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Journal of General Virology (1999), 80, 739–748. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Geographic distribution and evolution of Sindbis virus in Australia Leanne M. Sammels, Michael D. Lindsay, Michael Poidinger,† Robert J. Coelen‡ and John S. Mackenzie† Department of Microbiology, The University of Western Australia, Nedlands, Western Australia 6907, Australia The molecular epidemiology and evolution of Sindbis (SIN) virus in Australia was examined. Several SIN virus strains isolated from other countries were also included in the analysis. Two regions of the virus genome were sequenced including a 418 bp region of the E2 gene and a 484 bp region containing part of the junction region and the 5h end of the C gene. Analysis of the nucleotide and deduced amino acid sequence data from 40 SIN virus isolates clearly separated the Paleoarctic/ Ethiopian and Oriental/Australian genetic types of SIN virus. Examination of the Australian strains showed a temporal rather than geographic relationship. This is consistent with the virus having migratory birds as the major vertebrate host, as it allows for movement of virus over vast areas of the continent over a relatively short period of time. The results suggest that the virus is being periodically redistributed over the continent from an enzootic focus of evolving SIN virus. However, SIN virus strains isolated from mosquitoes collected in the south-west of Australia appear to represent a new SIN virus lineage, which is distinct from the Paleoarctic/Ethiopian and Oriental/Australian lineages. Given the widespread geographic dispersal of the Paleoarctic/ Ethiopian and Oriental/Australian lineages, it is surprising that the South-west genetic type is so restricted in its area of circulation. Nucleotide sequence data from the C gene of the prototype strain of the alphavirus Whataroa were also determined. This virus was found to be genetically distinct from the SIN virus isolates included in the present study ; however, it is clearly SIN-like and appears to have evolved from a SIN-like ancestral virus. Introduction Sindbis (SIN) and SIN-like viruses are the most widely distributed of all known arboviruses, with regular isolations occurring in four of the six zoogeographic regions of the world. Human SIN virus infection was considered a minor medical problem from a public health point of view until epidemics in South Africa in 1974 (McIntosh et al., 1976) and Author for correspondence : Leanne Sammels. Fax j61 8 9346 2912. e-mail lsammels!cyllene.uwa.edu.au † Present address : Department of Microbiology, The University of Queensland, Brisbane, Queensland 4072, Australia. ‡ Present address : Department of Biomedical and Tropical Veterinary Sciences, The James Cook University, Townsville, Queensland 4811, Australia. The GenBank accession numbers of the sequences reported in this paper are AF061205–AF061239 and AF061684–AF071715. 0001-5904 # 1999 SGM later in areas of Northern Europe in the mid 1980s (Skogh & Espmark, 1982 ; Lvov et al., 1984), often involving hundreds of cases, were reported. SIN virus is the most commonly isolated arbovirus from mosquitoes in Australia (Mackenzie et al., 1994). Despite this, human disease has only been reported on two occasions in Australia (Doherty et al., 1969 ; Guard et al., 1982), although seroepidemiological studies suggest that frequent subclinical infections do occur (Boughton et al., 1984 ; Doherty, 1973 ; Kanamitsu et al., 1979 ; Liehne et al., 1976). Strain variation or the presence of clinically similar diseases or antigenically related alphaviruses may explain the low incidence of clinical infections in humans in Australia. In a study of genetic identity among 15 strains of SIN virus using RNA–RNA hybridizations, Rentier-Delrue & Young (1980) found that two distinct geographic types of SIN virus could be distinguished. These authors suggested that divergent evolution had occurred between European\African and Indian\Far Eastern\Australian types. A subsequent study of antigenic variation among SIN virus isolates from Paleoarctic, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 HDJ L. M. Sammels and others Ethiopian, Oriental and Australian areas demonstrated antigenic diversion between viruses from the Paleoarctic\ Ethiopian and Oriental\Australian regions (Olson & Trent, 1985). Genetic studies, by RNase T1 mapping, indicated that the primary structures of RNA from viruses isolated in the four zoogeographic regions were distinct from one another (Olson & Trent, 1985). An RNase T1 mapping study of a limited number of Australian SIN virus isolates indicated that the virus may exist throughout Australia