Nucleotides from positions 278 to. 2550 in the DEN-3 genome encoding the prM/M and E genes were ... Purified DNAs were then used as templates in asymmetric PCRs to ...... Virus Information E'cchange Newsletter 8, 2-3. GUBLER, D. J. ...
Journal of General Virology (1994), 75, 65 75. Printedin Great Britain
65
Molecular evolution and epidemiology of dengue-3 viruses Robert S. Lanciotti,* Joyce Grant Lewis, Duane J. Gubler and Dennis W. Trent Division of Vector-Borne Infectious Diseases, National Center For Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, P.O. Box 2087, Fort Collins, Colorado 80522, U.S.A.
The nucleic acid sequences of the pre-membrane/ membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90 %. The similarity among deduced amino acids was between 95 % and 200 %, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylo-
genetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
Introduction
generally leads to a mild, self-limiting febrile illness (DF). However, in some DF cases, vascular and haemostatic abnormalities occur, which may progress to haemorrhage and shock (DHF/DSS). The precise mechanism(s) by which dengue viruses cause severe haemorrhagic disease is not well understood. One theory suggests that secondary infection with a heterologous dengue serotype can lead to severe disease (Halstead, 1988). In this model, circulating antibodies to the primary infecting virus are thought to complex with the heterologous secondary infecting virus and also enhance virus infection of mononuclear cells (Halstead, 1988). An alternative explanation is that genetic evolution of the virus within each of the serotypes has resulted in generation of more virulent or epidemic strains of dengue viruses (Rosen, 1977). Epidemiological analysis of dengue epidemics reveals that some dengue virus strains are associated with mild epidemics with low occurrences of DHF and inefficient virus transmission, whereas others are involved in severe epidemics with a high incidence of DHF/DSS and rapid virus transmission (Gubler et al., 1981). Previous studies of dengue virus molecular evolution described intra-serotypic antigenic variation in DEN-3 (Russell & McCown, 1972). Strains from Puerto Rico and Tahiti were shown to be antigenically and bio-
Dengue viruses (family Flaviviridae, genus Flavivirus) are plus-sense ssRNA viruses that cause dengue fever (DF) and dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) in humans. These viruses are maintained in a human-mosquito transmission cycle, primarily in tropical urban areas, by Aedes aegypti and are currently endemic in most tropical areas of the world (Gubler, 1988). The earliest recorded epidemic with truly compatible dengue disease-like illness occurred in Philadelphia in 1780 (Rush, 1789). From then until the 1950s, epidemics of DF occurred irregularly throughout tropical regions worldwide. After World War II, however, epidemic DHF emerged in Southeast Asia and has subsequently spread to the Pacific islands and Americas (Gubler, 1991). Dengue viruses are serologically classified into four antigenically distinct serotypes (DEN-1, DEN-2, DEN-3 and DEN-4). Infection with any of the serotypes
The nucleotide sequence data reported in this paper have been submitted to the GenBankdatabase and assignedaccessionnumbers Ll1422 to Ll1620. 0001-1856 © 1994SGM
66
R. S. Lanciotti and others
Table 1. Description of DEN-3 viruses compared by sequence analysis GenBank accession no.
Geographic origin
Year
Strain
Passage history*
Fiji India Indonesia Indonesia-a Indonesia-b Indonesia Malaysia-a Malaysia-b Malaysia Malaysia-a Malaysia-b Mozambique-a Mozambique-b Philippines Philippines Philippines Puerto Rico Puerto Rico Samoa Sri Lanka Sri Lanka Sri Lanka Sri Lanka Sri Lanka-a Sri Lanka-b Sri Lanka Sri Lanka-a Sri Lanka-b Sri Lanka-c Sri-Lanka-d Sri Lanka-e Tahiti Tahiti Thailand Thailand Thailand Thailand Thailand Thailand Thailand
1992 1984 1973 1978 1978 1985 1974 1974 1980 1981 1981 1985 1985 1956 1983 1984 1977 1963 1986 1981 1982 1982 1983 1985 1985 1989 1991 1991 1991 1991 1991 1965 1989 1962 1973 1975 1977 1980 1986 1987
29472 1416 228761 1280 1275 85-159 1300 1301 26041 29586 233 1558 1559 H-87 168.AP-2 059. AP-2 1340 PR6 1696 1326 1336 1333 1363 1594 1586 260698 2783 271242 3171 3198 1863 1327 2167 5987 CH3489D73-1 205lD75-279 1308 D80-260 D86-007 MK315
C6/36-1 C6/36-1 Mosq.-1 C6/36-2 C6/36-? C6/36-1 C6/36-? C6/36-? C6/36-2 C6/36-1 C6/36-1 C6/36-1 C6/36-2 [C6/36; SMB]-355 C6/36-? C6/36-1 C6/36-1 SMB-13 C6/36-1 C6/36-1 C6/36-? C6/36-? C6/36-? C6/36-1 C6/36-1 C6/36-? C6/36-1 C6/36-1 C6/36-1 C6/36-1 C6/36-1 C6/36-2 Mosq.-1 C6/36-2 C6/36-? C6/36-2 C6/36-1 C6/36-2 C6/36-? C6/36-?
