coat protein of Potyvirus species reported from Australia; Dianella chlorotic mottle virus and Kennedya virus Y should also be fully sequenced because only partial CP sequences are available. 107


Recently, Reed et al., (2005) used a shotgun sequencing approach to clone and sequence ~12kbp of the Nucleorhabdovirus Maize mosaic virus. This method proved to be relatively easy costing $0.38/nt compared to $0.50/nt using the more traditional primer walking method. The method also took less time (127h) compared to primer walking (217h). However purified vRNA is required which is a limitation of this method. Full genome sequencing of HarMV, and possibly other Australian-only subgroup viruses, could be undertaken using this method.

4.4.10 Conclusions A newly discovered virus from Hardenbergia comptoniana and H. violacae and the first WA isolates from Passiflora species were partially sequenced and characterised. The Potyvirus from Hardenbergia was a new species in the genus Potyvirus. The high degree of sequence diversity over isolates taken from a small geographic range indicates that HarMV was not transported to the SWAFR since European colonisation in the early 1800s, rather it co-evolved with indigenous wild plants over a long time period. The pattern of diversity suggests that the region where the city of Perth now stands is close to the point of origin of the species.

The classification status of several Passiflora-infecting Potyviruses was also investigated and several changes are suggested. The status of PWV should be revised because comparison of the CP genes shows that the isolates form three distinct groups, indicating three species. The group containing PWV isolates from WA and one from Qld should retain the name PWV, while the NSW group should be renamed as proposed by Adams et al., (2005b). The name Passiflora mosaic virus (PfMV) is suggested for these isolates. PaVY and PFVY appear to be the same species based on limited sequence information; further sequencing is required to confirm this. The Thai group of two isolates is clearly a different species and the name Passion fruit mosaic Thailand virus (PFMTV) is suggested for this species.

The phylogeny study of other Potyviruses showed a grouping together of Australian-only Potyvirus sequences including HarMV PWV, PfMV, ClVY, HiVY, S1VY and S2VY, a grouping that has not previously been reported. It is reasonable to suggest a common ancestor for these viruses. Recombination was detected within HarMV isolates, as was seen in two isolates from MU campus, where a very recent recombination event was shown. An earlier event was also observed between two isolates.

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V: OPTIMISATION OF PRINTING METHODS

Chapter 5: Optimisation of microarray printing methods

5.1 Introduction DNA microarrays rely on stable binding of DNA in spots fixed to the surface of glass slides or other flat surfaces. Glass or polymer slides are commonly used because they exhibit low autofluorescence, are flat, and can be modified easily to bind DNA at a high capacity (Hessner et al., 2003b).

In order for the non-reactive glass surface to bind DNA, slides must first be

treated to derivitise the glass. They are coated with a chemical so that a functional group: e.g. amino group, aldehyde group, epoxy group, or others, can adhere to the slide surface. Spots of DNA probes can then be fixed to this functionalised surface.

Glass or polymer slides functionalised with a variety of chemical groups to bind DNA probes are available commercially, but in-house preparations using a coating of PLL can be used as a more cost-effective option (Schena et al., 1995).

5.1.1 Creating arrays on PLL coated slides Cleaned microscope slides coated in a layer of PLL can bind DNA in discrete features in a microarray. Before printing coated slides with probes, slides are aged for several weeks which improves DNA retention on the slides. Slides cannot be stored indefinitely before printing and slides stored for over 12 weeks suffered a 50% decrease in relative fluorescence compared to four-week old slides (Hessner et al., 2004a).

Commercial microarray slides that come pre-activated are supplied with an optimised protocol designed to be used with them, however the protocol for using slides created in-house must first be optimised. Treatments, such as the printing buffer used, immobilisation conditions and slide chemistry, each affect the final quality of the microarray (Petersen et al., 2001; Wang et al., 2003; Hessner et al., 2004a), and optimisation is important to ensure the fabrication of high quality microarrays.

Printing buffer A printing buffer is required in which to suspend the oligonucleotide or cDNA probes before printing. The choice of printing buffer used affects the shape of the spot and the quantity of DNA immobilised (Diehl et al., 2001; Hessner et al., 2003a; Hessner et al., 2004b; Wrobel et al., 2003). Apart from commercially available printing buffers, other printing buffer solutions 109


used in microarray experiments include: 50% DMSO, 3x SSC and 150mM sodium phosphate (NaPO4).

Betaine is also included in many printing buffers as it improves the morphology of the probes by increasing the viscosity of the printing buffer/probe mix causing a longer drying time of the spot on the array. Diehl et al., (2001), found that the inclusion of 1.5M betaine in printing buffers increased the binding efficiency of probe molecules and improved spot homogeneity. Betaine increases the retention of cDNA probes (Hessner et al., 2003a) and oligonucleotide probes (Hessner et al., 2004b). Increasing spot drying time provides a longer time for the probe to bind which leads to more equal binding of DNA across the spot (Diehl et al., 2001).

DNA concentration The concentration of DNA in the printing buffer is also significant and must be optimised because the sensitivity of a microarray can be limited by the amount of free capture probe available to bind the target. Factors include: a) the amount of probe immobilised, and b) the physical orientation of the probe. Too little probe can result in faint spots, reducing the accuracy of estimates of differential gene expression (Yue et al., 2001; Hessner et al., 2004a). Conversely, too much probe can result in features with “comet tails” or overcrowded features with too much probe immobilised in a small area. This overcrowding may limit the amount of labelled target able to bind because of steric hindrance (Beaucage et al., 2001).

Immobilisation of probes The PLL surface creates a positive charge which attracts and binds the negatively charged phosphate backbone of DNA by ionic bonding.

This binding is relatively weak and is

strengthened either by thermal baking or exposure to UV radiation to create covalent bonds. Thermal baking is carried out either at 42oC for ~16 hours, or at 80oC 1-4 hours. When using UV crosslinking for probe immobilisation, Massimi et al., (2003) recommend 60mJ for immobilisation on PLL slides, but up to 450mJ has been used (Wang et al., 2003).

Slide Blocking Excess free groups on the slide surface should be blocked before hybridisation is undertaken. The free groups can bind the fluorescently labelled solutions during hybridisations and contribute to high slide background. The method of blocking depends on the slide surface used and involves removing any free active sites on the slide surface able to bind DNA, typically by reducing the free groups to non-reactive derivatives.

Reducing agents such as sodium

borohydride and succinic anhydride can be used on amino and aldehyde coated slides to reduce 110


background. Any unbound DNA remaining on the slide surface should also be removed by washing prior to blocking to prevent free DNA re-adhering locally to unblocked active sites (Massimi et al., 2003).

5.1.2 Pre-activated slide chemistries Many slides available commercially have already been derivitised and only require probes to be printed and immobilised. The advantage of these slides is that the user is supplied with slides of high uniformity and optimised protocols for the printing and immobilisations of probes. This considerably simplifies the printing process and reduces the time for optimisation of array production.

5.1.3 Probe intensity and morphology The shape of spots is an important consideration when printing microarrays. Spots should be round in shape with an even distribution of probe within the spot or feature. Uneven or ‘doughnut’ shaped spots can result from a rapid drying of probes on the slide following arraying. Comet tails on an array can be caused by an excess of probe in solution during suspension or inefficient probe immobilisation. During further washing and immobilisation steps, the DNA becomes smeared across the surface of the slide, and this results in comet tails (Best et al., 2003). These occur as bright tails on the slide starting at the site of printing which weaken as the distance increases from the site of printing and can prevent accurate quantification of probe fluorescence.

The level of background that occurs on slide surfaces is also important. Quantification of probe intensity is influenced by background. Background local to the probe is subtracted from raw fluorescence values to correct for any non-specific signal. The drying of probe solutions on the array during hybridisation or insufficient washing can cause excessively high background fluorescence on microarray slides.

Black-holes or signal reversal can then occur when

background fluorescence is higher than probe fluorescence which causes probe spots to appear black in a region of high background (Best et al., 2003).

The causes of some microarray problems include: insufficient removal of unincorporated nucleotides during labelling, the slide drying during hybridisation or washing, and insufficient washing post-hybridisation. Hybridisations must be carried out in a humid environment to prevent drying of slides. Typically, glass cover slips are used and hybridisation chambers and disposable adhesive cover slips are also available (Gene Frames; Ab Gene, Surrey UK).

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5.1.4 Gene expression versus diagnostic arrays The aim when using microarrays as a diagnostic tool is to simultaneously detect the presence or absence of pathogen-specific nucleic acid. This is unlike the use of microarrays for gene expression, the accurate quantification of fluorescence is not so important. Using pre-activated slide chemistries for diagnostic arrays offers the advantage of a supplied optimised protocol and their use produces high quality arrays. The disadvantage of this option is that it is relatively expensive, making it impractical for routine use. The development of an in-house slide which offers significant cost savings is therefore reasonable. .

5.1.5 Aims The overall aim of the following experiments was to determine if ‘in-house’ microarray slides would be as reliable as commercially available slides, when printed with probes for virus detection and diagnosis. The outcomes of this work would enable us to determine if cost savings could be made in developing microarrays as a tool for highly parallel screening of plant samples for virus infection.

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5.2 Methods and materials 5.2.1 Printing and immobilising PLL slides

Slides were coated with PLL (DeRisi 2003b), aged for four weeks, and printed using a GeneTac G3 Robotic Workstation (Genomic Solutions) as described in Chapter 2.

Seven

oligonucleotide probes for CMV and BYMV detection were printed onto PLL-coated slides. A probe specific for each of the three RNA molecules of the CMV genome, a probe specific for the Cucumovirus genus (Probe names: CMV1, CMV2, CMV3 & Cucumovirus), two to detect the actin gene (positive controls) and a probe for BYMV (negative control) were printed. A water and print buffer mix was also printed to ensure sufficient cleaning of the print pins between probe transfers and to determine the level of slide background. The sequences of these probes are described in Table 3.2.

Seven oligonucleotide probes were printed in six different printing buffers. The buffers used were: 50% DMSO, 3x SSC, 3x SSC with 1.5M betaine, 150mM NaPO4, 150mM NaPO4 with 1.5M betaine and 2x Array It buffer. Probes were printed at four concentrations on the array by diluting in water before adding print buffer. Probes were printed at concentrations of 200, 300, 400 and 500ng/µL. The seven probes and water control were printed on 30 replicate slides as a grid of six columns by four rows (Figure 5.1).

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Figure 5.1: Layout of the array printed on PLL slides with seven probes printed at four concentrations to test and compare six printing buffers. A. Names of probes within each grid representing a single probe sequence: 1, Actin F; 2, Water; 3, CMV1; 4, CMV2; 5, CMV3; 6, Cucumo; 7, BYMV; 8, Actin R. B. Replicate grids showing the probe concentrations and print buffers used for each. See Table 3.3 for probe sequences.

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Immobilising probes on slides Crosslinking by exposure to UV light and thermal baking were used to immobilise probes on PLL slides. Four UV crosslinking energies were used (70, 150, 250 and 400 mJ). Crosslinking was carried out in a Bio-Rad DNA Crosslinker. Baking was at 80oC for 2 h or 42oC for 12-16 h. Baking was carried out in a humidity chamber which was made by placing saturated NaCl solution (100g NaCl in 50mL ddH2O) at the bottom of a two-shelf chamber. To maintain a humid environment for the 42oC incubation, chambers were sealed with Parafilm™ during immobilisation. Three replicates of each method of immobilisation were prepared.

Blocking microarray slides Printed PLL microarray slides were blocked by treatment with sodium borohydride (NaBH4) to reduce any unoccupied positions on the slide surface and reduce background. The treatment was carried out as described below (Massimi et al., 2003). • • • • • •

wash 0.1% SDS for 1 min with shaking (repeat) wash ddH2O for 1 min with shaking wash 5min in 300mL phosphate buffered saline, 90mL EtOH (100%) and 1g NaBH4 with occasional shaking wash 0.1% SDS for 1min with shaking and repeat twice wash ddH2O for 1min with shaking centrifuge 10min @ 1200 rpm to dry

Slide scanning and data analysis Once immobilised, the microarray slides were tested by hybridising Cy3 labelled random nonamers to the array using the protocol supplied by the LSMAF (Chapter 2). Random nonamers hybridise to immobilised DNA on the slide surface and allow an estimation of the amount of DNA retained during printing to be made. Microarray slides were then scanned using a GeneTac GT UC4 Ver. 3.11 (Genomic Solutions) at a resolution of 5µm. Gain and background settings of Gain 55.0 and Black 0.0 were used to scan slides. Further analysis on the slides was carried out using GeneTAC™ Integrator Microarray Analysis Software, Version 3.3.0 to quantify fluorescence readings and allow comparison of the different variables tested. Local background intensity was estimated in a concentric circle surrounding the spot (Yang & Speed 2003) and subtracted from spot intensity. The raw data of the median fluorescence of probes were analysed with Microsoft Excel XP ver. 10. For example to determine the optimal probe concentration for printing, the values of all probes printed at 200, 300, 400 and 500ng/µL across the entire set of 12 slides were averaged (except the values for features printed with only water and buffer as baseline controls).

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5.2.2 Printing PowerMatrix slides Full Moon BioSystems (FMB; Sunnyvale, CA) PowerMatrix slides (FMB #OS25) were printed according to the manufacturer’s protocol. Probes were resuspended in 2xFMB Oligo printing buffer (FMB, #SPB01) to a final concentration of 1µg/µL. A 20µL aliquot of each probe solution was loaded into a 384 well plate and centrifuged to ensure contents were at the base of each well. Arrays were printed using a GeneTAC GC 3000 Robotic Workstation as described in Chapter 2.

Following printing, microarray slides were immobilised using the recommended protocol of incubation at 65-75% humidity for 12 h in a humidity chamber, then dried in air and stored in a desiccator. Before hybridisation, slides were pre-treated following the recommended protocol. This involved incubation in pre-treatment solution (2xSSC/0.2%SDS/0.1% BSA) pre-heated to 55oC for 30 min, followed by rinsing thoroughly in ddH2O and air drying. An array was designed to detect and differentiate closely related species and strains of Potyvirus (GenPoty array), which contained 15 virus-specific probes and a positive control probe specific to the 18S rDNA gene of plants (provided by Dr Neil Boonham; CSL, York, UK). Probes for six strains of HarMV (I-VIII), PWV, PFVY, BCMV, BYMV, SMV, TuMV and ZYMV were designed in the coat protein region of the genome. This was because of an availability of sequence information within this region. Probes were designed as described in Chapter 3 (Table 3.7). Probes were printed on the array in an 8x2 grid with four replicate grids printed across the array (Figure 5.2).

Replicate 1 1 9

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Figure 5.2: Layout of microarray slides showing positions of probes; Potyvirus specific probe (blue), HarMV specific probes (red), other viruses (green) and 18s rRNA positive control (yellow). Probe names: 1, GenPoty; 2, PWV1; 3, PFVY1; 4, HarMV-All; 5, HarMV-I; 6, HarMV-II; 7, HarMV-V; 8, HarMV-VI; 9, HarMV-VII; 10, HarMV-VIII; 11, BCMV; 12, BYMV; 13, SMV; 14, TuMV; 15, ZYMV; 16, 18SrRNA. See Table 3.7 for the sequences of probes.

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5.2.3 Virus extraction, labelling and hybridisation Cloned fragments of HarMV and PWV were used as template for labelling reactions. BYMVMI was provided by Prof. Roger Jones (DAFWA) and cDNA of isolate 14A of PFVY was provided by Dr Stephen Wylie (Murdoch University/DAFWA).

Fluorescent labelling of PCR amplicons PCR amplicons were fluorescently-labelled by incorporation of Cy3-dCTP and hybridised to the array. Recombinant plasmids containing the full CP and 3’ UTR for six isolates (MU-1C, MU2A, MU-3A, Sb-3, Sb-19 and PWV Gld-1) were used and first strand cDNA was used for the remaining isolates (MR-13, He-2, BYMV-MI and PFVY). PCR amplicons were fluorescently labelled as follows: •

•

Combine: 10µL 10x PCR buffer, 10µL 25mM MgCl2, 2µL 2.5mM dNTP’s (1.0mM dCTP), 1.5µL Cy3-dCTP, 1µL Taq DNA polymerase (Fisher Biotech), 1µL LegPotyF primer, 1µL LegPotyR primer, 2µL template (cDNA or plasmid, see above) and 71.5µL H2O. Mix and thermocycle as follows: 94oC for 10s then 94oC for 15s 30 cycles 50oC for 20s 72oC for 60s 72oC for 7min, store at 14oC.

An aliquot of the PCR was run in a 1% agarose gel to confirm amplification as described in Section 2.5.

Purification and hybridisation PCR reactions were purified by ethanol precipitation and purified DNA was resuspended in 12µL of ddH2O. The concentration of eluted labelled DNA and the incorporation of fluorescent nucleotides were measured using a Nanodrop UV-Vis spectrometer (Nanodrop Technologies). Probe solutions were denatured at 95oC for 5 minutes, chilled on ice and centrifuged. A 10µL volume of denatured probe solution was mixed with 110µL of hybridisation solution (5xSSC with 1% SDS) and hybridised to the slide in a 21x22mm Gene Frame (#AB1043, AB Gene) to prevent drying of the probe on the slide. Slides were incubated in 100% humidity overnight at 35oC. Following hybridisation, Gene Frames were removed and slides washed in pre-warmed (35oC) washing solution (2xSSC) for 5 minutes. Slides were then rinsed with ddH2O and dried and scanned using the GeneTac UC4 microarray scanner.

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5.3 Results 5.3.1 Optimising PLL slides Probe morphology The choice of printing buffer substantially affected the morphology of microarrays on PLL slides (Figure 5.3). A 50% DMSO (Figure 5.3A) print buffer solution gave spots that were bright and round, however spots were ‘doughnut-shaped’ (i.e. uneven distribution of DNA with more toward the edges of the spot). Print buffers without betaine (3x SSC and 150mM NaPO4, Figure 5.3B & D) gave features which were faint and irregularly shaped, but the addition of betaine greatly improved spot morphology and intensity (Figure 5.3C & E). The commercial buffer (2x Array It™, Telechem) (its contents were not stated) also gave bright regular shaped features (Figure 5.3F), which showed a more even distribution of probe throughout the feature. The morphology of probes on the array was not affected by the concentration of probes, or the method of immobilisation.

Figure 5.3: Optimisation of printing buffer for printing probes. Print buffers tested were: A; Probes printed in 50% DMSO, B; Probes printed in 3x SSC, C; Probes printed in 3xSSC and 1.5M betaine, D; Probes printed in 150mM NaPO4, E; Probes printed in 150mM NaPO4 and 1.5M betaine, F; Probes printed in 2x Array It™ print buffer (Telechem). Arrays were hybridised with Cy3 random nonamers. Slides were scanned at Gain 42.0 and Black 10.0.

DNA retention The amount of probe retained on the array following immobilisation was estimated by hybridising Cy3 labelled random nonamers to the array. Quantification of fluorescence showed that all three variables (print buffer, probe concentration and method of immobilisation) affected the amount of probe DNA retained on the array. The six print buffers tested had a large impact on the amount of fluorescence seen (Figure 5.4).

The choice of printing buffer largely

determined how much probe DNA was retained on the array and was available for hybridisation. Using a print buffer of 3x SSC with 1.5M betaine gave the highest fluorescence on the PLL arrays, followed by 50% DMSO, 150mM NaPO4 with betaine and 2x Array It also 117


produced high fluorescence. The 150mM NaPO4 and 3x SSC printing buffers were the least effective because the slides showed the lowest fluorescence. Average Water Actin F Actin R BYMV CMV1 CMV2 CMV3 Cucumo

Mean fluorescent intensity

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Figure 5.4: Effect of six printing buffers on mean fluorescence of seven probes. The fluorescence of water with each print buffer was background. Data represents means from 72 hybridisations with the standard deviation shown as bars.

As probe concentration was increased the average fluorescence also tended to increase (Figure 5.5). At 200ng/µL of probe, the median fluorescence was lowest (6595 median fluorescent intensity) and at 500ng/µL the median fluorescence was highest (7176 median fluorescent intensity). However, this trend was not found for all of the individual probes tested and in particular the fluorescence for ActinR decreased 7% (5818 to 5375) and CMV2 decreased 3% (4203 to 4045) when the probe concentration was increased. Apart from these results, the fluorescence of the remaining probes increased as probe concentration was increased, especially for the CMV3 probe, which increased 21% (8818 units to 10671 units) from the lowest to the highest DNA concentration. The probe sequence also had a major affect on fluorescence, for instance CMV3 and Cucumo probes were highly fluorescent, whereas Actin F and CMV2 were weakly fluorescent.

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V: OPTIMISATION OF PRINTING METHODS 20000 200ng/uL 300ng/uL 400ng/uL 500ng/uL

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16000 14000 12000 10000 8000 6000 4000 2000 0 Actin F

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Figure 5.5: The effect of DNA probe concentration on fluorescence of microarray probes tested at four concentrations (200, 300, 400 and 500ng/µL). The fluorescence of the negative (water) control and the mean of the seven probes (not including water) is also shown. Data represents the mean of 108 hybridisations with the standard deviation shown as bars.

The third variable tested was the method of immobilisation, in which measurements were made of DNA retention using four UV crosslinking energies and two methods of baking. The mean fluorescence measured from each of the six immobilisation methods was compared (Figure 5.6) for each of the print buffers tested, because this variable was previously shown to effect fluorescent intensity (Figure 5.4). To reduce the variation observed for different probes only measurements of the Cucumo probe were used.

The results of using different immobilisation conditions (Figure 5.6) showed that overnight incubation at 42oC gave the highest mean fluorescence (average of 13316 units), while fluorescence values from the other methods ranged from 10941 to 12406 units. The choice of buffer was important and different printing buffers caused wide variation in the mean fluorescence: the results showed that SSC/betaine worked well with all immobilisation methods and in particular for overnight incubation at 42oC, with a mean of 20494 units. For the other print buffers including DMSO, NaPO4 with betaine and Array It buffer, also worked well with most of the immobilisation methods. The energy level of UV crosslinking had a large impact on the fluorescence, since with SSC increasing the crosslinking energy doubled the fluorescence obtained from 4902 to 10,000 units. However for DMSO the opposite was found (14601 to 9122 units). Overnight incubation at 42oC provided the highest fluorescent values of those tested with the SSC and betaine printing buffer.

