Displaying publications 1 - 20 of 36 in total

  1. Escaffre O, Borisevich V, Vergara LA, Wen JW, Long D, Rockx B
    J. Gen. Virol., 2016 05;97(5):1077-86.
    PMID: 26932515 DOI: 10.1099/jgv.0.000441
    Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.
  2. Wang HJ, Liu L, Li XF, Ye Q, Deng YQ, Qin ED, et al.
    J. Gen. Virol., 2016 07;97(7):1551-6.
    PMID: 27100268 DOI: 10.1099/jgv.0.000486
    Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.
  3. Wei Chiam C, Fun Chan Y, Chai Ong K, Thong Wong K, Sam IC
    J. Gen. Virol., 2015 Nov;96(11):3243-3254.
    PMID: 26276497 DOI: 10.1099/jgv.0.000263
    Chikungunya virus (CHIKV), an alphavirus of the family Togaviridae, causes fever, polyarthritis and rash. There are three genotypes: West African, Asian and East/Central/South African (ECSA). The latter two genotypes have caused global outbreaks in recent years. Recent ECSA CHIKV outbreaks have been associated with severe neurological disease, but it is not known if different CHIKV genotypes are associated with different neurovirulence. In this study, the neurovirulence of Asian (MY/06/37348) and ECSA (MY/08/065) strains of CHIKV isolated in Malaysia were compared. Intracerebral inoculation of either virus into suckling mice was followed by virus titration, histopathology and gene expression analysis of the harvested brains. Both strains of CHIKV replicated similarly, yet mice infected with MY/06/37348 showed higher mortality. Histopathology findings showed that both CHIKV strains spread within the brain (where CHIKV antigen was localized to astrocytes and neurons) and beyond to skeletal muscle. In MY/06/37348-infected mice, apoptosis, which is associated with neurovirulence in alphaviruses, was observed earlier in brains. Comparison of gene expression showed that a pro-apoptotic gene (eIF2αK2) was upregulated at higher levels in MY/06/37348-infected mice, while genes involved in anti-apoptosis (BIRC3), antiviral responses and central nervous system protection (including CD40, IL-10RA, MyD88 and PYCARD) were upregulated more highly in MY/08/065-infected mice. In conclusion, the higher mortality observed following MY/06/37348 infection in mice is due not to higher viral replication in the brain, but to differentially expressed genes involved in host immune responses. These findings may help to identify therapeutic strategies and biomarkers for neurological CHIKV infections.
  4. Mok L, Wynne JW, Grimley S, Shiell B, Green D, Monaghan P, et al.
    J. Gen. Virol., 2015 Jul;96(Pt 7):1787-94.
    PMID: 25748429 DOI: 10.1099/vir.0.000112
    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.
  5. AbuBakar S, Wong PF, Chan YF
    J. Gen. Virol., 2002 Oct;83(Pt 10):2437-42.
    PMID: 12237425
    Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
  6. Chan YP, Koh CL, Lam SK, Wang LF
    J. Gen. Virol., 2004 Jun;85(Pt 6):1675-84.
    PMID: 15166452
    Hendra virus (HeV) and Nipah virus (NiV) are members of a new genus, Henipavirus, in the family paramyxoviridae. Each virus encodes a phosphoprotein (P) that is significantly larger than its counterparts in other known paramyxoviruses. The interaction of this unusually large P with its nucleocapsid protein (N) was investigated in this study by using recombinant full-length and truncated proteins expressed in bacteria and a modified protein-blotting protein-overlay assay. Results from our group demonstrated that the N and P of both viruses were able to form not only homologous, but also heterologous, N-P complexes, i.e. HeV N was able to interact with NiV P and vice versa. Deletion analysis of the N and P revealed that there were at least two independent N-binding sites on P and they resided at the N and C termini, respectively. Similarly, more than one P-binding site was present on N and one of these was mapped to a 29 amino acid (aa) C-terminal region, which on its own was sufficient to interact with the extreme C-terminal 165 aa region of P.