L11422 Ll1424 L11425 L11426 t Ll1428 L11429 t ~ Ll1427 t L11430 t L11423 L11432 # L11434 L11433 L11435 Ll1431 t i t Ll1436 t L11437 L11438 t t t t L11439 L11619 L11440 L11620 + t t Ll1441 Ll1442
* C6/36, Aedes albopictus cell line; Mosq., Toxorhynchites mosquitoes; SMB, suckling mouse brain. The number of passages is given where known. ~ Virus was subjected to partial sequencing (see text) and sequence data were not submitted to GenBank. :~ A combination of the cell line and mouse brain was used to passage the virus.
logically different from Asian DEN-3 viruses. Dengue viruses of all serotypes were genetically classified into topotypes by T1 RNase fingerprinting (Trent et al., 1990). Recently, limited nucleic acid sequencing of DEN1 and DEN-2 virus RNA verified strain variation within a serotype, allowing viruses to be classified into genetically distinct groups within the serotype (Rico-Hesse, 1990; Blok et al., 1989, 1991; Lewis et al., 1993). To study the molecular evolution and epidemiology of DEN-3 viruses, molecular analysis of DEN-3 premembrane/membrane (prM/M) and envelope (E) genes was performed. Sequence variation within the structural genes has enabled classification of DEN-3 viruses into
four discrete genetic lineages or subtypes. Analysis of the distribution of DEN-3 viruses worldwide has permitted identification of epidemiological patterns, suggesting that some virus genotypes are associated with either mild or severe forms of dengue disease. Methods Virus strains. The DEN-3 viruses used in this study (Table 1) were obtained from the collection at the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colo., U.S.A. Virus seeds consisted of either cell culture medium from C6/36 cells infected with low-passage virus or laboratory-infected Toxorhynchites mosquitoes.
Evohttion of dengue-3 viruses
67
Table 2. Oligonucleotide primers used for amplification and sequenchlg Primer* P278~c CP788 P722 P992 CP 1072 P 1259 CP 1559 P 1685 CP1819 P1908 CP2190 CP25505
Sequence
Genome positiont
5' CGCGGATCCACAGCAGGAGTCTTGGCTAGATGGGG 3' 5' TCCAAGCTCCTTCAGATGACATCCA 3' 5' GCTCCCCATGTCGGCATGGGACTGG 3' 5' T G G G T T G A C G T G G T G C T C G A G C A C G G 3' 5' C T G A A G C T C T A T G T C C A G C G T G G G 3' 5' G G C A A G G G A A G C T T G G T G A C A T G C G C 3' 5' TGGTAACGGCAGGTCTAGGAACCATTG 3' 5' CTAGGATCTCAAGAAGGAGCAATGCA 3" 5' C A T C C C T T T G A G T T T C A A T T T G T C C A T 3' 5' AAGGGGAAGATGCACCCTGCAAGATTCC 3' 5' TCCCAAGCTGTGTCTCCCAGAATGGCCAT 3' 5' CGCGGATCCATGGCTGTTGCCACTCTTTTGGGGGA 3'
278 to 788 to 722 to 992 to 1072 to 1259 to 1585 to 1685 to 1819 to 1908 to 2190 to 2550 to
303 764 746 1017 1049 1284 1559 1710 1793 1935 2162 2525
* Primer names beginning with ' P ' indicate a genome (plus)-sense orientation; names beginning with "CP" indicate a complementary sense orientation. t The genome positions are given according to the DEN-3 published sequence (Osatomi & Sumiyoshi, 1990). :~ The first nine bases of this primer consist of a three-base spacer followed by the BamHI restriction endonuclease recognition sequence.
R N A extraction. Viral RNA was isolated from virus in the culture medium of infected C6/36 cells or mosquitoes using the acid-guanidine isothiocyanate procedure described previously (Lanciotti et al., 1992), Selection andsynthesis of oligonucleotide primers. The DEN-3 primers used for amplification and/or sequencing (Table 2) were designed based on published dengue virus sequences with the aid of the OLIGO sequence analysis program (National Biosciences, Mason et al., 1987; Duebel et al., 1990; Osatomi & Sumiyoshi, 1990; Zhao et al., 1986). Oligonucleotides were synthesized using an Applied Biosystems automated synthesizer using phosphoramidite chemistry and then were purified on NENSORB columns (DuPont). Amplification o f DEN-3 RNA. Nucleotides from positions 278 to 2550 in the DEN-3 genome encoding the p r M / M and E genes were amplified using a reverse transcriptase (RT)-PCR. Two primer pairs were used in separate reactions to generate two overlapping dsDNA products covering this region (fragment 1, residues 278 to 1819; fragment 2, residues 1685 to 2550). The RNA was first transcribed to give cDNA using RT and downstream primers CP1819 or CP2550, followed by PCR amplification in the presence of the upstream primers P278 or P1685 (Lewis et al., 1992). DNA sequencing. Nucleic acid sequence analysis was performed either by ~manual" sequencing or on an automated Applied Biosystems 373A DNA sequencer. DNA fragments 1 and 2 (see above) generated by RT and PCR reactions were agarose gel-purified using the Bio 101 Gene-Clean kit. Purified DNAs were then used as templates in asymmetric PCRs to generate ssDNA copies representing both strands of the original DNA fragment (Gyllensten, 1989). Single-stranded DNA was sequenced in standard chain-termination sequencing reactions using 3~S-labelled dATP and the Sequenase kit (US Biochemical). Primers were positioned to 'walk" the region to be sequenced. For automated sequencing, gel-purified dsDNA fragments 1 and 2 were directly sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems). The primers used for sequencing were identical to those used for single-stranded sequencing and are shown in Table 2. Briefly, 0.5 pmol of each DNA fragment was combined with 1.0 pmol of primer and sequenced in a reaction cocktail containing the four dye-labelled dideoxynucleotide terminators. Cycle sequencing parameters used were as described in the manufacturer's protocol.