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V: OPTIMISATION OF PRINTING METHODS 25000 Average NaPO4 DMSO SSC/betaine

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Figure 5.6: A comparison of different probe immobilisation methods. Probe fluorescence from slides immobilised by UV crosslinking or baking. Slides were immobilised by 70, 150, 250 or 400mJ of UV energy; or by thermal baking at 42oC for 16h or at 80oC for 1h. The method of immobilisation was compared for the Cucumo probe printed with each printing buffer at four concentrations (200, 300, 400 and 500ng/µL) and hybridised with Cy3 random nonamers. Data represents the mean of 12 replicates with the standard deviation represented by bars.

5.3.2 Printing arrays on Power Matrix slides A Power Matrix slide printed with the GenPoty Array was hybridised with Cy3-labelled random nonamers and the scanned image showed bright spots indicating the immobilisation of substantial amounts of probe DNA (Figure 5.7). All the probe spots were visible on the array; but as on the PLL surface, many were doughnut shaped. Note: A technical fault with the Integrator software used for quantifying the fluorescence prevented making quantatitive comparisons to the PLL coated slides.

Figure 5.7: 5µm resolution scan of a PowerMatrix slide hybridised with Cy3labelled random nonamers containing Potyvirus probes (Fig 5.2). Slides were printed at 1µg/µL in 1x Oligo buffer and immobilised by incubating at 42oC for 16hours. Scanning settings of gain of 65.0 and black of 10.0 were used.

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5.3.3 Detection and discrimination of Potyvirus species To test the GenPoty array for selectivity and specificity, cDNA from ten Potyvirus isolates was amplified using specific or polyvalent primers and fluorescently-modified nucleotides. The actin gene of H. comptoniana was fluorescently labelled and hybridised to the array as a negative control. Agarose gel electrophoresis of PCR products confirmed the presence of amplicons of the expected size (Figure 5.8) and spectrophotometry confirmed incorporation of fluorescent nucleotides (Table 5.1). Amplicons were therefore suitable for hybridising to the GenPoty array. M

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Figure 5.8: Amplification of target Potyvirus sequences.. M: 5µL 100bp molecular weight marker (Promega); 1: MU-1C; 2: MU-2A; 3: MU-3A; 4: He-2; 5: MR-13; 6: Sb-3; 7: Sb-19; 8: PWV-Gld 1; 9: PFVY-14A; 10: BYMV-MI; 11: Actin. Isolates of HarMV were amplified using HarMV CP F&R primers and BYMV-MI, PFVY and PWV were amplified using LegPoty F&R (Table 3.4). The actin negative control was amplified by Actin F&R primers. Recombinant plasmid templates were used for isolates MU-1C, MU-2A, MU-3A, Sb-3, Sb-19 and Gld-1; and first strand cDNA was used for He-2, MR-13, BYMV-MI, PFVY and actin. Table 5.1: Assessment of yield and purity of test virus amplicons for hybridisation

Isolate MU-1C MU-2A MU-3A He-2 MR-13 Sb-3 Sb-19 PWV-Gld 1 PFVY-14A BYMV-MI Actin

Concentration Cy3 incorporation Purity (ng/µL) (pmol/µL) (260:280) 186.2 104.0 84.4 160.4 153.7 176.9 124.4 243.6 67.7 169.2 74.9

18.5 17.1 13.2 27.6 21.3 16.4 15.2 15.8 5.9 10.4 7.3

1.79 1.78 1.78 1.72 1.78 1.77 1.83 1.81 1.76 1.86 1.73

Scanned images showed the GenPoty array could be used to identify all ten Potyvirus isolates (Figure 5.9). The seven isolates of HarMV tested hybridised to the GenPoty and HarMV probes. Isolates Gld-1 (PWV), 14A (PFVY) and MI (BYMV) also hybridised to the GenPoty probe as well as to virus-specific probes appropriate for each virus. However, all the isolates tested hybridised to BCMV-1, SMV-1 probes to most isolates tested hybridised to the ZYMV-1 probe (Figure 5.9).

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In addition probes designed to differentiate strains of HarMV did not do so (Figure 5.9). Amplicons of MU-1C, MU-2A and Sb-3 failed to hybridise to any of the strain-specific probes, while non-specific hybridisation occurred to one or more strain-specific probes. For instance MU-3A (Figure 5.9C) hybridised to probes specific for HarMV strain groups I, II and V; however hybridisation to group V was expected. Therefore identification of HarMV strains using the GenPoty array was not successful.

Figure 5.9: Scanned images of the GenPoty array at 5µm resolution when hybridised with fluorescent amplicons from ten virus templates. A: MU-1C amplicon, B: MU-2A amplicon, C: MU-3A amplicon, D: He-2 amplicon, E: MR-13 amplicon, F: Sb-3 amplicon, G: Sb-19 amplicon, H: PWV-Gld 1 amplicon, I: PFVY-14A amplicon, J: BYMV-MI amplicon. Slides were scanned at Gain 42.0 and Black 10.0. See Figure 5.2 for slide layout.

An amplicon of the actin gene of H. comptoniana was hybridised to the GenPoty array as a negative control and was used to check that the fluorescence observed was not the result of nonspecific hybridisation or insufficient washing. The results (Figure 5.10) showed very little cross-hybridisation of the actin gene to the virus-specific probes, which confirmed the specific nature of the hybridisations in Figure 5.9.

Figure 5.10: Actin gene negative control slide showing faint hybridisation to the GenPoty array. The slide was hybridised with a fluorescently labelled amplicon of the actin gene and scanned at 5µm resolution. The slide was scanned at Gain 42.0 and Black 10.0.

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5.3.4 Probe design The effectiveness of each probe in hybridising to each virus isolate was evaluated. Pairwise comparisons of each probe/template combination (Table 5.2) were compared to the results obtained in hybridised arrays (Figure 5.9). Where the probe/template shared more then 78% homology hybridisation was observed on the array.

This resulted in some unintended

combinations hybridising, such as all virus isolates hybridising to the BCMV, SMV and ZYMV probes. These probe/template combinations shared 78-85% homology. An exception to this observation was the six HarMV strain specific probes (I-VIII) which showed 100% homology to their most complementary template but did not always hybridise together. For instance only background fluorescence was seen on the array between four isolates (MU-1C, MU-2A and MR-13) and the most complementary strain specific probe (Figure 5.9a, b and d). Other isolates (MU-3A, He-2, Sb-3 and Sb-19) showed hybridisation to incorrect strain specific probes. For instance MU-3A hybridised to HarMV-I, II, V and VIII; however it was only expected to hybridise to HarMV-V. Where hybridisation occurred to the strain specific probes there were at least 21 out of 51 homologous nucleotides; however many of the templates tested showed 100% homology to at least one strain specific probe and were expected to show hybridisation. These results indicate that the behaviour of these probe test combinations cannot be explained only by the level of nucleotide homology.

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Table 5.2: Nucleotide identities between microarray probes and the amplified templates hybridised to the GenPoty array.

Species and strains of Potyvirus used as templates for labelling C Probe HarMV PWV PFVY BYMV Microarray Probe A length MU-1C MU-2A MU-3A He-2 MR-13 Sb-3 Sb-19 Gld-1 14A MI 23 GenPoty B 23/23 23/23 23/23 23/23 23/23 23/23 23/23 23/23 21/23 23/23 26/51 25/51 PWV 51 35/51 35/51 32/51 34/51 34/51 32/51 29/51 51/51 53 29/53 27/53 26/53 25/53 24/53 28/53 29/53 29/53 29/53 48/53 PFVY 51 46/51 42/51 28/51 51/51 51/51 51/51 51/51 51/51 51/51 51/51 HarMV-All B 51 44/51 36/51 39/51 38/51 35/51 25/51 51/51 41/51 41/51 24/51 HarMV-I 51 35/51 34/51 30/51 34/51 24/41 23/51 36/51 39/51 51/51 40/51 HarMV-II 51 39/51 36/51 36/51 24/51 44/51 49/51 40/51 40/51 23/51 46/51 HarMV-V 51 35/51 32/51 32/51 23/51 39/51 42/51 40/51 34/51 40/51 51/51 HarMV-VI 51 29/51 27/51 22/51 28/51 27/51 35/51 30/51 35/51 35/51 51/51 HarMV-VII 51 40/51 35/51 34/41 29/51 41/51 40/51 35/51 51/51 21/51 37/51 HarMV-VIII 50 39/50 39/50 41/50 39/50 41/50 39/50 39/50 39/50 38/50 33/50 BCMV 51 26/51 26/51 25/51 23/51 25/51 26/51 22/51 25/51 27/51 45/51 BYMV 46 39/46 39/46 36/46 36/46 37/46 39/46 37/46 37/46 38/46 32/46 SMV 49 29/49 29/49 30/49 30/49 32/49 29/49 29/49 32/49 29/49 32/49 TuMV 52 42/52 42/52 41/51 41/51 41/51 42/52 43/52 42/52 44/52 35/52 ZYMV 18S rRNA 50 19/50 20/50 20/50 18/50 18/50 17/50 24/50 20/50 22/50 19/50 A: Probe sequences are provided in Table 3.7. B: Probes contained degenerate positions and the values given are the maximum identity between probe and target. C: Virus Names; BYMV, Bean yellow mosaic virus; HarMV, Hardenbergia mosaic virus; PFVY, Passiflora foetida virus Y; PWV, Passion fruit woodiness virus. Underlined cells represent the intended templates to which each probe was expected to hybridise. Bold cells represent where hybridisation between template and probe was observed on the array (Figure 5.9).


5.4 Discussion

5.4.1 Arrays on PLL slides The optimisation experiments showed that some array preparation variables had a greater effect on the quality of the microarrays than others. Printing buffer composition strongly affected both the morphology and intensity of spots, whereas different probe concentration only had a minor effect. Unexpectedly, large differences were seen in the fluorescence results from each immobilised probe, and this was independent of the printing or immobilisation method. Replicate measurements of the same variables also differed widely, which resulted in the large standard deviations observed (Figure 5.4, 5.5 and 5.6). The variables that gave optimum PLL slides were when probes were printed at 500ng/µL in a printing buffer of 3x SSC with 1.5M betaine added. Following printing slides should be immobilised at 42oC for at least 16 hours and then blocked with sodium borohydride.

Printing buffer The choice of print buffer had the greatest effect on the intensity and morphology of hybridised probes of all the variables tested in this study. Of the buffers tested 3x SSC with 1.5M betaine produced arrays with the most intense spots on the PLL slides. The 50% DMSO, 150mM NaPO4 with betaine and the Array It buffers also produced arrays of good quality, but on average the features were only 85% as intense (8903 compared to 7500 mean fluorescent units, Figure 5.4). DNA retention on the slide is important for array production and impacts on the quality and reproducibility of data from hybridisations (Hessner et al., 2003a). Low DNA retention reduces the fluorescence of scanned arrays (Diehl et al., 2001; Yue et al., 2001) and in diagnostic applications could result in false negatives for what should be positive samples.

The results confirmed previous observations that the addition of betaine greatly improves the fluorescence and morphology of arrayed probes, when it was added to NaPO4 and SSC printing buffers. Betaine increases the viscosity and reduces the rate of evaporation (Rees et al., 1993) of solutions. This effect enhances the binding of probes to PLL surfaces (Diehl et al., 2001; Hessner et al., 2003b) thereby increasing the fluorescence produced by hybridisation. Also, the higher DNA retention can increase the number of slides which can be printed from a single library amplification (Diehl et al., 2001) and this can reduce array fabrication costs.

Other combinations of print buffers have been tested and one of the most promising comprises 15% DMSO with 1.5M betaine (Hessner et al., 2003a; Hessner et al., 2004b). Compared to six other buffers tested: including 3x SSC with 1.5M betaine, 3x SSC, 50% DMSO, water and 125


1.5M betaine; this buffer gave the greatest probe retention (over 90% compared to ~60% for SSC with betaine) on PLL slides (Hessner et al., 2003a). This equates to an increase in probe intensity on slides which was also found in these experiments. A comparison of print buffers with a number of slide surface chemistries showed that the optimal print buffer varied with surface chemistry and that a formamide/betaine/nitrocellulose buffer was optimal for GAPSII slides (Corning) while a SSC with betaine buffer performed better on QMT Epoxy slides (Quantifoil Micro Tools) (Wrobel et al., 2003).

PLL slides were not tested in these

experiments. Work by others on PLL slides have used SSC/betaine (Diehl et al., 2001) and DMSO/betaine (Hessner et al., 2003a; Hessner et al., 2003b; Hessner et al., 2004b) as printing buffer. While it would be interesting to compare additional printing buffers, given the variation found in these experiments, these buffers may not work as effectively on the slides used in this study.

Different printing buffers also impacted greatly on the morphology of spots on the array. Probes printed in SSC and NaPO4 produced features that were small, irregular and only faintly fluorescent after hybridisation, however the same probes printed in SSC and NaPO4 with betaine were larger, round and bright (Figure 5.3). The probes printed in DMSO and Array It™ also showed bright round shaped features. Probes printed in the four buffers (DMSO, SSC/betaine, NaPO4/betaine, Array It™) all produced ‘doughnut’ shaped features on the array, which is caused by a variation in DNA concentration across the spot (Diehl et al., 2001). The presence of doughnut spots is important in gene expression studies because it influences the quantification of probe intensity (Yang & Speed 2003), but this is probably less important in diagnostic studies, where the desired result is only the presence or absence of a pathogen. Nevertheless highly irregular probes, like the ones seen using SSC or NaPO4 buffer, may be mistaken for dust or print artefacts. The addition of betaine or DMSO to spotting buffers has been reported to help reduce doughnut shaped features (Diehl et al., 2001) by increasing solution viscosity. Adding betaine to print buffers improved DNA retention (as above) and probe morphology (Figure 5.3) but doughnut features were still present. The homogeneity of features could have been enhanced by increasing the humidity in the microarrayor during printing (Best et al., 2003), which increases the time taken for the drying of buffers and so improves homogeneity of DNA distribution (Diehl et al., 2001). The size of the features printed limits the density or number of probes than can be printed on an array; however this is not of concern in the case of a plant virus array because currently only a few tens to hundreds of probes are used. A maximum of 180 probes have been used in a plant virus microarray study (Abdullahi et al., 2005), and at the density achieved here arrays of up to 5000 probes could be prepared. This would easily allow probes for all known plant viruses to be included, along with redundancy and controls probes.

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Probe immobilisation Thermal baking and UV crosslinking are both methods used frequently for DNA immobilisation. The method of immobilisation also impacted on the amount of DNA retained on the microarray slides, as measured by the fluorescence produced from random nonamer hybridisations. Baking slides at 42oC for 16 hours produced the highest mean fluorescence on average for the six print buffers tested. In particular the combination of SSC/betaine print buffer using immobilisation at 42oC worked well (Figure 5.6). The protocol used to coat PLL slides (Massimi et al., 2003) suggested UV crosslinking at 60mJ was the optimal method and that baking was not appropriate. Our results did not agree with this conclusion because baking at 42oC retained more probe DNA. Also the amount of fluorescence produced on hybridisation was dependent on the print buffer used, as expected, and matched what was seen in Figure 5.4. For instance whilst probe immobilisation at 42oC/16hr was optimal for SSC with betaine; 400mJ of UV crosslinking was optimal for SSC without betaine. Also increasing UV energy did not always improve DNA retention and the opposite was found when using DMSO, where DNA retention decreased.

The reported energy applied to crosslinking used for immobilising probes to PLL slides varies from low (60-200mJ: Diehl et al., 2001; Yue et al., 2001; Taylor et al., 2003; Hessner et al., 2004a) to high (200+mJ: Wang et al., 2003). Different slide chemistries require different methods for optimum probe attachment; however commercial slide chemistries show differences in the optimum amount of UV energy for probe attachment (Wang et al., 2003). Combinations of the two methods have also been reported (Hedge et al., 2000; Wrobel et al., 2003) and it might be useful to test combined methods on the slides developed in this thesis. In general, the best methods found by differing researchers for immobilisation of probes to PLL slides varies with the study and this probably results from other experimental differences such as the probe type (cDNA or oligonucleotide, modified or unmodified) and printing buffer used. These results confirm the need to optimise printing parameters when developing new methods such as those presented here.

Probe concentration Increasing the concentration of probe DNA in the printing buffer generally increased the resulting fluorescence on the array, and this indicates a higher retention of probe. The mean fluorescence was highest with probes printed at 500ngµL.

However over the four

concentrations tested (200, 300, 400 & 500ng/µL) the amount of probe retained increased by only 10%, while the amount of probe printed increased by 250%, indicating saturation was reached. The amount of DNA retained during immobilisation on PLL is approximately 30% (Hessner et al., 2003a) with the remaining DNA lost during washing steps. Increasing the probe 127


concentration thus does usually increase the amount of DNA retained on the slide, but most DNA appears to be lost in washing steps.

There are conflicting reports on the effect of probe concentration in microarray research. For gene expression studies low concentration (6.25ng/µL) causes underestimation of differential gene expression (Yue et al., 2001). In contrast, Wang and co-workers (2003b) showed that there was no significant change in expression ratios when dilute (6.25µM) probes were tested. This is less relevant for our work in which the aim is simply to determine the presence or absence of vRNA in plant samples, and not to compare and quantify transcript levels between samples. Printing probes at dilute concentrations will decrease the fluorescent intensity of spots on the array (Diehl et al., 2001; Hessner et al., 2004b; Ramdas et al., 2004); but in a non-linear manner i.e. the increase in total fluorescence is less than the increase in probe concentration. Decreasing probe concentration either had no effect (Taylor et al., 2003) or increased probe fluorescence (Wang et al., 2003); the latter only for some probes. This suggests a sequence effect possibly caused by steric hindrance (Shchepinov et al., 1997), at high concentrations. Yue et al., (2001) also observed variations in the fluorescence produced from probes printed at the same concentration and suggested that individual probes may differ in their efficiency of attachment to explain this result. There may be a similar explanation to our observation that some probes (i.e. CMV3 and Cucumo) were four to five times more fluorescent on the array (Figure 5.4 & 5.5) than others (i.e. Actin F and CMV2).

In the above published studies a wide range of variables were compared such as probe type (cDNA or Oligo), print buffer (including betaine or not) and slide chemistry (amine, PLL etc). The differences in methods are likely to have a large impact on the effect of applied probe concentration. For instance, inclusion of betaine improves DNA retention and therefore would allow satisfactory retention of probes at lower concentrations (Diehl et al., 2001). With the use of 3% DMSO/1.5M betaine buffer more than 70% of the probe was retained on the slide, compared to only 30% probe retention when 50% DMSO or 3X SSC were used (Hessner et al., 2003a). The inconsistent results of these studies confirm the need for optimisation of a method to an individual laboratory because changes in methodology can be detrimental to array quality.

5.4.2 Comparison of slide surfaces Using both the PLL and FMB proprietary slides, DNA was immobilised on a microarray for virus testing.

Qualitative observations of the two surfaces showed arrays printed on the

PowerMatrix surface had higher and more consistent probe fluorescence when hybridised with Cy3 random nonamers, compared to the optimised PLL slides. This can be seen in Figure 5.7 where the GenPoty array shows several saturated spots, seen in white, while the CMV array 128


(Figure 5.3) has several probes only faintly visible (i.e. CMV2 and Actin F). The PowerMatrix surface thus appears to provide more reliable and consistent detection of virus species and strains, which can be seen in Figure 5.7, and therefore their use is recommended for virus diagnostic arrays.

Wide disparity in the quality of arrays produced by using differing slide chemistries has been reported (Hessner et al., 2004a; Taylor et al., 2003; Wrobel et al., 2003). This reflects the problems of altering one variable on another. So while slide chemistry can impact on array quality, equally the choice of printing buffer and immobilisation method can conceal or increase differences. This problem is highlighted by a comparison of 14 slide chemistries in which the chemistry had a considerable effect on the amount of probe DNA retained (Hessner et al., 2004a). However in this case, all methods were compared using a single print buffer and immobilisation method. The results of this project and published research have shown that the printing buffer and immobilisation method can have a ranged effect on array quality and if experiments are repeated with different print buffers it is likely the results would change. Studies which have looked at these variables simultaneously (Taylor et al., 2003; Wrobel et al., 2003), rather than linearly, should provide better methods for array fabrication.

There was a limit to the extent of array optimisation that could be done in this thesis, since the aim of this section was limited to investigating cost-effective methods of array fabrication. Towards this goal arrays were fabricated using un-modified oligonucleotide probes, printed at a low concentration (200ng/µL) on slides produced in-house. However it turned out that this produced microarrays of low quality compared to the PowerMatrix surface. Therefore the results in general do not support the use of cheaper, home made slides because the array quality was significantly poorer, leading to less accurate results using diagnostic arrays.

One issue that was not solved was that a fault in the Integrator software prevented quantification of probe fluorescence and as a result quantitative comparisons could not be made. Whilst array images could have been quantified by alternative methods, comparisons would not have been meaningful and therefore this option was not followed. The time constraits of the project also meant that repairs to the software could not be completed in time. Quantitative comparisons of the slide chemistries would allow better comparisons of the two but are not essential.

5.4.3 Virus identification by microarray The GenPoty array was used to identify four species of Potyvirus including HarMV, which was detected and characterised as described in Chapter 4.

Species specific probes were used to

identify isolates of Potyvirus from four species; but cross-reactivity was observed. The BCMV, 129


SMV and ZYMV probes cross-hybridised to all isolates tested despite none of the isolates tested belonging to these species. This cross reactivity was due to the high homology between the probe/template combinations which were identified in Table 5.2, which is not surprising because all the viruses tested here (except BYMV) belong to the BCMV group of Potyviruses, which have high nucleotide homology (Adams et al., 2005b). The remaining species-specific probes (HarMV-All, PWV-1, PFVY-1 and BYMV-1) could be used to detect and discriminate these four species. The limited number of probes included in the GenPoty array allowed discrimination of four species, however a more practical tool for diagnostics should include more species specific probes, particularly for economically important viruses.

This is an

important area to develop in future work.

Probe design Aspects of probe design that were limiting in the GenPoty array were identified by testing with Potyvirus isolates. High sequence identity was found between the BCMV, SMV and ZYMV probes and all templates used (over 78%, Table 5.2) and this is probably why cross reactivity was seen on the array (Figure 5.9). The long length oligonucleotide probes (~50mers) used here have been shown previously to provide better signal intensities than short length probes (Ramdas et al., 2004). However non-target sequences which share more than 75% homology to long oligonucleotide probes can hybridise (Kane et al., 2000) causing cross-reactivity. The probes used on the GenPoty array were designed in the core of coat protein (Table 3.7) which is conserved (Shukla et al., 1994) allowing species specific probes to be designed; however by using the N-terminal CP or 3’ UTR higher discrimination could be achieved. On the other hand it will be harder to design species specific probes from this region because of the higher sequence diversity.