as a single topotype (Brand, 1989). The aim of this study was to examine the geographic distribution and evolution of SIN virus in Australia by determining the nucleotide sequence of regions of the E2 and C genes of a number of SIN virus strains isolated in various geographic locations in Australia. Molecular epidemiological studies of another Australian alphavirus, Ross River (RR) virus, have shown that distinct genetic types of the virus are associated with geographic origin (Faragher et al., 1985 ; Lindsay et al., 1993 ; Sammels et al., 1995). Thus, this study was undertaken to better understand the genetic variation in alphaviruses and to identify potential mechanisms of spread of these viruses in Australia. A limited number of SIN virus strains isolated in other countries, including Egypt, South Africa, Malaysia and Papua New Guinea (PNG), was also included in the study to examine the genetic variation of SIN virus in different zoogeographic regions of the world. An isolate of Whataroa (WHA) virus, an alphavirus belonging to the Western equine encephalitis antigenic complex, which is enzootic in parts of New Zealand (Miles, 1973), was also included. Neutralization tests have indicated that WHA virus is antigenically related to SIN and SIN-like viruses (Calisher et al., 1988 ; Karabatsos, 1975). Recently, nucleotide sequence analysis has shown that WHA virus is SIN-like, although its sequence diverges by about 20 % from SIN virus (Weaver et al., 1997). nucleotide variability. Therefore, a second, more highly conserved region of the SIN virus genome was targeted for sequencing (Strauss et al., 1984). Sequence data of the 3h end of the highly conserved junction region and the 5h end of the C gene were obtained for all the selected SIN virus isolates. RT–PCR of cDNA. Details of the methods used to amplify a 482 bp cDNA fragment of the SIN virus E2 gene and a 510 bp cDNA fragment containing part of the junction region and the 5h end of the C gene have been described previously (Sellner et al., 1992). Briefly, a 0n5 µl aliquot of tissue culture supernatant was digested with 5 µg proteinase K in a total volume of 12n5 µl for 30 min at 42 mC. Proteinase K was inactivated by heating to 94 mC for 5 min. The proteinase K reaction mix was then combined with the reagents for a one-step RT–PCR reaction in a total volume of 25 µl. Each reaction contained 20 pmoles each primer (SIN9170* and SIN9652 for the E2 gene or SIN7575* and SIN8086 for the C gene), 2 U Taq DNA polymerase, 0n5 U AMV reverse transcriptase, 100 µM dNTPs, 2 mM MgCl , 2 µg yeast tRNA and 5 µl 5i reaction # buffer resulting in final concentrations of 67 mM Tris–HCl (pH 8n8), 50 mM KCl, 6 µM EDTA and 0n1 % Triton X-100, 0n5 mM DTT. The tubes were overlaid with 60 µl paraffin oil and incubated in a thermal cycler for 30 min at 42 mC followed by 94 mC for 5 min (to activate and then inactivate the AMV reverse transcriptase) and then 35 cycles of 94 mC for 30 s, 60 mC for 30 s and 72 mC for 60 s, with a final extension at 72 mC for 7 min. Nucleic acid sequencing and analysis. Nucleic acid sequence analysis was performed on an automated Applied Biosystems 373A DNA sequencer. Both strands of the amplified cDNA fragments were sequenced using primers that were identical to those used in the RT–PCR reaction. The purified dsDNA was sequenced on an automated sequencer using the Prism DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and the cycle sequencing parameters used were as described in the manufacturer’s protocol. Multiple nucleotide and amino acid sequence alignments were performed with the CLUSTAL V program (Higgins & Sharp, 1989). Aligned sequences underwent phylogenetic analysis using programs from the PHYLIP package (Felsenstein, 1989). Briefly, distances were calculated using the Kimura two-parameter method and trees were calculated using the neighbour-joining method. Bootstrap resampling of 1000 replicates was used to place confidence values on groupings within trees. Methods Virus stocks. All SIN virus strains used in this study are listed in Table 1. Virus stocks were prepared on Vero cell (Monkey kidney ; ATCC CCL 81) monolayers at 37 mC at an m.o.i. of 0n1 p.f.u. per cell. At 48 h post-infection, cell culture supernatants were harvested, clarified by centrifugation at 4000 g for 30 min and stored at k70 mC. Design and synthesis of oligonucleotide primers. All oligonucleotide primers used in this study were synthesized on an automated DNA synthesizer (380B ; Applied Biosystems) to hybridize to specific locations of the SIN virus genome, according to the reported sequence (Strauss et al., 1984). The primers are identified according to the nucleotide site at which the 3h end