1887

Abstract

Rabbit hemorrhagic disease virus (RHDV), a member of the family comprising positive-stranded RNA viruses, is a highly virulent pathogen of rabbits. Until recently, studies into the molecular mechanisms of RHDV replication and pathogenesis have been hindered by the lack of an culture system and reverse genetics. This study describes the generation of a DNA-based reverse genetics system for RHDV and the subsequent investigation of the biological role of the RHDV VP2 protein. The full-length RHDV genome was assembled as a single cDNA clone and placed under the control of the eukaryotic human cytomegalovirus promoter. Transfection of cells with the DNA clone resulted in a clear cytopathic effect and the generation of infectious progeny virions. The reconstituted virus was stable and grew to titres similar to that of the parental virus. Although previous reports have suggested that the minor structural protein (VP2) of other caliciviruses is essential for the production of infectious virions, using the DNA-launch-based RHDV reverse genetics system described here it was demonstrated that VP2 is not essential for RHDV infectivity. Transfection of cells with a cDNA clone of RHDV lacking VP2 resulted in the generation of infectious virions. These studies indicate that the presence of VP2 could reduce the replication of RHDV, suggesting that it may play a regulatory role in the life cycle of RHDV.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.2008/003525-0
2008-12-01
2019-11-17
Loading full text...

Full text loading...

/deliver/fulltext/jgv/89/12/3080.html?itemId=/content/journal/jgv/10.1099/vir.0.2008/003525-0&mimeType=html&fmt=ahah

