Bacterial artificial chromosome-based reverse genetics system for cloning and manipulation of the full-length genome...

Avian infectious bronchitis virus (IBV) causes highly contagious respiratory reproductive and renal system diseases in chickens, and emergence of serotypic variants resulting from mutations in the viral S gene hampers vaccine management for IBV infection. In this study, to facilitate the molecular analysis of IBV pathogenesis and the development of a new-generation IBV vaccine, we established a reverse genetics system (RGS) for cloning the full-length cDNA of the IBV C-78E128 attenuated strain in a bacterial artificial chromosome (BAC). The BAC-cloned C-78E128 cDNA generated infectious viruses with biological properties of the parental C-78E128 strain with regard to an avirulent phenotype, tissue tropism and induction of virus neutralizing (VN) antibody in vivo. To assess the feasibility of genetic manipulation of the IBV genome using the BAC-based RGS, the S gene of the BAC-cloned C-78E128 cDNA was replaced with that of the IBV S95E4 virulent strain, which differs from the C-78E128 strain in serotype and tissue tropism, by bacteriophage lambda Red-mediated homologous recombination in Escherichia coli (E. coli). The resultant S gene recombinant virus was found to be avirulent and fully competent to induce a serotype-specific VN antibody against the S95 strain; however, the S gene recombinant virus did not fully recapitulate the tissue tropism of the S95E4 strain. These data imply that serotype-specific VN immunogenicity, but not tissue-tropism and pathogenicity, of IBV is determined by the viral S gene. The IBV BAC-based RGS that enables cloning and manipulation of the IBV virus genome entirely in E. coli provides a useful platform for the molecular analyses of IBV pathogenesis and the development of rationally designed IBV recombinant vaccines.