DNA sequence analysis. Overlapping nucleic acid sequences obtained from individual sequencing reactions were combined for analysis and editing with the aid of the DNASTAR program. Prior to phylogenetic analysis, DEN-3 virus nucleic acid sequences were aligned with each other and with the DEN-2 Jamaica virus, using the multiple sequence alignment CLUSTAL (D. Higgins, Heidelberg, Germany). DNA parsimony analysis was performed using either the heuristic or the branch and bound algorithm of PAUP (D. Swofford, Champaign, 111., U.S.A.). Parsimony analysis using other algorithms within PAUP or different analysis programs such as PHYLIP yielded nearly identical phylograms.
Results Nucleic acid and deduced amino acid sequences The nucleic acid sequences of the prM/M and E genes from 23 DEN-3 viruses were determined and submitted to GenBank. Fig. 1 displays the nucleic acid sequence of five representative DEN-3 viruses: prototype strain H-87 and one virus from each of the four newly proposed DEN-3 genetic subtypes (see below). Nucleic acid sequence similarity among the DEN-3 viruses ranged from 90% to 99%. The average sequence similarity within a DEN-3 subtype was 97.6 % for nucleic acids and 98.8 % for amino acids. The average similarity between the DEN-3 subtypes was 93.5 % and 97-0 % for nucleic and amino acids, respectively. Approximately 15% of the nucleic acid changes resulted in an amino acid change. The deduced amino acid sequence of the prM/M and E proteins of all 23 virus isolates revealed that the proteins were highly conserved and had a similarity between 95% and 100%. The majority of amino acid changes occurred in clusters common to several DEN-3 virus isolates (Fig. 2). The nucleic acid sequences of the prototype strain H-87, sequenced in this study, and the
68
R. S. Lanciotti and others
H-87 Fiji-92 Thailand-87 Sri Lanka-9l Puerto Rico-77
1010 1020 103U 1040 1050 1060 1070 1080 1090 1100 CCGCTGAAGCCATTTTACCTGAATAT GGAACCCTCGGGCTAGAATGCTCACCACGGACAGGTTTGGATTTCAATGAAATGATTTTATTGACAATGAAGAA , . ,T . . . . . . . . . C, .G . . . . . . . . . . . . . . . . . T ........... T ............... C.A . . . . . . . . . . . . . . . . . . . . G.............. ...T ...... T,.C..G ................. T.. ............................................................... • ,A.. , ....... C. ,G . . . . . . . . . . . . . . . . . T ............................................... C...C.A ........... • .A . . . . . . . . . . C..G ................. T ............................................... C..GC .............
H-87 F~i-92 Thmland-87 Sfi Lanka-91 Pue~o Rico-77
1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 CAAA•CAT•GATGGTA•ATAGACAAT••TTCTTTGA•TTACCCCTACCATGGACATCAGGAGCTACAACA•AAACA•CAACTTGGAACAGGAAAGAG•TT .............................. T ...... C . G , .TT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C..... T,A .......... ..................... G.......... C....................................... G........................... ..................................... C .... T ................................ G ..... C ........... G. . . . . . ..................................... C.... T ...................................... C........... G. . . . . . 1210
1220
1230
1240
1250
1260
1270
1280
1290
1300
H-87 F~i-92 Thmlmad-87 Sri Lanka-91 Puerto Rico-77
CTTGTGACATTTAAAAATGCACATGCAAAAAAGCAAGAAGTAGTTGTCCTTGGATCACAAGAGGGAGCAATGCATACAGCACTGACAGG•GCTACAGAGA
H-87 Fiji-92 Thailand-87 Sri Lanka-9 Puerto Rico-77
TcCAAAcCT CAGGAGGCA•AAGT A T T TT T GCGGGGCACT T AAAA T GT AGACT CAAGA T GGACAAAT T GAAACT CAAGGGGA T GA0CT A T GCAATGT GCTT
........... ........... ........... ...........
C..... C.............. A........... A ........... C............................................ C..... C.............. A ....................... C..... C........ G. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1310
H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77 H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
1320
H-87 Fiji-92 Thailand-87 Sri Lanka-9 l Puerto Rico-77 H-g7 Fiji-92 Thailand-87 Sri Lanka-9 l Puerto Rico-77
.T ....
A .........
......
A ...............
.T ....
T .....................
Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
1350
T .....
1370
1380
1390
1400
Jeloelm'*l*llllnOilggle'llG'OIIl''lllUOlellgle''OllOlOl•ll
C .................
G ...........
C ............................. C .....
1360
A.oTC
.......
C .....
T ...............
G ...............................
T ...............
G .............................
T..A
.....
T...C..G
AC
..............................
C
1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 GAATAC~TTTG~TT~AA~AAAGAAGT~T~CGAAA~GCA~CATG~ACAATA~TCATTAA~GTTGAGTACA/~AAGAT~CACC~T~CAAGATTCCT ....
G ....
C ....................
T ........
A ........
G ......................................
T ............
°°'•oo•'°°••-•°``••°••'''`••.•`°••`••••••••••••'•'--o••o'°°R••°````°o•••••oo'--•o••°•°-T•••°'••••°-• .............................. A ............................ G . , .C . . . . . . . G............... AGGC . . . . . C . . oC . . . . A . . . . . . . . T..T ................. G . . . . . T . . . . . AA . . . . . . . . . . . . . . . . . . . . . . . . .
T ............ T ............
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 TTCTCCACGGAG•ATGGA•AAGGGAAAGCTCACAATGG•AGACTGATCACAGCCAATCCAGTGGTGA•CAAGAAGGAGGAGCCTGTCAACATTGAGG•TG ............................. C ....................... T . . C . o oA,A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. • ...°e.oA.°ol.o..°.,oooo,.lo,.,.... D,°To., j...., j•o•0o,eCoe....°o,tao°°.•owmao Jl Jo.o---oo°to~°oo~°'e ........