References

  1. Bertolotti-Ciarlet, A., Crawford, S. E., Hutson, A. M. & Estes, M. K. ( 2003; ). The 3′ end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein. J Virol 77, 11603–11615.[CrossRef]
    [Google Scholar]
  2. Boga, J. A., Martin Alonso, J. M., Marin, M. S., Casais, R, López Vázquez, A., Machin, A. & Parra, F. ( 1999; ). Genome organisation and gene expression strategies of rabbit haemorrhagic disease virus. Recent Res Devel Virol 1, 107–119.
    [Google Scholar]
  3. Chang, K.-O., Sosnovtsev, S. S., Belliot, G., Wang, Q., Saif, L. J. & Green, K. Y. ( 2005; ). Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae. J Virol 79, 1409–1416.[CrossRef]
    [Google Scholar]
  4. Chaudhry, Y., Skinner, M. A. & Goodfellow, I. G. ( 2007; ). Recovery of genetically defined murine norovirus in tissue culture using a fowlpox virus expressing T7 RNA polymerase. J Gen Virol 88, 2091–2100.[CrossRef]
    [Google Scholar]
  5. Glass, P. J., White, L. J., Ball, J. M., Leparc-Goffart, I., Hardy, M. E. & Estes, M. K. ( 2000; ). Norwalk virus open reading frame 3 encodes a minor structural protein. J Virol 74, 6581–6591.[CrossRef]
    [Google Scholar]
  6. Glass, P. J., Zeng, C. Q. & Estes, M. K. ( 2003; ). Two non-overlapping domains on the Norwalk virus ORF3 protein are involved in the formation of the phosphorylated 35K protein and ORF3–capsid protein interactions. J Virol 77, 3569–3577.[CrossRef]
    [Google Scholar]
  7. Gregg, D. A., House, C., Meyer, R. & Berninger, M. ( 1991; ). Viral haemorrhagic disease of rabbits in Mexico: epidemiology and viral characterization. Rev Sci Tech 10, 435–451.
    [Google Scholar]
  8. Kaiser, W. J., Chaudhry, Y., Sosnovtsev, S. V. & Goodfellow, I. G. ( 2006; ). Analysis of protein–protein interactions in the feline calicivirus replication complex. J Gen Virol 87, 363–368.[CrossRef]
    [Google Scholar]
  9. Liu, S. J., Xue, H. P., Pu, B. Q. & Qian, S. H. ( 1984; ). A new viral disease in rabbits. Anim Husb Vet Med 16, 253–255.
    [Google Scholar]
  10. Liu, G. Q., Zhang, Y. Y., Ni, Z., Yun, T., Sheng, Z. T., Liang, H. L., Hua, J. G., Li, S. M., Du, Q. Y. & Chen, J. P. ( 2006; ). Recovery of infectious rabbit hemorrhagic disease virus from rabbit after direct inoculation with in vitro-transcribed RNA. J Virol 80, 6597–6602.[CrossRef]
    [Google Scholar]
  11. Meyers, G. ( 2003; ). Translation of the minor capsid protein of a calicivirus is initiated by a novel termination-dependent reinitiation mechanism. J Biol Chem 278, 34051–34060.[CrossRef]
    [Google Scholar]
  12. Meyers, G., Wirblich, C. & Thiel, H.-J. ( 1991; ). Genomic and subgenomic RNAs of rabbit hemorrhagic disease virus are both protein linked and packaged into particles. Virology 184, 677–686.[CrossRef]
    [Google Scholar]
  13. Meyers, G., Wirblich, C., Thiel, H. J. & Thumfart, J. O. ( 2000; ). Rabbit hemorrhagic disease virus: genome organization and polyprotein processing of a calicivirus studied after transient expression of cDNA constructs. Virology 276, 349–363.[CrossRef]
    [Google Scholar]
  14. Mitro, S. & Krauss, H. ( 1993; ). Rabbit hemorrhagic disease: a review with special reference to its epizootiology. Eur J Epidemiol 9, 70–78.[CrossRef]
    [Google Scholar]
  15. Morales, M., Barcena, J., Ramirez, M. A., Boga, J. A., Parra, F. & Torres, J. M. ( 2004; ). Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on(−)sense genomic RNA: mapping of a subgenomic promoter. J Biol Chem 279, 17013–17018.[CrossRef]
    [Google Scholar]
  16. Mutze, G., Cooke, B. & Alexander, P. ( 1998; ). The initial impact of rabbit hemorrhagic disease on European rabbit populations in South Australia. J Wildl Dis 34, 221–227.[CrossRef]
    [Google Scholar]
  17. Nowotny, N., Bascunana, C. R., Ballagi-Pordany, A., Gavier-Widen, D., Uhlen, M. & Belak, S. ( 1997; ). Phylogenetic analysis of rabbit haemorrhagic disease and European brown hare syndrome viruses by comparison of sequences from the capsid protein gene. Arch Virol 142, 657–673.[CrossRef]
    [Google Scholar]
  18. Oehmig, A., Buttner, M., Weiland, F., Werz, W., Bergemann, K. & Pfaff, E. ( 2003; ). Identification of a calicivirus isolate of unknown origin. J Gen Virol 84, 2837–2845.[CrossRef]
    [Google Scholar]
  19. Ohlinger, V. F., Haas, B., Meyers, G., Weiland, F. & Thiel, H.-J. ( 1990; ). Identification and characterization of the virus causing rabbit hemorrhagic disease. J Virol 64, 3331–3336.
    [Google Scholar]
  20. Parra, F., Boga, J. A., Marin, M. S. & Casais, R. ( 1993; ). The amino terminal sequence of VP60 from rabbit hemorrhagic disease virus supports its putative subgenomic origin. Virus Res 27, 219–228.[CrossRef]
    [Google Scholar]
  21. Sosnovtsev, S. V. & Green, K. Y. ( 2000; ). Identification and genomic mapping of the ORF3 and VPg proteins in feline calicivirus virions. Virology 277, 193–203.[CrossRef]
    [Google Scholar]
  22. Sosnovtsev, S. V., Belliot, G., Chang, K. O., Onwudiwe, O. & Green, K. Y. ( 2005; ). Feline calicivirus VP2 is essential for the production of infectious virions. J Virol 79, 4012–4024.[CrossRef]
    [Google Scholar]
  23. Thumfart, J. O. & Meyers, G. ( 2002; ). Feline calicivirus: recovery of wild-type and recombinant viruses after transfection of cRNA or cDNA constructs. J Virol 76, 6398–6407.[CrossRef]
    [Google Scholar]
  24. Wirblich, C., Meyers, G., Ohlinger, V. F., Capucci, L., Eskens, U., Haas, B. & Thiel, H. J. ( 1994; ). European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other calicivirus. J Virol 68, 5164–5173.
    [Google Scholar]
  25. Wirblich, C., Sibilia, M., Boniotti, M. B., Rossi, C., Thiel, H. J. & Meyers, G. ( 1995; ). 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity. J Virol 69, 7159–7168.
    [Google Scholar]
  26. Wirblich, C., Thiel, H. J. & Meyers, G. ( 1996; ). Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies. J Virol 70, 7974–7983.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.2008/003525-0
Loading
/content/journal/jgv/10.1099/vir.0.2008/003525-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error