a case report of astrovirus-associated progressive ...

Next generation sequencing for diagnosis and tailored therapy: a case report of astrovirus-associated progressive encephalitis

SUPPLEMENTAL DATA TABLE OF CONTENTS

METHODS ................................................................................................................................................ 2 High throughput sequencing and bioinformatics analysis .................................................................. 2 RT-PCR confirmation of astrovirus detection in case patient’s brain biopsy ...................................... 2 Draft assembly and genome finishing ................................................................................................. 3 Genome annotation ........................................................................................................................... 3 Phylogenetic analysis .......................................................................................................................... 3 Definition of a generic PCR amplification system for VA1/HMO-C related astroviruses ................... 4 DETAILED RESULTS .................................................................................................................................. 5 REFERENCES .......................................................................................................................................... 11

Figure S1. Building of the complete genome of HAstV-VA1/HMO-C-PA. ............................................................... 6 Figure S2. Sequence alignment of the 3’ end of astroviruses belonging to the VA1/HMO-C clade. ..................... 7 Figure S3. RT-PCR detection of HAstrV-VA1/HMO-C-PA in brain biopsy.. .............................................................. 8

Table S1. PCR and RT-PCR conducted on CSF and brain biopsy ............................................................................. 9 Table S2. Description of the contigs obtained from the brain biopsy and their match with known astroviruses . 9 Table S3. RT-PCR and 5’/3’ RACE primers used for genome finishing. ................................................................. 10

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METHODS High throughput sequencing and bioinformatics analysis Total RNA was extracted from the brain biopsy with TRIzol / chloroform (TRIreagent and Chloroform from Sigma Aldrich), using a FastPrep system (MP Biomedicals) and purified on a RNeasy Mini Spin column treated by DNase I (Qiagen) to digest genomic DNA, as recommended. cDNA synthesis was performed with the Superscript III reverse transcriptase (SuperScript III First Strand Synthesis System Kit from Life Technologies, Saint Aubin, France) using random hexamers. Ligation of cDNA and nucleic acid amplification were done with the Whole Transcriptome Amplification kit (Qiagen) using the bacteriophage Phi29 polymerase, as described previously [1]. Reads were generated on Ion Proton (PathoQuest SAS, Paris, France) with a sequencing depth of 75.6×106 reads of 146 nt in length. Sequences were trimmed and filtered according to their quality score and length (>100nt) (proprietary algorithms, PathoQuest SAS, Paris, France). After human genome sequence subtraction (from NCBI build 37.1/assembly hg19) using BWA aligner (v. 0.7.9, “mem” algorithm) and Paracel LLC nucleotide blast (default parameters), reads were de novo assembled using CLC Genomics assembly cell (v. 4.22.107090, Cambridge, USA), and contigs and singletons were assigned to a given taxonomy using nucleotide alignments results (Paracel LLC nucleotide blast, default parameters) and a proprietary tools for fast taxonomy retrieval (PathoQuest SAS, Paris, France). Bioinformatics analysis was performed on the PathoQuest IT infrastructure using a fully automated workflow dedicated to an in silico detection of known and unknown viruses. After sequencing, raw data were analyzed in less than 4 hours. RT-PCR confirmation of astrovirus detection in case patient’s brain biopsy To confirm the presence of sequences belonging to an astrovirus, a pair of primers Astro-255-Fw 5’ATCACCACAAGAGGCGTAG-3’ (forward) and Astro-255-Rv 5’-AACCCCCTTCAACCTCATC3’ (reverse) was defined in the longest contig found (related to astrovirus VA1). We also used the published couple of primers SF0073 5’-GATTGGACTCGATTTGATGG-3’ (forward) and SF0076 5’-CTGGCTTAACCCACATTCC-3’ (reverse) that was initially developed to target a region in ORF 1b which is conserved among Human astroviruses 1-8, MLB1, and some other animal stains of astroviruses

[2],

as

well

as

a

generic

couple

of

primers

PA-SG-VA1-Fw

5’-

GCAAGGGGATTTTGCCGTAA-3’ and PA-SG-VA1-Rv 5’-GGGTCAATCTGTAGCGATGC-3’ developed in this study for the detection of clade VA1/HMO-C astroviruses (see also below: Definition of a generic PCR amplification system for VA1/HMO-C related astroviruses). Total RNA extracted from the brain biopsy was analyzed by qRT-PCR in a 20µl reaction volume that contained 1X Master Mix, 1X RT Mix and 300nM of the forward and reverse primers respectively (KAPA SYBR FAST One-Step qRT-PCR Kit, Clinisciences). The qRT-PCR program was 42°C for 5 min, 95°C for 3 min then 45 cycles at 95°C for 10 sec, 60°C for 20 sec and 72°C for 1min (Figure S3).