.....
A ...............................................
A. . . . . . . . . . .
C ...........
T ...........................
1620
A..T .....
1630
1640
T ....................
T..C ....................
1650
1660
T ..........
A ......................
1670
1680
1690
1700
AACCT CCT•TTGGGGAAA•TAATATAGTAATTGGAATTGGAGACAAAGCCCTGAAAATCAA•TGGTACAGGAAGGGAAG•TCGATTGGGAAGATGTTCGA
-°o•••`••-•••••••`•--°•••`o•'•--•-•••o•°•°o--•°o•oT°o•-•°-----•o°•°o°A•--A°•°•••o•••••°°-°°••'-•••-...................... C . . . . . G. . . . . . . . . . . . . . . . . . . . . T ....... T .......... .............................................. C...T .................. ...................... C°°T..G ....... C............. T...G...T ..........
A .............................. A ............ T ................. A...A ...........................
1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 GGCCACTGCCAGAGG'rGCAAC,UCGCATGGCCATcTrGGGAGACACAGCCTGGGACTTTGGATCAGTGGGTGGTGTTTTGAATTCATTAGGGAAAATGGTC o. y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A ........... A ........... AG . . . . . . . G .............,.....o.....oo ..........
o.o,o....o..o.ooo.oooo...o.o.o..,0,o....o..o,,.o.C,Aoo......oo.......o..G
........................................
.........
T ....................
C ....
T ...........................
T .............................
C ........ C .....
G ....................
T..A
C ........
G
........
G
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 CACCAAATATTTGGGAGTGCTTACAC&GCCCTATTTAGTGGAGTCTCCTGGATAATGAAAATTGGAA.i.&GGTGTCCTCTTAACCTGGATAGGGTTGAATT ooooooooooooI•Aooooooooooo.ooooooooooooo.o.o..-o~,oooooooooo oooooeoooo.oooooaTo~ogolaooo--II**°°l"°° ...........
C..A
...........
C..A ....................
.....
C .................
G .....
1910 H-87
1340
IOiillOlplllWetl.elOOOOOl•m••a*llOeaOllell
1610 H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
1330
G................. C ......................... G................. C..... G................... G................................... C..... A. G................................... C.......
.................
1920
G ..............
C ........... To . T . . . . .
1930
C .....
1940
A ....................................................
T...G.G ....................
T . . . . . Go .T . . . . . . . . . . . . . . . . A.T ........ T ..............
T ...........................
1950
1960
1970
CN4NLAACACTTCTATGTCATTTTCATGCATTGCGATAGGAMCATTACACTCTATCTGGGAGTCGTGGTGCAAGCT ,,o,oooooooooo....oooooooooooeoooToo.ooooooeoooo~eooooooooo,.I JCooooooIoooo,. .............
.C . . . . . . . . .............
C .............................
T ...................
A..C ................................................. C ...................
T ............
C ...........
Fig. 1, For legend see opposite.
A ....
C ......
A ......
CT . . . . .
A ......
CT ........
G...
C.
Evolution of dengue-3 viruses
69
prM H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77 H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
H-87 Fiji-92 Thailand- 87 Sri Lanka-91 Puerto Rico-77 H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
,~ 10 20 30 40 50 60 70 80 90 100 TTCCACTTAACTTCACGAGATGGAGAGCCGCGCATGATTGTGGGGAAGAATGAAAGAGGAA/~L4TCCCTACTTTTTAAGACAGCCTCTGG/~ T ~ C A TGT ..... Y..G ........ G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C......................... ........
G ........
C .........................................
G .......................
T ................
110 120 130 140 150 1~ 1~ 180 1~ 200 GCA~CT~TA~CCAT~ATTTGGGAGAGATGTGTGAT~CG~T~CTTACAAATGCCCC~TTACCG~GTGGAGCCTG~TTGACTGCTG ................... .......
C ............................................
T ....................
T ....
A ...............
~
.............
T ........
G ..........
A ....................
C .......................
C.,T
.....
N 210 220 ~0 240 250 2~ 2~ , 280 299 300 GTGC~CCTTA~TCGA~TG~TGA~T~GG~TGCAATAAGCTG~GCATAGACGC~T~GA~T~GTGGCGTTAGCTCCC~TGTCGGC ...........................
C..C
.....
G ....................
C .................
G ........................
•••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•••••••••••••••••••••••••••••••.••••.••••• .........
......
~ .....
TT.G
A ....................
G .............................
CeeA ....................
.....................................................
T..C
T .........
.................
G ...............
310 320 330 340 350 360 370 380 390 400 ATGGGACTGGACACACGCACTCAAACCTGGATGTC•GCTGAAGGAGCTTGGAGACAAGTCGAGAAGGTAGAGACATGGGCCCTTAGGCACCCAGGGTTTA ........
A ...........
C ...................................
G ........................
T ................
C.
~°~`°`~'~°°°~'°~'`~`~°~°~°''B*``°~°~`j~e`~°~o`a~°`°``~°e`Teee~Ie~e~jee~eo~e geaoO~oeeeeee~meeeee~eeeoe'meI*~e~eeeoee~e~*-ee~*e~ee~ee~ee~eee~-~eee--~-e~e~ ..............
G..A
.........................................
T ........
G ...........
TT ..................
lZ
410 H-87 /3iji-92 Thailand-87 Sfi Lanka-91 Puerto Rico-77 H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
420
H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
..........
