1887

Abstract

In order to obtain attenuated live vaccine candidates of porcine reproductive and respiratory syndrome virus (PRRSV), a series of deletions was introduced at the 3′ end of the viral genome using an infectious cDNA clone of the Lelystad virus isolate. RNA transcripts from the full-length cDNA clones were transfected into BHK-21 cells. The culture supernatant of these cells was subsequently used to infect porcine alveolar macrophages to detect the production of progeny virus. It is shown that C-terminal truncation of the nucleocapsid (N) protein, encoded by ORF7, was tolerated for up to six amino acids without blocking the production of infectious virus. Mutants containing larger deletions produced neither virus nor virus-like particles containing viral RNA. Deletion analysis of the 3′ UTR immediately downstream of ORF7 showed that infectious virus was still produced after removal of seven nucleotides behind the stop codon of ORF7. Deletion of 32 nucleotides in this region abolished RNA replication and, consequently, no infectious virus was formed. Serial passage on porcine alveolar macrophages demonstrated that the viable deletion mutants were genetically stable at the site of mutation. In addition, the deletions did not affect the growth properties of the recombinant viruses , while their antigenic profiles were similar to that of wild-type virus. Immunoprecipitation experiments with the six-residue N protein-deletion mutant confirmed that the truncated protein was indeed smaller than the wild-type N protein. The deletion mutants produced in this study are interesting candidate vaccines to prevent PRRS disease in pigs.

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2001-11-01
2019-09-18
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References

