Isolation,Identification And Primary Study Of Vaccine Candidate Strains Of Infectious Bronchitis Virus

Infectious bronchitis(IB),caused by Infectious bronchitis virus(IBV),is one of the highly-contagious respiratory and acute diseases in chickens.IB was first erupted in the1930s.Nowadays,it has become popular all over the world,which has brought huge losses to poultry industry.Frequent occurrence of immune failure was caused by poor cross-protection between multiple serotypes of IBV.Therefore,it is significant to investigate the epidemic strain of IBV and screen for the ideal strain as a vaccine candidate strain for studying the immunogenicity and cross-immune protection of IBV.First,we isolated and identified 9 strains of IBV from the pathogenic clinical samples sent to provinces of Shandong,Anhui,Jiangsu,etc.between 2017 to 2018 and studied their genetic variation.The genetic evolution analysis of the S1 gene sequence showed that 7strains of the 9 IBV strains belong to genotype I represented by QX-like,2 strains belonged genotype VI.The homology of nucleotide and amino acid sequences between 7 IBV and QX reference strains was 94.8%?97%and 93.6%?96.1%respectively.The homology of nucleotide sequence and deduced amino acid sequence of S1 gene of two isolates of genotype VI was lower,67.4%?68%and 60.9%?62%respectively.Compared with the S 1 gene of vaccine strain H120,9 IBV isolates showed extensive point mutation,gene insertion and deletion,the homology of nucleotide and amino acid sequences was only 65.4%?78.1%and58.4%?77.3%,and the amino acid sequences of the cleavage sites were also quite different,indicating that the genotypes of the prevalent strains and the conventional vaccine strains in China were different and the homology was low.Based on the background information of the isolates,the genetic evolution analysis of S1 gene and the biological characteristics of the isolates,selected strain JS-96 and strain AH-48 which represented by QX-like and genotype VI respectively for pathogenicity study.10-day-old SPF chickens were infected with 10^5.0EID₅₀/0.1 m L of challenge dose,result showed that JS-96 was highly pathogenic,the mortality rate was 65%within 14 days infection.The diseased chickens showed mild respiratory symptoms and obvious pathological changes such as kidney enlargement and spotted kidney.The mortality rate of AH-48 challenging group was 17.5%,the diseased chickens showed typical respiratory symptoms of IBV.Severe tracheal catarrhal mucus and other pathological changes were observed by dissection.The results of antibody detection in chickens after challenge showed that the positive antibody production time JS-96 strain was 3-7 days earlier than AH-48 strain.Highly pathogenic strain JS-96 and commercial vaccine strain M41 were selected to study the immunogenicity and cross-protection,according to the regulation of immune efficacy test in national inactivated IBV vaccine.The 21-day-old SPF chickens were inoculated with monovalent inactivated oil emulsion vaccine through muscle,35 days after immunization were challenged different genotypes of IBV strains by intranasal and eye dropping.The results showed that the chickens from JS-96 inactivated vaccine immunization group were attacked by different strains.The finding showed that it caused slight symptoms,the morbidity was 10%?20%,and the mortality was 0?20%,there were significant differences in the morbidity and mortality between the groups immunized with M41inactivated vaccine(p<0.05),which were 0?60%and 0?30%respectively.The results of antibody test showed that antibody was produced 7 to 14 days after immunization,and showed an upward trend with time.The antibody titers of chickens immunized with JS-96were higher after 21 days of second immunization,which had better immunogenicity.The virus isolation rate of M41 group and JS-96 group was 0?50%and 0?20%respectively.The protective effect of JS-96 inactivated vaccine against QX-type IBV was significantly higher than that of non-immune challenge group(p<0.01),and the protective rate against heterologous strains was 80%?90%.The protective effect of JS-96 inactivated vaccine on genotype VI IBV was higher than that of commercial strain M41 inactivated vaccine,which could provide reference for the prevention and control of genotype VI IBV.The results showed that conventional vaccine strain M41 could not completely protect the current epidemic strains.The JS-96 inactivated vaccine isolates had good matching with the current IBV epidemic strains,high protection rate and significantly reduced the risk of horizontal transmission.This study provides a theoretical basis for the screening of better immunogenic strains and the development of candidate strains of multivalent IBV vaccine.