Isolation And Identification Of Avian Infectious Bronchitis Virus Shanxi Local Strains And Analysis Of S1 Gene Sequences

Infectious bronchitis virus(IBV)is a highly infectious pathogen that can cause diseases in the chicken’s respiratory,digestive and reproductive systems.Infectious bronchitis(IB)is common in most places where poultry is raised in the world.The disease affects the growth of chicks and the egg production of laying hens.This is one of the most important causes of economic losses in the poultry industry.In this study,we isolated virus strains from the tissues of sick chickens suspected of IBV-infected in Shanxi Province.Three isolates were identified by electron microscopy,hemagglutination test,chicken embryo pathogenicity test,NDV interference test,RT-PCR and animal regression test.The genetic variation of S1 gene sequences of the isolates was analyzed.In this test,diseased tissues were treated and inoculated to 9~11 days old SPF chicken embryos.Chicken embryo allantoic fluid was cultured for five generations.Under the electron microscope,the virus structure of the three isolates was consistent with the coronavirus.The three isolates were named as CK/CH/SX/BEI,CK/CH/SX/A and CK/CH/SX/ZJQ.The results of the hemagglutination test showed that the three isolates were unable to agglutinate 1% chicken red blood cells.But after I type of phosphoesterase C treatment,the allantoic fluid of chicken embryos from three isolates could agglutinate 1% chicken red blood cells.The results of chicken embryo pathogenicity test showed that the fifth generation allantoic fluid of three isolates could hinder the development of 9~11 days old chicken embryos and dwarf embryos appeared.The results of the NDV interference test showed that three isolates can interfere with the replication of NDV,which was consistent with the biological characteristics of IBV strains.Using specific IBV M gene primers for RT-PCR amplification,the M gene nucleic acid fragments were obtained in the same size as expected,with a size of about 803 bp.After sequencing,Blast showed that all three isolates were IBV.Three IBV isolates infected10-day-old chicks without IBV maternal antibody.Three days later,the chicks developed sneezing,breathing with their mouths,and other respiratory symptoms.Diseased chickens and dead chickens were mainly affected by respiratory and renal diseases.The S1 gene sequences of the three IBV isolates were analyzed.Using specific IBV S1 gene primers for RT-PCR amplification,the S1 gene nucleic acid fragments were obtained in the same size as expected,with a size of about 1850 bp.After sequencing,Blast showed that all three isolates were IBV.The nucleotide sequence was uploaded to the NCBI and three registration numbers were obtained.Analysis of the deduced amino acid sequence of the S1 gene showed that the three isolates had continuous multipoint mutations in seven regions compared with H120,and the amino acid mutations were larger in 53~102 and 115~156.The amino acid and nucleotide sequence homology between the IBV reference strains and the three isolates were 71.1%~98.0% and 74.9%~98.2%,respectively.The nucleotide sequence homology between the three isolates and the vaccine strains was 77.0%~79.0%.The amino acid and nucleotide sequence homology between the three isolates and our seven strains were 77.2%~96.1% and 79.6%~97.2%,respectively.The Neighbor-Joining Tree method of MEGA5.0software was used to construct the phylogenetic tree of S1 gene.Forty five IBV strains can be divided into seven groups.The three isolates isolated in this study were distributed in group QX-type.The vaccine strains were mainly distributed in group Mass-type.Groups QX-type to TW-type were mainly composed of strains isolated in different regions at domestic in recent years,and form relatively independent evolutionary groups,which were relatively distantly related to Group Mass-type.Therefore,IBV can still occur when chickens had been vaccinated.In this study,three IBV strains were isolated from chickens suspected to be infected with IBV in Shanxi.The isolates are distantly related to existing vaccine strains.The isolates are closely related to some IBV strains that are prevalent in China.This study provides a reference for vaccine development and lays a theoretical foundation for the prevention of IB.