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Draft assembly and genome finishing Due to partial redundancy, 14 contigs were selected and aligned to the closest astrovirus strain, astrovirus VA1, for genome drafting. The resulting draft assembly covered 4,090 nt out of 6,586 nt (62.1%

of

astrovirus

VA1).

The

complete

genome

of

HAstrV-VA1/HMO-C-PA

(

Figure S1) was elaborated from the initial draft by a combination of RT-PCR (Superscript III reverse transcriptase with random hexamers, Invitrogen, and Phusion High-Fidelity DNA Polymerase, New England BioLabs), and 5’/3’ Rapid Amplification of cDNA Ends (RACE) (5’/3’Race kit 2nd generation and Expand High Fidelity PCR system, both Roche). Primers were designed to generate a series of ten inter-contigs RT-PCR amplicons (p1 to p10) with an average size of 400 bp, plus six additional supporting RT-PCR reactions (p5r-1, p1-3, p3-4, p4-6, p8-9, pDel). Virus-specific primers for 5′ RACE were 5′-CACGCTCAAGCTCATGTCTG-3′ (SP1) for reverse transcription, 5′CACCAACAATGGCAGTAGGT-3′ (SP2) for the first PCR with Oligo dT-Anchor primer, and 5′CCCAATCATTAAGTCCCGGT-3′ (SP3) for the nested PCR with PCR Anchor Primer. Primers for 3′ RACE were 5′-CAACCCAATTGACAGTCGCC-3′ (SP5) and PCR Anchor Primer for the first PCR and 5′-CCGTAATATGGCAGGTTGGTAG-3′ (SP6) and PCR Anchor Primer for the nested PCR. All primers used are summarized in Table S3. PCR products were characterized on 2% agarose gel, purified with the Nucleospin Gel & PCR Clean-Up kit (Macherey-Nagel) and then sequenced in both directions using Sanger chemistry (Eurofins Cochin, Paris, France). In the case of 3’RACE analysis, 4 independent PCRs have been performed starting from the same cDNA source. An additional pDel PCR system, encompassing the putative deletion of 42 bases, was used on a second and independently synthetized cDNA material in order to secure the deletion result. The resulting HAstrV-VA1/HMO-C-PA complete genome is 6,547 bases long (GenBank accession number KM401565). Genome annotation ORFs were predicted using NCBI ORF Finder and supported by local nucleotide alignments with annotated astrovirus VA1 (FJ973620) and SG (GQ891990). Pfam version 27 (EMBL-EBI) was used 3

to define the trypsin-like peptidase domain from the putative ORF1a translation. The ribosomal slippery heptamer (AAAAAAC) and the potential stem-loop structure, both conserved in all astroviruses and which together constitute a proposed frameshift mechanism (-1 frame) for the translation of the RNA-dependent RNA polymerase (ORF1b) non-structural protein [3], were identified manually. A putative promoter sequence initiating subgenomic RNA synthesis, which encodes

the

capsid

precursor

(ORF2),

UUUGGAGGGGAGGUCCAAAGCAAAGCCAUGGC

(start

was codon

identified underlined)

