Exploration Of Genetic Evolution And Attenuation Of Domestic Infectious Bronchitis Virus

Avian infectious bronchitis(IB)is an acute infectious disease,which is caused by infectious bronchitis virus(IBV),Because of its special replicating and RNA polymerase proofreading mechanism,genome of IBV is quite easy to mutate and recombine,causing severe challenges to the prevention of this epidemic disease.This study systematically analyzed the genetic evolution of IBV isolates in China,compared the pathogenicity of domestic prevalent QX-like isolate YN and its attenuated strain aYN,and analyzed possible attenuating-related gene mutations.A series of full-length IBV genome colone were constructed and the reverse genetic system has been successfully established,laid the foundation for the subsequent exploration of the attenuating mechanism of IBV.Recent years outbreaks of IB happened occasionally in most provinces of China.According to the gene biological analysis of Chinese IBV sequences uploaded to NCBI in the last 20 years,apparent differences have been found in the gene sequences of new strains comparing with Mass-type strains.14 isolates in total were identified as recombinants by RDP4,most probably generated through the recombination between QX and other genotype strains.The IBV strains in China was found to evolve continually at a rate of approximately 10-5 substitutions/site/year.We also found that although purifying selection was the main evolutionary pressure in IBV,S1 gene is under the greatest positive selection pressure,the corresponding regions may be more susceptible to mutations and can change virus phenotypes.Under the influence of multiple selection pressures,the compositional structure of domestic IBV genotypes is changing,the proportion of QX-like strains is increasing gradually.Developing vaccines against new genotype strains is of great significance to control the epidemic of IB in China.To determine the utility of YN strain as a vaccine,we serially passaged it in SPF embryonated eggs for 118 generations,and compared the pathogenicity of attenuated strains aYN and its parental strain YN,aYN strain showed significant lower replication ability and pathogenicity,confirming that we have obtained a QX-type attenuated virus strain with well-immunity.By comparing the complete genome between the two strains,we found that they share a high similarity of 99.7%.Most of the mutations occurred in the gene lab,spike gene and 81 nt deletion in the gene 5a,suggesting a potential role in the loss of viral pathogenicity.In order to explore whether the natural deletion of 5a gene would affect the virulence and pathogenicity of IBV,the full-length cDNA clone of YN strain wIBV-YN was constructed based on Vaccinia virus recombination system.Using deletion and other molecular methods we further reconstructed the cDNA clone of YN strain,obtained wIBV-YN/del5a(81 nt deletion of 5a coding region)and vvIBV-YN/EGFP(replace the 5a gene with the EGFP gene).Subsequently,we successfully rescued the IBV YN and YN/EGFP strain using T7 promotor in-vitro transcription technology.The above-mentioned molecular cloning based on the full-length construction of IBV genome and successful establishment of the reverse genetic system laid the foundation for the subsequent exploration of the attenuating molecular mechanism of IBV.