Phylogenetic analysis of African horse sickness virus segment 10: sequence variation, virulence characteristics and cell exit

  • L.-A. Martin
  • A. J. Meyer
  • R. S. O’Hara
  • H. Fu
  • P. S. Mellor
  • N. J. Knowles
  • P. P. C. Mertens
Conference paper


African horse sickness virus (AHSV) genome segment 10 encodes the non-structural proteins NS3/NS3a, which is involved in release of virus from cells. Full length segment 10 cDNAs were amplified by reverse transcription-polymerase chain reaction, from isolates of AHSV serotypes 2, 3, 4, 5, 7, 8 and 9. These cDNAs were cloned, sequenced and their phylogenetic relationships analysed. High levels of sequence homology were detected in segment 10 from some isolates of different serotypes, confirming that they could be grouped on this basis (serotypes 4, 5, 6 and 9 (group α); serotypes 3 and 7 (group β); serotypes 1, 2, and 8 (group γ)). However, data from bluetongue virus (the prototype orbivirus) indicate that the AHSV serotype is determined exclusively by the structural outer coat proteins VP2 and VPS, encoded by genome segments 2 and 5 respectively. Therefore, as a direct consequence of genome segment reassortment between AHSV strains from different serotypes, the differences observed in segment 10 do not give a reliable indication of virus serotype. Segment 10 of AHSV 3 (virulent) and AHSV 3att (attenuated) were also analysed. These strains, together with AHSV 8, have been used to study of the genetic basis of virulence using reassortment (O’Hara et al., this publication). Virus release studies, using Culicoides cell cultures, indicate that differences in segment 10 of AHSV 3att and 8 can influence the timing of virus release from the infected cell.