by

as direct

comparison with previous studies [4,5]. Phylogenetic analysis Twenty eight genomes of astroviruses, including the novel strain HAstrV-VA1/HMO-C-PA, were aligned with ClustalW. A phylogenetic tree was constructed in Geneious version 8.1.5 using a Tamura-Nei, Neighbor-Joining model, with no outgroup. The GenBank accession numbers used are as follows: Human astrovirus 1 (HAstV1), KF211475; HAstrV2, KF039911; HAstV4, DQ344027; HAstrV5, DQ028633; HAstrV6, GQ495608; HAstrV8, AF260508; Turkey, NC_002470; Sheep, Y15937; Duck, KJ020899; Bovine, NC_024297; Murine, NC_018702; Feline, NC_022249; Porcine, NC_019494; Rabbit, JF729316; Wild-boar, NC_016896; Mink, NC_004579; MLB1, NC_011400; MLB2, NC_016155; MLB3, JX857870; VA1, NC_013060; VA2, NC_018669; VA3, NC_019026; VA4, NC_019027; SG, GQ891990; BF34, KF859964; HAstV-VA1/HMO-C-UK1, KM358468; HAstV-VA1/HMO-C-UK1(a), KJ920196 and HAstrV-VA1/HMO-C-PA, KM401565. Definition of a generic PCR amplification system for VA1/HMO-C related astroviruses A pair of primers PA-SG-VA1-Fw 5’-GCAAGGGGATTTTGCCGTAA-3’ and PA-SG-VA1-Rv 5’GGGTCAATCTGTAGCGATGC-3’ was defined in a region of ORF2 conserved among astroviruses HAstrV-VA1/HMO-C-PA, SG and VA1, i.e. encompassing most known neurotropic astroviruses at the time of the work. After cDNA synthesis (Superscript III, random hexamers, Invitrogen), PCR was performed using 1X Phusion HF Buffer, 200µM dNTP, 500nM of each of the primers and 0.02U/µl Phusion DNA Polymerase. The PCR program was 98°C for 30 sec, 35 cycles at 98°C for 10 sec, 65°C for 30 sec, 72°C for 30 sec, 72°C for 7 min. Amplification product were then characterized on 2% agarose gel, purified with the Nucleospin Gel & PCR Clean-Up kit (Macherey-Nagel) and sequenced (Eurofins Cochin, Paris, France), confirming the detection of HAstrV-VA1/HMO-C-PA in the brain biopsy.

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DETAILED RESULTS Brain biopsy total RNAs were subjected to NGS with the aim of identifying the transcripts and, for RNA viruses, the genome, of a potential encephalitis-associated pathogen. Among a total of 75.6 million reads, 15 contigs (Table S2) and 12 singletons had top-scoring hits for sequences belonging, within the Astroviridae family, to a clade distant from Human astrovirus 1 to 8. Members of this distant clade are the VA1 (FJ973620) [6], SG (GQ891990) [7], HAstV-VA1/HMO-C-UK1 (KM358468) [8] and HAstV-VA1/HMO-C-UK1(a) (KJ920196) [9] strains. Total reads coverage encompassed more than 60% of the astrovirus VA1 genome (Figure 2A). No sequence of other pathogens was found. The presence of an astrovirus genome in the brain biopsy was confirmed by a PCR system defined within the longest sequencing contig (primers Astro-255-Fw and Astro-255-Rv defined in ORF 1a/1ab, see Figure 2A & S2). The complete genome was obtained by a combination of inter-contig PCRs and 5’/3’ RACE experiments (Figure S1). Phylogenetic analysis confirmed high levels of homology between the genomes of the VA1/HMO-C clade (Figure 2A & 2B). Yet, a major difference was noted, i.e. a 42 bases deletion in the capsid gene, as compared to other strains of this group (Figure S2). This novel strain was named HAstrV-VA1/HMO-C-PA, for Paris strain, and the annotated sequence has been deposited in GenBank (accession number KM401565). In contrast to brain biopsy, all PCR assays conducted on CSF samples with primers Astro-255-Fw and Astro-255-Rv were negative. Furthermore, PCRs performed with the published primers SF0073 and SF0076 (ORF 1ab, see Figure 2A), which were intended to target a wide range of human astroviruses [2], failed at detecting HAstrV-VA1/HMO-C-PA in both brain biopsy and CSF samples. Such inability to detect this novel astrovirus strain is likely the consequence of the number of mismatches between the primers and the target (4 mismatches with SF0073 and 3 with SF0076, data not shown). Therefore, we developed a more generic PCR targeting a conserved region among the C-terminal part of the capsid gene of HAstrV-VA1/HMO-C-PA, astrovirus VA1 and astrovirus SG (PA-SG-VA1-Fw and PA-SG-VA1-Rv), , which was successfully used to detect HAstrV-VA1/HMO-C-PA in the brain biopsy sample. This generic PCR should help for future detection of VA1/HMO-C clade-related astroviruses, as exemplified by the good level of sequence conservation within the primers binding zone observed after the two recent HAstrV-VA1/HMO-C-UK1 and HAstrV-VA1/HMO-C-UK1(a) cases have been reported (see alignment in Figure S2).