of the primer was designed to anneal. All primers have complementary-sense orientation except those marked with an asterisk which have plus-sense orientation. Initially, the E2 gene was chosen for sequencing because the structural genes are known to be more variable than the non-structural genes in alphaviruses and also because the E2 gene carries the major antigenic determinants. However, numerous attempts to produce cDNA of the region for several SIN virus isolates failed, suggesting considerable HEA Results The 41 SIN virus strains and the WHA isolate (M78) included in the analysis, with their location and host of origin, are listed in Table 1. The SIN virus strains were isolated from four different zoogeographic regions of the world over a 40 year period. Thirty-five of these strains were isolated from mosquitoes trapped in various geographic locations in Australia over a 33 year period. The published sequence of the prototype strain of Ockelbo virus, Edsbyn82 (Shirako et al., 1991), was obtained from GenBank and included in the analysis. Nucleotide sequencing studies in the E2 gene The nucleotide sequences of a 418 bp region of the E2 gene were determined for 32 SIN virus isolates. Five strains of virus, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 Table 1. Details of Sindbis and SIN-like virus isolates Abbreviations used : PNG, Papua New Guinea ; QLD, Queensland, Australia ; NSW, New South Wales, Australia ; Vic, Victoria, Australia ; WA, Western Australia, Australia ; NT, Northern Territory, Australia. Isolate name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 EgAR339 EgAR338 SAAR86 M78‡ MRE16 MK6962 M78 MRM39 16700 RRD764 RRD800 AS19016 BB19153 NL26287 RB27266 BH2907 MM840 BH40503 OR6 OR339 OR374 OR405 OR437 OR499 OR922 WK465 K2673 CX184 K2588 K1961 K2924 K3141 K3028 K3057 K10390 K12710 SW6562 SW20055 SW25713 SW33029 V620 V634 Location Cairo, Egypt Cairo, Egypt South Africa New Zealand Sarawak, Malaysia Joinjakaka, northern PNG New Zealand Kowanyama, Cape York Peninsula, far north QLD Charleville, south-central QLD Ross River Dam, north coast QLD Ross River Dam, north coast QLD Lachlan River, south-central NSW Griffith, south-central NSW Narran Lake, north-central NSW Redbank Weir, south-west NSW Barmah Forest, north-west Vic Lake Moodemere, Vic Barmah Forest, north-west Vic Ord River, NE Kimberley, WA Ord River, NE Kimberley, WA Ord River, NE Kimberley, WA Ord River, NE Kimberley, WA Ord River, NE Kimberley, WA Ord River, NE Kimberley, WA Ord River, NE Kimberley, WA Camballin, W Kimberley, WA Lake Argyle, NE Kimberley, WA Ord River, NE Kimberley, WA Billiluna, SE Kimberley, WA Billiluna, SE Kimberley, WA Parrys Ck, NE Kimberley, WA Parrys Ck, NE Kimberley, WA Nullagine, Pilbara, WA Nullagine, Pilbara, WA Derby, SW Kimberley, WA Billiluna, SE Kimberley, WA Southern suburbs, Perth, south-west WA Peel region, south-west WA Leschenault region, south-west WA Peel region, south-west WA Darwin, north-central coast NT Maratanka, north-central NT Date collected Source (vector or host) 1953 1953 1954 1962 1975 Feb. 1966 1962 Apr. 1960 1974 Feb. 1991 Feb. 1991 Feb. 1979 Feb. 1979 Apr. 1981 Nov. 1981 Feb. 1974 Feb. 1974 Jan. 1984 6\6\72 30\3\74 2\4\74 6\4\74 Mar.\Apr. 1974 Mar.\Apr. 1974 June\July 1976 3\8\79 22\4\82 13\6\82 21\4\89 24\4\89 25\4\89 25\4\89 17\2\90 19\2\90 6\4\93 20\4\93 6\2\90 20\1\92 13\10\92 5\10\93 6\3\84 15\3\84 Culex univittatus Unavailable Culex species Culex pervigilans Unavailable Ficalbia flavens Culex pervigilans Culex annulirostris Unavailable Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Anopheles amictus Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Aedes normanensis Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Aedes eidsvoldensis Culex annulirostris Culex annulirostris Aedes pseudonormanensis Aedes pseudonormanensis Culex annulirostris Culex annulirostris Culex annulirostris Culex annulirostris Aedes camptorhynchus Aedes camptorhynchus Culex annulirostris Aedes normanensis Passage history* Supplier† 14ismb, 1iVero 12ismb, 1iVero 4ismb, 1iVero Unavailable 6imosq, 1ismb, 1iVero 3ismb, 1iVero 3ismb, 1iBHK 1ismb, 1iVero 3ismb, 1iVero 1iC6\36, 1iBHK 1iC6\36, 1iBHK 1ismb, 1iVero 1ismb, 1iVero 1ismb, 1iVero 1ismb, 1iVero 1ismb, 1iVero 1ismb, 1iVero 1ismb, 1iVero 2ismb, 1iVero 2ismb, 1iVero 2ismb, 1iVero 2ismb, 1iVero 2ismb, 1iVero 2ismb, 1iVero 2ismb, 1iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 2iC6\36, 2iVero 1iC6\36, 3iVero 1iC6\36, 3iVero 1iC6\36, 3iVero 1iC6\36, 3iBHK 1iC6\36, 3iBHK 1iC6\36, 3iBHK IM IM IM DP IM IM DP DP DP BK BK IM IM IM IM IM IM IM UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA UWA RW RW HEB * Passage history, history of each isolate upon arrival in this laboratory. C6\36, C6\36 clone of Aedes albopictus cells ; smb, suckling mouse brain ; Vero, Vero (African green monkey kidney) cells ; and BHK, baby hamster kidney cells. † UWA, Arbovirus Laboratory, Department of Microbiology, University of Western Australia, Nedlands, Australia ; RW, Richard Weir, Berrimah Agricultural Centre, NT, Australia ; IM, Ian Marshall, Department of Biochemistry, Australian National University, Canberra, ACT, Australia ; DP, Debbie Phillips, Laboratory of Microbiology and Pathology, State Health Laboratory, Department of Health, QLD, Australia ; and BK, Brian Kay, Queensland Institute of Medical Research, QLD, Australia. ‡ M78 is a strain of WHA virus. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 Evolution of Sindbis virus No. L. M. Sammels and others Fig. 1. Sequences of the 137 amino acid residues deduced from the nucleotide sequences of a 418 nucleotide region of the E2 gene of 33 SIN virus isolates described in Table 1. The dots indicate identity with the prototype strain EgAR339, while letters indicate amino acid substitutions. The N-glycosylation sites are underlined. including the WHA isolate (M78) and the four strains of SIN virus isolated in the south-west region of Western Australia (WA), failed to produce cDNA in an RT–PCR reaction. Several attempts to produce cDNA from these strains using different reaction conditions were performed without success. The sequences obtained for the prototype strain of SIN virus, EgAR339, and the South African strain, SAAR86, were identical to the published E2 gene sequences of these two isolates (Davis et al., 1986 ; Russell et al., 1989). The nucleotide sequences of each strain were aligned and compared with the SIN prototype EgAR339 sequence. When all of the strains were included in pairwise comparisons, the nucleotide sequence variability of the 418 bp segment of E2 was high, with a maximum divergence of 26n1 %. The majority of the nucleotide substitutions occurred at the third position of the codon and 72 % were silent. The maximum nucleotide divergence was much lower (5n1 %) when only Australian strains were included. Interestingly, in pairwise comparisons of the Malaysian (MRE16) and PNG (MK6962) strains with the Australian strains, the maximum nucleotide divergence was only 3n9 and 4n1 %, respectively, suggesting a close genetic relationship between SIN virus isolates from these different geographic areas. Malaysia lies within the Oriental zoogeographic region while PNG is part of the Australian zoogeographic region (Wallace, 1962) ; therefore this group of isolates was designated the Oriental\Australian genotype. The isolates from Egypt, South Africa and Sweden also shared high nucleotide conservation in pairwise comparisons with a maximum nucleotide divergence of 6n5 % ; the group was designated the Paleoarctic\Ethiopian genotype. Within the Australian SIN strains, there were several nucleotide fixations associated with time of isolation. No HEC nucleotide fixations observed in the Australian SIN virus isolates correlated with geographic location, host or passage history. The divergence in nucleic acid sequence observed between the Paleoarctic\Ethiopian and Oriental\Australian strains was also reflected in the encoded amino acid sequence alignments (Fig. 1). There were 37 different amino acid substitutions, of which 17 were conservative and 20 were non-conservative changes. The amino acid sequence divergence between the two groups of isolates was 13–16 %. The amino acid sequences within the Paleoarctic\Ethiopian and Oriental\Australian genotypes were highly conserved with maximum variations of 4n4 and 3n6 %, respectively. The phylogenetic tree constructed from the E2 nucleotide sequence data (Fig. 2) defined two clusters of SIN virus isolates corresponding to the Paleoarctic\Ethiopian and Oriental\ Australian genotypes identified from the nucleotide and deduced amino acid alignments. Within the Paleoarctic\ Ethiopian genotype, the South African and Ockelbo strains were more closely related to each other than they were to the two Egyptian isolates. All of the isolates within the Oriental\ Australian genotype, including the Malaysian and PNG isolates, were closely related, sharing greater than 95 % nucleotide sequence. Due to the high nucleotide sequence divergence between the Paleoarctic\Ethiopian strains and the Oriental\Australian strains, the branch lengths within the Oriental\Australian cluster of isolates were short, making it difficult to examine the relationships between the strains within this genotype. Therefore, the analysis was repeated without the data from the Paleoarctic\Ethiopian strains in order to produce a tree which would demonstrate