Another option for better design of species specific probes is to use shorter probes. Many diagnostic arrays have used short length (~20mer) probes and this is particularly true for discriminating the highly conserved rRNA genes of fungi and other higher organisms (e.g. Loy et al., 2002; Nicolaisen et al., 2005; Lievens 2006; Nicolaisen & Bertaccini 2007). Probes of approximately 20 nucleotides can discriminate single nucleotide polymorphisms, particularly when these are centrally located (Lievens 2006). While the sensitivity of these probes is lower (Ramdas et al., 2004) they maybe useful if an amplification step is included during the labelling reaction.

Contrary to expectations high identity between a probe and an isolate did not always correlate with subsequent high fluorescent intensity on the GenPoty array (Figure 5.9). The failure of isolates to hybridise to any probes, or to unexpected probes, was not because of insufficient 130


homology. The CP of all isolates was known from Chapter 4 and probes shared 100% homology to at least one isolate of HarMV. Strains of Potato virus M (PVM), Potato virus S (PVS) and PVY were differentiated by 40mer oligonucleotide probes (Bystricka et al., 2005). Further experimentation is needed to check whether this result would translate to differentiating strains of HarMV.

More sequence specific probes would greatly improve the usefulness of microarrays for virus diagnostics by enabling simultaneous genus, species and strain level identification. Additional experimentation on probe design, and in particular validation of a number of probes for each strain/species of interest is needed, with probes tested against a wide range of templates. Those showing the required specificity and selectivity under the hybridisation and washing conditions of interest could be useful in diagnostic arrays. It is particularly important to do this research for viruses where both very damaging strains and more benign strains can be present in imported plant material. In biosecurity and quarantine situations a microarray based diagnostic tool could be very useful.

5.4.4 Conclusions When printing microarrays a complex interaction occurs between the probe, print buffer, slide surface and immobilisation method. Therefore when developing microarrays there is a need to optimise each aspect to ensure that high quality arrays are produced. Purchasing pre-activated slides is a more expensive option, but probably provides savings in time and effort because an optimised protocol is supplied. Therefore, unless large scale applications are envisaged, preactivated slides which produce high quality arrays should be used. Work developing the GenPoty array also highlights the potential for simultaneous identification of viruses at the species and strain levels, although this aim was not fully realised. More work, especially on aspects of probe design such as probe length and sequence, could improve microarray based diagnostic methods. Nevertheless, this study has provided a sound basis for further work on virus diagnostic arrays and with further research will contribute to methods for virus diagnostics using microarrays.

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VI: AMPLIFICATION METHODS FOR LABELLING

Chapter 6: Optimising methods of nucleic acid amplification for microarray-based virus detection

6.1 Introduction The optimisation of printing and immobilising of microarrays is described in the previous chapter: the next requirement was to develop methods for fluorescent labelling of plant and vRNAs. The traditional method for labelling on microarrays uses reverse transcriptase to make first-strand cDNA. Labelled nucleotides are incorporated into the cDNA strand by a RNAdependent DNA polymerase, and there is no amplification of cDNA molecules. As indicated previously, amplification for studies in which patterns of gene expression are compared, should conserve relative transcript abundance following amplification (Nygaard & Hovig 2006). However, for detection or diagnostic assays, such as for viruses, amplification of the target molecule before hybridisation may be required to increase the sensitivity or provide clear evidence for presence or absence of the virus to be detected.

The amount of target RNA required for consistent detection in microarrays can be up to 100µg of total RNA, although less purified mRNA can be used.

Amplification of target RNA to

increase detection by microarrays was first proposed in studies where the sample size was limited, such as in tissue biopsy and cancer studies (Nygaard & Hovig 2006). The use of poly dT primers (dT50) to prime cDNA synthesis of all mRNAs prevents reverse transcription of other RNAs, thereby enriching for mRNA transcripts. This is not an option for virus diagnostic studies because some virus genomes are not polyadenylated, e.g. the Cucumoviruses.

Although PCR can be used to amplify small regions of DNA for virus diagnostics, virus sequence data is needed for primer design. PCR amplification of conserved regions, such as the 16S rRNA gene has been used to identify and classify bacterial species (Greisen et al., 1994). Similarly conserved 18S rRNA genes have also been used to identify fungi (Atkins & Clark 2004) and nematodes (Powers 2004). Unfortunately, such conserved motifs are not present in plant virus genomes.

Ideally, methods for amplification of viral nucleic acid in infected samples should work without previous knowledge of the infecting agent/s. This makes PCR unrealistic because multiplexing using specific primers for all possible candidate viruses is impractical. A maximum of eight virus targets have been multiplexed in a single reaction (Sanchez-Navarro et al., 2005). A generic amplification method to increase the amount cDNA within a sample would improve the

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sensitivity of microarray diagnostics, without requiring previous knowledge of the infecting agent/s.

Several methods for random amplification of nucleic acids are available: Random amplification PCR (rPCR): This approach makes use of short primers, often random hexamers or octamer primers, for amplification. The primers may be used unmodified as in Vora et al., (2004), or can be modified to include 5’ adaptor sequences such as used by Burton et al., (2005), Wang et al., (2002), or Agindotan & Perry (2007). The reaction is carried out under conditions similar to conventional PCR, but with lower annealing temperatures. The advantage of using adaptor primers is the increased melting temperature of additional thermocycling rounds, and the adaptor can be used for specific priming in subsequent amplification rounds.

Three prime end amplification or single primer amplification:

This method is similar in

principle to the rPCR but only a single modified heel primer is employed, which is used by the DNA polymerase to direct linear amplification of template DNA (Dixon et al. 1998 and Smith et al. 2003). Rolling circle amplification: Φ29 DNA polymerase was evaluated as an amplification method by Vora et al., (2004) for microarray use. This method uses the Φ29 polymerase to amplify circular templates exponentially. Vora and co-workers (2004) tested this system on bacterial DNA samples and found a 10-20 times increase in bacterial genome copy number as tested by real time PCR. Rolling circle amplification was also used by Haible et al., (2006) to amplify Geminiviruses, and by Gronenborn (2004) and Stanley (2004), to amplify members of the Nanoviridae.

In the case of both viruses the amplification product was not used for

hybridisation to a microarray. The method is also limited to detecting circular templates, and since the majority of plant viruses have linear genomes, the approach is limited to detection of circular DNA viruses and satellite sequences.

Klenow based amplification: In a study of amplification methods (Vora et al., 2004) the Klenow fragment of DNA polymerase was used to amplify and fluorescently label samples of total nucleic acid for microarray detection of E. coli. While Klenow amplification used on its own lacked sensitivity, a tandem Klenow + Klenow approach (i.e. the product of one amplification reaction is purified and used as the template in a second Klenow amplification reaction) improved the limit of detection up to 1000 fold, and increased sensitivity of all tandem approaches tested. A PCR using short random hexamers (N6mer) primers was also used with or without tandem Klenow amplification.

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The aim of the work in this chapter was to compare labelling methods for plant virus detection using microarrays. The work described here was done whilst I was at CSL, York, UK, under the supervision of Dr Neil Boonham. A high titre virus (PVX) and a low titre virus (PVY) were used to compare any effect of virus titre on the sensitivity of detection. Three amplification methods were used to test the sensitivity of each method and the results from these methods were then compared with a commercially-available indirect fluorescence labelling kit. The amount of virus nucleic acid present using each method was determined by real time PCR and this value was used to determine the amount of target DNA required for detection of viruses by microarray.

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6.2 Materials and methods

6.2.1 Microarray slides The microarray slides used were CEL Vantage Aldehyde Slides (CEL Associates, USA) preprinted with 45-50 base oligonucleotide probes. Probes were printed in three replicate groups across the array (Figure 6.1). The names and sequences of probes are listed in Table 3.3. Probes for PVX (green) and PVY (red) were included along with probes for a number of viruses that were not tested for (white). These probes for additional viruses were used to check for cross hybridisation. The positive control probe was a ubiquitous 18S rRNA fragment used to detect host nucleic acids and to check labelling and hybridisation conditions. Probes were printed in the array so that scanned images could be orientated correctly to identify positive probes. The irregular shape of the replicate groups and location of positive control probes (#1&23) provided a point of reference for orientating the images.

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Figure 6.1: Layout of microarray slides showing 18s rRNA positive controls (yellow), PVX probes (green) and PVY probes (red) in three replicate patches. Probes for viruses and viroids not tested (white). Names of probes and sequences are provided in Table 3.3.

6.2.2 Microarray hybridisation and scanning Purified fluorescently labelled target solutions were hybridised to slides in a volume of 120µL. Cy3 labelled solutions (green) were used for PVX and Cy5 labelled solutions (red) were used for PVY in all labelling reactions. For two colour hybridisations each reaction was eluted in 60µL to make a total of 120µL. Target solutions were first incubated at 95oC for 3 min to denature the DNA, and then rapidly chilled on ice for up to 3 min. Hybridisations were carried out using 21x22mm Gene Frames (AbGene Ltd, Surrey UK) with a volume of 120µL to make an airtight seal around slides to prevent drying during hybridisations. Hybridisations were carried out overnight in a humid environment at 42oC.

The following day slides were washed to remove non-specific binding. This was carried out using pre-warmed wash buffers (30oC). Slides were washed in each buffer for 5 min before transferring to the next buffer. The three buffers used were; 1: 2x SSC, 0.1% SDS, 2: 1x SSC, 3: 0.1x SSC. Slides were then dried with compressed nitrogen and transferred to a light-proof container, until scanning. 135


Microarray slides were scanned using a GenePix 4000B scanner (Molecular Devices) at a photomultiplier tube (PMT) gain of 800 and laser power of 100%. Local background intensity was estimated in a concentric circle surrounding the spot (Yang & Speed 2003) and subtracted from spot intensity. Data analysis was carried out using GenePixPro v3.0. Graphs and tables were drawn using Microsoft Excel 2002 Pro.

6.2.3 RNA extraction and precipitation RNA was extracted from infected and healthy Nicotiana tabacum leaf tissues using an RNeasy Mini Kit modified to include a chloroform extraction and lithium chloride precipitation as described in Chapter 2. Following elution, RNA was diluted ten fold and checked for integrity on a spectrophotometer. RNA was used in microarray experiments only when the concentration was ~1000µg/mL with a 260:280 ratio of at least 1.7, preferably 1.8.

RNA was then dispensed into aliquots (typically 30µg) and precipitated with 0.1 volumes of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol. The RNA precipitate was collected by centrifugation for 10 min at 20,000 xg, washed twice with 200µL 70% ethanol and centrifuged for 3 min at 20,000 xg. Pelleted RNA was then dried at room temperature to remove traces of ethanol before being used in microarray experiments or stored at -20oC until use.

6.2.4 Gel Electrophoresis Gels were run in 1xTBE buffer at 90-110V for 60-90 min, with 5µL of Hyperladder I (#BIO33025, Bioline) DNA size standard.

6.2.5 CyScribe Post-Labelling Kit (Indirect Labelling) For indirect microarray labelling a CyScribe Post-Labelling Kit (#RPN5660, Amersham, UK) was used for amino allyl labelling of cDNA and CyDye coupling. This was carried out following manufacturer’s protocols except that a QiaQuick PCR cleanup kit (Qiagen) was used for all purification steps (Section 2.7.2).

RNA (30µg) was combined with 1µL random hexamers and 1µL oligo dT primers for each reaction, and made up to 11µL with nuclease free H2O. This was heated to 70oC for 5 min and cooled at room temperature for 10min. Then, 4µL 5x CyScript buffer, 2µL 0.1M DTT, 1µL 20x dNTP mix, 1µL AA dUTPs, 1µL CyScript reverse transcriptase was added. This was mixed gently and incubated at 42oC for 90 min. 136


RNA was degraded from the cDNA by adding 2µL 2.5M NaOH, mixed and incubated for 15 min at 37oC. To neutralise, 10µL of 2M HEPES free acid was added. A QiaQuick cleanup kit removed nucleotides and degraded RNA (Section 2.7.2), and cDNA was eluted in 60µL of 0.1M sodium bicarbonate (pH 9.0) buffer.

Reactive CyDye NHS ester was then coupled to the amino-allyl cDNA by resuspending a single freeze-dried dye aliquot in the amino-allyl cDNA solutions (60µL of 0.1M sodium bicarbonate), incubated at room temperature for 90 min in a light-proof container.

Excess CyDye was quenched by adding 15µL 4M Hydroxylamine to each reaction and incubated in the dark for 15min. A QiaQuick PCR cleanup kit removed excess CyDye. Purified cDNA was then eluted in 60µL of Hybridisation Buffer #1 (Ambion) and hybridised to slides as outlined above.

6.2.6 Burton’s random amplification method The method devised by Burton et al. (2005) was tested as a randomly primed amplification method for plant virus detection. This method uses a primer (Random A) with eight N’s at its 3’ end and a 5’ adaptor sequence complementary to the primer Random B, which is used for subsequent amplification reactions. Two rounds of PCR are used to amplify and label virus cDNA from starting total RNA. Sequences of oligonucleotide primers are shown in Table 3.1.

Following the modified method of Burton et al., (2005) (Lab protocol, N. Boonham, pers. Comm.. 2006) a combination of the Omniscript and Sensiscript reverse transcription kits (Qiagen) were used to make first strand cDNA. This was done as follows: • • •

Combine: 2µL 10x buffer, 2µL 5mM dNTP’s, 2µL Random A primer (40µM), 0.5µL RNase Inhibitor, 0.5µL Omniscript reverse transcriptase, 0.5µL Sensiscript reverse transcriptase, 1.0µL template RNA (total RNA) and 6.5µL RNase free H2O. Mix and incubate at 37oC for 1h, then heat to 95oC for 3min. Cool to 4oC then add: 1.5µL 25mM MgCl2, 0.5µL 100mM DTT, 1.0µL Klenow fragment (Bioline) and 1µL 3mM dNTP’s. Mix and incubate at 37oC for 30min, then cool to 4oC. Dilute with 45µL molecular biology grade water.

The second round involved a randomly-primed PCR using the tagged sequence Random Primer B and diluted template from Round A. This was carried out as follows: •

Combine: 10µL 10xTaq DNA polymerase buffer, 8µL 25mM MgCl2, 2µL 10mM dNTP’s, 1µL 100µM Random Primer B, 1µL Taq DNA polymerase (Fermentas), 15µL diluted template from round A and 63µL H2O.

137


•

94oC for 10s, then 94oC for 30s 40oC for 30s 50oC for 30s 72oC for 60s 72oC for 2min, store at 14oC.

Mix and thermocycle as follows: 35 cycles

A 10µL aliquot of Round B amplicon was run on a 1% agarose gel to confirm amplification and the intensity of the smear was used to decide the amount of dilution for round C. Template DNA was diluted 1 in 10 or used undiluted. A second round of PCR with fluorescent dNTP’s was used to label the products. This was carried out as follows: •

•

Combine 10µL 10x Taq DNA polymerase buffer, 8µL 25mM MgCl2, 2µL dNTPs (2.5mM dATP, dTTP, dGTP, 1.0mM dCTP), 1.5µL Cy3 dCTP (Amersham), 1µL 100µM Random Primer B, 1µL Taq DNA polymerase (Fermentas), 10µL diluted template from round B and 66.5µL H2O. Mix and thermocycle as follows: 94oC for 10s, then 94oC for 30s 25 cycles 40oC for 30s 50oC for 30s 72oC for 60s 72oC for 2min, store at 14oC.

The PCR product from the final round of amplification was purified with a QiaQuick PCR cleanup kit (Qiagen). Purified labelled cDNA was then eluted in 60µL of Hybridisation Buffer #1 (Ambion) and hybridised to slides as above.

6.2.7 Klenow based random amplification Two methods for random amplification devised by Vora et al., (2004) were tested. They were based on random primed PCR using Taq DNA polymerase and Klenow fragment DNA polymerase for amplification. These two methods were used together in tandem as either a random PCR followed by Klenow amplification, or as Klenow amplification followed by another round of Klenow amplification.

rPCR Amplification • • • •

Reverse transcription was carried out using the Omniscript/Sensiscript method (Section 6.2.6) except that random nonamers were used instead of Random A primer. 1µL of total RNA (approx. 1µg) from plants infected with PVX or PVY was used for template The reaction was purified using QiaQuick PCR cleanup kit. DNA was eluted in 30µL RNase-free water. Combine 5µL 10x PCR buffer, 5µL 25mM MgCl2, 2µL 10mM dNTP’s, 1µL 100µM N9mer, 20µL first strand (fs) cDNA template, 1µL Taq DNA polymerase (Fermentas) and 16µL H2O.

138


•

94oC for 5min, then 94oC for 30s 25oC for 2min 72oC for 1min then store at 14oC.

Mix and thermocycle as follows: 35 cycles

A 10µL aliquot of first strand cDNA was kept and run on a 1% agarose gel.

Klenow Amplification • • • • •

The reverse transcription was carried out using the Omniscript/Sensiscript method (Section 6.2.6) using 1µL of PVX or PVY infected total RNA (approx. 1µg). Reaction was purified using QiaQuick PCR cleanup kit as described before. DNA eluted in 30µL molecular biology grade water. Combine: 5µL 10x PCR buffer, 5µL 25mM MgCl2, 2µL 10mM dNTP’s, 1µL 100µM N9mer, 20µL fs cDNA template, 1µL 2mM DTT and 15µL water. Mix well and heat denature at 95oC/5min and cool at room temperature. Add 1µL Klenow DNA polymerase I (Promega, USA) and mix before incubating at 37oC/4h. Store at 4oC.

Following rPCR or Klenow amplification, DNA was again purified using the QiaQuick PCR cleanup kit and eluted in 45µL H2O. A second round of Klenow amplification was then carried out as described above except that 35µL of purified 1st round amplification product was used as template, no water was added to the reaction and 1µL aa-dUTP/dNTP* mix was used (* 10mM each dATP, dCTP, dGTP + 6mM aa-dUTP + 4mM dTTP).

This second round Klenow

amplification reaction was again purified using the QiaQuick PCR cleanup kit and eluted in 70µL of 0.1M NaHCO3 (pH 9.0). Reactive CyDye NHS ester was then coupled to the purified DNA using the CyScribe post labelling method (Section 6.2.5), and hybridised to the arrays.

6.2.8 Genus-specific primer amplification Genus-specific primers for Potexvirus (van der Vlugt & Berendsen 2002) and Potyvirus (Gibbs & Mackenzie 1997) were modified to include a 5’ adaptor sequence. The adaptor sequence was used for amplification and labelling reactions as described previously (Section 6.2.6). The primers of Gibbs & Mackenzie 1997 (potyvirid 1 & 2) were modified by excluding the restriction enzyme sites at the 5’ end (Table 3.1).

Amplicons were then fluorescently-labelled with Cy3 dCTP and hybridised to microarrays. Total RNA extractions were used as template during 1st strand cDNA. This was synthesised using the Sensiscript/Omniscript method (Section 6.2.5) except Potyvirid 1 or Potex1RC primers were used instead of Random A primer.

139


PCR amplification was then carried out using primer Potyvirid 2 or Potex5 for 10 cycles before Random primer B was added and cycling continued for 30 cycles. This was carried out as below: •

Combine: 10µL 10x PCR buffer, 8µL 25mM MgCl2, 2µL 10mM dNTP’s, 1µL Taq DNA polymerase (Fermentas), 1µL 100µM Potyvirid 2 primer/Potex5 and 15µL diluted Round A cDNA template. • Mix and thermocycle as follows: 94oC for 10s, then 94oC for 30s 10 cycles 40oC for 30s 50oC for 30s 72oC for 60s o 72 C for 5min during which add 1µL 100µM Random B primer, then: 94oC for 30s 30 cycles 40oC for 30s 50oC for 30s 72oC for 60s 72oC for 2min, store at 14oC. A 10µL aliquot was then run on a 1% agarose gel to confirm amplification. A second round of PCR was carried to further amplify and fluorescently label the PCR amplicon. This was carried out as for Burton’s random amplification method (Section 6.2.6) except that the Round B PCR product was used undiluted. This product was purified with a QiaQuick PCR cleanup kit and hybridised to the microarray.

6.2.9 Real Time PCR The amount of virus template before and after amplification was estimated using real time PCR or reverse transcription real time PCR. Real time PCR was carried out on virus templates (PVX & PVY) and Cytochrome oxidase (COX) as described in Section 2.5.3 using protocols supplied by Dr Neil Boonham of CSL. COX was used as control to estimate the effect of amplification methods on plant mRNA’s.

An outline of the amplification methods used in this research project is included in Figure 6.2.

140

CyScribe (non-amplified control)

RNA Reverse transcription

First strand cDNA (amino allyl labelled) Conjugate NHS-Cy3

Labelled DNA (including vDNA)

Burton labelling RNA Reverse transcription Incorporate ‘tagged’ primers

fs cDNA (Round A) PCR using ‘tagged’ primers

Round B DNA 2nd PCR using ‘tagged’ primers and fluorescent nucleotides

(labelled DNA)

Round C DNA

fs cDNA

(with Klenow pol.)

Klenow amp.

Reverse transcription

RNA

Klenow Labelling

rPCR (using

N6mers)

2nd Klenow amplification (with Klenow polymerase and fluorescent nucleotides)

(including vDNA)

Labelled DNA

Microarray and real time PCR analysis

Genus-specific primers

RNA

Reverse transcription Incorporate ‘tagged’ genus specific primers

fs cDNA (Round A)

PCR using ‘tagged’ primers

Round B DNA

2nd PCR using ‘tagged’ primers and fluorescent nucleotides

(labelled DNA)

Round C DNA

Figure 6.2: Overview of the three amplification methods and non-amplified control which were tested to improve microarray sensitivity All methods were analysed by real time PCR and microarrays. The concentration of virus (PVX and PVY) and ubiquitous plant control (COX) were measured by real time PCR. Fluorescently labelled amplicons were hybridised to microarrays containing virus specific probes.


6.3 Results The results of the real time PCR and microarray hybridisations for each of the random amplification methods are presented here. These were compared to the real time PCR and microarray hybridisations for the CyScribe Post labelling method (no amplification).

