Isolation And Full-length Sequencing Of Two Avian Infectious Bronchitis Viruses,and Cloning And Expression Of The S1gene

Infectious bronchitis(Infectious bronchitis,IB),caused by avian infectious bronchitis virus(infectious bronchitis virus,IBV),is an acute and highly contagious diseases of poultry industry.The genome of IBV is variable and susceptible to recombination and the genotype and serotype of IBV are complicated.The cross-protective efficacy among different serotypes of IBV is poor.Notably,the genetic recombination/mutagenesis generally results in the emergence of the novel genetic variants or immune rescape mutants,which make it more difficult to prevent the IBV.In this study,two isolates of IBV were isolated from clinical samples collected from IBV vaccinated chicken flocks,followed with the whole genome sequencing and cloning of the S1 gene,which pave the way for further study of potential immune evasion mechanism and vaccine selection for IBV.1.Isolation and identification of two isolates of infectious bronchitis virusIn this study,clinical samples were collected from suspected IBV chicken flocks.And the tissue homogenates were inoculated into chicken embryos for virus isolation.The inocauted chicken embryos showed as dwarfing and curling at day 5 post inoculation,but the allantoic fluid showed no heamagglutination activity.Then the RNA of the allantoic fluid was extracted and reversely transcribed to cDNA for further amplification of IBV S1 gene.Data showed that IBV S1 gene could be efficiently amplified from two samples and confirmed by sequencing.The two IBV isolates were designated as IBV34 and IBV216 respectively.Sequence homology analysis revealed that IBV34 and IBV216 both belonged to QX type based on the S1 sequence.Notably.The IBV216 S1 gene has a 12bp deletion.which is different from IBV34 and the other IBV strains from GenBank.The isolated IBV34 and IBV216 from the vaccinated chicken flocks may provide materials for further study on the immune evasion mechanism of IBV.2.Whole genome sequence analysis for the two isolates of infectious bronchitis virusTo study the potential mutation and recombination,the IBV34 and IBV216 genome were amplified.Cloned into TA vector and sequenced.The genome of IB V34 and IB V216 was 27647bp and 27654bp in full-length respectively(not including the sequnces of 5' cap and 3' poly A tail).And showed 96.6%and 96.7%identity with the genome of QX type strain YX10 respectively.Recombination analysis revealed that three recombination sites in IBV34 were predicted.Which located in 5567-8121 bp,8672-11925bp and 22159-23945bp respectively.Similar to IBV34,three recombination sites located in 5564-7886bp,8402-11923bp and 22148-23934bp were also predicted in IBV216.Further analysis indicated that IBV34 and IBV216 were possibly derived from the recombination of QX type and TC07-2 type of IBV.3.Cloning and expression of Slgene for the two isolates of infectious bronchitis virusTo further study the antigenicity of Slgene of IBV34 and IBV216 which isolated from the IBV vacciated chicken flocks.The Slgene of IBV34 and IBV216 was amplified and cloned into the linear vector pGEX-6p-1 and pcDNA3.1 respectively,and the corresponding recombinant plasmid was designated as pGEX-6P-1-IBV34-S1 and pGEX-6P-1-IBV216-S1.And pcDNA3.1-IBV34-S1 and pcDNA3.1-IBV216-S1 respectively.Then,the recombinant plasmids pGEX-6P-1-IBV34-S1 and pGEX-6P-1-IBV216-S1 were transformed into BL-21 competent cells for prokaryotic expression.SDS-PAGE analysis showed that GST-S1 could be efficiently expressed as soluble protein.Immunofluorescence assay showed that the chicken sera against QX type IBV could efficiently recognize the S1 protein expressed in LMH cells transfected with pcDNA3.1-IBV34-S1.But not with pcDNA3.1-IBV216-S1.This result sugges that the deletion of the four amino acids(62-65aa)in S1 protein from IBV216 may affect the antigenicity and immunoractivity of S1 protein.