Isolation And Identification Of Infectious Bronchitis Virus Isolated In2011~2012and S1Gene Sequence Analysis Of All Isolated Strains

Abstract: Infectious Bronchitis (IB) is an acute and highly infectious respiratorydisease which is caused by the avain infectious bronchitis virus (IBV).Since break outin the1930s,the disease has been one of the major infectious virus in theworldwide.IBV has many serotrypes,different serotypes have less or no protection,andthis characteristic has brought great difficulties to the prevention and treatment ofIBV.17IBV strains were isolated and identificated from the test materials collectedfrom Beijing,Shandong,Liaoning,Guangxi,Jiangsu and Heibei provinces during2011~2012.Suspected diseased chicken tissues treated by conventional methods and wereinoculated in9-day-old to11-day-old specific-pathogen-free (SPF) chicken embryosfor5blind passage.All IBV isolates can cause apparent growth inhibition in chickembryo after a blind passage.11of the17isolates was purified by limited dilution for3times（F1~F3)，the purified virus was innoculated in chicken embryos，denoted byF4generation. The hemagglutination titer of F4generation was in the range of26~29after treated by1%trypsin. The F4generation can interfere with the replication ofNDV when inoculated in chicken embryos with La Sota (attenuated vaccine of NDV）.The result of animal regression test for HC13strain show that:HC13strain can causerespiratory and kidney disease,and caused45%mortality rate.The S1gene of17IBV isolates was sequenced and submitted to National Centerfor Biotechnology Information(NCBI)， and got the corresponding GenBanknumbers.The result of sequence alignment showed that：the nucleotide and amino acidhomology between the17isolates were84.7%~99.8%，82.6%~99.4%;the nucleotideand amino acid homology between isolates and52reference strains were73.2%~99.6%,70.0%~99.8%;the nucleotide and amino acid homology betweenisolates and vaccine strain H120were76.9%~82.2%;compared with vaccine strainH120,point mutations, gene insertion or deletion can be observed at many positions inthe S1gene of isolates.A phylogenetic analysis of17isolates and52reference strains was made and the result showed that：69IBV strains could be divided into nine evolutionary group，14of the17isolates belonged to phylogenetic group I，the S protein cleavage recognitionsites of phylogenetic group I was HRRRR；isolate HC18and4/91belonged tophylogenetic group II；17isolates and vaccine strain H120,M41,28/86belonged todifferent phylogenetic groups，and the evolutionary relationship between them wasrelatively far.Results of cross neutralization test showed that vaccine strain M41and isolatedstrain HC10and HC13belonged to different serotypes. Serological crossneutralization test and Homology analysis showed that homologous can reflect theperformance of the relevant antigen to some extent，but changes in gene sequencedoes not necessarily indicate changes in antigenic.S1gene is the main immunogen gene of IBV，S1protein can induce the body toproduce neutralizing antibody、hemagglutination inhibition antibody、cross-reactivityELISA antibody and immune response of cell-mediated，so the body can be protected.In this experiment，S1gene of isolates in the past two years was studied and somereference was provided for the research of IBV prevalence and the development ofIBV vaccine.