Studies on molecular mechanisms of virulence of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the etiological agent of a highly contagious immunosuppressive disease in young chickens. Spontaneous enhancement of virulence has occurred without significant changes in antigenicity of circulating classical virulent strains. The sites on the viral genome involved in such virulence variations and the underlying molecular mechanisms are poorly understood. Since the rate of viral replication influences virus virulence, attempts were made to map the regulatory sequences involved in IBDV genome expression and replication. A series of chimeric genes containing various lengths of the 5′ noncoding sequences of IBDV segment A were constructed and analysed for promoter activity by measuring their capacity to direct the expression of the luciferase reporter gene. Progressive deletions of the 5′ noncoding region of 131 bp of segment A of IBDV and transient transfection studies identified a minimal region of 32 bp that had maximal promoter activity without any enhancer activity. Attempts were also made to isolate virus variants able to escape neutralizing antibody. A partial neutralization resistant mutant of IBDV was isolated by serially passaging a QC2 strain of IBDV in Vero cells in the presence of polyclonal antibody, and its phenotypic and genotypic characteristics were studied. The selected mutant had similar growth characteristics as the parental virus but had a smaller plaque morphology. Its genome segment A had an additional Pst I restriction site in the major antigenic region. There were four and three nucleotide exchanges in the 3′ noncoding regions of segments A and B respectively and none in the 5′ noncoding regions. Moreover, less severe pathology in the bursa of Fabricius and higher virus neutralization titres. Such a variant may be useful in the development of attenuated vaccines of IBDV. Furthermore, efforts were made to develop a reverse genetics system for IBDV by constructing two independent full-length cDNA clones containing segments A and B of OH strain of IBDV. Infectious progeny viruses were recovered in Vero cells transfected with combined transcripts of segment A and segment B of these cDNA clones. Moreover, transfection of Vero cells with full-length segment A transcript and a partial length segment B transcript derived from a partial length cDNA construct of segment B without its 3′ noncoding sequences also produced infectious virus that was almost indistinguishable from the wild-type IBDV. The 3′ noncoding sequences that were absent in the partial length cDNA construct of segment B were regenerated in the genomic RNA of the progeny infectious virus indicating the functional significance of this region in establishing a productive infection. (Abstract shortened by UMI.).