  7. Shafee N, AbuBakar S
    J. Gen. Virol., 2003 Aug;84(Pt 8):2191-5.
    PMID: 12867651
    Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17.5 and 51.5 % of cells expressing NS3 serine protease and NS2B-NS3(185) serine protease precursor protein [NS2B-NS3(185)(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3(185)(pro). NS2B-NS3(185)(pro) was detected as NS2B-NS3(185)(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His(51) with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3(185)(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3(185)(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.
  8. Kho CL, Tan WS, Tey BT, Yusoff K
    J. Gen. Virol., 2003 Aug;84(Pt 8):2163-8.
    PMID: 12867648
    The nucleocapsid protein (NP) of Newcastle disease virus expressed in E. coli assembled as ring- and herringbone-like particles. In order to identify the contiguous NP sequence essential for assembly of these particles, 11 N- or C-terminally deleted NP mutants were constructed and their ability to self-assemble was tested. The results indicate that a large part of the NP N-terminal end, encompassing amino acids 1 to 375, is required for proper folding to form a herringbone-like structure. In contrast, the C-terminal end covering amino acids 376 to 489 was dispensable for the formation of herringbone-like particles. A region located between amino acids 375 to 439 may play a role in regulating the length of the herringbone-like particles. Mutants with amino acid deletions further from the C-terminal end (84, 98, 109 and 114 amino acids) tended to form longer particles compared to mutants with shorter deletions (25 and 49 amino acids).
  9. Saito A, Kono K, Nomaguchi M, Yasutomi Y, Adachi A, Shioda T, et al.
    J. Gen. Virol., 2012 Mar;93(Pt 3):594-602.
    PMID: 22113010 DOI: 10.1099/vir.0.038075-0
    The antiretroviral factor tripartite motif protein 5 (TRIM5) gene-derived isoform (TRIMCyp) has been found in at least three species of Old World monkey: rhesus (Macaca mulatta), pig-tailed (Macaca nemestrina) and cynomolgus (Macaca fascicularis) macaques. Although the frequency of TRIMCyp has been well studied in rhesus and pig-tailed macaques, the frequency and prevalence of TRIMCyp in cynomolgus macaques remain to be definitively elucidated. Here, the geographical and genetic diversity of TRIM5α/TRIMCyp in cynomolgus macaques was studied in comparison with their anti-lentiviral activity. It was found that the frequency of TRIMCyp in a population in the Philippines was significantly higher than those in Indonesian and Malaysian populations. Major and minor haplotypes of cynomolgus macaque TRIMCyp with single nucleotide polymorphisms in the cyclophilin A domain were also found. The functional significance of the polymorphism in TRIMCyp was examined, and it was demonstrated that the major haplotype of TRIMCyp suppressed human immunodeficiency virus type 1 (HIV-1) but not HIV-2, whilst the minor haplotype of TRIMCyp suppressed HIV-2 but not HIV-1. The major haplotype of TRIMCyp did not restrict a monkey-tropic HIV-1 clone, NL-DT5R, which contains a capsid with the simian immunodeficiency virus-derived loop between α-helices 4 and 5 and the entire vif gene. These results indicate that polymorphisms of TRIMCyp affect its anti-lentiviral activity. Overall, the results of this study will help our understanding of the genetic background of cynomolgus macaque TRIMCyp, as well as the host factors composing species barriers of primate lentiviruses.
  10. Voon K, Chua KB, Yu M, Crameri G, Barr JA, Malik Y, et al.
    J. Gen. Virol., 2011 Dec;92(Pt 12):2930-6.
    PMID: 21849518 DOI: 10.1099/vir.0.033498-0
    We previously described three new Malaysian orthoreoviruses designated Pulau virus, Melaka virus and Kampar virus. Melaka and Kampar viruses were shown to cause respiratory disease in humans. These viruses, together with Nelson Bay virus, isolated from Australian bats, are tentatively classified as different strains within the species Pteropine orthoreovirus (PRV), formerly known as Nelson Bay orthoreovirus, based on the small (S) genome segments. Here we report the sequences of the large (L) and medium (M) segments, thus completing the whole-genome characterization of the four PRVs. All L and M segments were highly conserved in size and sequence. Conserved functional motifs previously identified in other orthoreovirus gene products were also found in the deduced proteins encoded by the cognate segments of these viruses. Detailed sequence analysis identified two genetic lineages divided into the Australian and Malaysian PRVs, and potential genetic reassortment among the M and S segments of the three Malaysian viruses.