the phylogeny of the Oriental\Australian Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 Evolution of Sindbis virus Fig. 2. Phylogenetic tree showing the genetic relationships based on partial sequence of the E2 gene of the SIN virus isolates included in this study. Numbers above branches show bootstrap values. Vertical distances are arbitrary ; horizontal branch lengths are proportional to mutation distance as indicated (bar, 0n1 substitutions per nucleotide). strains more clearly (Fig. 3). In this phylogenetic tree, the original 1960 Australian SIN virus isolate was located on a separate branch (designated group A) and the remaining isolates could be divided into three clusters, designated groups B, C and D. The clustering of the isolates did not correlate with geographic origin, mosquito species or passage history. However, there was a very strong temporal relationship between the strains included in each group. Group B contained eight strains, including the PNG strain isolated in 1966 and all of the strains isolated in Australia in the 1970s (except strain OR922). The PNG isolate was somewhat distinct from the other isolates although the maximum nucleotide divergence between the strains in group B was only 1n7 %. The 16 isolates within group C were also highly conserved with a maximum divergence of 1n2 %. The group included strains isolated in the Pilbara and Kimberley regions of WA, north- and south-central New South Wales (NSW), north-west Victoria (Vic), central and north-coastal Northern Territory (NT). All of the strains were isolated between 1976 and 1990. Group D contained four Australian SIN virus strains, which shared at least 99n5 % nucleotide sequence identity, and the somewhat distinct Malaysian strain. Each of these groups contained isolates Fig. 3. Phylogenetic tree showing the genetic relationships based on partial sequence of the E2 gene of the SIN virus isolates from the Oriental/Australian genotype. The location (MAL, Malaysia ; see legend to Table 1 for other abbreviations) and year of isolation of each strain are listed. Numbers above branches show bootstrap values and genetic groupings (A–D) are indicated on the right hand side. Vertical distances are arbitrary ; horizontal branch lengths are proportional to mutation distance as indicated (bar, 0n01 substitutions per nucleotide). which shared identical nucleotide sequences in the E2 gene fragment despite being isolated in widely separated geographic regions of Australia. Nucleotide sequence studies of the C gene The sequences obtained for the majority of the strains included 48 nucleotides of the junction region, which are untranslated, and 436 nucleotides of the 3h end of the capsid gene which encode the first 145 amino acids of the capsid protein. The RT–PCR reactions on the SIN virus strains isolated in the south-west region of WA consistently produced a cDNA product approximately 200 bp shorter than that produced from all other isolates. Several attempts to produce cDNA fragments of the correct size from these strains using different reaction conditions were performed without success. These shorter products were subsequently sequenced and the data included 48 untranslated nucleotides of the junction region and 232 nucleotides of the C gene, encoding 77 amino acid residues. Pairwise comparisons of the nucleotide sequences revealed a high degree of genetic divergence with a maximum of 38 % Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 HED L. M. Sammels and others Fig. 4. Sequences of the 145 amino acid residues deduced from the nucleotide sequences of a 436 nucleotide region of the C gene from 37 SIN virus isolates and the WHA virus isolate (M78) described in Table 1. The dots indicate identity with the prototype strain EgAR339, while letters indicate amino acid substitutions. between the Australian 1960 strain MRM39 and the WHA isolate (M78). The sequences in the untranslated junction region were highly variable, although the three in-phase termination codons were maintained in all strains except the four south-west WA isolates, which have only two in-phase stop codons. Pairwise comparisons clearly distinguished the Paleoarctic\Ethiopian and Oriental\Australian isolates. In addition, a third genotype of strains comprising the four isolates from the south-west of WA (designated the Southwest genotype) could be distinguished in the pairwise comparisons. The nucleotide sequence of the WHA (M78) virus isolate was substantially different to the sequence of all the other strains. The level of nucleotide divergence between the Paleoarctic\Ethiopian and the Oriental\Australian isolates was 24–26n5 %. The ranges of divergence between the nucleotide sequences