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Short Communication |
1 Oxford University Clinical Research Unit, 190 Ben Ham Tu, Ho Chi Minh City, Vietnam
2 Hospital for Tropical Diseases, 190 Ben Ham Tu, Ho Chi Minh City, Vietnam
3 Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands
4 Centre for Tropical Medicine, Oxford University, UK
Correspondence
Menno D. de Jong
dejongmd{at}gmail.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number of the sequence reported in this paper is EU350586.
The sequence of the primers used in this study is shown in Supplementary Table S1, available with the online version of this paper.
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). Years later, antigenic characterization showed that this virus belonged to the Semliki Forest virus (SFV) complex, but was antigenically distinct from SFV. Based on these analyses, the virus was classified as a new member of the genus Alphavirus, and named Me Tri virus (MTV) after the village from where the mosquitoes carrying the virus were originally collected (Ha et al., 1995). Serological studies showed the presence of antibodies against MTV in cattle, pigs, horses, monkeys as well as humans in Vietnam, with relatively high seroprevalence in young patients with clinical encephalitis (9/65 cases, 15 %), suggesting an aetiological role (Ha et al., 1995).
Based on a high degree of homology of a partial nucleotide sequence of the envelope glycoprotein gene (E1), it has recently been argued that MTV represents an isolate of SFV rather than a separate species (Powers et al., 2001). However, classification based on partial sequences may result in misclassification, since this does not rule out the possibility that new viruses emerge as a result of recombination events between different viruses. This is strikingly illustrated by the fact that another Alphavirus, Western equine encephalitis virus (WEEV), arose by recombination of the capsid and non-structural genes from an Eastern equine encephalitis virus (EEEV)-like virus and the remaining genes from a Sindbis virus (SINV)-like virus (Hahn et al., 1988; Weaver et al., 1993). Of note, based on antigenic analyses, WEEV was initially misclassified as belonging to the SINV group.
We analysed the complete genome sequence of MTV to elucidate its genetic relationship with SFV and other alphaviruses, and to enable definitive classification of this virus. The virus was propagated in Vero cells from lyophilized stock virus derived after five passages in suckling mice brain in the 1970s, and the complete genome was sequenced by primer-walking strategies. In brief, RNA was extracted from culture supernatants (QIAamp viral RNA kit; Qiagen), followed by the synthesis of full-length cDNA using random hexamer primers (Roche Diagnostics) and reverse transcriptase (Superscript III; Invitrogen). Overlapping fragments of the genome were amplified by specific primers designed on the basis of a reference SFV sequence (GenBank accession no. NC_003215 [GenBank] ). Primer sequences are shown in Supplementary Table S1, available with the online version of this paper. The 5'-terminal region was sequenced using a 5' RACE reaction kit (Invitrogen), following the manufacturer's instructions. The 3'-terminal region was amplified using oligo-dT- and virus-specific primers. PCR products were sequenced directly or after cloning, using either a Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) or a Dye Terminator Cycle Sequencing kit (Beckman Coulter) in ABI377 (Applied Biosystems) or CEQ 8000 (Beckman Coulter) automated sequencers, respectively. In case of differences between MTV and SFV reference sequences, amplification and sequencing procedures were repeated with the same or different primers, and only confirmed changes were considered unique changes in MTV.
Excluding the 5' cap and poly(A) tail, the complete genome of MTV was 11 820 nt with a composition of 20.1 % U, 26.1 % C, 27.1 % A and 26.7 % G. The genetic location of non-structural and structural proteins was predicted with the ZCURVE_V program (Guo & Zhang, 2006), and showed a genetic structure characteristic of alphaviruses. The 5' and 3' non-translated regions (NTR) were composed of 208 and 521 nt, respectively. The open reading frame (ORF) of the non-structural proteins initiated at position 208–300 and terminated at position 7504–7506, encoding a polyprotein of 2432 aa from which the individual non-structural proteins (nsP1–nsP4) are formed. As in most other alphaviruses, this ORF was interrupted by an opal codon located at the nsP3/nsP4 junction (nt position 5641–5643). Therefore, translation is predicted to generate two non-structural polyproteins: a major product (nsP1–3) and a minor one (nsP1–4) generated by occasional read through of this opal codon. The subgenomic RNA (26S RNA) consisted of a 39 nt 5'-NTR corresponding to the 26S promoter, and was 4315 nt long, encoding a 1253 aa polyprotein from which the individual structural proteins (capsid, E1–3, 6K) are formed.
