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Journal of Virology, December 2001, p. 11974-11982, Vol. 75, No. 24
Molecular and Cell Biology
Program,1 Center for Agricultural
Biotechnology of University of Maryland Biotechnology
Institute,2 and Virginia-Maryland
Regional College of Veterinary Medicine,3
University of Maryland, College Park, Maryland 20742
Received 4 April 2001/Accepted 20 August 2001
Infectious bursal disease viruses (IBDVs), belonging to the family
Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and
nonstructural proteins, whereas segment B encodes the viral
RNA-dependent RNA polymerase (VP1). To identify the molecular
determinants for the virulence, pathogenic phenotype, and cell tropism
of IBDV, we prepared full-length cDNA clones of a virulent strain,
Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of
segments A and B between the attenuated vaccine strain (D78) and the
virulent IM or GLS variant strain. Using the cRNA-based
reverse-genetics system developed for IBDV, we generated five chimeric
viruses after transfection by electroporation procedures in Vero or
chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the
recovered viruses in vivo, we inoculated 3-week-old chickens with D78,
IM, GLS, or chimeric viruses and analyzed their bursae for pathological
lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1
coding sequences of the virulent strain IM were substituted for the
corresponding region in the vaccine strain failed to induce hemorrhagic
lesions in the bursa. In contrast, viruses in which the VP2 coding
region of the vaccine strain was replaced with the variant GLS or
virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions
in the bursa, as seen with the variant or classical virulent strain,
respectively. These results show that the virulence and
pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of
the chimeric viruses containing VP2 sequences of the virulent strain
could not be recovered in Vero or CEF cells but was recovered in
embryonated eggs, suggesting that VP2 contains the determinants for
cell tropism. Similarly, one of the chimeric viruses containing the VP1
segment of the virulent strain could not be recovered in Vero cells but
was recovered in CEF cells, suggesting that VP1 contains the
determinants for cell-specific replication in Vero cells. By comparing
the deduced amino acid sequences of the D78 and IM strains and their
reactivities with monoclonal antibody 21, which binds specifically to
virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues Gln at
position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the
virulence, cell tropism, and pathogenic phenotype of virulent IBDV.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.11974-11982.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Determinants of Virulence, Cell Tropism,
and Pathogenic Phenotype of Infectious Bursal Disease Virus
*
Corresponding author. Mailing address: Center for
Agricultural Biotechnology, 6126 Plant Sciences Building, University of Maryland, College Park, MD 20742. Phone: (301) 405-4777. Fax: (301)
314-9075. E-mail: vakharia{at}umbi.umd.edu.
Present address: Wyeth-Lederle Vaccines and Pediatrics, Marietta,
PA 17547.
Journal of Virology, December 2001, p. 11974-11982, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.11974-11982.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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