of strains in the South-west genotype and isolates of the Paleoarctic\Ethiopian and Oriental\ Australian genotypes were 16n8–19n4 and 25n4–28n9 %, respectively, suggesting that the South-west genotype is more closely related to the Paleoarctic\Ethiopian isolates than to the Oriental\Australian isolates. The WHA virus isolate (M78) appeared to be equally divergent from the Paleoarctic\ Ethiopian, Oriental\Australian and South-west isolates with sequence divergence values of 33–34, 33n5–34n9 and 35– 35n4 %, respectively. The level of nucleotide divergence observed between isolates within each of the three main genotypes was very low indicating high nucleotide conservation in this region of the capsid gene. The maximum divergence between the four HEE strains included in the Paleoarctic\Ethiopian genotype was 4 %. The Oriental\Australian isolates showed a maximum divergence of 5n7 %. The South-west isolates, which were all obtained from mosquitoes trapped in the south-west region of WA between 1990 and 1993, shared greater than 99 % nucleotide sequence identity. The deduced amino acid sequence alignments (Fig. 4) included 143 residues of the capsid protein of all the SIN virus strains except those belonging to the South-west genotype, from which only 77 amino acids were identified. There was a total of 92 amino acid substitutions, of which 28 were conservative and 64 were non-conservative changes. The alignment indicated a highly variable region between residues 57 and 96 with numerous substitutions, although there were substitutions scattered throughout the entire length of the capsid fragment. The amino acid divergence values between M78 and the Paleoarctic\Ethiopian, Oriental\Australian and South-west genotypes were 31n2–32n6, 28–29n4 and 36 %, respectively. The maximum amino acid variation between the isolates of the Oriental\Australian genotype was 6n9 %. The variations between the Oriental\Australian and Paleoarctic\ Ethiopian genotypes and between the Oriental\Australian and South-west genotypes were 16–21n7 and 20n8–26 %, respectively. The amino acid variation between the Paleoarctic\ Ethiopian and South-west genotypes was somewhat lower at 9–11n7 %. The phylogenetic tree constructed from the nucleotide sequence data (Fig. 5) defined three clusters of SIN virus isolates corresponding to the Paleoarctic\Ethiopian, Oriental\ Australian and South-west genotypes. The WHA virus isolate Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 Evolution of Sindbis virus Fig. 5. Phylogenetic tree showing the genetic relationships based on partial sequence of the C gene of the SIN virus isolates and the WHA virus prototype strain included in this study. Numbers above branches show bootstrap values. Vertical distances are arbitrary ; horizontal branch lengths are proportional to mutation distance (bar, 0n1 substitutions per nucleotide). (M78), with its unique nucleotide and amino acid sequences, was located on a separate branch of the tree. The tree also indicated that isolates belonging to the South-west genotype were more closely related to strains from the Paleoarctic\ Ethiopian genotype than they were to those of the Oriental\ Australian genotype. All of the remaining Australian SIN virus isolates were grouped within the Oriental\Australian genotype. The analysis was repeated without data from the Paleoarctic\Ethiopian and South-west genotypes to produce a tree which would demonstrate the phylogeny of the Oriental\ Australian strains more clearly (Fig. 6). The analysis placed the 1960 Australian SIN virus isolate MRM39 on a separate branch of the tree (designated group A) and the remaining isolates were divided into three clusters, groups B, C and D. Group B contained the 1966 PNG isolate and nine strains isolated from various geographic locations in Australia during the 1970s. The nucleotide sequences of the Australian isolates within this group were highly conserved with a maximum nucleotide divergence of less than 0n5 %. The PNG strain was approximately 1n5 % divergent from the Australian isolates. Group C contained 15 isolates, most of which were isolated in the late 1970s and the 1980s from widely separated geographic Fig. 6. Phylogenetic tree showing the genetic relationships based on partial sequence of the C gene of the SIN virus isolates from the Oriental/Australian genotype. The location (MAL, Malaysia ; see legend to Table 1 for other abbreviations) and year of isolation of each strain are listed. Numbers above branches show bootstrap values and genetic groupings (A–D) are indicated on the right hand side. Vertical distances are arbitrary ; horizontal branch lengths are proportional to mutation distance (bar, 0n01 substitutions per