title: Establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain H120.
abstract: Infectious bronchitis virus (IBV) strain H120 was successfully rescued as infectious clone by reverse genetics. Thirteen 1.5-2.8 kb fragments contiguously spanning the virus genome were amplified and cloned into pMD19-T. Transcription grade complete length cDNA was acquired by a modified "No See'm" ligation strategy, which employed restriction enzyme Bsa I and BsmB I and ligated more than two fragments in one T4 ligase reaction. The full-length genomic cDNA was transcribed and its transcript was transfected by electroporation into BHK-21 together with the transcript of nucleocapsid gene. At 48 h post transfection, the medium to culture the transfected BHK-21 cells was harvested and inoculated into 10-days old SPF embryonated chicken eggs (ECE) to replicate the rescued virus. After passage of the virus in ECE five times, the rescued H120 virus (R-H120) was successfully recovered. R-H120 was subsequently identified to possess the introduced silent mutation site in its genome. Some biological characteristics of R-H120 such as growth curve, EID50 and HA titers, were tested and all of them were very similar to its parent strain H120. In addition, both R-H120 and H120 induced a comparable titer of HA inhibition (HI) antibody in immunized chickens and also provided up to 85% of immune protection to the chickens that were challenged with Mass41 IBV strain. The present study demonstrated that construction of infectious clone from IBV vaccine strain H120 is possible and IBV-H120 can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach.
authors.affiliation: School of Life Science, Sichuan University, Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Chengdu, Sichuan 610064, China.
authors.forename: Ying Shun
authors.initials: YS
authors.lastname: Zhou
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hong Ning
authors.initials: HN
authors.lastname: Wang
authors.forename: Wen Qiao
authors.initials: WQ
authors.lastname: Fan
authors.forename: Xin
authors.initials: X
authors.lastname: Yang
authors.forename: An Yun
authors.initials: AY
authors.lastname: Zhang
authors.forename: Fan Ya
authors.initials: FY
authors.lastname: Zeng
authors.forename: Zhi Kun
authors.initials: ZK
authors.lastname: Zhang
authors.forename: Hai Peng
authors.initials: HP
authors.lastname: Cao
authors.forename: Cheng
authors.initials: C
authors.lastname: Zeng
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D002642:Chick Embryo; D002645:Chickens; D018076:DNA, Complementary / Q000235:genetics; D016679:Genome, Viral; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000276:immunology*; D010063:Ovum / Q000276:immunology; D059386:Reverse Genetics / Q000379:methods*; D014158:Transcription, Genetic; D014162:Transfection; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D018076:DNA, Complementary; D014613:Vaccines, Attenuated; D014765:Viral Vaccines