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Figure S1. Building of the complete genome of HAstV-VA1/HMO-C-PA . Sequencing contigs (black lines c1 to c14), inter-contigs RT-PCR (red lines p1 to p10), additional supporting RT-PCR (red lines p5r-1, p1-3, p3-4, p4-6 and p8-9, pDel) and 5’/3’ RACE (blue lines) are depicted in the context of the novel HAstV-VA1/HMO-C-PA genome coordinates (grey line, upper part, from 1 to 6,547 bases).

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Figure S2. Sequence alignment of the 3’ end of astroviruses belonging to the VA1/HMO-C clade. Alignment of astroviruses SG (GQ891990), VA1 (NC_013060), HAstV-VA1/HMO-C-UK1 (KM358468), HAstV-VA1/HMO-C-UK1(a) (KJ920196) and HAstV-VA1/HMO-C-PA (KM401565) was done with ClustalW. The coordinates (right part) indicate the genomic position of the last nucleotide of the line. Stars (*) highlight which nucleotides are shared by the five sequences, and bring out a deletion of 42 nucleotides in the C terminal part of the capsid gene of HAstV-VA1/HMO-C-PA. Underlined are the position of primers PA-SG-VA1-Fw/Rv developed in this study for the detection of HAstV-VA1/HMOC-PA and other VA1/HMO-C clade-related astroviruses, and primers SG/VA1-Fw/Rv used by Quan and colleagues for the detection of astrovirus SG in a XLA boy with encephalitis [7].

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Figure S3. RT-PCR detection of HAstrV-VA1/HMO-C-PA in brain biopsy. Agarose gel (2%) showing positive amplification of astrovirus HAstrV-VA1/HMO-C-PA in the brain biopsy with primers Astro255-Fw/Astro-255-Rv and PA-SG-VA1-Fw/PA-SG-VA1-Rv, but not with primers SF0073/SF0076. W: Water; M: 50 bp ladder.

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Table S1. PCR and RT-PCR conducted on CSF and brain biopsy Viral screening Parvovirus EBV CMV Adenovirus HSV-1 HSV-2 VZV HHV6 HHV8 Enterovirus Astrovirus Respiratory viruses Measles Mumps JC Virus

CSF negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative

Brain biopsy negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative

Table S2. Description of the contigs obtained from the brain biopsy and their match with known astroviruses Family

Astroviridae

Astroviridae Astroviridae Astroviridae

Main organism (Number of contigs)

Astrovirus human/SZ908 /China/2008 (5) HMO Astrovirus C (3) Astrovirus SG (1) Astrovirus VA1 (6)

Main GI (# contigs)

# contigs

# reads in contigs

Average contigs identity

Average contig length

Max contig length

254032065 (5)

5

21

94.87%

221

316

261597215 (3)

3

35

95.61%

423

602

1

3

97.22%

216

216

6

44

96.93%

319

635

296932862 (1) 237856656 (6)

9

Table S3. RT-PCR and 5’/3’ RACE primers used for genome finishing. Primer

Sequence (5’-3’)

Application

Product size (bp)

Position in complete genome

p1 Fw p1 Rv p2 Fw p2 Rv p3 Fw p3 Rv p4 Fw p4 Rv p5 Fw p5 Rv p6 Fw p6 Rv p7 Fw p7 Rv p8 Fw p8 Rv p9 Fw p9 Rv p10 Fw p10 Rv p5r-1 Fw p5r-1 Rv p1-3 Fw p1-3 Rv p3-4 Fw p3-4 Rv p4-6 Fw p4-6 Rv p8-9 Fw p8-9 Rv pDel Fw pDel Rv SP1 SP2 SP3 SP5 SP6