nucleotide). locations within Australia. The Malaysian strain (MRE16) was most closely related to these isolates although it was located on a separate branch of the tree. The maximum divergence between the isolates in group C was 1n9 %. The four strains included in group D, two from northern Queensland (QLD) in 1991 (RRD764 and RRD800) and two isolated in Kimberley (WA) in 1993, shared 99 % nucleotide sequence identity. The phylogeny inferred from the C gene sequences (Fig. 6) and E2 nucleotide sequences (Fig. 3) of the Oriental\Australian SIN virus isolates was very similar. Discussion Our results show that at least two distinct genotypes of SIN virus are circulating in Australia. One of these, the Oriental\Australian genotype, circulates throughout most of Australia and in PNG and Sarawak, Malaysia, whereas a second, previously unrecognized genotype appears to be endemic in a distinct geographic region located in the temperate south-west of Australia. The results confirm earlier reports that significant genetic diversity exists between isolates of SIN virus from the Paleoarctic\Ethiopian and Oriental\Australian zoogeographic regions (Norder et al., 1996 ; Olson & Trent, 1985 ; Rentier- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 HEF L. M. Sammels and others Delrue & Young, 1980). The isolates contained within each of the two genotypes were closely related genetically. One genotype included most of the Australian isolates and the single strains from PNG and Malaysia. These were isolated over a 33 year period and were obtained from widely distributed geographic locations including many areas of the Australian continent. Although all of the isolates within this genotype were genetically similar, four genetic subtypes (groups A–D) were identified within the panel of isolates. There is an obvious temporal relationship between isolates in the different groups which suggests that SIN virus strains circulating across most of the Australian continent have been replaced at various points through time. The first strain of SIN virus isolated in Australia, MRM39, was obtained from mosquitoes trapped in far northern Queensland in 1960. The genotype of MRM39 was not observed in any other SIN virus isolates examined, although no Australian strains isolated between 1960 and 1973 were available for inclusion in this study. The results indicated that the group B genetic type predominated across most of Australia during the 1970s. The close genetic relationship observed between these strains and the 1966 PNG isolate suggests that this genetic type could have been introduced into Australia from PNG. Virus strains isolated in the late 1970s to 1990, from widely separated geographic locations in Australia, were identified as members of group C. Similarly, the most recent isolates included in the study, collected in 1991 and 1993, were separated into group D. The strain isolated in Malaysia in 1975, MRE16, was closely related to the Australian strains in groups C and D. These results suggest that the genetic type represented by group C may have been introduced into Australia from the Oriental region in the 1970s, when group B was still the predominant genetic type, and subsequently replaced group B across the Australian continent. The emergence of group D as the dominant genetic type may have occurred as a result of genetic drift leading to a significant growth advantage or by the reintroduction of a new strain from the Oriental region. The temporal clustering of the isolates in the different genetic types, represented by groups A–D, indicates that the virus may be maintained in an enzootic focus from where it is rapidly spread to other regions of Australia. This study could not determine whether the enzootic focus is located within Australia or another geographic location, such as PNG or Malaysia. However, it appears that the virus is disseminated over vast geographic areas of Australia with isolates from widely separated locations sharing identical nucleotide sequences. Virus isolations and antibody studies have established that birds are the major vertebrate host in Australia (Doherty et al., 1966). Many species of birds migrate annually within the Australian continent between the north-west and south-east regions and north-east and south-east regions of Australia (Williams & Williams, 1990). Thus, it seems likely that such species may play an important role in widely disseminating SIN virus in Australia. HEG The similarity of the Malaysian SIN virus isolate to the Australian isolates in this study is of interest, as Malaysia is in the Oriental zoogeographic region. Rentier-Delrue & Young (1980) and Olson & Trent (1985), using a different Malaysian isolate of