The relationship between MTV and other alphaviruses was assessed by comparative and phylogenetic analyses using available complete genome sequences of alphaviruses. Pairwise comparison at the nucleotide level revealed that the degree of identity between MTV and other alphaviruses ranged from 43.9 to 96.2 %, with SFV showing the highest homology (Table 1). The same levels of homology were obtained when non-structural and structural gene sequences were compared separately. Phylogenetic trees based on complete genomes as well as on non-structural and structural polyprotein-coding genes, using neighbour-joining and minimum evolution methods, confirmed the close relatedness of MTV and SFV (Fig. 1).
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). The first combination included a forward primer starting at the 5' end of the insertion and a reverse primer targeted at the junction between the insertion and the rest of the genome and extending 3 nt into the 3' end of the insertion (Fig. 2a). The second combination consisted of a forward primer targeted at the 3' end of the insertion and extending 3 nt upstream from the junction and a reverse primer targeted upstream from the insertion at nt position 293–316 (Fig. 2a). To further increase the specificity of the PCR assays, cDNA was synthesized using specific primers targeted at position 924–904 of MTV/SFV. Single PCR products of expected sizes were obtained with each primer pair (Fig. 2b). The expected nucleotide sequences of the products were confirmed by cloning and sequencing (data not shown). Based on 98 % identity to a region of the MTV/SFV nsP2-coding gene, the insertion seemed to have resulted from homologous recombination. While homologous recombination is known to occur in alphaviruses (Weiss & Schlesinger, 1991), the biological relevance of this particular insertion is unclear at present and requires further research. Since our virus has undergone five passages in suckling mice brain, it cannot be ruled out that the recombination has occurred during these passages.
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; Ou et al., 1982), the precise mechanisms contributing to the diversity of the alphavirus 3'-NTR remain largely unclear. Likewise, the biological meaning of this diversity requires further study. Santagati et al. (1994) linked differences in RSEs between the L10 and A7 SFV strains to differences in replication rates of these two viruses in neurons. It should be noted that the L10 and A7 SFV variants are laboratory strains resulting from extensive passaging. Our MTV/SFV strain was isolated in suckling mice brain from mosquitoes followed by limited passages in suckling mice, hence our whole genome sequence may better represent naturally occurring virus. Comparison with other SFV strains isolated from mosquitoes or humans is desirable, but availability of SFV sequences is very limited at present.
In conclusion, analysis of the complete genome sequence confirmed that MTV is not a separate member within the SFV complex of the genus Alphavirus, but represents a variant of SFV. While this virus represents the first reported isolation of SFV outside Africa, further research is needed to determine whether and to what extent SFV continues to circulate in Vietnam, and whether it represents a significant cause of human disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ha, D. Q., Calisher, C. H., Tien, P. H., Karabatsos, N. & Gubler, D. J. (1995). Isolation of a newly recognized alphavirus from mosquitoes in Vietnam and evidence for human infection and disease. Am J Trop Med Hyg 53, 100–104.
Hahn, C. S., Lustig, S., Strauss, E. G. & Strauss, J. H. (1988). Western equine encephalitis virus is a recombinant virus. Proc Natl Acad Sci U S A 85, 5997–6001.
Hajjou, M., Hill, K. R., Subramaniam, S. V., Hu, J. Y. & Raju, R. (1996). Nonhomologous RNA-RNA recombination events at the 3' nontranslated region of the Sindbis virus genome: hot spots and utilization of nonviral sequences. J Virol 70, 5153–5164.
Ou, J. H., Trent, D. W. & Strauss, J. H. (1982). The 3'-non-coding regions of alphavirus RNAs contain repeating sequences. J Mol Biol 156, 719–730.[CrossRef][Medline]
Powers, A. M., Brault, A. C., Shirako, Y., Strauss, E. G., Kang, W., Strauss, J. H. & Weaver, S. C. (2001). Evolutionary relationships and systematics of the alphaviruses. J Virol 75, 10118–10131.
Santagati, M. G., Itaranta, P. V., Koskimies, P. R., Maatta, J. A., Salmi, A. A. & Hinkkanen, A. E. (1994). Multiple repeating motifs are found in the 3'-terminal non-translated region of Semliki Forest virus A7 variant genome. J Gen Virol 75, 1499–1504.
Weaver, S. C., Hagenbaugh, A., Bellew, L. A., Netesov, S. V., Volchkov, V. E., Chang, G. J., Clarke, D. K., Gousset, L., Scott, T. W. & other authors (1993). A comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related RNA viruses. Virology 197, 375–390.[CrossRef][Medline]
Weiss, B. G. & Schlesinger, S. (1991). Recombination between Sindbis virus RNAs. J Virol 65, 4017–4025.
Received 14 March 2008;
accepted 13 May 2008.
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