GCGCTCATCTTTACAATTGGATG TTTTCACCAACAACCACTCC CACATCAACAACAACATCCTGG CAACAACATGTCCGGCTGTA ATATGCTGTTAGCACCCGAA TCCTCAGTCCAAACTTTCCC TGGCTGAAACTGAACCACTTA GGCGGCTTTTCCTCACATTT AGCGTATAGGCGGTCATTTG GTACCATCATAACGCGTCCA GAGTTCGACTGGACGCGTTA GTTGTCACATAAAATAGGGGTT AGATGATAGGTTAACTACAACCC GT CCTCCCCTCCAAAGCCTAT C GAAATTTGTGAAGAAACAGCTA TAAAGAGGTCTCCAGGCCAT GA GTCAAGAAATTGCTAGGTAGAT TGTAGTGGTATTAGGGCCTGT CAG CAACCCAATTGACAGTCGCC TGAGTATTGTGCCCGCAAAG GTCAAAGCACGGATACAGGG TCTGGATCAAGACACACACCA TGTTAGTGCCACACTGGGTC TTTATGATGACAGCACCGCC AATTGATTCGGCTTGCTGTTG ACCCAAGCCTTAGTTGTTCTG AAGGCTTGGGTACTGGAGAG GCAAATCTCCCCTAAGACACG AGGAAGCACAAGGATGTTGC GGGCCTGTATTTGGTGCATT GGCATGTGGTATCGTGCTTT GCTGTACCCTCGGTCCTACT CACGCTCAAGCTCATGTCTG CACCAACAATGGCAGTAGGT CCCAATCATTAAGTCCCGGT CAACCCAATTGACAGTCGCC CCGTAATATGGCAGGTTGGTAG

Inter-contig

789

446-1234

Inter-contig

194

1274-1467

Inter-contig

375

1706-2080

Inter-contig

298

2565-2862

Inter-contig

567

2890-3456

Inter-contig

381

3428-3808

Inter-contig

429

3769-4197

Inter-contig

563

4361-4923

Inter-contig

180

5223-5402

Inter-contig

311

5734-6044

Additional supporting PCR Additional supporting PCR Additional supporting PCR Additional supporting PCR Additional supporting PCR Additional supporting PCR 5’RACE 5’RACE 5’RACE 3’RACE 3’RACE

515

111-625

670

1153-1822

761

1948-2708

824

2698-3521

668

4723-5390

281

6180-6461

N.A. N.A. 345 N.A. 477

479-498 (primer) 371-390 (primer) 326-345 (primer) 5734-5753 (primer) 6070-6091 (primer)

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REFERENCES 1. Cheval J, Sauvage V, Frangeul L, et al. Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples. J. Clin. Microbiol. 2011; 49:3268–3275. 2. Finkbeiner SR, Le BM, Holtz LR, Storch GA, Wang D. Detection of newly described astrovirus MLB1 in stool samples from children. Emerging Infect. Dis. 2009; 15:441– 444. 3. Lewis TL, Greenberg HB, Herrmann JE, Smith LS, Matsui SM. Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein. J. Virol. 1994; 68:77–83. 4. Jiang H, Holtz LR, Bauer I, et al. Comparison of novel MLB-clade, VA-clade and classic human astroviruses highlights constrained evolution of the classic human astrovirus nonstructural genes. Virology 2013; 436:8–14. 5. Phan TG, Nordgren J, Ouermi D, et al. New astrovirus in human feces from Burkina Faso. J. Clin. Virol. 2014; 60:161–164. 6. Finkbeiner SR, Li Y, Ruone S, et al. Identification of a novel astrovirus (astrovirus VA1) associated with an outbreak of acute gastroenteritis. J. Virol. 2009; 83:10836–10839. 7. Quan P-L, Wagner TA, Briese T, et al. Astrovirus Encephalitis in Boy with X-linked Agammaglobulinemia. Emerg Infect Dis 2010; 16:918–925. 8. Naccache SN, Peggs KS, Mattes FM, et al. Diagnosis of Neuroinvasive Astrovirus Infection in an Immunocompromised Adult With Encephalitis by Unbiased NextGeneration Sequencing. Clin. Infect. Dis. 2015; 9. Brown JR, Morfopoulou S, Hubb J, et al. Astrovirus VA1/HMO-C: An Increasingly Recognized Neurotropic Pathogen in Immunocompromised Patients. Clin. Infect. Dis. 2015;

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