H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
~50
T ..............................
460
470
C .............................
.TC ......................................
480
~;90
X
A .............
C ..........
A ........
C.C ....
C.G...T
C, ,T .............. ....
C .................
510 520 530 540 550 560 570 580 590 600 GAGATGTGTGGGAGTAGGAAACAGAGATTTTGTGGAAGGCCTATCGGGAGCCACGTGGGTTGACGTGGTGCTCGAGCACGGTGGGTGTGTGACTACCATG ............................................. A ..... T ...................................... l ..... T... ...... C................................ T..G ........ T........... T ........ T .................... C. . . . . . ............. A ......................... T ..... A ..... T ............................. C.................. ...... C........ G........................ T .......... T ........... A .............. T ........ C............ 620
630
~0
650
660
670
680
690
700
GCTAAGAACAAGCCCACGCTGGACATAGAGCTTCAGAAGACCGAGGCCACCCAACTGGCGACCCTAAGGAAGCTATGCATTGAGGGAAAAATTACCAACA ..................
T ....
T ........
C ......................................
A .....
G
T .....................
~ - - - - - - ~ - - ~ - ~ - T - - - - - - ~ - - ' ' - ~ - - - - - - - - - - ~ - - ° - - - - ' ~ - ~ - - ° - ~ - - ~ - ~ ` ~ - - ~ w ~ - ~ ` ~ - ~ ` ~ ° ~ `-••-•••••-••---`-T•`••-•••--•••---•---•••---•°--••°•-----------------••••--•-•----•--G-------------
710 7'20 730 7/,0 750 7'60 770 780 790 800 TAACAAC•GACT•AAGATGT••CACCCAAGGGGAAGCGATTTTACCTGAGGAGCAGGACCAGAACTACGTGTGTAAGCATACATACGTGGACAGAGGCTG ................
G..C
.......
T ........
G .....
.......
T ..............
...........................................................
.......
T ..............................
T .....
820
C ....................
G .........................................
T ...............
810
H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
440
•CATA•TAG•••TATTT•TT•CCCATTACATA••CACTTCCTT•ACCCA•AAA•T••TTATTTTTATACTATTAAT•CTG•TTACCCCATCCATGACAA• .A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C...... C.......... C.................
610
H-87 Fiji-92 Thailand-87 Sri Lanka-91 Puerto Rico-77
430
830
A ........
C ...........
T .....
G ....
G ............................................
A ...........
G ....
G ............................................
A ...........
840
850
860
870
880
890
T..
900
GGGAAACGGTTGTGGTTTGTTTGGCAAGGGAAGCTTGGTGACATGCGCGAAATTTCAATGTTTAGAATCAATAGAGGGAAAAGTGGTGCAACATGAGAAC ..................................
C ....
........................
A..A
• ..G
...........
•..G
...................................
A..G
....................
G ...................
.................................
C .......................
C .....
A . . G . . T .............. A ..... T ...............
C ......
G .........
C ................................
CC.G...C ....................... C.G . . . . . . . . . . . . . . . . . . . . . . . . . . .
T ........ T ........
910 920 930 940 950 960 970 980 990 1000 CTCAAATACACCGTCATCAT CACAGT••ACACAGGAGACCAACACCAGGTGGGAAATGAAACGCAGGGAGTTACGGCTGAGATAACATCCCAGGCATCAA ...oo..
Dml..
...........
....................
• .e.oo...e T ........
j a.......o......T..................., T ......................................
T ................
........................................................................
.............
m....,
C ........... A ....................
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C ....
T ..........
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Fig. 1. Comparison of the nucleotide sequence of the p r M / M and E genes from five DEN-3 viruses representing the four subtypes. The nucleotide sequence of H-87 displayed is not that reported by Osatomi & Sumiyoshi (1990), but was derived in this laboratory from the virus listed in Table l.
70
R. S. Lanciotti and others
IxM
G 10
20
30
40
50
60
M
~70
80
90
H-87
FHLTSRDGEPRM1VGKNERGKSLLFKTASG~NMCTL~AMDLGEMCDDTVTYKCPHITEVEPED~DCWCNLTST14V.rYGTCNQAGEHRR~KRSVALApHVG
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....
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260
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270
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see
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p.
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Evo/utiotl of dengue-3 viruses G
310 H-87
LKYTVI
TAHITI-B9
ooo ...........
1320
I TVHTGOOIKIVGNET • ......
330
34Q
350
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....
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MALAY-81
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...............
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.....................
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SAHOA" 86
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....
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....
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....
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R. S. Lanciotti and others
72
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Fig. 2. Comparison of the deduced amino acid sequence of the prM/M and E proteins from 23 DEN-3 viruses. Nqinked glycosylation
sites (G), cysteine residues involved in disulphide bridges (bold, underlined C), the proposed conserved flavivirus fusion domain (F and underlined), and membrane anchor domain (M and underlined) are noted in the sequence. Subtype ~
M
A
L
A
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K97(PHILIPPINBS19S6)-PHILIPPII(~$~.9SS PHILIPPIN'B$1964 MALAYSIA197411 MALAYSIA1960 INDOHRSIA19S5 INDONESIA197|| -- I 1974~ INDONRSIA1971 TAHITI1989 FIB 1992 INDONIISIA197S6 MALAYSIA1981| MALAYSIA19516 THAILAND1962 - T H A I L A H 1 D 1973 TNAILA~t975 THAILANDI9|6 -- 11 THAILAND19|7 THAILAND1980 THAILAND1977 - Sll LANKA981 - SRILANHA1982| SRILANHA195~b SRILANRA198JS SRILANKA195SS $RILA~KA19$$ SAMOA1956 INDIA19846 INDIA19., - III I SRILANKA19916 SRILANIA19914 SRILANKA1991e SRILANIA1991| MOZAMmQUR1985t ..... o.,9.b PUERTORICO1977 TAHITI1965 IV TT
~
_
Fig. 3. Cladogram generated by parsimony analysis of nucleic acid
sequences from the prM/M and E genes of 23 DEN-3 viruses and 1 kb portion from an additional 18 DEN-3 viruses. Parsimony analysis was performed by the heuristic branch swapping algorithm of P A U P .