6.3.1 Comparison of PVX and PVY virus titre The relative amounts of PVX and PVY nucleic acid in a sample of total tobacco RNA was estimated by microarray hybridisation and confirmed by real time PCR. A microarray slide was simultaneously hybridised with 30µg of PVX infected total RNA labelled with Cy3 (Green) and 30µg of PVY infected total RNA labelled with Cy5 (Red) (Figure 6.3). Labelling was carried out using the CyScribe Post-Labelling kit as described above.

Figure 6.3: Comparison of PVX-labelled cDNA (Green) to PVY-labelled cDNA (Red). 30µg of total RNA was used for each labelling reaction. Yellow probes (probes #1&23) represent transcripts present in each plant at approximately the same concentration. White probes (#9&11) represent pixels with a saturated fluorescent intensity. See Figure 6.1 for the layout of the array and Table 3.3 the sequence of probes.

From the dual hybridisation it is clear that the amount of PVX present is much higher than the amount of PVY present (i.e. probes 9, 11 & 12 are brighter than probes 13-22). Three of the four green PVX probes (second row) are expressed at high levels with white representing saturated pixels (Probes 9&11). The red PVY probes (13-22) fluoresce faint and this indicates that the amount of PVY was much lower. This difference is not due to the amount of total RNA used, or the labelling efficiency, since the intensities of 18S rRNA in each reaction (probe 1 & 23) is similar (top left and bottom bright spots), which indicates equal labelling with Cy3 and Cy5 18S rRNA probes.

A histogram which presents the quantified relative fluorescence of each probe on the array (corrected for background) is provided in Figure 6.4 which shows the relative fluorescence of the microarray probes. The PVX probes C#27 and PVX 4#1 clearly fluoresce higher than any of the PVY probes. The brightest of the PVY probes (PVY 59#1) is only ~2.8% as intense (1808 mean fluorescence units) compared to 64451 mean fluorescence units for PVX 4#1. The two 18S rRNA control probes show essentially the same fluorescence for both PVX and PVY labelled RNA. This indicates that there was no bias in the amount of RNA added, or in the labelling efficiencies with Cy3 and Cy5. Real time PCR of PVX and PVY RNA samples 142


confirms this result because the threshold cycle (Ct) values show that the amount of PVX is higher in both the RNA extractions and after labelling compared to PVY (Figure 6.5). A three cycle difference is approximately equal to a ten fold difference in template concentration (Lee et al., 2006). Therefore because a six cycle difference was observed it is reasonable to assume there was a 100 fold difference in the amount of virus RNA. The CyScript method also converts most of the RNA template available in the RNA extraction to first strand cDNA as seen by the similar Ct values for the RNA extraction and labelling reactions. PVX PVY

Mean Fluorescence minus background

60000

50000

40000

30000

20000

10000

co n C trol SV C d9 SV 7 d 2 PS 42 T PS V TV d 9 TS D 2 W 50 V TS 04 W #1 Pe V 0 pM 4# V 2 03 PV # 1 X PV 4# X 1 C PV # 2 X 6 C PV #2 X 7 1 PV 1# Y 1 5 PV 5# Y 1 5 PV 7# Y 1 5 PV 9# Y 1 5 PV 8# Y 1 9 PV 7# Y 1 PV 99 Y #1 1 PV 03 Y #1 10 PV 1 # Y 1 PV 54 Y #1 10 2# co 1 nt ro l

0

Probe Name

Figure 6.4: Fluorescence levels of PVX and PVY cDNA labelled by CyScribe Kit. Background corrected fluorescence for all probes on the array for both PVX (lilac) and PVY (magenta). Data represents measurements of three replicate patches from a single slide. 25

RNA Extraction CyScribe Post Labelling 20.3

20

20.3

19.5

19.2

15

Ct Value

13.4

12.7

10

5

0 PVX

PVY

COX

Real Time Assay

Figure 6.5: Real time PCR results for PVX, PVY and COX assay from both the RNA extraction (lilac) and following CyScribe labelling (magenta). Threshold cycle values (Ct) are above the column and represent the average of two replicate reactions.

143


6.3.2 Burton’s amplification method The first random amplification method tested was based on the method of Burton et al., (2005). Five different amounts of RNA were used as template for the reaction (from 0.25µg to 30µg) for both PVX and PVY.

Labelling reactions were hybridised to the microarray slides

containing PVX, PVY and non-target virus spots with the relative fluorescence measured for each spot. The labelled reactions were all analysed by real time PCR. The results from this method were then compared to the CyScribe post-labelling method. The scanned arrays are shown in Figure 6.6 and the mean relative fluorescence values for each probe are provided in Figure 6.7. The real time PCR results are in presented Figure 6.8.

For PVX, the lower template levels of RNA gave more intense PVX and control spots, especially at 0.25µg of RNA. At higher RNA concentrations, such as 10µg and 30µg of RNA, the PVX spots did not become brighter but the background on the slide increased, making it impossible to call the sample as PVX positive. For all PVX slides the 18S rRNA control probe (#1&23) was positive. In several of the slides other probes, such as PSTVd9 (#4) at 0.5µg of RNA, and TSWV04#2 (#7) at 30µg of RNA, were positive. The real time PCR results also show an increase in template concentration for PVX for each cycle of amplification; however the concentration of PVX was still much lower than that in the original RNA extraction as well as the CyScribe post-labelling method.

The method of Burton et al., (2005) improved the sensitivity of detection of PVX and PVY. For instance, the fluorescence seen on the array for probes PVX 4#1 (#9) and PVX C#27 (#11) were approximately as intense when using 0.25µg of RNA and Burton’s amplification method (Figure 6.7) as 30µg of RNA using the CyScript labelling method (Figure 6.4). The difference in fluorescence was ~22% between the methods; however there was a 120 fold difference in the amount of RNA added.

PVY was not detected accurately in any of the slides using any amount of RNA. The 18S rRNA probe was clearly positive, but no PVY-specific spots were bright enough to call as positive. In addition, spots for other viruses such as PVX, TSWV and PSTVd were positive. This can be seen in Figure 6.7 where the spot for PSTVd9 (#4) is strongly positive when 0.25µg of PVY RNA was used. The PVY and COX real time PCR results (Figure 6.8) also show an improvement in template concentration using Burton’s method, but the original RNA extraction and CyScribe methods still showed much higher template concentrations.

144

Figure 6.6: A comparison of the amount of virus infected RNA used as template using the amplification method of Burton et al., (2005). A-E: Slides hybridised with PVX labelled DNA, F-I: Slides hybridised with PVY labelled DNA. A: 0.25µg labelled PVX RNA, B: 0.5µg labelled PVX RNA, C: 0.9µg labelled PVX RNA, D: 10µg labelled PVX RNA, E: 30µg labelled PVX RNA, F: 0.25µg labelled PVY RNA, G: 1.5µg labelled PVY RNA, H: 10µg labelled PVY RNA, I: 30µg labelled PVY RNA. All PVX samples were labelled with Cy3 and all PVY samples with Cy5. Slides were scanned with a PMT gain of 800 and laser power of 100%. The sequences and locations of probes are provided in Figure 6.1 and Table 3.3.


Mean Fluorescence minus background

60000

50000

PVX 30ug RNA

PVX 10ug RNA

PVX 0.5ug RNA

PVX 0.25ug RNA

PVY 30ug RNA

PVY 10ug RNA

PVY 0.25ug RNA 40000

30000

20000

10000

C

co nt ro SV l d C SV 97 d 2 PS 42 TV PS d TV 9 TS D 2 50 W V TS 04 W #1 V Pe 0 pM 4#2 V 03 PV #1 X PV 4#1 X C PV #2 X 6 C PV #27 X 1 PV 1#1 Y 5 PV 5#1 Y 5 PV 7#1 Y 5 PV 9#1 Y 5 PV 8#1 Y 9 PV 7#1 Y PV 99# 1 Y 1 PV 03# 1 Y 10 PV 1#1 Y PV 54# 1 Y 10 2# 1 co nt ro l

0

Probe Name

Figure 6.7: A comparison of amount of RNA used for labelling showing the resultant average fluorescent intensity of microarray probes specific for PVX, PVY and non-target viruses. Four amounts of PVX containing RNA (30, 10, 0.5 & 0.25µg) and three amounts of PVY containing RNA (30, 10 & 0.25µg) were labelled using the method of Burton et al., (2005). Values represent the average of three replicate patches from a single slide. 40

40.0

PVX PVY COX

35.4 35 30.5 28.8 28.8

30

24.8

Ct Value

25

23.2 19.2

20

15

20.3 18.9

19.7

13.4

19.5

20.3

12.7

10

5

0

RNA Extraction

Burton Round A

Burton Round B

Burton Round C

CySribe Post Labelling

Figure 6.8: Threshold cycle values for all stages of labelling using Burton’s amplification method and the CyScribe post-labelling method. PVX, PVY and COX Ct values for RNA extraction, and three rounds of amplification are provided together with the results from the CyScribe labelling method. Ct values are given above each column and represent the average of two replicate reactions.

In general the Burton method gave higher non-specific binding, compared to the CyScript method. The non-specific binding is evident in Figure 6.7 where all the spots show a much higher level of fluorescence especially compared to the levels in Figure 6.4. Hybridised images of the slides (Figure 6.6) show spots only slightly brighter than background. Non-specific hybridisation occurred, for instance the PSTVd #9 (#4) and TSWV04#2 (#7) spots. 146


6.3.3 Amplification using Klenow labelling The second random amplification method used was based on a Klenow fragment DNA polymerase labelling reaction and random primed PCR, as reported by Vora et al., (2004). Single and tandem amplification reactions were used to determine the amount of PVX and PVY by real time PCR (Figure 6.9). Scanned microarrays of labelling reactions (Figure 6.10) are also included.

The real time PCR results showed that neither single nor tandem amplification methods worked for PVX or PVY. The cDNA had the highest titre of both methods with Ct values of 22.9 and 30.3 for PVX and PVY respectively. These Ct values are several cycles higher than those obtained for the single and tandem amplification methods.

The Ct values for the single

amplification methods (rPCR and Klenow) were also lower (indicating more of the template is present) than for the tandem amplification methods (rPCR/Klenow + Klenow/Klenow). A reduction in Ct indicates a reduction in template concentration.

This means that after

amplification there was less virus cDNA present. For instance the rPCR method had Ct values of 24.3 for PVX compared to the rPCR/Klenow where the Ct was 25.0. For PVY the results of the Klenow/Klenow amplification (32.5) were marginally better than rPCR/Klenow (34.2). Both tandem amplification methods failed to amplify PVY nucleic acid when compared to the initial cDNA, which has a Ct of 30.3. The same result was seen with COX which showed a reduction in the amount of nucleic acid from a Ct value of 31.4 in the original cDNA to a Ct value of 34.2 and 34.9 for the Klenow/Klenow and rPCR/Klenow reaction respectively. 40

34.89 34.19

35

31.43 30.25

32.64 31.65

31.73

Amplification method

34.17 32.50

29.82

30

27.51 25

PVX PVY COX

27.41

24.96

24.26 22.89

20

15

10

5

0 cDNA

rPCR

Klenow

rPCR/Klenow

Klenow/Klenow

Ct value

Figure 6.9: Real time PCR cycle threshold values for Klenow amplification of three stages of amplification. Ct values for cDNA synthesis step, both single amplification methods (rPCR & Klenow) and both tandem amplification methods (rPCR/Klenow & Klenow/Klenow) are shown above columns as an average of two replicate reactions. Results for PVX (lilac) and PVY (magenta) and COX (green) are given for each method.

147


The tandem amplification methods showed no virus signal for either method after hybridisation to microarray slides (Figure 6.10). This approach was repeated twice with no hybridisation signal obtained. Given the lack of amplification from the real time PCR results and the negative results from microarray hybridisations it was decided not to continue work on this method.

A

B Figure 6.10: Microarrays scanned for fluorescence after hybridisation with Klenow amplified total nucleic acid from PVX (Cy3) and PVY (Cy5) infected plants. A: DNA amplified by rPCR with a second round of Klenow amplification, B: DNA amplified by Klenow followed by a second round of Klenow amplification. Arrays were scanned with a PMT gain of 800 and a laser power of 100%.

6.3.4 Genus specific primer amplification The genus specific primers of van der Vlugt & Berendsen (2002), and Gibbs & Mackenzie A range of MgCl2 concentrations (1-

(1997) were tested in a random amplification method.

3mM) and two PCR buffers were optimised for use with genus specific primers. One buffer contained KCl and the other contained (NH4)2SO4.

Each primer pair was tested for

amplification efficiency at a range of MgCl2 concentrations (1-3mM) using both buffers. The amount of PVX, PVY and COX RNA was estimated by real time PCR in each case. Different amplification reactions were carried out for PVX and PVY and therefore a COX real time PCR assay was carried out for each virus. The values are given individually for PVX and PVY. Samples of each reaction were also analysed by gel electrophoresis (Figure 6.11).

850bp

M

1

2

3

4

5

M

6

7

8

9

10 M

Figure 6.11: Optimisation of PCR conditions for Potexvirus and Potyvirus fragment amplification under two buffer conditions: KCl and NH4SO4. M is 100bp DNA ladder (Bioline). Lane 1: fs PVX cDNA, 2: KCl buffered Round B (2.5mM MgCl2) of PVX, 3: (NH4)2SO4 buffered Round B (2.5mM MgCl2) of PVX, 4: KCl buffered Round C (2.0mM MgCl2) of PVX, 5: (NH4)2SO4 buffered Round C (2.0mM MgCl2) of PVX, 6: fs PVY cDNA, 7: KCl buffered Round B (2.5mM MgCl2) of PVY, 8: (NH4)2SO4 buffered Round B (2.5mM MgCl2) of PVY, 9: KCl buffered Round C (2.0mM MgCl2) of PVY, 10: (NH4)2SO4 buffered Round C (2.0mM MgCl2) of PVY.

148


The results showing the amounts of PVX DNA measured by real time PCR is shown in Figure 6.12. These results indicate that the optimisation of the PCR significantly affected the amount of PVX template present for labelling in round C.

Using genus specific primers for

amplification decreased the Ct of PVX from 26.6 in the cDNA labelling reaction (Round A) to 18.8 for 2.5mM MgCl2 with KCl based polymerase buffer (i.e. the amount of virus template increased). In Round C this was increased slightly to 22.9 using a KCl based polymerase buffer. PVX template was not detected (Ct value 40) when the (NH4)2SO4 buffer was used. The decrease in Ct of 3.7 cycles over the entire amplification method represents approximately a ten fold increase in PVX template concentration by this amplification method. 45 40

PVX KCl PVX NH4SO4 COX KCl COX NH4SO4

40.0 37.6 38.1

39.2

37.3 36.7

36.4

36.1

35 Threshold cycle number (Ct)

40.0 40.0

38.2 35.2 33.2

31.2 31.2

31.1

30 26.6 26.6

25

23.8 23.3

22.9 21.2

20

18.8

19.0

15 10 5 0 Round A

1.0mM

2.0mM

2.5mM

3.0mM

Round C

Figure 6.12: Real time PCR measurements of each stage of the genus specific primer amplification process for PVX. PVX and COX were amplified using potassium chloride and ammonium sulphate based PCR buffers. Average Ct values for duplicate reactions are given above each column. Round A represents the cDNA synthesis reaction, while 1.0mM to 3.0mM are Round B amplifications of differing MgCl2 concentrations. Round C is the final reaction when the DNA is fluorescently labelled and further amplified.

An unexpected result was that the Ct values of COX increased in each of the methods tested, and this indicates that virus templates were amplified preferentially to COX mRNA. In the first strand cDNA (Round A) the Ct for COX was 31.2 and this increased to between 35.2 and 38.2 depending on the reaction conditions; however at 2.0mM MgCl2 using (NH4)2SO4 polymerase buffer was undetectable. For round C the COX Ct was 39.2 for (NH4)2SO4 polymerase buffer and undetectable (40) for KCl. Over the three rounds the COX template was reduced by 13 Cts which represents a significant reduction in non-virus templates present in the reaction.

The concentration of MgCl2 was therefore important to the amplification efficiency of the reaction, but this effect depends on the sequence being amplified. This is indicated by the Ct value for PVX varying from 33.2 (2.0mM) to 18.8 (2.5mM) over the range of magnesium 149


chloride tested here. The concentration of COX, however, did not change significantly across same range of MgCl2 concentrations, meaning that PVX was preferentially amplified over plant cDNAs, represented by COX. The concentration of COX most likely decreased because of dilution effects from one round to the next. Of the two PCR polymerase buffers tested, the KClbased polymerase buffer worked best with Ct values lower than for the (NH4)2SO4 based polymerase buffer.

A similar improvement in template concentration was seen for PVY (Figure 6.13) where the Ct value decreased from 40, i.e. undetectable in first strand cDNA, to 22.0 when 2.0mM MgCl2 was used with (NH4)2SO4 polymerase buffer (Round B). In round C the Ct for PVY was slightly increased to 23.2 to indicate a lower PVY concentration. Using genus specific primers the concentration of virus template increased dramatically but the actual amplification could not be estimated with any accuracy because PVY was not detectable in the first strand cDNA (i.e. in Round A when the Ct value was 40). The amount of COX template also decreased during the virus amplification from 30.7 in the first strand cDNA to between 24.3 and 35.4 (Figure 6.13) in round B, depending on the buffer and MgCl2 concentration used. In round C this was further reduced to between 34.5 and 35.9, so over the three rounds of amplification the amount of COX was reduced by up to 5.3 cycles which represents approximately a 100 fold reduction. This is probably due to the dilution from one round to the next, which was also seen with PVX, with no amplification of COX template. 45 40

PVY KCl PVY NH4SO4 COX KCl COX NH4SO4

40.0 40.0

Threshold cycle number (Ct)

35

40.0

33.0 30.7 30.7 29.1

30

32.7

34.5

32.4

32.0

35.9

35.6

35.4

31.7

30.0

30.9

26.4 24.3

25

22.6

22.9

22.0

23.2

24.2

20 15 10 5 0 Round A

1.0mM

2.0mM

2.5mM

3.0mM

Round C

Figure 6.13: Real time PCR measurements of all each stage of the genus specific primer amplification process for PVY. PVY and COX were amplified using potassium chloride and ammonium sulphate based PCR buffers. Average Ct values for duplicate reactions are given above each column. Round A represents the cDNA synthesis reaction, while 1.0mM to 3.0mM are Round B amplifications of differing MgCl2 concentrations. Round C is the final reaction when the DNA is fluorescently labelled and further amplified.

150


The composition of the PCR buffer was also very important for PVY amplification with 2.0mM MgCl2 with (NH4)2SO4 polymerase buffer giving the lowest Ct value; however 2.0mM MgCl2 and KCl polymerase buffer also worked well (Ct 22.6). The KCl buffer worked well over a range of MgCl2 concentrations for both viruses and gave a slightly lower Ct value for PVY in round C (23.2 to 24.2).

In the final found of amplification (Round C) fluorescent nucleotides were included and the resultant amplicons were hybridised to the microarray. Both viruses were clearly detected (Figure 6.14).

For both KCl (Figure 6.14A & C) and (NH4)2SO4 Figure 6.14B & D)

polymerase buffers PVX and PVY spots were present; however for both PVX and PVY the (NH4)2SO4 buffer clearly worked better.

Figure 6.14: Scanned microarray images using Potexvirus and Potyvirus specific primers for amplification; A; PVX amplified with KCl PCR buffer, B; PVX amplified with (NH4)2SO4 PCR buffer, C; PVY amplified with KCl PCR Buffer, D; PVY amplified with (NH4)2SO4 PCR buffer.

The microarray images when scanned and quantified showed that PVX and PVY were detected accurately and there was no non-specific hybridisation (Figure 6.15). For PVX the ammonium sulphate buffer gave higher hybridisation signals than the potassium chloride buffer. This could be seen by eye on the slides (Figure 6.14). For PVY the potassium chloride buffer gave a more consistent signal for most probes (55#1, 57#1, 59#1, 58#1 & 103#1) that was higher than that under ammonium sulphate buffering conditions, except probe 103#1 (#19) which was higher using the ammonium sulphate buffer. The 18S rRNA probe (#1&23) was only positive in Figure 6.14D, which was unexpected as the genus specific primers should not have amplified any sequences which could hybridise to the probe.

151

VI: AMPLIFICATION METHODS FOR LABELLING PVX KCl PVX NH4SO4 PVY KCl PVX NH4SO4

40000

30000

20000

10000

0 co n C trol SV C d9 SV 7 d 2 PS 42 T PS Vd TV 9 TS D 2 W 50 V TS 04 W #1 V Pe 0 pM 4#2 V 03 PV #1 X PV 4# X 1 C PV #2 X 6 C PV #27 X 1 PV 1#1 Y 5 PV 5#1 Y 5 PV 7#1 Y 5 PV 9#1 Y 5 PV 8#1 Y 9 PV 7#1 Y PV 99# 1 Y 1 PV 03# 1 Y 10 1 PV #1 Y PV 54# 1 Y 10 2# 1 co nt ro l

Mean fluorescence of probe with background corrected

50000

-10000

Figure 6.15: Fluorescence levels of PVX and PVY PCR products labelled using either a potassium chloride or ammonium sulphate based buffer. The product was hybridised to a microarray (see Figure 6.14 for slide images). Data represent background corrected measurements of three replicated patches from a single slide. Controls are included of probes from host 18S rRNA genes. See Table 3.3 for probe sequences.

152


6.4 Discussion The four methods of microarray labelling tested showed clear differences in sensitivity of detection with three of them. CyScribe post-labelling, Burton’s method and genus-specific primers, allowed detection of the two plant viruses tested. The amplification methods increased the copy number of virus target molecules, but this did not always increase the sensitivity of the array.

6.4.1 CyScript post labelling The CyScript post labelling system from Amersham Biosciences was sensitive for both viruses tested and relatively easy to perform. When testing for low abundance viruses an efficient reverse transcriptase which converts the majority of vRNA into cDNA will improve the sensitivity of virus detection. The amount of virus template when measure by real time PCR (Figure 6.5) was close to the level seen in the original RNA extraction, which was indicated by the close Ct values between the RNA and CyScript samples. The concentration of PVX was also found to be much higher than PVY with real time PCR showing a six Ct difference between them. Differences in virus concentration in samples could make detection of a low-abundance virus difficult if it is swamped by the high-abundance virus. The real time PCR and microarray results on the CyScript post-labelling method confirmed the need to investigate amplification methods when using microarrays for detection of plant viruses.