Genome Segment Nonstructural Protein Versus Versus Versus Versus Versus Virus Release Bluetongue Virus 
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  1. 1.
    Burroughs JN, O’Hara RS, Smale CJ, Hamblin C, Walton A, Armstrong R, Mertens PPC (1994) Purification and properties of virus particles, infectious sub viral particles, cores and VP7 crystals of African Horse sickness virus serotype 9. J Gen Virol 75: 1849–1857PubMedCrossRefGoogle Scholar
  2. 2.
    Calisher CH, Mertens PPC (1998) Taxonomy of African horse sickness viruses. In: Mellor PS, Baylis M, Hamblin C, Calisher CH, Mertens PPC (eds) African horse sickness. Springer, Wien New York, pp 3–11 (Arch Virol [Suppl] 14 )Google Scholar
  3. 3.
    de Sa RO, Zenner M, Grubman MJ (1994) Phylogenetic analysis of segment 10 from African horse sickness virus and cognate genes from other orbiviruses. Virus Res 33: 157–165PubMedCrossRefGoogle Scholar
  4. 4.
    Eaton BT, Hyatt AD, Brookes SM (1990) The replication of bluetongue virus. Curr Top Microbiol Immunol 162: 89–118PubMedGoogle Scholar
  5. 5.
    Felsenstein J (1993) PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, 1993Google Scholar
  6. 6.
    Finney DJ (1964) Statistical method in biological assay, 2nd ed. Griffin, LondonGoogle Scholar
  7. 7.
    Fu H, Leake CJ, Mertens PPC, Mellor PS (1998) The barriers to bluetongue virus infection dissemination and transmission in the vector Culicoides variipennis ( Diptera: Ceratopogonidae ) (in preparation)Google Scholar
  8. 8.
    Hwang G, Yang Y, Chiou J, Li JK (1992) Sequence conservation among the cognate nonstructural NS3/3A protein genes of six bluetongue viruses. Virus Res 23: 11–161Google Scholar
  9. 9.
    Hyatt AD, Gould AR, Coupar B, Eaton BT (1991) Localization of the non-structural protein NS3 in bluetongue virus-infected cells. J Virol 72: 2263–2267CrossRefGoogle Scholar
  10. 10.
    Hyatt AD, Zhao Y, Roy P (1993) Release of bluetongue virus-like particles from insect cells is mediated by BTV non-structural protein NS3/NS3A. Virology 193: 592–603PubMedCrossRefGoogle Scholar
  11. 11.
    Jensen MJ, Cheney IW, Thompson LH, Mecham JO, Wilson WC, Yamakawa M, Roy P, Gorman BM (1994) The smallest gene of the orbivirus, epizootic hemorrhagic disease is expressed in virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus. Virus Res 32: 353–364PubMedCrossRefGoogle Scholar
  12. 12.
    Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266: 19867–19870PubMedGoogle Scholar
  13. 13.
    Laegried WW, Burrage TG, Stone-Marschat MA, Skrowronek AJ, Hussein A (1995) The S10 gene of African horse sickness virus is a major virulence determinat. Abstracts, Fifth International Symposium on double-stranded RNA viruses (P46)Google Scholar
  14. 14.
    Laviada MD, Roy P, Sanchez-Vizcaino JM, Casal JI (1995) The use of African horse sickness virus NS3 protein, expressed in bacteria, as a marker to differentiate infected from vaccinated horses. Virus Res 38: 205–218PubMedCrossRefGoogle Scholar
  15. 15.
    Lee JW, Roy P (1986) Nucleotide sequence of a cDNA clone of RNA segment 10 of bluetongue virus (serotype 10). J Gen Virol 67: 2833–2837PubMedCrossRefGoogle Scholar
  16. 16.
    Martin L-A, Mertens PPC (1998) Purification and characterisation of RNA. Methods Mol Biol (in press)Google Scholar
  17. 17.
    Mertens PPC, Burroughs JN, Walton A, Wellby MP, Fu H, O’Hara RS, Brookes SM, Mellor PS (1996) Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species. Virology 217: 582–593PubMedCrossRefGoogle Scholar
  18. 18.
    Mertens PPC, Burroughs JN, Anderson J (1987) Purification and properties of virus particles, infectious sub-viral particles and cores of bluetongue virus serotypes 1 and 4. Virology 170: 561–565CrossRefGoogle Scholar
  19. 19.
    Moss SR, Jones LD, Nuttall PA (1992) Comparison of the nonstructural protein, NS3, of tick-borne and insect-borne orbiviruses. Virology 187: 841–844PubMedCrossRefGoogle Scholar
  20. 20.
    Nicholas KB, Nicholas HB Jr (1996) Gene Doc: a tool for editing and annotating multiple sequence alignments. Distributed by the authorGoogle Scholar
  21. 21.
    O’Hara RS, Meyer AJ, Pullen L, Martin L-A, Mertens PPC (1998) Development of a mouse model system and identification of the individual genome segments of African horse sickness virus serotypes 3 and 8 involved in determination of virulence. In: Mellor PS, Mertens PPC, Baylis M, Hamblin C (eds) African horse sickness. Springer, Wien New York, pp 259–279 (Arch Virol [Suppl] 14 )Google Scholar
  22. 22.
    Page RDM (1996) TREEVIEW: An application to display phylogenetic trees on personal computers. CABIOS 12: 357–358PubMedGoogle Scholar
  23. 23.
    Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425PubMedGoogle Scholar
  24. 24.
    Sailleau C, Moulay S, Zientara S (1997) Nucleotide sequence comparison of the segments S10 of the nine African horse sickness virus serotypes. Arch Virol 142: 965–978PubMedCrossRefGoogle Scholar
  25. 25.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467PubMedCrossRefGoogle Scholar
  26. 26.
    Stoltz MA, van Staden V, Napier GB, Huismans H (1997) Unpublished dataGoogle Scholar
  27. 27.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680PubMedCrossRefGoogle Scholar
  28. 28.
    van Staden V, Huismans H (1991) A comparison of the genes which encode nonstructural protein NS3 of different orbiviruses. J Gen Virol 72: 1073–1079PubMedCrossRefGoogle Scholar
  29. 29.
    van Staden V, Stoltz MA, Huismans H (1995) Expression of nonstructural protein NS3 of African horse sickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells. Arch Virol 140: 289–306PubMedCrossRefGoogle Scholar
  30. 30.
    Wechsler SJ, McHolland LE, Tabachnick WJ (1989) Cell-lines from Culicoides-variipennis ( Diptera, Ceratopogonidae), support replication of bluetongue virus. J Invertebr Pathol 54: 385–393PubMedCrossRefGoogle Scholar
  31. 31.
    Zientara S (1995) Thesis, Laboratoire de Virologie Equine, CNEVAGoogle Scholar
  32. 32.
    Zientara S, Sailleau C, Moulay S, Crucière C (1995) Differentiation of African horse sickness viruses by polymerase reaction and segment 10 restriction patterns Vet Microbiol 47: 365–375Google Scholar
  33. 33.
    Zientara S, Sailleau C, Plateau E, Moulay S, Mertens PPC, Crucière C (1998) Molecular epidemiology of African horse sickness virus based on analyses and comparisons of genome segments 7 and 10. In: Mellor PS, Baylis M, Hamblin C, Calisher CH, Mertens PPC (eds) African horse sickness. Springer, Wien New York, pp 317–327 (Arch Virol [Suppl] 14 )Google Scholar

Copyright information

© Springer-Verlag/Wien 1998

Authors and Affiliations

  • L.-A. Martin
    • 1
  • A. J. Meyer
    • 1
  • R. S. O’Hara
    • 1
  • H. Fu
    • 1
  • P. S. Mellor
    • 1
  • N. J. Knowles
    • 1
  • P. P. C. Mertens
    • 2
  1. 1.Pirbright LaboratoryInstitute for Animal HealthWoking, SurreyUK
  2. 2.Pirbright LaboratoryInstitute for Animal HealthPirbright, Woking, SurreyUK

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