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.

The avian coronavirus spike protein

“…The cell culture adapted IBV strain Beaudette has also been the primary choice as backbone for the development of recombinant viruses (Casais et al, 2001;Fang et al, 2007;Youn et al, 2005). Only recently, a recombinant system based on the attenuated H120 vaccine strain was generated (Zhou et al, 2013). Recombinant viruses are excellent tools to dissect the contribution of each of the viral proteins to the various steps of the infection cycle.…”

Recombinant infectious bronchitis coronavirus H120 with the spike protein S1 gene of the nephropathogenic IBYZ strain remains attenuated but induces protective immunity

“…In considering the limitations of the live attenuated and inactivated IBV vaccines, reverse genetic IBV vaccines have been developed in recent years as they display increased safety and efficacy [28][29][30]. As reverse genetic technology is primarily based on the H120 and Beaudette strains [31][32][33], both of which are passagegenerated attenuated strains, the mechanism of virus virulence attenuation remains unclear, thereby posing difficulties in the development of attenuated vaccines using this technology. In addition to the function of host invasion and increasing genetic diversity, the IBV S1 subunit can induce virus neutralizing and cross-reactive antibodies [34].…”

Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure

“…Several studies have employed the reverse genetic system of IBV to study pathogenicity, tissue tropism, and functionality of accessory genes, as well as to explore the potential for a vector-based vaccine of IBV (Casais et al, 2003Armesto et al, 2009;Bentley et al, 2013). However, to the best of our knowledge, all existing reverse genetics systems for IBV were either based on the non-pathogenic Beaudette strain (Casais et al, 2001) or the Mass-type attenuated vaccine strains (Zhou et al, 2013;Beurden et al, 2017). While according to previous researches, the prevalent serotype of IBV circulating in China and many other countries is QX type (Monne et al, 2008;Zhao et al, 2016), and the QX-type strains typically share < 80% nucleotide identity with Mass-type strains.…”