One of the advantages of a microarray system over other molecular diagnostic methods such as ELISA and RT-PCR is the highly parallel testing of a single sample for many different virus species. Parallel detection was demonstrated for two viruses (PVX and PVY) on an array containing virus specific probes. The strategy of microarray based diagnostics could be applied to detect additional viruses in plants infected with two or more viruses at once.

6.4.2 Burton amplification method The method of Burton et al., (2005) was not suitable for virus amplification because at all RNA concentrations tested PVY was not identified clearly. The reason for this result is not clear because the real time PCR results (Figure 6.8) show that a comparable amount of PVY template was present (Ct of 19.7 for round C compared to 19.5) as obtained using CyScript post labelling, which gave clear hybridisation for several PVY probes (Figure 6.3). An equivalent amount of RNA for both methods showed non-specific hybridisation in Burton’s method. So while the fluorescent values are higher in Figure 6.7 than for Figure 6.4, a clear identification cannot be made. 153


An excess of RNA in labelling reactions may swamp the reaction because when 0.25µg of PVX RNA was added to labelling reactions the resultant fluorescent signal on the array was higher than when using 0.5µg or 10µg of RNA (Figure 6.7). Starting with 30µg of total RNA in the labelling reaction gave the highest fluorescence on the microarray, however this could be because enough vRNA was present for no amplification to be necessary. Burton et al., (2005) found they were able to detect as little as 2.8fg of chromosomal Bacillus anthracis DNA using purified pathogen DNA, but not from a complex sample containing a large excess of plant RNA, such as used in this study. In addition the targets of the array were rRNA gene transcripts which are present in high copy numbers in Eukaryote and Prokaryote genomes. The copy number may be from 1-15 copies in bacteria (Klappenbach et al., 2001) and is higher in fungi (~150 copies in Saccharomyces cerevisiae, Kobayashi et al., 1998) and plants (several thousand in Cucurbita pepo, Torres-Ruiz & Hemleben 1994) making detection of these targets much easier.

In contrast to PVX, the amount of PVY DNA was lower than the amount of 18S rRNA as seen in Figure 6.3 (quantified values in Figure 6.4) because the positive control probes 1&23 (18S rRNA) were much brighter than PVY specific probes (13-22). Again differences in target virus concentrations could make detection difficult for low titre viruses, such as PVY, as the amplification of vRNA may be swamped by more common RNA species such as non-coding RNAs (tRNA & rRNA).

The extent of this potential problem could be determined

experimentally by adding increasing amounts of host RNA to a known and constant amount of vRNA which is then amplified and labelled. If interference occurred, the fluorescent intensity of virus specific probes would decrease as the amount of host RNA increased, to show clearly if swamping of the reaction was occurring.

The reverse transcription reaction (Round A) method did not work as well as the CyScript reverse transcription. As can be seen in Figure 6.8 there is a large difference in the amounts of templates for PVX, PVY and COX between Round A and the CyScript Post labelling. Optimisation of this step may improve the outcome of the method.

The non-specific binding seen in the arrays is unlikely to be because of hybridisation conditions. The same conditions were used for both methods. These conditions were shown to be effective in previous microarray experiments (N. Boonham, 2006, pers comm.)

The most likely

explanation is that the two rounds of PCR made short extension products because of the random nonamer primers used in Round A. The random nonamers when extended may bind nonspecifically to the array giving the high level of background seen on other probes in the array (Figure 6.7). Changing the hybridisation conditions could improve the specificity of the array but would also reduce the sensitivity of the array. 154


6.4.3 Klenow amplification The Klenow based amplification method of Vora et al., (2004) did not detect either PVX or PVY (Figure 6.10).

Real time PCR (Figure 6.9) confirmed this result, although more

replicates should be completed. The protocol followed that of Vora et al., (2004) as far as possible in which it was used to detect E. coli in purified and environmental samples. Using this method, Vora et al., (2004), tested five target genes in E. coli where and found a 20-150 fold increase in copy number using Klenow amplification, and an 8-156 fold increase in target copy number using rPCR. While virus template was available for labelling (Figure 6.9), its concentration actually decreased following the ‘amplification’ reaction to a value that was lower than for the CyScribe post-labelling (Figure 6.4) and Burton (Figure 6.8) amplification methods.

Vora et al., (2004) also tested the effectiveness of the method on an environmental sample containing non-target DNA sequences. Water samples were spiked with E. coli and total DNA extracted and labelled as for laboratory cultures. The level of background, or non-target DNA, was estimated at 63 times that of the spiked E. coli DNA. Using this environmental sample E. coli was detected successfully, but there was a 10 to 1,000 fold decrease in sensitivity compared to results from purified laboratory cultures.

The titre of virus RNA in cells varies significantly between cell types, host plants and virus species tested (Hull 2002). For a virus of low titre, such as PVY, a large excess of noncoding RNA, such as rRNA, that can constitute up to 80% of cellular RNA (Ambion Technical Bulletin #151, www.ambion.com/techlib/tb/tb_151.html, accessed 21/08/07; Wong & Medrano 2005) may have swamped the reaction as suggested in above. If random nonamer primers were replaced with a poly dT primer, only mRNA and vRNA would be reverse transcribed, but the disadvantage of this approach is that only poly-adenylated viruses such as the Flexivirdae and Potyviridae would be detected. Agindotan & Perry (2007) used random hexamers to amplify CMV, PVY and PLRV between 100 and 1000 fold in the presence of host-derived non-virus sequences. This was used for hybridisation to nylon macroarrays similar to microarrays.

6.4.4 Genus specific primers The use of genus-specific primers, while not a true random amplification approach, did show potential for use in microarray diagnostics. Using degenerate primers for amplification is similar to using rRNA or mitochondrial gene sequences for species identification. Using genusspecific primers both PVX and PVY were successfully detected (Figure 6.14 & 6.15) and the reaction amplified virus nucleic acid preferentially to COX (Figure 6.12 & 6.13); a result not seen with any of the other methods tested. At present group-specific primers for all the 155


genera/families of virus do not exist. A number of genus specific primers are available (Table 1.1), which could be used in microarray labelling. Group specific primers are fickle and may not amplify all intended virus species therefore they may be of limited in combination with microarrays.

Considerable bioinformatics analysis still needs to be done on virus sequences to design effective primers that can be used to detect most virus groups. Group specific primers are usually designed from conserved regions of the genome, but redundancy of the genetic code and high level of diversity in virus genomes means that mismatches are inevitable. The most common method to circumvent this is to synthesise primers containing all possible nucleotide sequences, but this approach can reduce sensitivity and specificity (James et al., 2006). An alternative is to incorporate bases with reduced base-pair stringency such as deoxyinosine, which can bind to all four regular nucleotides so improving duplex stability.

The amount of amplification achieved for PVX (3.7 Cts, Figure 6.12) and PVY (16.8 Cts, Figure 6.13) using genus-specific primers is below the upper limits for PCR amplification. Up to 3x1011 fold amplification of specific mRNAs has been achieved using total RNA (Iscove et al., 2002). The lower amplification efficiency using genus specific primers may be because the reaction became saturated with non-virus DNA, or because the amplification reaction needed further optimisation. The presence of smearing seen in the gel (Figure 6.11) indicates nonspecific amplification products were produced using the Potyvirus specific primers. This was probably due to the polyT stretch in the downstream primer, which could anneal to both the mRNA and vRNA sequences.

Optimisation of the PCR reaction conditions did improve the amplification of virus cDNA templates, however further optimisation could be made by testing other Potyvirus-specific primers (eg Chen et al., 2001a; Hsu et al., 2005; Langeveld et al. 1991) including those designed to amplify fragments of undescribed Potyvirus from the indigenous flora of Western Australia (LegPoty primers, Table3.3).

Further optimisation of the buffer used for amplification is also required because the potassium chloride buffer worked well for the PVY, but not for PVX, for which the ammonium sulphate buffer gave more amplification (Figure 6.14 & 6.15). Of concern was the positive result for the 18S rRNA probe when a PCR buffer containing ammonium sulphate was used. The potyvirid primers should only amplified the NIb and CP of Potyviruses (Gibbs & Mackenzie 1997), but the real time PCR results show that COX template was still detectable in the labelling reaction for PVY (Round C, Figure 6.13) so 18S rRNA may also be amplified non-specifically.

156


Amplification of 18S rRNA was the only evidence seen of non-specific hybridisation and indicates the use of genus specific virus primers reduced cross hybridisation. In comparison, the Burton method, which was similar except random primers with adaptor sequences were used, showed non-specific hybridisation for the PSTVd #9 (#4) and TSWV04#2 (#7) probes when tested with PVY labelled DNA. The fluorescent intensity of the slides (Figure 6.15) was also very high with fluorescence approximately 80% as intense as for the CyScript Post labelling (Figure 6.4), however only 1µg of each virus RNA was used compared to 30µg for the CyScript method. In future, a greater range of template RNA amounts could be tested in a similar way to Burton’s amplification method.

The primers of van de Vlugt & Berendsen (2002), which were used on PVX samples, amplify a fragment of the RNA dependent RNA polymerase (RdRp) of approximately 735bp. However, the four PVX probes used in this study hybridise to the coat protein which is located 3000nt from the region amplified with the Potex primers. The fluorescence seen on PVX probes may have been the results of mispriming between the primers and the PVX genome. The primer sequences used in this study included adaptor sequences on the 5’ end which were used for subsequent amplification round, unlike in the original report (van der Vlugt & Berendsen, 2002), and the thermocycling conditions were also changed. These two changes may have caused mispriming. Sequencing of the PCR product in Figure 6.11 and alignments to both the probe sequences and PVX genome would determine whether non-specific binding occurred. If non specific priming is occurring the reaction could be modified to improve stringency (e.g. lower MgCl2 concentration) or perhaps other Potexvirus primers could be tested (Table 1.1). The primers of Gibbs & Mackenzie (1997) amplify approximately 1.7kbp of the NIb, CP and 3’ UTR of most Potyvirus species.

The probes on the array which were not in this region, and

would therefore not be expected to show hybridisation, were: PVY 97#1, PVY 99#1 and PVY 101#1 (Table 3.3). Of the remaining probes five show hybridisation, particularly PVY 103#1 and two (PVY 54#1 & PVY 102#1) did not show hybridisation. The reason for the lack of fluorescent signal from the two probes is not known, but it could be that the strain of PVY used did not match the sequence of the probes on the array, although this could be tested experimentally. Sequencing of the PVY isolate to verify the sequence of the CP and possibly NIb would answer this question.

6.4.5 A Multiplex polyvalent PCR A test using polyvalent primers such as Potyvirus or Potexvirus primers would be of use for diagnostics, however when testing samples for unknown viruses, multiple pairs of primers would be used. These primers would be run in parallel on an infected sample as it would be 157


very difficult to optimise a single multiplexed reaction for multiple pairs of polyvalent primers. In some cases sequencing of the product would be simpler than hybridisation to a microarray so this approach may only have limited use. . Gel-electrophoresis should also be carried out on reactions before labelling and hybridisations, to confirm amplification and prevent wastage of expensive microarray slides and fluorescent nucleotides. This would increase the time required for a test and require more operator input, but should still detect co-infections. Depending on the time and cost constraints it may be easier to clone and/or sequence bands from a polyvalent PCR to identify the infecting agents.

An emulsion PCR could potentially be used to multiplex a PCR containing many sets of polyvalent primers. Emulsion PCRs utilise a mixture of PCR reagents, including: multiple primer pairs, DNA polymerase and nucleotides, suspended in a water-in-oil emulsion. An average of one amplification reaction occurs in each aqueous droplet of the emulsion which allows the simple amplification of multiple loci in complex mixtures of nucleic acids, for instance in amplification of genomic libraries (Williams et al., 2006). The advantage of an emulsion PCR is that the template is isolated in a minimum volume and amplified separately, which avoids the problems of short-fragment bias (Polz & Cavanaugh 1998; Williams et al., 2006) and generation of non-specific fragments (Meyerhans et al., 1990) which are seen in traditional PCR. A combination of an emulsion PCR, using a number of polyvalent primer pairs, with a microarray, for detection of specific virus nucleic acids, could prove a practical diagnostic tool.

6.4.6 Other amplification approaches Other amplification approaches could also be tested for amplification and microarray hybridisation.

Long oligonucleotide probes called padlock probes allow exponential

amplification of multiple pathogen species, with a microarray hybridisation used for identification of specific pathogens. Target complementary sequences are included at the 5’ and 3’ ends which recognise adjacent sequences of the pathogen DNA (Szemes et al., 2005). A unique zip sequence identifier and universal primer sequences serve for microarray hybridisation and universal amplification respectively.

Upon hybridisation to the target

sequence the probe ends are in adjacent positions and are ligated enzymatically. This concept was shown by Szemes et al., (2005) to detect as little as 5pg of plant pathogen DNA at the genus, species and subspecies level, however no virus pathogens were included in the study. The highly degenerate nature of RNA virus genomes may make the identification of 20nt regions that are both conserved and unique for probe design difficult, but that difficulty is already faced in PCR primer and microarray probe design. 158


The three methods tested in this study all involved the amplification of target nucleic acids. Signal amplification methods, such as dendrimer technology (Stears et al. 2000) and tyromide signal amplification (Karsten et al. 2002), could also be tested. These methods improve the amount of signal produced by each hybridised copy of pathogen nucleic acid. An increase in the signal produced by a small amount of hybridisation decreases the amount of pathogen nucleic acid needed for detection and therefore improves the sensitivity of detection. Nucleic acid amplification could be used in combination with signal amplification methods to further improve the sensitivity of diagnostic microarrays.

Other amplification methods (reviewed by Nygaard & Hovig 2006) could be used such as single primer amplification (Smith et al., 2003), however most of these methods give linear amplification of nucleic acids, whereas the methods discussed here give exponential amplification and would therefore be expected to give better sensitivity. By incorporating PCR primer sites at both ends of cDNA, exponential amplification can be achieved from low amounts of starting material. First strand cDNA synthesis is primed with an oligo dT primer and terminal transferase is used to create an oligo dA tail. A universal oligo dT primer can then be used to amplify total mRNA exponentially (Isocove et al., 2002). A similar approach was used by Hertzberg et al., (2001) in microarray analysis. Rolling circle amplification using Φ29 polymerase is useful in amplification of circular virus genomes, such as the Geminiviruses (Haible et al., 2006) where it was used to detect 1-50pg of virus DNA, but it has not been demonstrated for viruses with linear genomes.

Possibly combining Padlock probes for

circularisation of virus templates with the amplification of RCA could be used as a diagnostic method (Mumford et al., 2006a).

6.4.7 Conclusions Comparisons of the amplification methods (Table 6.1) showed the use of genus specific primers gave the largest amplification of virus template (real time PCR results) and resulted in a large increase (634%) in PVY probe fluorescence on the microarray. The random amplification method of Burton et al., (2005) worked well to increase PVY fluorescence (434%); however non-specific binding was seen, especially for PVY. Using genus specific primers this nonspecific binding was eliminated and sensitive identification of both PVX and PVY was made using small amounts of RNA (~1µg). This method also preferentially amplified virus templates over host templates such as COX, however using genus specific primers would limit testing to particular virus species/genera at a time. In comparison, the CyScribe labelling method gave the lowest Ct values of any method tested and these were close to the values from RNA extractions (Figure 6.4), however the fluorescence of PVY specific probes was low and using genus159


specific primers for amplification improved PVY probe fluorescence.

The CyScript method

gave clear identification of PVX and PVY and could be used for labelling all RNA viruses simultaneously, if random primers were included in the labelling reaction.

160

64,479 5,395 34.544

12.7 19.5 20.3

(non amplified control)A A simple method with supplied protocol and optimised reagents Can label all RNA viruses simultaneously

CyScribe labelling Klenow/Klenow

-4.5 -2.2 -2.7

-D Complex and time consuming protocol. No hybridisation seen on microarrays caused by low virus concentration.

-4.6 -4 -3.5

Random approach, therefore no information about potential viruses needed.

rPCR/Klenow

Klenow labelling

Genus-specific primers

3.7 16.8 -8

High amplification efficiency Concentration of plant RNA’s reduced. Viruses detected using 1/30th the RNA of the CyScribe kit 9.5 9.1 4

Range of viruses amplified depends on the primers used

-22.5% 673% 42%

Non-specific hybridisation on microarray. Plant RNA’s amplified in addition to vRNA.

-18.7% 434% -94%

Random approach, therefore no information about potential viruses needed.

Burton et al., (2005) labelling

Table 6.1: Overview of the advantages and limitations of the amplification methods tested for microarray based plant virus diagnostics

Advantages of the method Real time PCR results B Change in Ct of PVX Change in Ct of PVY Change in Ct of COX Microarray results C Increased fluorescence of PVX Increased fluorescence of PVY Increased fluorescence of 18S rRNA

Limitations

No amplification of vRNA Expensive Large amount of purified RNA needed (30µg)

Virus Names: PVX, Potato virus X; PVY, Potato virus Y; COX, Cytochrome oxidase. A: The CyScribe method did not include an amplification step and therefore real time PCR and microarray results did not measure an increase in virus concentration. This method was used as a control to compare the efficiency of the three amplification methods. B: Measured as the increase in threshold cycle number between the cDNA and final round of labelling for each method. Negative results indicate a rise in Ct values which is caused by a decrease in template concentration. Values for the CyScribe labelling method are absolute values, not relative values. C: Measured as the increase in the percentage increase in mean fluorescent intensity of the most fluorescent virus specific probe. Values for the CyScribe labelling method are absolute, not relative values. Negative values indicate a decrease in fluorescence compared to the CyScript labelling method. D: No signal seen on arrays hybridised with Klenow amplified DNA (Figure 6.9).

VII: GENERAL DISCUSSION

Chapter 7: General discussion

7.1 Overview In a major aspect of this research, HarMV, a newly-discovered species of Potyvirus was found in a member of the indigenous flora of the SWAFR. HarMV was characterised biologically and genetically, and its phylogeny determined by comparison with other virus species. To extend plant virus diagnostics, the application of microarray technology to virus detection was studied. Microarray slides were designed and tested to identify six distinct strains of HarMV simultaneously and to distinguish this virus from other related and unrelated viruses.

7.2 Characterisation of Hardenbergia mosaic virus A new virus was found that infected Hardenbergia comptoniana. Based on comparison of its CP gene to those of other species in the genus it was identified as a new species of Potyvirus. The name Hardenbergia mosaic virus (HarMV) was proposed for this virus. Adams et al., (2005b) suggests 23-24% nucleotide diversity in the CP as a line of demarcation between Potyvirus species - the closest relative of HarMV was PWV which was less than 75.7% identical to HarMV at the nucleotide level. Importantly, using phylogenetic reconstructions, it was possible to show the existence of a group of six distinct species of Potyvirus that form an Australian subgroup within the larger BCMV group of the Potyviruses.

Genomic

recombination was identified both within isolates of HarMV, and between it and other species of Potyvirus. Recombination is an important mechanism for evolution in viruses (Wrobel & Holmes 1999). The high diversity found between HarMV isolates (mean 13.5%, max 20.4%); suggest that HarMV has been present in Australia since long before European colonisation of Australia in 1788 (eastern) and 1829 (western). The common lineage with other species of virus known only from Australia supports this conclusion. This work represents the first study into a native virus of the SWAFR and has clarified the Australian origin of six Potyvirus species (Figure 4.12).

Plants infected experimentally with HarMV included two species of lupins (Lupinus angustifolius and L. luteus): lupins are the major grain legume grown in WA, and are thus economically important and are used for stock feed. The finding of a common endemic virus that could infect this legume crop represents a possible threat to WA lupin growers. Symptoms of apical meristem death and reduced seed set were found in L. angustifolius and L. luteus infected with HarMV. It is not known whether HarMV infects lupin species naturally; but similar symptoms occur when lupins are infected by non-necrotic strains of BYMV, a common 162


virus in Western Australia (Cheng & Jones 1999). It is therefore quite possible that BYMV may have been confused inadvertently with HarMV, because field surveys were mainly based on recording plants with symptoms (R. Jones 2006 pers. comm.). In the future, lupin crops should be surveyed for natural HarMV infection, and this will clarify and quantify this potential threat to the Western Australian lupin industry.

Most studies on Potyvirus species have involved viruses of cultivated species in managed ecosystems. Rarely have natural ecosystems been examined despite the fact that most viruses are derived from them (Cooper & Jones 2006). This study on HarMV represents one of the first detailed studies on a population of a native virus in its natural ecosystem that has been relatively unaffected by man’s activities. Isolates of HarMV fell into eight clades which all showed high inter-clade (6.24 to 20.4% nt diversity) and small intra-clade diversity (0.0 to 3.65% nt diversity). Genetic diversity between isolates is likely to be a response to genetically diverse hosts. In this case virus diversity is currently limited to H. comptoniana, and so presumably virus diversity reflects the adaptation of virus isolates to genetic differences in H. comptoniana at different locations.

HarMV isolates could be differentiated by their experimental host ranges. This is consistent with other members of the genus Potyvirus, such as BYMV, where host specialisation by strains is evident (Wylie et al., 2008) across its worldwide range (Edwardson & Christie 1991). The CP diversity of Potyvirus species is up to 22-24% (Adams et al., 2005b); however unlike other highly diverse species, such as BYMV, HarMV is found in a relatively small geographic range, in a single undomesticated and presumably highly diverse host. The high diversity of virus species in a natural setting is probably a common occurrence, for instance this has been shown for KYMV (Skotnicki et al., 1996) and Potyviruses of South America (Shepherd et al., 1969; Jones & Fribourg 1979; Spetz et al., 2003). The high diversity of HarMV may provide future researchers with insights into virus evolution in a natural ecosystem.

Evolutionary pressures in wild systems lead either to virus resistance in hosts (e. g. resistance genes to potato viruses in Solanaceae in South America (Jones 1981)) or to virus strains which give moderate symptoms or are latent (symptomless) on host plants (Hull 2002). The existence of strains represents evidence of virus adaptation to a host or other factors. Interactions between strains of HarMV and populations of H. comptoniana would provide a rich area for future research. Viruses are also able to influence wild ecosystems by reducing fecundity; particularly when infection pressure is strong (Malmstrom et al., 2005; Cooper & Jones 2006).

The

incidence of HarMV was high at Wireless Hill Park and Margaret River (over 60% infection) and may have important implications in the ecology of ecosystems in the SWAFR.

