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

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

•Reverse genetics system to clone attenuated IBV C-78 full-length genome in a BAC.•Rescued virus from BAC-cloned C-78 show parental virus properties.•Genetic manipulation of BAC-cloned C-78 using Red-mediated recombination in E. coli.•Construction of S gene recombinant C-78 by swapping with the S ge...

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Published in:Current research in microbial sciences Vol. 3; p. 100155
Main Authors: Inayoshi, Yujin, Oguro, Shiori, Tanahashi, Erika, Lin, Zhifeng, Kawaguchi, Yasushi, Kodama, Toshiaki, Sasakawa, Chihiro
Format: Journal Article
Language:English
Published: Elsevier B.V 01-01-2022
Elsevier
Subjects:
Coronavirus
Infectious bronchitis virus
Recombinant IBV
Research Paper
Reverse genetics system
Spike protein
Vaccine
Reverse genetics system
Vaccine
Coronavirus
Spike protein
Recombinant IBV
Infectious bronchitis virus
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Summary:•Reverse genetics system to clone attenuated IBV C-78 full-length genome in a BAC.•Rescued virus from BAC-cloned C-78 show parental virus properties.•Genetic manipulation of BAC-cloned C-78 using Red-mediated recombination in E. coli.•Construction of S gene recombinant C-78 by swapping with the S gene of virulent IBV.•S gene determines neutralization specificity but not virulence and tissue-tropism. 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. [Display omitted]
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ISSN:2666-5174
2666-5174
DOI:10.1016/j.crmicr.2022.100155