  11. Cabauatan PQ, Melcher U, Ishikawa K, Omura T, Hibino H, Koganezawa H, et al.
    J. Gen. Virol., 1999 Aug;80 ( Pt 8):2229-37.
    PMID: 10466823
    The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses.
  12. Shirako Y, Yamaguchi Y
    J. Gen. Virol., 2000 May;81(Pt 5):1353-60.
    PMID: 10769079
    Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3' poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3' terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.
  13. Chua BH, McMinn PC, Lam SK, Chua KB
    J. Gen. Virol., 2001 Nov;82(Pt 11):2629-39.
    PMID: 11602774
    The complete nucleotide sequences are reported of two strains of echovirus 7, the prototype Wallace strain (Eo7-Wallace) and a recent Malaysian strain isolated from the cerebrospinal fluid of a child with fatal encephalomyelitis (Eo7-UMMC strain). The molecular findings corroborate the serological placement of the UMMC strain as echovirus 7. Both Eo7-Wallace and Eo7-UMMC belong to the species human enterovirus B and are most closely related to echovirus 11. Eo7-UMMC has undergone significant genetic drift from the prototype strain in the 47 years that separate the isolation of the two viruses. Phylogenetic analysis revealed that Eo7-UMMC did not arise from recombination with another enterovirus serotype. The molecular basis for the severely neurovirulent phenotype of Eo7-UMMC remains unknown. However, it is shown that mutations in the nucleotide sequence of the 5' untranslated region (UTR) of Eo7-UMMC result in changes to the putative structure of the 5' UTR. It is possible that these changes contribute to the neurovirulence of Eo7-UMMC.
  14. Chan YP, Chua KB, Koh CL, Lim ME, Lam SK
    J. Gen. Virol., 2001 Sep;82(Pt 9):2151-5.
    PMID: 11514724
    We have completely sequenced the genomes of two Nipah virus (NiV) isolates, one from the throat secretion and the other from the cerebrospinal fluid (CSF) of the sole surviving encephalitic patient with positive CSF virus isolation in Malaysia. The two genomes have 18246 nucleotides each and differ by only 4 nucleotides. The NiV genome is 12 nucleotides longer than the Hendra virus (HeV) genome and both genomes have identical leader and trailer sequence lengths and hexamer-phasing positions for all their genes. Both NiV and HeV are also very closely related with respect to their genomic end sequences, gene start and stop signals, P gene-editing signals and deduced amino acid sequences of nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, glycoprotein and RNA polymerase. The existing evidence demonstrates a clear need for the creation of a new genus within the subfamily Paramyxovirinae to accommodate the close similarities between NiV and HeV and their significant differences from other members of the subfamily.
  15. Druka A, Burns T, Zhang S, Hull R
    J. Gen. Virol., 1996 Aug;77 ( Pt 8):1975-83.
    PMID: 8760450
    Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.
  16. Fan Z, Dahal G, Dasgupta I, Hay J, Hull R
    J. Gen. Virol., 1996 May;77 ( Pt 5):847-54.
    PMID: 8609480
    The DNA genomes of isolates of rice tungro bacilliform virus from Bangladesh, India, Indonesia, Malaysia and Thailand were cloned and compared with that of the type isolate from the Philippines. Restriction endonuclease maps revealed differences between the isolates and cross-hybridization showed that they fell into two groups, those from the Indian subcontinent and those from south-east Asian countries. The genomes of isolates from the Indian subcontinent contained a deletion of 64 bp when compared with those from south-east Asia. The implications of this variation are discussed.