163


The SWAFR provides a unique opportunity to study native viruses in endemic flora that may be lost with increasing development. The rise in global trade threatens agricultural commodities with increased movement of plant material and the potential for the introduction of new strains and species of plant pathogens with potentially dire consequences. This work highlights the threat to cultivated plants such as Passiflora edulis, an introduced species which is affected by the native virus PWV, and may be distributed worldwide through world trade in plants and plant products. Such new encounters are expected to increase through global trade in plant products (Cooper & Jones 2006).

7.3 Development of microarray based methods In this project a new virus in the native legume H. comptoniana was identified using ELISA and PCR diagnostic methods. When dealing with an unknown or unexpected virus these methods were time consuming and highlighted the need for approaches with a broader scope for diagnosis of viruses where little or no information is available on them. Microarrays were investigated for this reason. This required investigation of different methods for slide printing and labelling. Others have used microarrays for plant virus detection and their methods are summarised and compared with our methods in Table 7.1. A virus diagnostic microarray was also used in identifying Broad bean wilt virus 2 (BBWV-2) in Digitalis spp. samples that could not be identified to the species level by other methods (Mumford et al., 2006b). The method of Pasquini et al., (2007) and Abdullahi et al., (2005), appear to be the best of these methods because they detected and discriminated virus strains and were combined with simple labelling protocols (similar to CyScript method, Chapter6). More work on improving sensitivity, such as the method of Agindotan & Perry (2007), could decrease the limit of detection.

Microarray based methods have potential applications in plant biosecurity, preventing the introduction of damaging viruses to areas they are not present. They could include ‘commodity based chips (e.g. banana or potato pathogens), or even a universal virus microarray for detection of all known plant viruses. The results of our research are thus relevant for uptake of this technology, and strain ID may allow better biosecurity and quarantine policy implementation in case of disease introductions.

164

2005

1 (CMV)

2005

5 (4 replicates)

4 (PVX, PVY, PVA, PVSA, PVSO)

2003

cDNA (0.1-0.8kbp)

8 (3 replicates

6 (PVA, PVS, PVX, PVY, PLRV, PMTV)

2003

FAST Slide (Schleicher & Schuell)

cDNA (0.5-0.7kbp)

9 (4 replicates)

9 (ZGMMV, CGMMV, CFMMV, KGMMV, TMV, PMMoV, PVX, CMV, ZYMV)

UltraGAPS (Corning)

70mer oligonucleotides

19 (12 replicates)

1 (six strains of PPV)

Pasquini et al.,

2005 6 (PLRV, PVMO, PVMI, PVYO, PVYNTN, PVSA, PVSO, PVA, PVX)

5 (6 replicates)

cDNA (0.7-1.2kbp)

poly-L-lysine

Table 7.1: Comparison of published methods for microarray based plant virus diagnostics. Abdullahi et al., Bystricka et al., Deyong et al., Boonham et al., Bystricka et al.,

(this study) 12, plus Synchytrium endobiotichumA (PVY, PVS, PVV, PVA, PVT, APLV, APMV, TSV, PSTVd, PBRSV, PYVV, PVX) 29

24mer oligonucleotides

poly-L-lysine

GenPoty Array

8 (BCMV, BYMV, HarMV, PWV, PFVY, SMV, TuMV, ZYMV)

180 (8 replicates) 40mer oligonucleotides

Aldehyde-coated glass

baking at 80oC

baking at 80oC

-

(2007)

16 (4 replicates) 50mer oligonucleotides poly-L-lysine

Lee et al., 2003

Number of probes used 45 to 52mer oligonucleotides Epoxy-coated glass (MWG Biotech)

Methods

Type of probes PowerMatrix Slides (FMB)

Viruses testedB

Type of slide

UV crosslinking

UV crosslinking

-

-

-

-

succinic anhydride

succinic anhydride

Cy3-dCTP

incubation at 75% humidity for 12h Bovine serum albumin

Cy3-dCTP

Cy3 – NHS or Cy5 - NHS

Alexa-fluor 647 & 546

Cy3-dCTP

-

2-5µg

30µg

Cy3-dUTP or Cy5dUTP 10µg

Cy3-dCTP or Cy5dCTP 0.5-40µg

Cy3 dCTP or Cy5 dCTP -

-

CyDye incorporation into PCR product using CMV specific primers

Immobilisation method Slide blocking Fluorescent label RNA amount

indirect labellingcoupled to aa-dUTP by ImPromII reverse transcriptase

indirect labelling with Superscript III reverse transcriptase and aminoallyl dUTP

direct labellingAmersham CyScribe first strand cDNA labelling kit

direct labelling with Superscript II reverse transcriptase

CyDye incorporation into PCR product using Potyvirus specific primers

-

Fluorescent PCR amplification with virus species specific primers

Labelling method

NaOH incubation

RNase A

NaOH incubation

QiaQuick PCR kit

NaOH incubation

-

QiaQuick PCR kit

50µg direct labellingAmersham CyScribe first strand cDNA labelling kit NaOH incubation Autoseq G50 columns 16h at 55oC

QiaQuick PCR kit

QiaQuick PCR kit

2h at 40oC

QiaQuick PCR kit

ethanol cleanup

see Ideker et al., 2003

20h at 55oC

23h at 55oC

12h at 42oC

16h at 42oC

Microcon 30 minicolumns 4h at 42oC

RNA removal Target purification Hybridisation

A: S. endobioticum is a quarantine-status fungal pathogen of potato (Abdullahi et al., 2005). B: Virus names: APLV, Andean potato latent virus; APMoV, Andean potato mottle virus; BCMV, Bean common mosaic virus; BYMV, Bean yellow mosaic virus; CFMoMV, Cucumber fruit mottle mosaic virus; CGMMV, Cucumber green mottle mosaic virus; CMV, Cucumber mosaic virus; HarMV, Hardenbergia mosaic virus; KGMMV, Kyuri green mottle mosaic virus; PBRSV; Potato black ringspot virus; PFVY, Passiflora foetida virus Y; PLRV, Potato leaf roll virus; PMMoV, Pepper mild mottle virus; PMTV, Potato mop-top virus; PSTVd, Potato spindle tuber viroid; PVA, Potato virus A; PVM, Potato virus M; PVS, Potato virus S; PVT, Potato virus T; PVV, Potato virus V; PVX, Potato virus X; PVY, Potato virus Y; PWV, Passion fruit woodiness virus; PYVV, Potato yellow vein virus; SMV, Soybean mosaic virus; TMV, Tobacco mosaic virus; TSV, Tobacco streak virus; TuMV, Turnip mosaic virus; ZGMMV, Zucchini green mottle mosaic virus; ZYMV, Zucchini yellow mosaic virus.


7.3.1 Cost effective methods for microarray slides The high cost of microarrays means that cost-effective methods warrant further research and towards this aim a method for coating glass microscope slides in PLL was investigated. A commercial microarray slide, PowerMatrix (FMB), was compared to the PLL slides. The PowerMatrix slides were superior to the PLL slides because they retained more probe DNA and showed less inter-slide variability. The in-house method of creating PLL-based microarrays proved to be less reliable and gave inconsistent results.

Arrays on glass surfaces are relatively expensive to produce and only a small number of samples can be processed simultaneously (Call et al., 2005; Mumford et al., 2006a). New techniques are in development which offer higher sample through-put and lower costs such as the Array-Tube™ microchip (Borel et al., 2008), fibre optic arrays (Epstein et al., 2003) and liquid bead arrays (Spiro et al., 2000). These methods are reviewed by Call et al. (2005) who noted that bead-based arrays are of particular promise. Identification of pathogens and viruses (Spiro et al., 2000; Cowan et al., 2004) has been demonstrated using liquid bead arrays; but no reports of plant virus identification have been published. The advantage of bead-arrays, such as the Luminux system (Luminux, Austin) is the higher sample throughput and use of automated 96 well sample extraction systems which help reduce assay costs (Call et al., 2005). At present, in principle up to 100 targets can be detected simultaneously, and this suits the small crop and/or genus specific arrays which have been developed (Table 7.1), including the GenPoty Array developed from research in this project.

Of promise is research that shows probe

sequences are transferable from alternative array formats (Cowan et al., 2004).

Any

improvements in costs and throughput will increase the applications of array techniques in diagnostics and the approaches here indicate what form these improved methods will take.

7.3.2 Strain specific microarrays In this research project, microarrays were developed with the aim of using them to detect new and unexpected species of Potyviruses such as HarMV and PWV, which have potential to cause damage in economically important crops. HarMV was used as a model because six highly distinct strains were available to test. An aim of this thesis was to determine whether microarrays can distinguish between closely-related Potyvirus species and strains, given they have up to 20% intra species diversity and 55% inter species diversity in the CP gene (Adams et al., 2005b)? To this end a microarray was designed to detect six strains of HarMV and seven other Potyvirus species, as a proof-of-concept approach.

A microarray was designed to detect and distinguish between isolates from six strains of HarMV and seven other Potyvirus species. Isolates were distinguishable at the species level 166


from hybridisation to genus and strain specific probes, but not to the HarMV strain level. Strain specific probes showed perfect homology to the strains they were designed to detect, but some did not show fluorescence when hybridised with perfectly complementary strain DNA (e.g. probe HarMV-VIII; Table 5.2). The probe sequence was also checked for secondary structures but importantly only across the probe sequence. Secondary structures of surrounding regions may be responsible for the failure of some probes to hybridise (Chandler et al., 2003; Lane et al., 2004) and insufficient diversity may explain the cross reactivity of probes in this study. Better testing for secondary structure could help improve probe results; however it would be wise to test several probes for each desired target empirically to find the best performing sequences (Bystricka et al., 2005). Some strains of plant viruses have been differentiated successfully by microarray (Boonham et al., 2003; Bystricka et al., 2005; Deyong et al., 2005; Pasquini et al., 2007); however probes designed in silico do not always behave as expected. Probe efficiency or sensitivity cannot be accurately predicted, and a lack of correlation between sequence complementarity and fluorescent intensity has been shown (Abdullahi et al., 2005). Therefore it is not based solely on sequence homology (Binder et al., 2004) and empirical testing of several probes is often necessary to find good probes.

Microarrays for pathogen detection require reliable probes for diagnosis of virus pathogens to the species and strain level. The aspects of probe design that are most important for selectivity are not known and therefore more information about how probe length and probe/target homology affect sensitivity and selectivity is needed. For instance while shorter probes are more selective in target binding (Lievens 2006) they have reduced sensitivity (Ramdas et al., 2004) which may make them unsuitable with methods of fluorescent labelling that do not involve amplification. An investigation into the effect of probe length on diagnostic arrays was beyond the scope of this project but this is an area for future research. Better use of probe design programs may also help improve probe performance, such as the EXIQON software (http://oligo.lnatools.com/expression/) used by Pasquini et al., (2007). While programs for probe design are good and could have been utilised more in this project, empirical testing is still necessary. The results of systematically studying probe length and probe/target homology will be beneficial in future arrays and will enable faster development of better probes. This will allow future arrays to diagnose virus isolates to the strain and species level.

7.3.3 Labelling methods for increased sensitivity Amplification is widely used to increase nucleic acid concentrations and improve sensitivity in virus diagnostics. While the limit of detection by a microarray has been estimated at 10 copies of a gene per cell (Kane et al., 2000) the large amounts of RNA used (up to 30µg per assay) in microarrays means this represents a higher copy number per assay than detected by real time 167


PCR. Labelling during reverse transcription is best suited for target molecules present at high concentration within plant tissue (Boonham et al., 2007) and therefore high titre viruses are relatively easy to detect by microarray. Non-amplified samples of PVX and PVY were tested using equivalent amounts of total RNA, the results of which showed there was a 35 fold difference in the fluorescence of virus specific probes on the array (Figure 6.4). For viruses present at low concentrations in a sample the low sensitivity of microarray methods raises the possibility of false negative results.

Virus genome fragments from different strains of four virus species (HarMV, PWV, BYMV and PFVY) were either amplified by RT-PCR or cloned and used on the GenPoty array to test probes for species and strain specificity (Chapter 5). Although such templates are unsuitable for diagnostic samples, they can be used to confirm specificity of probes immobilised on the slide. An aim of the research in this project was to evaluate amplification methods for virus targets to increase the sensitivity of the microarray.

Amplification methods have been

suggested for improving sensitivity of microarray experiments, as outlined in Chapter 6. Of the methods tested in this project two showed amplification (the method of Burton et al., 2005 and using genus specific primers); but non-specific hybridisation was found with the method of Burton et al., (2005) (Table 6.1).

The sensitivity of microarray methods without amplification has been estimated at a level equivalent to ELISA (Boonham et al., 2003), whereas Lee et al., (2003) showed virus detection for total RNA samples diluted up to 1 in 200. Also Abdullahi et al., (2005) found as little as 0.5µg of total RNA from potato infected with APLV was necessary for detection, but the actual amount of APLV RNA needed was not determined. This raises the possibility that viruses might not be detected in imported material, which could increase the likelihood of disease introductions. Therefore amplification methods that improve sensitivity of detection are well worth researching.

Pre-amplification methods for improving sensitivity of microarray experiments were outlined in Chapter 6. Primers for universal gene targets such as 16S rRNA genes are commonly used in phytopathology (e.g. Anderson et al., 2006; Franscois et al., 2006; Nicolaisen & Bertaccini 2007). Arrays of probes in universal genes can be amplified and hybridised to arrays to improve the sensitivity of microarray methods to levels comparable to PCR and agarose gel electrophoresis (Call et al., 2001). However this approach is not applicable to virus diagnostics because of the lack of conserved sequences across families. Selective amplification of virus nucleic acid can be done by PCR with virus-specific primers, with the array used to identify amplicons as was done for Cucumber mosaic virus (Deyong et al., 2005) and potato viruses

168


(Bystricka et al., 2003). PCR based labelling methods work well for identifying expected viruses, but are unsuitable for samples containing unknown or unexpected viruses.

The effectiveness of amplification is best measured by an increase in the signal (i.e. virus nucleic acid) compared to noise (i.e. plant RNAs).

The use of genus-specific primers

successfully amplified virus nucleic acid while the plant control (COX) reduced in concentration over the two rounds of amplification. As a draw back the number of viruses detected was reduced to only members of the family Potyviridae. There appears to be a trade off between the amplification efficiency and the range of detectable viruses. Alternatively whole genome amplification methods (WGA) can also be used. Nygaard & Holvig (2006) review current WGA methods. The advantage of WGA methods is that they can be used to detect many species of pathogen and are potentially useful for detecting unknown or unexpected species in samples (Boonham et al., 2007).

Methods such as WGA cause only weak

amplification of virus nucleic acid but can be used to detect a wide range of viruses (Mumford et al., 2006a); whereas more specific amplification, such as PCR, only allows a small number of viruses to be detected. For instance a WGA method (Agindotan & Perry 2007) showed 100 to 1000 fold amplification of virus nucleic acid, but using this relatively long and complex procedure the sensitivity remained at ELISA like levels. This trade off between sensitivity of detection and the range of detectable viruses is undesirable but seems unavoidable.

When virus RNA is limiting, amplification methods can be used to increase it. Iscove et al., (2002) found that amplification of up to 3x1011 fold preserved abundance relationships using 10pg of total RNA starting material. In plant virus diagnostics much larger starting amounts of RNA have been used (e.g. 50µg in Boonham et al., 2003). Therefore when using large amounts of nucleic acids only limited amplification may occur before saturation occurs (Saiki et al., 1988). As the relative transcript abundance is preserved (Iscove et al., 2002), only a small amount of virus amplification may occur. This is especially true for low abundance targets such as the phloem limited PLRV (Waterhouse et al., 1988). Further investigation into amplification efficiencies is needed especially when larger amounts of RNA are used. Methods should be confirmed by real time PCR for both high and low titre viruses as well as host genes.

The methods studied here did show an improvement in sensitivity, but problems such as nonspecific binding were observed (Figure 6.6). All the methods tested were aimed to increase the amount of target for binding, however signal amplification methods such as tyramide signal amplification and dendrimer labelling could improve sensitivity (Nygaard & Holvig 2006). These methods increase the signal produced by each hybridised target molecule allowing more sensitive detection. Methods of enriching for the target species, such as vRNA, would also increase the sensitivity of the array, e.g. dsRNA extractions, Immunocapture or subtractive 169


hybridisation. While not demonstrated for diagnostics subtractive hybridisation was used to examine changes in gene expression caused by virus infection (Munir et al., 2004).

7.4 Future work The results of this research project identified the new native virus HarMV which warrants further investigation. Infection of lupin species represents a possible threat to lupin production and survey work for natural infection of HarMV should be carried out. Finding the natural vector/s of this virus will also be needed to properly asses the risk. Surveys of WA natives for both HarMV and other native virus species are also recommended as this region presents an ideal location to investigate viruses in relatively undisturbed natural vegetation. Further surveys of WA natives for virus infection will be investigated as part of an Australian Research Council project with further studies on HarMV planned for a future PhD student.

The methods used here for probe design were limiting and this was reflected in the nonperformance of HarMV strain specific probes. To realise the potential of microarrays better probes for strain distinction are needed, and this represents an important area of future research. The secondary structure and other important properties of probes can affect their performance (Lane et al., 2004) and by testing several probes for a required pathogen the most efficient may be identified. This could also lead to improved predictive ability for probe performance. Better bioinformatics tools may also be needed for microarray probe design. Both longer (Pasquini et al., 2007) and shorter (Deyong et al., 2005) probes have been used for strain differentiation. The increased sensitivity of longer probes (Ramdas et al., 2004) makes them a more promising approach. Also needed are probes for economically important species of Potyvirus such as PVY and PPV which can be included on the GenPoty array to create a test useful in diagnosing unknown species of Potyvirus quickly and accurately. Virus diagnostic microarrays can offer time and cost savings over current methods, such as PCR and sequencing. Current methods took several days to identify PWV in Passiflora spp, and longer still for HarMV.

Work on labelling methods to increase sensitivity of detection was carried out in collaboration with Dr Neil Boonham (CSL, York, UK) as part of ongoing collaboration with the Plant Biotechnology Research Group. Methods of WGA warrant investigation in collaboration with microarray methods, such as the Φ29 polymerase method of Vora et al., (2004) or the rPCR method of Iscove et al., (2002). The target independent methods of amplification should also be tested, especially the randomly primed PCR method of Agindotan & Perry (2007), as a genus specific method greatly limits the number of viruses which can be diagnosed simultaneously.

170


7.5 Conclusions This research project achieved the following outputs: •

HarMV represents a new species of Potyvirus which shows high nucleotide diversity in its host species and was related most closely to other Potyviruses found only in Australia.

•

HarMV’s experimental host range was narrow but it infects several species important to agriculture

•

Investigation of array fabrication lead to the conclusion that commercial microarray slides were more consistent, showed higher probe retention, and were easier to use.

•

In-house PLL slides were affected by many of the variables tested and were inconsistent. Their use is therefore not recommended in biosecurity or quarantine situations where reliability is critical.

•

Four species of Potyvirus, including HarMV, were identified using species-specific microarray probes. Strain differentiation, which was a key aim of this project, was not achieved.

•

Amplification methods increase concentration of virus nucleic acid.

Non-specific

amplification of all nucleic acids suffers from lower overall amplification efficiency than more specific methods, such as PCR. The disadvantage of targeted amplification methods, however, is that the ability to detect unknown or unexpected viruses is lost.

A microarray could be used in situations where unknown or unexpected viruses are present. Because of the large number of possible viruses, methods like PCR are unsuitable, and a microarray could of greater use than these methods. I envisage that biosecurity and quarantine are likely to be the main areas of application of this technology, where large arrays containing potentially hundreds of virus specific probes could be used. Also more specific applications exist, such as in plant breeding, were more tailored arrays for known plant virus genera/species would be beneficial. Arrays used in these situations would probably contain probes only for the most important pathogens or each crop.