1963 Puerto Rico virus revealed a large number of nonconservative amino acid substitutions which may be related to their high passage history (Table 1). All DEN3 viruses possess the coding sequences for amino acids postulated to play a major role in processing and folding of the E protein (Henchal & Putnak, 1990). The 12 cysteine residues, predicted N-linked glycosylation sites, proteolytic cleavage sites, membrane anchor and translocational signal sequences present in flavivirus prM/M and E proteins were identified in all DEN-3 virus isolates
(Henchal & Putnak, 1990). In addition, the 14 amino acid region from amino acid positions 97 to 111 in the E protein, predicted to form the flavivirus fusion domain, was conserved.
Phylogenetic analysis of DEN-3 isolates Parsimony analysis of the prM/M and E gene sequences of 23 DEN-3 viruses produced a cladogram with four distinct lineages. To confirm these relationships, a portion of the E gene (approx. 1 kb) from an additional 18 virus isolates was sequenced. Parsimony analysis results from the complete prM/M and E sequences of 23 DEN-3 viruses combined with partial sequence data from these additional 18 viruses clearly confirmed the existence of the four DEN-3 subtypes (Fig. 3). Subtype I comprises viruses from Indonesia, Malaysia and the Philippines, and recent isolates from the South Pacific islands; subtype II comprises Thailand viruses: subtype III comprises viruses from Sri Lanka, India, Africa and the 1986 Samoa virus; subtype IV comprises Puerto Rico and the 1965 Tahiti viruses. To analyse the ancestral relationships among the DEN-3 viruses, the complete prM/M and E data from 23 DEN-3 viruses were used to generate a phylogram in which the distances of the horizontal lines are proportional to the number of nucleotide changes between the DEN-3 viruses (Fig. 4). The phylogram indicates that subtype IV viruses are the most distant, with their node emerging directly from the DEN-2 outgroup. Parsimony analysis of the deduced amino acid sequence of the prM and E proteins generated a similar phylogram (data not shown) in which the DEN3 viruses were subdivided into four subtypes.
Evohltion o f dengue-3 viruses
73
Subtype 2,= H 8 7 (PHILIPPINES 1 9 5 6 ) a= P H I L I P P I N E S 1 9 8 3 =~-r M A L A Y S I A 1 9 7 4 I=F :1 laJ~L I N D O N E S I A 1 9 8 8
I I~
~=
INDONES,A
1978
P-~ MALAYSIA 1981 Ii I N D O N E S I A 1 9 7 3 *~lg~ T A H I T I 1 9 9 8 ---~ FIJ11992 THAILAND 1962 :1 • THAILAND 1973 ~ THAILAND 1988 THAILAND 1987 7 SRI L A N K A 1 9 8 1 -9 SRI L A N K A 1 9 8 5 5r1"l_~ S A M O A 1 9 8 6 I~L~ I N D I A 1 9 8 4 z.I I~z S i l l L A N K A 1 9 8 9 ~1 SRI L A N K A 1 9 9 1 / h2 M O Z A M B I Q U E 1 9 8 5 =1 UERTO RICO 1977 TAHITI 1988 el P U E R T O R I C O 1 9 6 3
III
IV DEN-2
Discussion Genetic evolution among the p r M / M and E structural protein genes of DEN-3 viruses has occurred independently within geographical regions where the viruses are endemic. In spite of this evolution, the p r M / M and E proteins have retained an amino acid sequence similarity greater than 95 % over the 36 year period studied. These data suggest that domains responsible for predicted flavivirus protein architecture a n d / o r biological function are strictly conserved. It is likely that the random mutation rate for dengue virus R N A is similar to that of other R N A viruses (Holland et al., 1982); however, the mutations in the DEN-3 virus genome appear to be buffered by selective pressures. Dengue viruses, and other arthropod-borne viruses, seem to have a genetic stringency imposed on them because they replicate in both arthropod and vertebrate hosts (Weaver et al., 1991). Previous studies to analyse the genetic diversity among DEN-3 viruses were based on cross-neutralization assays (Russell & McCown, 1972) and R N A fingerprinting (Trent et al., 1990). Neutralization assays suggested a unique subtype of DEN-3 had circulated in the Caribbean basin in 1963 (Russell & McCown, 1972). Our data confirmed this observation and place the 1963 Puerto Rico and the 1965 Tahiti viruses together in subtype IV (Fig. 3). In addition, the phylogram (Fig. 4) clearly shows that subtype IV viruses are distantly related to all other DEN-3 viruses and suggests that they share a closer genetic relationship with a common ancestor of the dengue viruses. Oligonucleotide fingerprint analysis of DEN-3 viruses identified five genetic topotypes (Trent et al., 1990). The data presented agree with this classification, except that the nucleic acid sequence and parsimony analysis combine viruses from the Philippines
Fig. 4. Phylogramgenerated by parsimony analysis of nucleic acid sequences from prM/M and E genes of 23 DEN-3 viruses. The numbers displayed above the horizontal lines correspond to the number of nucleotide substitutions at phylogenetically informative sites occurring between the terminal virus and the ancestral node.