SIN virus, have also demonstrated that the Malaysian isolate was more closely related to Australian isolates than to other isolates from the Oriental zoogeographic region. The Oriental and Australasian zoogeographic regions are separated by the Wallace Line in the Indo–Australian archipelago, and seroepidemiological studies have indicated that the alphavirus Chikungunya virus and the flavivirus Japanese encephalitis (JE) virus may be restricted to the Oriental zoogeographic region, whereas the alphavirus RR virus and flavivirus Murray Valley encephalitis (MVE) virus are restricted to the Australian zoogeographic region (Mackenzie et al., 1994). Thus, the finding that SIN virus isolates from Malaysia and Australia are genetically similar may indicate that the apparent restricted distribution of these arboviruses (Chikungunya, RR, JE and MVE viruses) may be less rigid than previously believed. Indeed, KUN virus, a flavivirus which is widely distributed in Australia, has also been isolated from mosquitoes collected in Malaysia (Bowen et al., 1970) and JE virus has recently been isolated in the Torres Strait of north-eastern Australia (Hanna et al., 1995). Many species of birds migrate annually between south-east Asia and northern Australia (Williams & Williams, 1990) providing an opportunity for virus spread between these geographic regions by movement of viraemic birds. The identification of a new distinct genetic type of SIN virus circulating in the south-west region of WA was an unexpected result. At present, we have determined the nucleotide sequence of 48 bases of the junction region and 232 bases of the C gene from these strains. Nevertheless, enough data were obtained to indicate that these isolates represented an independent enzootic focus in south-west Australia. Analysis of the nucleotide sequences indicated that strains from the South-west genotype are more closely related to Paleoarctic\Ethiopian strains of SIN virus isolates than to those from the Oriental\Australian region. This finding suggests that SIN virus circulating in the south-west region of Australia may have been introduced, perhaps by a viraemic migratory bird or travelling human, and evolved under the selective pressures of the ecological conditions in this region. Many of the endemic species of birds in the south-west of WA are territorial and do not move over very large distances (Slater, 1974 a, b). It is possible that SIN virus circulating in the south-west of Australia has adapted to and is being maintained in the region by these more sedentary bird species. Alternatively, the virus may have adapted to a new transmission cycle utilizing vertebrate hosts with limited mobility, such as small mammals including marsupials. A survey of avian and mammalian species common to the south-west region for antibody to SIN virus may help to identify potential major hosts of the virus in this region. Surveillance of arbovirus activity in the south-west region of WA has been undertaken Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 07 May 2017 01:06:31 Evolution of Sindbis virus since May 1987 and has involved regular collections of mosquitoes at various locations. In that time, there have been over 200 isolations of RR virus but only twelve isolations of SIN virus. All SIN virus isolates were obtained from mosquitoes trapped between February 1990 and December 1996 (M. D. Lindsay & C. A. Johansen, unpublished results). The focus of this program is the surveillance of saltmarsh-breeding mosquito species located in coastal environments where large outbreaks of RR virus disease occur. Consequently, saltmarsh mosquitoes in the genus Aedes comprise over 80 % of the adult mosquito population in the region in which surveillance is carried out. The low number of isolates of SIN virus compared to RR virus may indicate that these species are not major vectors of SIN virus in the region. More extensive genetic and antigenic characterization of these unique SIN strains and other SIN strains isolated from mosquitoes collected in the southwest region of WA is currently in progress. The genetic information obtained from the WHA virus prototype strain (M78) confirms previous antigenic and genetic studies which indicated that the virus was distantly related to SIN and SIN-like viruses (Calisher et al., 1988 ; Weaver et al., 1997). The results of phylogenetic analysis suggest that WHA may have evolved in isolation from an ancestral SIN-like virus which may have originally been introduced into New Zealand by the arrival of viraemic birds. We thank those researchers who provided many of the virus isolates used in the study (listed in Table 1). 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