171

APPENDICES

Appendix 1: BLAST search results of HarMV isolate MU-1C coat protein showing the 100 closest matching sequences in GenBank. Max Total Query Max E score score coverage value ident

Accession

Description

AJ430527.1

Passionfruit woodiness virus partial genomic RNA for polyprotein, genomic RNA

641

641

90%

1e-180

79%

U67149.1

Passion fruit partial cds

woodiness

virus

coat

protein

gene,

637

637

85%

2e-179

80%

U67150.1


woodiness

virus

coat

protein

gene,

632

632

85%

7e-178

80%

U67151.1


woodiness

virus

coat

protein

gene,

627

627

85%

3e-176

79%

AF228515.1 Clitoria virus Y polyprotein gene, partial cds

619

619

84%

4e-174

79%

Y11774.1

Peanut stripe virus RNA for NIb protein and coat protein, isolate T7

603

603

86%

3e-169

79%

Y11776.1


600

600

86%

4e-168

79%

Y11772.1


600

600

86%

4e-168

79%

Y11771.1


600

600

86%

4e-168

79%

AJ132158.1

Peanut stripe virus polyprotein isolate Indonesia I15

gene

3'

terminus,

600

600

87%

4e-168

79%

AJ132156.1


gene

3'

terminus,

600

600

86%

4e-168

79%

AJ132155.1


gene

3'

terminus,

600

600

87%

4e-168

79%

Peanut stripe virus polyprotein AJ132154.1 isolate Indonesia I11

gene

3'

terminus,

600

600

87%

4e-168

79%

AJ132153.1


gene

3'

terminus,

600

600

86%

4e-168

78%

AJ132152.1

Peanut stripe virus isolate Indonesia I9

polyprotein

gene

3'

terminus,

600

600

87%

4e-168

79%

Peanut stripe virus AJ132151.1 isolate Indonesia I7

polyprotein

gene

3'

terminus,

600

600

87%

4e-168

79%

AJ132150.1


polyprotein

gene

3'

terminus,

600

600

87%

4e-168

79%

AJ132149.1


polyprotein

gene

3'

terminus,

600

600

86%

4e-168

78%

AJ132148.1


polyprotein

gene

3'

terminus,

600

600

87%

4e-168

79%

Peanut stripe virus AJ132147.1 isolate Indonesia I2

polyprotein

gene

3'

terminus,

600

600

87%

4e-168

79%

598

598

84%

1e-167

79%

DQ098901.1 Siratro 2 virus Y polyprotein gene, partial cds Peanut stripe virus AJ132146.1 isolate Indonesia I1

polyprotein

gene

3'

terminus,

596

596

87%

5e-167

78%

DQ367846.1 Peanut stripe virus coat protein gene, partial cds

594

594

86%

2e-166

78%

Z21700.1

Peanut stripe virus 370 capsid protein

594

594

86%

2e-166

78%

Y11775.1

Peanut stripe virus RNA for NIb protein and coat protein, isolate 95/399

594

594

86%

2e-166

78%

Y11773.1


594

594

86%

2e-166

78%

U34972.1

Peanut stripe virus mRNA polyprotein mRNA, complete cds

594

594

86%

2e-166

78%

U05771.1

Peanut stripe virus, complete genome

594

594

86%

2e-166

78%

Bean common mosaic virus partial gene for AJ889245.1 polyprotein, genomic RNA, strain Peanut stripe virus

590

590

86%

2e-165

78%

Peanut stripe virus clone SN-Nib3 pol protein (pol) gene, partial cds

590

590

86%

2e-165

78%

Peanut stripe virus clone SN-Nib2 pol protein (pol) AF200623.1 gene, partial cds

590

590

86%

2e-165

78%

590

590

86%

2e-165

78%

590

590

86%

2e-165

78%

590

590

86%

2e-165

78%

590

590

86%

2e-165

78%

AF200624.1

AF191748.1

Peanut stripe virus severe polyprotein gene, partial cds

AJ132157.1


necrotic

strain

gene

3'

terminus,

Peanut stripe virus AJ132145.1 isolate China W2

polyprotein

gene

3'

terminus,

Peanut stripe virus isolate China W

polyprotein

gene

3'

terminus,

AJ132144.1

172

APPENDICES Accession

Max Total Query Max E score score coverage value ident

Description

AY968604.1 Peanut stripe virus polyprotein gene, complete cds

585

585

86%

9e-164

78%

585

585

86%

9e-164

78%

581

581

86%

1e-162

78%

581

581

86%

1e-162

78%

AF073380.1 Peanut stripe virus polyprotein gene, partial cds

572

572

86%

5e-160

78%

DQ098900.1 Siratro 1 virus Y polyprotein gene, partial cds

569

569

85%

7e-159

77%

AF228516.1 Hibbertia virus Y polyprotein gene, partial cds

565

565

85%

8e-158

78%

562

562

86%

1e-156

78%

560

560

78%

3e-156

79%

X63559.1

Peanut stripe virus gene for capsid protein

AJ132143.1

Peanut stripe virus isolate China G

AF063222.1

Peanut stripe virus strain Ts clone TS4 polyprotein gene, partial cds

L11890.1

Bean common mosaic protein gene, 3' end

polyprotein

virus

gene

(strain

3'

terminus,

Mexican)

coat

AF079116.1 Peanut stripe virus coat protein gene, partial cds Bean common mosaic virus (strain US5) coat protein gene, 3' end

560

560

86%

3e-156

77%

AY575773.1 Blackeye cowpea mosaic virus, complete genome

545

545

86%

8e-152

77%

AY112735.1 Bean common mosaic virus strain NL1, complete genome

545

545

86%

8e-152

77%

Blackeye cowpea AF395678.1 partial cds

545

545

86%

8e-152

77%

545

545

86%

8e-152

77%

L19473.1

mosaic

Bean common mosaic AF045066.1 gene, partial cds

virus

virus

polyprotein

strain

GGSSA

gene,

polyprotein

L21767.1

Bean common mosaic virus (strain Puerto Rico) coat protein gene, 3' end

545

545

86%

8e-152

77%

L21766.1

Bean common mosaic virus (strain NL4) coat protein gene, 3' end cds

545

545

86%

8e-152

77%

AB012663.1

Azuki bean mosaic virus RNA for polyprotein (NIb-CP region), partial cds

545

545

86%

8e-152

77%

542

542

86%

9e-151

77%

Bean common mosaic virus (strain NL1) coat protein gene, complete cds

542

542

86%

9e-151

77%

L15332.1

Bean common mosaic virus coat protein gene, 3' end

542

542

86%

9e-151

77%

U37073.1

Bean common mosaic gene, partial cds

540

540

86%

3e-150

77%

DQ054366.1

Bean common mosaic virus polyprotein precursor, gene, partial cds

536

536

86%

4e-149

77%

S66251.1

polymerase, coat protein [bean common mosaic virus BCMV, NL1, Genomic, 945 nt, segment 1 of 2]

536

536

86%

4e-149

77%

Y17823.1

Blackeye cowpea mosaic potyvirus cp gene and partial nuclear inclusion gene b, strain Florida

536

536

86%

4e-149

77%

Z15057.1

Bean common (partial)

536

536

86%

4e-149

77%

L19474.1

Bean common mosaic virus (strain US7) coat protein gene, 3' end

536

536

86%

4e-149

77%

U37075.1


virus

strain

536

536

86%

4e-149

77%

Bean common mosaic AF083559.1 mRNA, partial cds

virus

NIb

533

533

86%

5e-148

77%

533

533

86%

5e-148

77%

Bean common mosaic AF045065.1 gene, partial cds L15331.1

mosaic

virus

virus

virus

strain

GGSUS

strain

gene

US3

polyprotein

coat

for

coat

protein

coat

protein

protein/coat

protein

U72204.1

Cowpea aphid-borne mosaic protein gene, complete cds

L19472.1

Bean common mosaic virus coat protein (strain NL2) mRNA, 3' end

533

533

86%

5e-148

77%

L12740.1

Bean common mosaic virus coat protein mRNA, complete cds

533

533

86%

5e-148

77%

U23564.1

Dendrobium mosaic partial cds

531

531

86%

2e-147

77%

AF328759.1

Bean common mosaic virus polyprotein mRNA, partial cds

531

531

81%

2e-147

78%

AF328751.1

Bean common mosaic virus isolate TLAXCALA polyprotein mRNA, partial cds

531

531

81%

2e-147

78%

U37072.1

Bean common mosaic virus strain US10 coat protein gene, partial cds

527

527

86%

2e-146

76%

S66253.1

polymerase, coat protein [blackeye cowpea mosaic virus BlCMV, W, Genomic, 945 nt, segment 1 of 2]

526

526

86%

7e-146

76%

U37074.1


524

524

86%

2e-145

76%

DQ897639.1

Bean common mosaic virus from siratro coat protein gene, partial cds

517

517

86%

4e-143

76%

517

517

86%

4e-143

76%

Bean common mosaic AY863025.1 gene, complete cds

virus

Nl7

protein

potyvirus

virus

virus

NIb

coat

protein/coat

protein

isolate

strain

strain

US4

RU-1

gene,

Chihuahua

coat

protein

polyprotein

AJ312438.1

Bean common mosaic virus cowpea isolate Y, complete genome

515

515

86%

1e-142

76%

U37077.1


515

515

86%

1e-142

76%

virus

strain

RU1

173

coat

protein

APPENDICES Max Total Query Max E score score coverage value ident

Accession

Description

AJ312437.1

Bean common mosaic virus cowpea isolate R, complete genome

509

509

86%

5e-141

76%

AJ293276.1

Bean common polyprotein

509

509

86%

5e-141

76%

S66252.1

polymerase, coat protein [bean common mosaic virus BCMV, NY15, Genomic, 945 nt, segment 1 of 2]

504

504

86%

2e-139

76%

AF328756.1


504

504

81%

2e-139

77%

AF361337.1

Bean common mosaic virus isolate 93/65 polyprotein gene, partial cds

504

504

86%

2e-139

76%

L19539.1

Bean common mosaic virus coat protein mRNA sequence, 3' end

504

504

86%

2e-139

76%

502

502

84%

8e-139

76%

mosaic

virus

partial

mRNA

isolate

Ceratobium mosaic potyvirus strain AF022444.1 polyprotein (NIb/CP) gene, partial cds

for

viral

Guanajuato

CerMV-6

DQ860147.1

Florida passionflower potyvirus LAJ-2006 coat protein gene, partial cds

499

499

86%

1e-137

75%

AY656816.1

Wisteria vein mosaic virus isolate Beijing, complete genome

499

499

91%

1e-137

75%

Wisteria vein mosaic virus WVMV-BJ coat protein (CP) AY519365.1 gene, partial cds

499

499

91%

1e-137

75%

495

495

81%

1e-136

76%

495

495

81%

1e-136

76%

495

495

81%

1e-136

76%

493

493

84%

4e-136

75%

491

491

86%

1e-135

76%

AF328757.1


isolate

Guanajuato

AF328755.1


isolate

Guanajuato

AF328753.1


isolate

Ceratobium mosaic potyvirus strain AF022446.1 polyprotein (NIb/CP) gene, partial cds U60100.1

Zacatecas CerMV-19

Azuki bean mosaic virus polyprotein gene, partial cds

Passion fruit AY461662.1 partial cds

woodiness

virus

coat

protein

gene,

488

488

53%

2e-134

84%

Wisteria vein mosaic virus polyprotein gene, partial AF484549.1 cds

488

488

87%

2e-134

75%

DQ112219.1

Passiflora foetida virus Y polyprotein gene, partial cds

482

482

85%

8e-133

75%

AJ628754.1

Soybean mosaic virus partial polyprotein gene for NIb and coat proteins, genomic RNA, isolate WH2

480

480

87%

3e-132

74%

AJ628753.1

Soybean mosaic virus partial polyprotein gene for NIb and coat proteins, genomic RNA, isolate WH1

480

480

87%

3e-132

74%

Zucchini yellow mosaic potyvirus polyprotein gene, AF014811.2 complete cds

479

479

84%

9e-132

75%

Soybean mosaic virus strain 746 coat protein gene, partial cds

479

479

86%

9e-132

75%

Watermelon mosaic virus partial gene for polyprotein AJ579494.1 encoding coat protein CP, genomic RNA, isolate BAR99.3

479

479

86%

9e-132

75%

Zucchini yellow mosaic virus (ZYMV-S) mRNA for coat protein

479

479

84%

9e-132

75%

AY216487.1

X62662.1

174

APPENDICES

BLAST search results of HarMV isolate MU-1C partial NIa/NIb showing the 100 closest matching sequences in GenBank. Accession

Description

Max score

Total score

AJ012651.1

Bean common mosaic virus gene encoding polyprotein for partial NIa and NIb protein

324

324

98%

4e-85

70%

AY216010.1

Soybean mosaic virus strain G7, complete sequence

307

307

98%

3e-80

69%

AY138897.1

Bean common mosaic gene, complete cds

306

306

98%

1e-79

69%

AY282577.1

Bean common mosaic necrosis polyprotein mRNA, complete cds

306

306

98%

1e-79

69%

U19287.1

Bean common mosaic gene, complete cds

306

306

98%

1e-79

69%

necrosis

virus virus

necrosis

polyprotein strain

virus

NL-3

polyprotein

Max Query E coverage value ident

AJ310200.1

Soybean mosaic virus genomic RNA for polyprotein

304

304

98%

4e-79

69%

AY216987.1

Soybean mosaic virus strain precursor, mRNA, complete cds

302

302

98%

1e-78

69%

AF241739.1

Soybean mosaic virus G7 polyprotein gene, complete cds

302

302

98%

1e-78

69%

AY864314.2

Bean common mosaic necrosis virus strain NL-3 K polyprotein gene, complete cds

300

300

98%

5e-78

69%

AB100443.1

Soybean mosaic virus genomic RNA, complete genome, isolate:Aa15-M2

298

298

98%

2e-77

69%

AB100442.1

Soybean mosaic virus genomic RNA, complete genome, isolate:Aa

298

298

98%

2e-77

69%

AY294045.1

Soybean mosaic virus strain G7H, complete genome

293

293

98%

7e-76

69%

AY294044.1

Soybean mosaic virus strain G5, complete genome

293

293

98%

7e-76

69%

AJ312439.1

Soybean genome

291

291

98%

2e-75

69%

S42280.1

SMV genome [soybean mosaic virus SMV, strain G2, Genomic RNA Complete, 9588 nt]

284

284

98%

4e-73

68%

AJ507388.2

Soybean mosaic virus gene for polyprotein, genomic RNA, strain P

273

273

98%

7e-70

68%

AB246773.1

East Asian Passiflora virus genomic RNA, complete genome, strain: AO

271

271

98%

2e-69

68%

AF469171.1

Calla lily latent gene, partial cds

268

268

88%

3e-68

69%

AJ628750.1

Soybean mosaic virus RNA, isolate HZ1

266

266

98%

1e-67

68%

D00507.2

Soybean genome

266

266

98%

1e-67

68%

AY437609.1

Watermelon genome

259

259

88%

1e-65

69%

D00717.1

Soybean mosaic virus genomic RNA, C' terminal of CI protein, NIa proteinase, N'terminal of NIb protein

253

253

61%

6e-64

72%

DQ399708.1

Watermelon mosaic virus strain WMV-CHN, complete genome

251

251

88%

2e-63

68%

AY112735.1

Bean common genome

246

246

89%

9e-62

68%

mosaic

mosaic

virus

mosaic

severe

virus

virus

polyprotein

strain,

polyprotein

polyprotein N

virus

mosaic

G7d

genomic strain

virus

complete

precursor,

gene,

genomic

RNA,

complete

WMV-Fr,

complete

strain

NL1,

complete

AY575773.1

Blackeye cowpea mosaic virus, complete genome

244

244

89%

3e-61

68%

AJ619757.1

Soybean mosaic virus gene for polyprotein, genomic RNA

244

244

98%

3e-61

67%

AB218280.1

Watermelon mosaic virus genome, strain:WMV-Pk

242

242

88%

1e-60

68%

DQ054366.1


228

228

89%

2e-56

68%

AM039800.1

Fritillary virus Y complete virion genome, isolate Pan'an

224

224

98%

3e-55

67%

AJ312437.1

Bean common mosaic complete genome

219

219

95%

1e-53

67%

AF348210.1

Cowpea aphid-borne mosaic virus polyprotein gene, complete cds

217

217

98%

4e-53

67%

AJ312438.1

Bean common mosaic complete genome

212

212

89%

2e-51

67%

D13913.1

Watermelon mosaic virus 2 (isolate USA) gene for polyprotein precursor, partial cds

208

208

88%

2e-50

67%

AY863025.1

Bean common mosaic virus strain RU-1 polyprotein gene, complete cds

208

208

94%

2e-50

67%

AY656816.1

Wisteria vein complete genome

206

206

98%

8e-50

66%

AY968604.1

Peanut stripe virus polyprotein gene, complete cds

196

196

82%

1e-46

67%

U34972.1

Peanut stripe complete cds

172

172

89%

2e-39

66%

genomic

virus

mosaic

virus

RNA,

polyprotein

virus

virus

virus

mRNA

cowpea

cowpea

complete precursor,

isolate

isolate

isolate

Y,

Beijing,

polyprotein

175

R,

mRNA,

APPENDICES Accession

Description

Max score

Total score


U05771.1

Peanut stripe virus, complete genome

172

172

89%

2e-39

66%

AY279000.1

Zucchini yellow mosaic virus strain KR-PS complete genome

161

161

98%

3e-36

65%

AY278999.1

Zucchini yellow mosaic virus strain KR-PE complete genome

161

161

98%

3e-36

65%

AY278998.1

Zucchini yellow mosaic virus strain KR-PA complete genome

156

156

98%

1e-34

65%

AF014811.2

Zucchini yellow mosaic potyvirus polyprotein gene, complete cds

152

152

80%

2e-33

66%

AY626825.4

Zantedeschia mild mosaic virus strain TW, complete genome

150

150

78%

5e-33

66%

L29569.1

Zucchini yellow mosaic virus polyprotein; P1 protease; helper component-protease; P3 protein; cylindrical inclusion protein; P6K; VPg; NIa protease; NIb replicase, and capsid protein

149

149

92%

2e-32

65%

AJ429071.1

Zucchini yellow mosaic strain A, genomic RNA

138

138

98%

3e-29

65%

AJ515911.1

Zucchini yellow mosaic virus gene for polyprotein, genomic RNA, isolate WM

136

136

98%

1e-28

64%

X68509.1

Zucchini Yellow Mosaic Virus-S RNA

134

134

80%

4e-28

66%

AJ298033.1

Dasheen mosaic virus genomic RNA for polyprotein gene, isolate M13

134

209

72%

4e-28

69%

X96665.1

Soybean mosiac virus mRNA for NIb and coat protein

131

131

45%

5e-27

68%

AY188994.1

Zucchini yellow mosaic virus strain B*, complete genome

123

123

98%

7e-25

64%

EF062583.1

Zucchini yellow mosaic virus AG, complete genome

118

118

98%

3e-23

64%

EF062582.1

Zucchini yellow mosaic virus NAT, complete genome

118

118

98%

3e-23

64%

AY864851.2

Impatiens flower break potyvirus polyprotein gene, partial cds

114

226

82%

4e-22

73%

L31350.1

Zucchini yellow mosaic virus polyprotein, complete cds; P1 protease; P2 HC-protease; cylindrical inclusion protein; protease; replicase; capsid protein

111

111

98%

5e-21

64%

DQ124239.1

Zucchini yellow complete genome

105

105

98%

2e-19

64%

virus

mosaic

polyprotein

virus

isolate

gene,

Kuchyna,

AF048981.1

Dasheen mosaic virus polyprotein gene, partial cds

105

195

82%

2e-19

67%

AJ316229.2

Zucchini yellow mosaic virus gene for polyprotein, genomic RNA, isolate WG

104

104

79%

7e-19

64%

AF200624.1

Peanut stripe virus clone (pol) gene, partial cds

104

104

56%

7e-19

66%

AM422386.1

Zucchini yellow mosaic virus gene for polyprotein, genomic RNA

102

102

79%

2e-18

64%

AF127929.2

Zucchini yellow complete genome

100

100

79%

8e-18

64%

AB188115.1

Zucchini yellow mosaic virus genomic RNA, complete genome, isolate:Z5-1

95.1

95.1

22%

4e-16

72%

AJ307036.2

Zucchini yellow mosaic virus gene for polyprotein, genomic RNA, isolate CU

95.1

95.1

79%

4e-16

64%

AJ316228.2

Zucchini yellow mosaic virus gene for polyprotein, genomic RNA, isolate SG

91.5

91.5

79%

4e-15

64%

AB188116.1

Zucchini yellow mosaic virus genomic RNA, complete genome, isolate:2002

89.7

89.7

22%

2e-14

71%

82.4

82.4

40%

2e-12

66%

64.4

64.4

18%

6e-07

70%

mosaic

SN-Nib3

virus

pol

protein

isolate

TW-TN3

DQ645729.1

Zucchini yellow mosaic virus polyprotein gene, partial cds

isolate

ZYMV

C-16

D89545.1

Bean yellow mosaic virus genomic polyprotein, partial cds, isolate:90-2

RNA

for

DQ851496.1

Banana bract mosaic virus, complete genome

51.8

51.8

9%

0.004

76%

DQ986288.1

Tobacco etch virus isolate TEV7DA, complete genome

51.8

51.8

11%

0.004

73%

AF023848.1

Peanut mottle virus strain M, complete genome

51.8

51.8

19%

0.004

67%

M15239.1

Tobacco etch virus RNA, complete genome

51.8

51.8

11%

0.004

73%

M11458.1

Tobacco etch virus (highly (HAT)) complete genome

51.8

51.8

11%

0.004

73%

DQ821939.1

Basella rugose mosaic virus isolate BR, complete genome

48.2

48.2

7%

0.047

78%

DQ821938.1

Basella rugose mosaic virus isolate AC, complete genome

48.2

48.2

7%

0.047

78%

DQ641248.1

White lupin complete cds

48.2

48.2

6%

0.047

80%

AJ865077.1

Shallot yellow stripe virus partial polyprotein, genomic RNA, isolate ZQ1

48.2

48.2

12%

0.047

70%

M92280.1

Plum pox virus strain M isolate SK 68, complete genome

48.2

48.2

9%

0.047

74%

DQ851494.1

Peace lily mosaic virus isolate Haiphong, complete

46.4

46.4

7%

0.16

80%

mosaic

virus

aphid

transmissible

polyprotein

176

gene

gene, for

APPENDICES Description

Max score

Total score

U38621.1

Tobacco vein mottling virus VAM-resistancebreaking strain (TVMV-S), complete sequence

46.4

46.4

5%

0.16

83%

AC187013.2

Canis Familiaris chromosome 21, clone XX-155I13, complete sequence

44.6

44.6

4%

0.57

90%

AC125021.12

Mus musculus chromosome complete sequence

44.6

44.6

3%

0.57

93%

Accession


genome

1,

clone

RP24-212P5,

DQ174243.1

Carrot virus Y polyprotein gene, partial cds

44.6

44.6

8%

0.57

75%

AJ865076.1

Shallot yellow isolate ZQ2

44.6

44.6

7%

0.57

76%

BX957220.1

Methanococcus segment 2/5

44.6

44.6

4%

0.57

90%

AY162218.1

Papaya ringspot complete genome

44.6

44.6

4%

0.57

88%

U47033.1

Bean yellow complete cds

44.6

44.6

8%

0.57

76%

CU464047.1

Pan troglodytes chromosome X clone CH251-079P7 map Xq28, complete sequence

42.8

42.8

7%

2.0

77%

XR_022706.1

PREDICTED: Pan troglodytes similar to Myotubularin 1 (MTM1), mRNA

42.8

42.8

7%

2.0

77%

PREDICTED: Macaca mulatta similar to eukaryotic XR_010788.1 translation initiation factor 4A, isoform 1 (LOC698387), mRNA

42.8

42.8

4%

2.0

86%

stripe

virus

maripaludis virus

mosaic

mosaic

S2

type

potyvirus

virus

complete

genome,

complete

genome;

P

from

Thailand,

polyprotein

AY192568.1

Bean yellow genome

42.8

42.8

5%

2.0

81%

AC115290.5

Mus musculus BAC clone RP23-65M10 from chromosome 10, complete sequence

42.8

42.8

5%

2.0

83%

AC122816.4

Mus musculus BAC clone RP23-235N10 from chromosome 6, complete sequence

42.8

42.8

5%

2.0

83%

AC122864.3

Mus musculus BAC complete sequence

42.8

42.8

3%

2.0

92%

BX510334.6

Zebrafish DNA sequence from clone linkage group 7, complete sequence

42.8

42.8

4%

2.0

86%

BX663604.9

Zebrafish DNA sequence from clone CH211-222K9 in linkage group 7, complete sequence

42.8

42.8

4%

2.0

86%

AF002223.2

Homo sapiens chromosome X multiple clones map q28, complete sequence

42.8

42.8

7%

2.0

77%

CR591300.1

full-length cDNA clone CS0DM012YD08 of Fetal liver of Homo sapiens (human)