and Indonesia into one monophyletic lineage whereas fingerprinting distinguished these two groups. The other topotypes defined by oligonucteotide fingerprint analysis (Thailand, Sri Lanka and Caribbean) are similar to those described here. The data presented here increase our understanding of the epidemiology and worldwide movement of DEN-3 viruses. Epidemiological information suggested that the DEN-3 virus responsible for the Mozambique epidemic may have been introduced from India (Gubler et al., 1986). Genetic analysis of virus isolates from these two areas indicate that the Mozambique and Indian DEN-3 viruses belong to the same genetic subtype, along with isolates from Sri Lanka. Epidemics of DEN-3 in Puerto Rico (1963) and Tahiti (1965) caused classical D F with no confirmed cases of D H F (Neff et al., 1967; Laigret et al., 1967). Antigenic analysis indicated that the Tahiti and Puerto Rico DEN-3 viruses were similar, suggesting that the Caribbean strain of DEN-3 had been introduced into Tahiti (Russell & McCown, 1972). Silent DEN-3 transmission apparently continued in Tahiti until 1969, when another small outbreak of D F occurred (Saugrain et al., 1970). The DEN-3 epidemic in Puerto Rico in 1977 also caused only D F (Lopez-Correa et aL, 1978). Genetic data indicate that the 1963 "and 1977 Puerto Rico DEN-3 isolates are closely related, suggesting the virus was also maintained in a silent transmission cycle. It is noteworthy that subtype IV viruses, in addition to being genetically distinct from all other DEN-3 viruses, have never been associated with D H F epidemics. The recent (1989) D H F epidemic in Tahiti and the South Pacific islands (Chungue et al., 1990) was caused by a subtype I virus which appears to have been introduced from Indonesia, Malaysia or the Philippines. The introduction of a subtype previously associated with D H F (Gubler et
74
R. S. Lanciotti and others
al., 1979), concurrent with a change in severity of disease, suggests that subtypes I and IV DEN-3 virus possess distinct biological properties. Epidemiological observations such as those described above have led to the suggestion that epidemic strains of DEN-3 exist (Gubler et aI., 1979, 1981). In Indonesia and Thailand the frequency of DEN-3 isolations from DHF cases increased abruptly in 1976 and 1987 respectively, suggesting that an epidemic strain of DEN-3 had been introduced into these areas (Nisalak, 19891). The phylogenetic data, however, indicate that new viruses were not introduced into Indonesia or Thailand; rather, the endemic viruses had undergone some genetic drift. The disease pattern in Sri Lanka and India is unique in that a long period of endemicity of classical DF with no confirmed DHF cases was followed by DHF epidemics occurring in Sri Lanka (1989) and then in India (1990) (Vitarana, 1990; Chakravarti et al., 1990). DEN-3 viruses isolated during the 1989 to 1991 DHF epidemic in Sri Lanka are classified within subtype III, along with viruses isolated in the early 1980s before the occurrence of DHF. These viruses, however, are grouped on separate distal branches of the cladogram and phylogram (Fig. 3 and Fig. 4). As in Indonesia and Thailand, the data indicate that a new subtype was not introduced into Sri Lanka. However, a measurable and consistent genetic change occurred in the DEN-3 viruses present in Sri Lanka between 1985 and 1989. Whether these changes have altered the virulence of these viruses is not known. DEN-3 viruses have undergone independent evolution which has resulted in four genetic subtypes within the serogroup. The classification of viruses isolated from future dengue outbreaks will be possible using the data presented. The phylogenetic data, together with the epidemiological observations, suggest that genetic differences among (and within) the subtypes may result in differences in virus virulence and/or epidemic potential. Changes in the E protein in particular could alter host cell susceptibility thereby affecting virus replication and disease severity. Additional research on the biology of representative members of the four DEN-3 subtypes should increase understanding of the molecular basis of dengue virus virulence. We thank the following colleagues at the Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colo., U.S.A.: Dr Richard M. Kinney for assisting with the sequence analysis software and Mr Kenneth Robbins for preparing the primers.
References BLOK, J., SAMUEL,S., Gmas, A. J. & VITARANA,U. T. (1989). Variation of the nucleotide and encoded amino acid sequences of the envelope gene from eight dengue-2 viruses. Archives ~?f Virology 105, 39-53.