  1. Allende, R., Lewis, T. L., Lu, Z., Rock, D. L., Kutish, G. F., Ali, A., Doster, A. R. & Osorio, F. A. ( 1999; ). North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. Journal of General Virology 80, 307-315.
    [Google Scholar]
  2. Cavanagh, D. ( 1997; ). Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Archives of Virology 142, 629-633.
    [Google Scholar]
  3. Cologna, R. & Hogue, B. G. ( 1998; ). Coronavirus nucleocapsid protein–RNA interactions. Advances in Experimental Medicine and Biology 440, 355-359.
    [Google Scholar]
  4. Dea, S., Gagnon, C. A., Mardassi, H., Pirzadeh, B. & Rogan, D. ( 2000; ). Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Archives of Virology 145, 659-688.[CrossRef]
    [Google Scholar]
  5. de Vries, A. A., Chirnside, E. D., Bredenbeek, P. J., Gravestein, L. A., Horzinek, M. C. & Spaan, W. J. ( 1990; ). All subgenomic mRNAs of equine arteritis virus contain a common leader sequence. Nucleic Acids Research 18, 3241-3247.[CrossRef]
    [Google Scholar]
  6. Groot Bramel-Verheije, M. H., Rottier, P. J. M. & Meulenberg, J. J. M. ( 2000; ). Expression of a foreign epitope by porcine reproductive and respiratory syndrome virus. Virology 278, 380-389.[CrossRef]
    [Google Scholar]
  7. Hsue, B., Hartshorne, T. & Masters, P. S. ( 2000; ). Characterization of an essential RNA secondary structure in the 3′ untranslated region of the murine coronavirus genome. Journal of Virology 74, 6911-6921.[CrossRef]
    [Google Scholar]
  8. Masters, P. S. & Sturman, L. S. ( 1990; ). Functions of the coronavirus nucleocapsid protein. In Coronaviruses and their Diseases , pp. 235-238. Edited by D. Cavanagh & T. D. K. Brown. New York:Plenum Press.
  9. Meulenberg, J. J. & Petersen-den Besten, A. ( 1996; ). Identification and characterization of a sixth structural protein of Lelystad virus: the glycoprotein GP2 encoded by ORF2 is incorporated in virus particles. Virology 225, 44-51.[CrossRef]
    [Google Scholar]
  10. Meulenberg, J. J. M., de Meijer, E. J. & Moormann, R. J. M. ( 1993a; ). Subgenomic RNAs of Lelystad virus contain a conserved leader–body junction sequence. Journal of General Virology 74, 1697-1701.[CrossRef]
    [Google Scholar]
  11. Meulenberg, J. J., Hulst, M. M., de Meijer, E. J., Moonen, P. L., den Besten, A., de Kluyver, E. P., Wensvoort, G. & Moormann, R. J. ( 1993b; ). Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192, 62-72.[CrossRef]
    [Google Scholar]
  12. Meulenberg, J. J. M., Petersen-den Besten, A., De Kluyver, E. P., Moormann, R. J. M., Schaaper, W. M. M. & Wensvoort, G. ( 1995; ). Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology 206, 155-163.[CrossRef]
    [Google Scholar]
  13. Meulenberg, J. J., Bos-de Ruijter, J. N., van de Graaf, R., Wensvoort, G. & Moormann, R. J. ( 1998a; ). Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. Journal of Virology 72, 380-387.
    [Google Scholar]
  14. Meulenberg, J. J., van Nieuwstadt, A. P., van Essen-Zandbergen, A., Bos-de Ruijter, J. N., Langeveld, J. P. & Meloen, R. H. ( 1998b; ). Localization and fine mapping of antigenic sites on the nucleocapsid protein N of porcine reproductive and respiratory syndrome virus with monoclonal antibodies. Virology 252, 106-114.[CrossRef]
    [Google Scholar]
  15. Molenkamp, R. & Spaan, W. J. ( 1997; ). Identification of a specific interaction between the coronavirus mouse hepatitis virus A59 nucleocapsid protein and packaging signal. Virology 239, 78-86.[CrossRef]
    [Google Scholar]
  16. Molenkamp, R., van Tol, H., Rozier, B. C. D., van der Meer, Y., Spaan, W. J. M. & Snijder, E. J. ( 2000; ). The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription. Journal of General Virology 81, 2491-2496.
    [Google Scholar]
  17. Murtaugh, M. P., Elam, M. R. & Kakach, L. T. ( 1995; ). Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Archives of Virology 140, 1451-1460.[CrossRef]
    [Google Scholar]
  18. Narayanan, K., Maeda, A., Maeda, J. & Makino, S. ( 2000; ). Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells. Journal of Virology 74, 8127-8134.[CrossRef]
    [Google Scholar]
  19. Peng, D., Koetzner, C. A. & Masters, P. S. ( 1995; ). Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination. Journal of Virology 69, 3449-3457.
    [Google Scholar]
  20. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  21. Snijder, E. J., van Tol, H., Pedersen, K. W., Raamsman, M. J. & de Vries, A. A. ( 1999; ). Identification of a novel structural protein of arteriviruses. Journal of Virology 73, 6335-6345.
    [Google Scholar]
  22. van Berlo, M. F., Horzinek, M. C. & van der Zeijst, B. A. ( 1982; ). Equine arteritis virus-infected cells contain six polyadenylated virus-specific RNAs. Virology 118, 345-352.[CrossRef]
    [Google Scholar]
  23. van Nieuwstadt, A. P., Meulenberg, J. J., van Essen-Zanbergen, A., Petersen-den Besten, A., Bende, R. J., Moormann, R. J. & Wensvoort, G. ( 1996; ). Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion. Journal of Virology 70, 4767-4772.
    [Google Scholar]
  24. Wensvoort, G., Terpstra, C., Boonstra, J., Bloemraad, M. & Van Zaane, D. ( 1986; ). Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Veterinary Microbiology 12, 101-108.[CrossRef]
    [Google Scholar]
  25. Wootton, S. K. & Yoo, D. (1999). Structure–function of the ORF7 protein of porcine reproductive and respiratory syndrome virus in the viral capsid assembly. In Proceedings of the International Symposium on PRRS and Aujeszky’s Disease, pp. 37–38. 21–22 June 1999, Ploufragan, France.
  26. Yang, S. X., Kwang, J. & Laegreid, W. ( 1998; ). Comparative sequence analysis of open reading frames 2 to 7 of the modified live vaccine virus and other North American isolates of the porcine reproductive and respiratory syndrome virus. Archives of Virology 143, 601-612.[CrossRef]
    [Google Scholar]
  27. Yuan, S., Murtaugh, M. P. & Faaberg, K. S. ( 2000; ). Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection. Virology 275, 158-169.[CrossRef]
    [Google Scholar]
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