42.8

42.8

7%

2.0

77%

42.8

42.8

5%

2.0

81%

42.8

42.8

3%

2.0

92%

42.8

42.8

4%

2.0

88%

clone

isolate

GDD,

mRNA,

RP23-142M19

Mouse DNA sequence from clone BX005167.12 chromosome X, complete sequence AC116115.11

Mus musculus chromosome complete sequence

6,

AL732470.7

Mouse DNA sequence from clone chromosome X, complete sequence

complete

from

DKEY-4D11

RP23-247K19

clone

in

on

RP23-60P18,

RP23-38D3

177

6,

on

Bun-9

0 0.61 0.85 0.85 14.0 14.5 14.2 14.2 12.5 9.04 13.1 13.3 13.1 13.8 12.9 13.3 13.2 13.1 13.4 13.4 17.0 17.1 17.0 17.0 18.1 18.1 17.2 17.5 18.1 18.0 25.2 25.9 25.2 26.0 25.8 28.8 28.5 28.5 27.5 26.7 26.6 27.2 MU-1C

1.10 0 0.24 0.24 13.4 13.9 13.6 13.6 12.1 8.42 12.5 12.7 12.5 13.2 12.3 12.7 12.6 12.5 12.8 12.8 16.5 16.6 16.5 16.5 17.5 17.7 16.7 17.0 17.6 17.5 24.5 25.3 24.5 25.4 25.2 28.5 28.0 28.2 27.2 26.5 26.3 27.0 Med-7

1.47 0.4 0 0.24 13.7 14.2 13.8 13.8 12.3 8.67 12.7 12.9 12.7 13.4 12.6 12.9 12.8 12.7 13.1 13.1 16.7 16.9 16.7 16.7 17.5 17.7 17.0 17.2 17.8 17.7 24.8 25.5 24.8 25.6 25.4 28.7 28.2 28.5 27.5 26.7 26.5 27.2 -

Med-7

Wel-1

1.47 0.4 0.0 0 13.7 14.2 13.8 13.8 12.3 8.42 12.5 12.7 12.7 13.4 12.3 12.7 12.6 12.5 12.8 12.8 16.7 16.9 16.7 16.7 17.7 18.0 16.7 17.0 17.6 17.5 24.5 25.3 24.5 25.4 25.2 28.5 28.2 28.2 27.2 26.5 26.3 27.0 -

Wel-1

WHP-2

6.59 5.49 5.86 5.86 0 1.94 1.33 1.21 10.2 10.4 10.8 11.0 10.8 11.2 11.3 11.3 11.0 10.8 11.0 11.0 15.6 15.5 15.6 15.6 20.1 20.4 17.1 17.2 18.1 18.3 25.6 25.7 25.5 25.0 25.6 27.9 28.0 28.1 29.0 27.3 27.0 27.2 -

WHP-2

He-2

7.69 6.59 6.96 6.96 2.91 0 2.30 2.18 11.0 11.8 11.5 11.8 11.5 11.8 12.5 12.5 12.1 12.0 12.2 12.2 16.6 16.4 16.6 16.6 20.9 21.1 17.8 17.9 18.7 19.0 26.7 26.8 26.3 26.1 26.7 28.3 29.0 29.0 29.8 28.0 27.9 27.9 -

He-2

KiP-1

6.59 5.49 5.86 5.86 0.36 2.55 0 1.58 10.2 11.0 10.8 11.0 10.8 11.2 11.5 11.6 11.3 11.0 11.4 11.4 16.2 16.1 16.2 16.2 20.2 20.5 17.0 17.1 18.2 18.4 25.9 26.1 25.8 25.1 25.9 28.2 28.6 28.6 28.9 28.0 27.8 27.9 -

KiP-1

He-1

6.59 5.49 5.86 5.86 1.09 2.91 0.73 0 10.6 10.5 10.7 10.9 10.7 11.1 11.4 11.4 11.2 10.9 11.2 11.2 15.6 15.5 15.6 15.6 20.0 20.2 16.7 16.9 17.7 17.9 25.7 25.8 25.6 25.1 25.7 28.0 28.4 28.4 29.3 27.8 27.5 27.6 -

He-1

Cgt-1

8.79 8.42 8.06 8.06 5.84 6.93 5.84 6.20 0 11.0 10.3 11.0 10.1 10.8 11.7 12.0 12.2 11.4 11.4 11.4 15.6 15.7 15.6 15.6 19.6 19.8 18.0 18.3 18.4 18.6 25.2 24.9 24.9 24.8 24.8 28.2 27.4 29.4 28.7 27.9 27.9 27.5 -

Cgt-1

MU-2A

5.49 4.76 5.13 5.13 5.84 6.96 5.13 5.13 6.23 0 6.72 6.96 6.84 7.57 5.49 5.86 5.86 5.37 5.86 5.86 15.6 15.5 15.4 15.4 17.8 18.1 17.7 18.0 18.9 19.1 24.3 25.2 24.1 24.7 25.0 28.1 27.5 28.0 28.0 26.6 26.5 27.1 -

MU-2A

MU-3A

6.23 5.49 5.86 5.86 4.36 6.55 4.36 4.73 4.38 1.83 0 1.33 1.33 1.70 3.76 3.77 3.76 3.27 3.76 3.76 15.9 15.8 15.7 15.7 19.5 19.8 18.2 18.4 19.0 19.3 25.3 26.3 25.3 25.3 26.2 27.4 26.9 28.0 29.3 26.7 26.1 27.2 -

MU-3A

BB-6

6.59 5.86 6.23 6.23 4.73 6.91 4.73 5.09 4.74 2.20 0.36 0 1.45 1.82 3.64 3.65 3.64 3.15 3.64 3.64 16.3 16.2 16.1 16.1 19.6 19.9 18.6 18.6 19.2 19.4 25.0 25.9 25.0 24.7 25.8 27.4 27.0 27.7 28.7 26.4 25.8 26.9 -

BB-6

WHP-1

6.59 5.86 6.23 6.23 4.00 6.18 4.00 4.36 4.01 2.20 0.36 0.73 0 0.36 3.64 3.65 3.64 3.15 3.64 3.64 15.7 15.6 15.5 15.5 19.3 19.5 18.3 18.6 19.2 19.4 25.3 26.1 25.3 25.1 25.9 27.5 26.8 28.2 28.9 25.9 25.3 26.2 -

WHP-1

Can1-1

8.06 7.33 7.69 7.69 5.11 6.57 5.11 5.47 5.11 3.66 1.46 1.82 1.09 0 4.01 4.01 4.01 3.53 4.01 4.01 16.4 16.3 16.2 16.2 19.7 20.0 18.7 19.0 19.6 19.8 25.8 26.5 25.8 25.6 26.4 27.7 27.3 29.0 29.3 26.0 25.8 26.3 -

Can1-1

MU-1A

6.23 5.49 5.86 5.86 4.73 6.91 4.73 5.09 5.47 1.47 1.09 1.45 1.45 2.55 0 0.24 1.58 0.97 1.45 1.45 16.6 16.4 16.3 16.3 19.2 19.4 17.7 17.9 18.9 18.4 25.5 26.4 25.7 25.5 26.1 27.3 26.6 27.7 28.0 26.4 25.8 26.9 -

MU-1A

Can1-2

6.59 5.86 6.23 6.23 4.74 6.57 4.74 5.11 5.47 1.83 1.09 1.46 1.46 2.55 0.73 0 1.58 0.97 1.46 1.46 16.9 16.8 16.7 16.7 19.2 19.5 17.8 18.0 18.7 18.5 25.6 26.5 25.8 25.6 26.2 27.1 26.6 28.1 28.1 26.2 25.9 26.6 -

Can1-2

Co-1

6.23 5.49 5.86 5.86 4.73 6.55 4.73 5.09 6.20 1.83 1.82 2.18 2.18 3.28 1.45 1.46 0 1.09 1.58 1.58 16.6 16.4 16.3 16.3 19.5 19.8 18.4 18.7 18.9 18.7 25.6 26.6 25.8 25.6 26.4 27.2 27.2 27.7 27.9 26.1 25.3 26.6 -

Co-1

Med-1

5.86 5.13 5.49 5.49 4.36 6.55 4.36 4.73 5.11 1.10 0.73 1.09 1.09 2.19 0.36 0.36 1.09 0 0.48 0.48 16.1 15.9 15.8 15.8 19.3 19.5 17.5 17.7 18.4 18.2 25.2 25.9 25.5 25.0 25.8 27.2 26.8 27.6 28.0 26.2 25.6 26.7 -

Med-1

BB-1

5.86 5.13 5.49 5.49 4.36 6.55 4.36 4.73 5.11 1.10 0.73 1.09 1.09 2.19 0.36 0.36 1.09 0.0 0 0.0 16.1 15.9 15.8 15.8 19.6 19.9 17.3 17.6 18.3 18.1 25.3 26.1 25.6 25.1 25.9 27.2 27.2 27.6 28.2 26.3 25.7 26.8 -

BB-1

BB-2

5.86 5.13 5.49 5.49 4.36 6.55 4.36 4.73 5.11 1.10 0.73 1.09 1.09 2.19 0.36 0.36 1.09 0.0 0.0 0 16.1 15.9 15.8 15.8 19.6 19.9 17.3 17.6 18.3 18.1 25.3 26.1 25.6 25.1 25.9 27.2 27.2 27.6 28.2 26.3 25.7 26.8 -

BB-2

Sb-3

8.42 8.06 8.42 8.42 9.85 11.0 9.85 9.85 11.0 9.16 8.76 9.12 8.76 9.85 9.12 9.85 9.85 9.12 9.12 9.12 0 0.36 0.49 0.49 18.5 18.6 16.1 16.6 17.0 17.0 24.3 25.1 24.6 24.5 24.9 26.5 27.7 29.8 29.3 28.0 27.7 28.5 -

Sb-3

Sb-15

8.42 8.06 8.42 8.42 9.85 11.0 9.85 9.85 11.0 9.16 8.76 9.12 8.76 9.85 9.12 9.85 9.85 9.12 9.12 9.12 0.0 0 0.36 0.36 18.5 18.6 16.3 16.8 17.3 17.3 24.6 25.3 24.8 24.7 25.2 26.5 27.5 29.6 29.3 28.1 27.9 28.6 -

Sb-15

Sb-5

8.42 8.06 8.42 8.42 9.85 11.0 9.85 9.85 11.0 9.16 8.76 9.12 8.76 9.85 9.12 9.85 9.85 9.12 9.12 9.12 0.0 0.0 0 0.0 18.6 18.7 16.2 16.7 17.2 17.2 24.5 25.2 24.7 24.6 25.1 26.4 27.6 29.7 29.2 28.0 27.7 28.5 -

Sb-5

Appendix 2: Percentage differences of Australian-only sub group isolates. Nucleotide values are given in bold and amino acids in italics.

Bun-9 MU-1C Med-7 Wel-1 WHP-2 He-2 KiP-1 He-1 Cgt-1 MU-2A MU-3A BB-6 WHP-1 Can1-1 MU-1A Can1-2 Co-1 Med-1 BB-1 BB-2 Sb-3 Sb-15 Sb-5 Sb-6 Sb19-1 Sb19-4 MR-13 MR-21 MR-3 MR-30 PWV-299 PWV Car-1 PWV CoP-1 PWV Gld-1 PWV Ku-1 PWV-TB* PWV-S* PWV-M* S1VY HiVY S2VY CliVY PWV-Culne PWV-Newry PWV-UniSy PWV-K*

MU-1C

*Isolates of PWV (TB, S, M & K) for which no nucleotides sequences are available.

Bun-9

Sb-6

Sb19-1

11.0 10.3 10.6 8.42 10.9 12.0 11.3 10.6 13.1 10.6 10.6 10.9 10.9 11.7 10.6 11.0 10.9 10.6 10.6 10.6 10.6 10.6 10.6 10.6 0 0.24 17.5 17.7 17.0 17.1 25.2 25.9 25.2 25.8 25.6 27.4 30.3 28.8 29.5 28.0 27.6 28.7 -

Sb19-1

Sb19-4

11.4 10.6 11.0 10.6 11.3 12.4 11.6 10.9 13.5 11.0 10.9 11.3 11.3 12.0 10.9 11.3 11.3 10.9 10.9 10.9 11.0 11.0 11.0 11.0 0.36 0 17.6 17.8 17.1 17.2 25.5 26.2 25.5 26.1 25.8 27.4 30.6 29.0 29.6 28.0 27.8 28.7 -

Sb19-4

MR-13

9.89 9.52 9.89 9.89 9.45 10.9 9.45 9.45 11.3 9.89 9.45 9.82 9.09 10.22 9.09 9.49 9.82 9.09 9.09 9.09 7.66 7.66 7.66 7.66 10.6 10.9 0 0.48 5.05 5.05 26.2 26.5 26.1 26.0 26.1 27.6 29.4 27.7 29.7 26.8 26.6 27.3 -

MR-13

MR-21

9.89 9.52 9.89 9.89 9.45 10.9 9.45 9.45 11.3 9.89 9.45 9.82 9.09 10.22 9.09 9.49 9.82 9.09 9.09 9.09 7.66 7.66 7.66 7.66 10.6 10.9 0.0 0 5.29 5.29 26.5 26.7 26.4 26.2 26.4 27.8 29.6 27.5 29.5 26.8 26.6 27.3 -

MR-21

MR-3

10.3 9.89 10.3 10.3 9.82 11.2 9.82 9.82 11.7 10.26 9.82 10.18 9.45 10.58 9.45 9.12 10.18 9.45 9.45 9.45 8.03 8.03 8.03 8.03 10.9 11.3 0.72 0.72 0 0.72 26.4 27.1 26.4 26.2 26.7 27.4 29.4 27.9 28.5 27.1 27.0 28.0 -

MR-3

MR-30

9.89 9.52 9.89 9.89 9.45 10.9 9.45 9.45 11.3 9.89 9.45 9.82 9.09 10.22 9.09 9.49 9.82 9.09 9.09 9.09 7.66 7.66 7.66 7.66 10.6 10.9 0.36 0.36 0.36 0 26.8 27.6 26.8 26.7 27.2 27.9 29.2 28.0 28.8 27.3 27.2 28.3 -

MR-30

PWV-299

16.1 15.8 16.1 16.1 18.2 18.8 18.2 18.2 19.0 16.9 16.7 17.1 16.7 17.2 17.1 17.5 17.5 17.1 17.1 17.1 16.4 16.4 16.4 16.4 16.0 16.4 17.3 17.3 17.7 17.3 0 6.21 2.27 5.97 6.45 22.9 24.1 24.1 22.3 23.9 23.5 23.9 -

PWV-299

PWV Car-1

17.2 16.9 17.2 17.2 18.6 19.6 18.6 18.6 19.3 18.0 17.1 17.5 17.1 17.5 17.5 17.9 18.2 17.5 17.5 17.5 16.8 16.8 16.8 16.8 17.1 17.5 17.7 17.7 18.1 17.7 4.66 0 5.97 4.90 0.96 24.0 23.7 24.2 22.5 24.7 24.0 24.1 -

PWV Car-1

PWV CoP-1

16.9 16.5 16.9 16.9 18.9 19.6 18.9 18.9 19.7 17.6 17.5 17.8 17.5 17.9 17.8 18.3 18.2 17.8 17.8 17.8 17.2 17.2 17.2 17.2 16.4 16.7 18.1 18.1 18.4 18.1 2.15 5.73 0 6.21 6.21 22.9 24.0 23.2 21.7 23.7 23.3 23.4 -

PWV CoP-1

PWV Gld-1

17.2 16.9 17.2 17.2 18.6 20.3 18.6 18.6 19.0 18.0 17.1 17.5 17.1 18.3 17.8 17.9 18.2 17.5 17.5 17.5 16.8 16.8 16.8 16.8 17.1 17.5 16.6 16.6 17.0 16.6 6.09 6.81 7.17 0 5.14 23.2 23.7 23.7 21.8 23.8 23.1 23.5 -

PWV Gld-1

PWV Ku-1

16.9 16.5 16.9 16.9 18.2 19.2 18.2 18.2 19.0 17.6 16.7 17.1 16.7 17.2 17.1 17.5 17.8 17.1 17.1 17.1 16.4 16.4 16.4 16.4 16.7 17.1 17.3 17.3 17.7 17.3 5.38 2.15 6.45 7.53 0 24.3 24.0 24.2 22.8 25.0 24.3 24.4 -

PWV Ku-1

PWV-TB*

16.0 15.6 16.0 16.0 17.1 17.5 17.1 17.1 18.6 16.7 16.0 16.4 16.0 17.1 16.4 17.1 16.7 16.4 16.4 16.4 16.0 16.0 16.0 16.0 15.2 15.6 16.4 16.4 16.7 16.4 1.12 5.20 2.23 5.20 5.95 0 -

PWV-TB*

PWV-S*

16.0 15.6 16.0 16.0 17.1 17.5 17.1 17.1 19.0 16.7 16.4 16.7 16.4 17.5 16.4 17.1 16.7 16.4 16.4 16.4 16.4 16.4 16.4 16.4 14.9 15.2 16.4 16.4 16.7 16.4 1.86 5.58 2.60 5.58 6.32 1.12 0 -

PWV-S*

PWV-M*

16.7 16.4 16.7 16.7 17.8 18.2 17.8 17.8 19.3 17.5 16.7 17.1 16.7 17.8 17.1 17.8 17.5 16.4 17.1 17.1 16.7 16.7 16.7 16.7 15.6 16.0 17.1 17.1 17.5 17.1 4.46 6.69 5.20 7.06 7.43 7.43 4.09 0 -

PWV-M*

S1VY

18.7 18.3 18.7 18.7 19.3 20.7 19.3 19.3 20.8 19.1 18.9 19.3 18.9 19.3 19.3 18.6 18.6 17.1 18.9 18.9 19.7 19.7 19.7 19.7 18.9 19.3 19.9 19.9 19.5 19.9 14.7 14.7 15.1 14.7 15.4 15.4 13.4 13.4 0 22.8 24.5 23.7 20.3 19.8 20.9 -

S1VY

HiVY

18.7 18.3 18.7 18.7 20.4 22.1 20.4 20.0 21.5 19.4 19.3 19.6 19.3 20.1 19.6 19.7 20.0 18.9 19.3 19.3 18.3 18.3 18.3 18.3 20.4 20.7 19.5 19.5 19.9 19.5 15.8 15.8 16.2 16.2 16.6 16.6 14.1 14.5 15.1 0 26.6 27.7 24.6 23.9 24.7 -

HiVY

S2VY

20.2 20.2 20.5 20.5 19.0 19.7 19.0 18.6 20.4 19.4 17.9 18.3 17.9 19.0 18.3 18.6 17.9 19.3 17.9 17.9 21.2 21.2 21.2 21.2 17.9 18.3 19.7 19.7 20.1 19.7 16.1 17.2 16.1 15.7 17.9 17.9 14.9 16.0 17.9 19.0 0 20.2 24.1 23.6 24.2 -

S2VY

CliVY

19.8 19.4 19.8 19.8 19.6 20.3 19.6 19.3 19.7 20.2 18.9 19.3 18.9 19.3 19.3 19.7 19.3 19.3 19.3 19.3 19.7 19.7 19.7 19.7 19.3 19.6 19.5 19.5 19.9 19.5 15.5 15.8 15.5 16.2 16.6 16.6 14.5 15.2 18.4 19.4 13.5 0 24.5 23.7 23.9 -

CliVY

PWV-Culne

19.1 18.7 19.1 19.1 18.2 17.8 18.2 18.2 18.6 18.7 17.5 17.8 17.1 17.2 17.8 17.9 17.1 17.8 17.8 17.8 19.0 19.0 19.0 19.0 18.2 18.6 18.8 18.8 19.1 18.8 12.9 13.3 13.3 13.6 14.0 14.0 12.3 12.6 15.4 15.8 16.4 18.0 0 2.13 2.32 -

PWV-Culne

PWV-Newry

19.4 19.1 19.4 19.4 18.6 19.2 18.6 18.6 19.7 19.1 17.8 18.2 17.5 17.9 18.2 18.6 17.1 18.2 18.2 18.2 19.3 19.3 19.3 19.3 18.2 18.6 19.1 19.1 19.5 19.1 13.3 12.9 13.6 14.0 13.6 13.6 12.6 13.0 15.4 16.6 17.2 18.4 2.48 0 4.02 -

PWV-Newry

PWV-UniSy

19.1 18.7 19.1 19.1 18.2 17.8 18.2 18.2 18.6 18.7 17.5 17.8 17.1 17.2 17.8 17.9 17.1 17.8 17.8 17.8 19.0 19.0 19.0 19.0 18.6 18.9 18.8 18.8 19.1 18.8 12.9 13.6 13.3 14.7 14.3 14.3 12.3 13.0 15.4 15.5 16.1 18.0 2.09 2.09 0 -

PWV-UniSy

PWV-K*

18.0 17.6 18.0 18.0 17.1 17.8 17.1 17.1 18.3 17.6 16.4 16.7 16.0 16.4 16.7 17.2 15.6 16.7 16.7 16.7 17.9 17.9 17.9 17.9 16.7 17.1 18.1 18.1 18.4 18.1 11.5 12.2 11.9 13.3 12.2 12.2 10.8 11.5 13.7 14.8 15.3 16.9 2.52 2.88 2.52 0

PWV-K*

Bun-9 MU-1C Med-7 Wel-1 WHP-2 He-2 KiP-1 He-1 Cgt-1 MU-2A MU-3A BB-6 WHP-1 Can1-1 MU-1A Can1-2 Co-1 Med-1 BB-1 BB-2 Sb-3 Sb-15 Sb-5 Sb-6 Sb19-1 Sb19-4 MR-13 MR-21 MR-3 MR-30 PWV-299 PWV Car-1 PWV CoP-1 PWV Gld-1 PWV Ku-1 PWV-TB* PWV-S* PWV-M* S1VY HiVY S2VY CliVY PWV-Culne PWV-Newry PWV-UniSy PWV-K*

Appendix 2: (Continued) Percentage differences of Australian-only sub group isolates. Nucleotide values are given in bold and amino acids in italics.

8.42 8.06 8.42 8.42 9.85 11.0 9.85 9.85 11.0 9.16 8.76 9.12 8.76 9.85 9.12 9.85 9.85 9.12 9.12 9.12 0.0 0.0 0.0 0 18.6 18.7 16.2 16.7 17.2 17.2 24.5 25.2 24.7 24.6 25.1 26.4 27.6 29.7 29.2 28.0 27.7 28.5 -

Sb-6

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