  17. Guo J, Kitamura T, Ebihara H, Sugimoto C, Kunitake T, Takehisa J, et al.
    J. Gen. Virol., 1996 May;77 ( Pt 5):919-27.
    PMID: 8609488
    The JC polyomavirus (JCV) is ubiquitous in humans infecting children asymptomatically, then persisting in renal tissue. Since JCV DNA can be readily isolated from urine, it should be a useful tool with which to study the evolution of DNA viruses in humans. We showed that JCV DNA from the urine of Japanese, Taiwanese, Dutch and German patients can be classified into A and B types, based upon restriction fragment length polymorphisms (RFLPs). This work was extended in the present study. We established multiple JCV DNA clones from the UK, Spain, Italy, Sweden, South Korea, People's Republic of China, Malaysia, Indonesia, Mongolia, India, Sri Lanka, Saudi Arabia, Ethiopia, Kenya, Zambia, South Africa and Ghana. Using type-specific RFLPs, most clones except the four clones from Ghana were classified as either type A or B. We constructed a molecular phylogenetic tree for the Ghanaian clones and several representative type A and B clones. According to the phylogenetic tree, the Ghanaian clones constituted a major new group, tentatively named type C. From the findings presented here and elsewhere, the following conclusions were drawn: (i) type A is prevalent only in Europe; (ii) type B is found mainly in Asia and Africa; and (iii) type C is localized to part of Africa. Our findings should help to clarify how JCV evolved in humans.
  18. Debnath NC, Tiernery R, Sil BK, Wills MR, Barrett AD
    J. Gen. Virol., 1991 Nov;72 ( Pt 11):2705-11.
    PMID: 1940867
    Defective interfering (DI) particles of the flavivirus West Nile (WN) were generated after as few as two high multiplicity serial passages in Vero and LLC-MK2 cells. Six cell lines (Vero, LLC-MK2, L929, HeLa, BHK-21 and SW13) were used to assay interference by DI particles in a yield reduction assay. Interference was found to vary depending on the cell type used. The highest levels of interference were obtained in LLC-MK2 cells, whereas no detectable effect was observed in BHK-21 and SW13 cells. The ability of DI virus to be propagated varied depending on the cell line used; no detectable propagation of DI virus was observed in SW13 cells. Optimum interference was obtained following co-infection of cells with DI virus and standard virus at a multiplicity of 5. Interference between DI and standard viruses occurred only when they were co-infected or when cells were infected with DI virus 1 h before standard virus. Investigation of heterotypic interference by DI particles of WN virus strains from Sarawak, India and Egypt revealed that interference was dependent on the strain of WN virus or flavivirus used as standard virus. A measure of the similarity between five strains of WN virus and other flaviviruses was made on the basis of interference by DI viruses, and was found to be similar to that based on haemagglutination inhibition tests using a panel of monoclonal antibodies.
  19. Chen WR, Tesh RB, Rico-Hesse R
    J. Gen. Virol., 1990 Dec;71 ( Pt 12):2915-22.
    PMID: 2273391
    Forty-six strains of Japanese encephalitis (JE) virus from a variety of geographic areas in Asia were examined by primer-extension sequencing of the RNA template. A 240 nucleotide sequence from the pre-M gene region was selected for study because it provided sufficient information for determining genetic relationships among the virus isolates. Using 12% divergence as a cutoff point for virus relationships, the 46 isolates fell into three distinct genotypic groups. One genotypic group consisted of JE virus isolates from northern Thailand and Cambodia. A second group was composed of isolates from southern Thailand, Malaysia, Sarawak and Indonesia. The remainder of the isolates, from Japan, China, Taiwan, the Philippines, Sri Lanka, India and Nepal, made up a third group. The implications of these findings in relation to the epidemiology of JE are discussed. Results of this study demonstrate that the comparison of short nucleotide sequences can provide insight into JE virus evolution, transmission and, possibly, pathogenesis.
  20. Lanciotti RS, Lewis JG, Gubler DJ, Trent DW
    J. Gen. Virol., 1994 Jan;75 ( Pt 1):65-75.
    PMID: 8113741
    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 100%, 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 phylogenetic 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.
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