BLOK, J., GraBS, A. J., McWILLIAM, S. M. & V~TARANA, U. T. (1991). NSt gene sequences from eight dengue-2 viruses and their evolutionary relationships with other dengue-2 viruses. Archives of Virology 118, 209-223. CHAKRAVARTI,S. K., MUKHERJEE,K. K., DE, P. N., BHATTACHARJEE, N. & CHAKRABORTY, M.S. (1990). Dengue haemorrhagic fever (DHF) outbreak in Calcutta. Report fi'om the Department of Virology, School of' Tropical Medicine, Calcutta, India. CHUNGUE, E., SPIEGEL, A., ROUX, J., LAUDON, F. & CARDINES, R. (1990). Dengue 3 in French Polynesia: preliminary data. Virus
hTfbrmation Exchange News'letter 7. DUEBEL, V., KINNEY, R.M. & TRENT, D.W. (1990). Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. Virology 155, 365-377. GUBLER, D. J. (1988). Dengue. In The Arboviruses: Epidemiology and Ecology, vol. 2, pp. 223-260. Edited by T. P. Monath. Boca Raton: CRC Press. GUBLER, D. J. (1991). Dengue haemorrhagic fever: a global update. Virus Information E'cchange Newsletter 8, 2-3. GUBLER, D. J., SUHARYONO,W., SUMARMO,WULUR, H., JAHJA, E. & SAROSO, J, S. (1979). Virological surveillance for dengue haemorrhagic fever in Indonesia using the mosquito inoculation technique. Bulletin o["the Worhl Health Organization 57, 931-936. GUBLER, D. J., SUHARYON0,W., LUBIS, I., ERAM, S. & GUNARSO,S. (1981). Epidemic dengue 3 in central Java, associated with low viremia in man. American Journal of' Tropical Medicine and Itygiene 30, 1094~1099. GUBLER, D.J., SATHER, G.E., KUNO, G. & CABRAL, J. R. (1986). Dengue 3 virus transmission in Africa. American Journal of Tropical Medicine and Hygiene 35, 1280-1284. GYLLENSTEN, U. (1989). Direct sequencing of in vitro amplified DNA. In PCR Technology: Principles and Applications for DNA Amplification, pp. 45-60. Edited by H. A. Erlich. New York: Stockton Press. HALSTEAD, S.B. (1988). Pathogenesis of dengue: challenges to molecular biology. Science 239, 476M81. HENCHAL, E. A. & PUTNAK, J. R. (1990). The dengue viruses. Clinical Microbiology Reviews 3, 37(~396. HOLLAND,J., SPINDLER,K., HORODYSKI,F., GRABAU,E., NICHOL,S. & VANDEPOL, F. (1982). Rapid evolution of RNA genomes. Science 215, 1577-1585. LAIGRET, P.J., ROSEN, L. & SCHOLLHAMMER, G. (1967). Sur une 6pid6mie de dengue survenue a Tahiti en 1964. Relations avec les fievres h6morragiques' de sud-est Asiatique. Bulletin de la Socidtd de Pathologic Exotique 60, 339 353. LANCIOTTI, R.S., CALISHER, C.H., GUBLER, D.J., CHANG, G. & VORNDAM, A.V. (1992). Rapid detection and typing of clinical samples by using reverse transcriptase-polymerase chain reaction, Journal o/" Clinical Microbiology 30, 545 551. LEWIS, J, G., CHANG, G.-J., LANCIOTTI, R. S. & TRENT, D. W. (1992). Direct sequencing of large flavivirus PCR products for analysis of genome variation and molecular epodemiological investigations. Journal of Virological Methods' 38, 11 24. LEWIS,J. G., CHANG, G.-J., LANCIOTTL R. S., K~NNEY, R. K., MAYER, L. W. & TRENT, D. W. (1993). Phylogenetic relationships of dengue 2 viruses: correlation with epidemiology. Virology (in press). LOPEZ-CORREA, R. H., CLINE, B. 1., RAMIREZ-RONDA,C., BERMUDEZ, R., SATrrER, G.E. & KUNO, G. (1978). Dengue fever with hemorrhagic manifestations: a report of three cases from Puerto Rico. American Journal o[' Tropical Medicine and Hygiene 27, 1216 1224. MASON, P.W., MCADA, P.C., MASON, T.W. & FOURN1ER, M.J. (1987). Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. Virology 161, 262267. NEEF, J. M., MORRIS, L., GONZALEZ-ALCOVErt, R., COLEMAN, P. H., LYSS, S. B. & NEGRON, H. (1967). Dengue fever in a Puerto Rican community. American Journal of Epidemiology 86, 162-184. NISALAK, A. (1989). Dengue virus isolations from Bangkok, Thailand, 196~88. Virus btformation Exchange Newsletter 6, 117. OSATOML K. & SUMIYOSHI,H. ( 199% Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology 176, 643-647.
Evolution o f dengue-3 viruses
RICO-HESSE, R. (1990). Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology 174, 47%493. ROSEN, L. (1977). The emperor's new clothes revisited, or reflections on the pathogenesis of dengue hemorrhagic fever. American Journal of Tropical Medicine and ttygiene 26, 337-343. RUSH, B. (1789). An account of the bilious remitting fever as it appeared in Philadelphia in the summer and autumn of the year 1780. Medical Inquiries and Observations 104-117. RUSSELL, P. K. & McCOwN, J. M. (1972). Comparison of dengue-2 and dengue-3 virus strains by neutralization tests and identification of a subtype of dengue-3. American Journal of Tropical Medicine and Hygiene 21, 97-99. SAUGRAIN, P. J., ROSEN, L., OUT1N-FABRE,D. & MOREAU, J.-P. (1970). Une r6cente 4piddmie d'arbovirose du type dengue a Tahiti: comparaisons avec l'6pid4mie de 1964. Bulletbz de la Soci&O de Pathologie Exotique 63, 63~642.
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TRENT, D. W., MANSKE, C. L., FOX, G. E., CHU, M. C., KLIKS, S. C. & MONATH, T.P. (1990). The molecular epidemiology of dengue viruses; genetic variation and microevolution. Applied giralagy Research 2, 293 315. VITARANa, T. (1990). Dengue haemorrhagic fever. Ceylon Medical Journal 35, 83-87. WEAVER, S.C., SCOTT, T.W. & RICO-HESSE, R. (199l). Molecular evolution of eastern equine encephalomyelitis virus in North America. Virology 182, 774-784. ZHAO, B., MACKOW, E., BUCKLER-WHITE,A., MARKOFF, L., CHANOCK, R. M., LA[, C. & MAKINO, Y. (1986). Cloning full length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins. Virology 155, 77 88.
(Received 27 May 1993; Accepted 2 September 1993)
