Preliminary Cultivation And Screening Of Infectious Bronchitis Attenuated Vaccine Candidate Stains

Avian infectious bronchitis?IB?is an acute and highly contagious respiratory disease caused by avian infectious bronchitis virus?IBV?.Because of the high variability of IBV,new serotype and genotype strains are constantly emerging.At present,commercial Massachusetts serotype vaccines,such as H120 and H52 which mainly used in China,have little protective effect for the mainly epidemic strains in China.It is important to prevent the occurrence and epidemic of IB,through investigating local IBV epidemical strains,evolution and cultivating the live attenuated vaccine.In this study,16 strains of IBV were isolated from different areas of Guangdong Province between 2016 and 2017.Studies of biological characterristics was showed that 16viruses could cause dwarfing of chicken embryos after subculture.Sequence analysis of the S1 genes were showed that there have numbers of mutations,substitution and insertion of S1 genes in the 16 isolates.Comparison with domestic and foreign strains was indicated that one isolates was in the same group with 2992/02 and J2,and five isolates were in the same gene group whit the epidemic stain LX4.Ten isolates might form a new genotype which was a Chinese new genotype in recent years.A representative new genotype strain GDTS13 was selected and the whole genome sequence was determined.The main structural genes such as S1 gene,M gene and N gene were analyzed by genetic evolution.The homology between GDTS13 and the main reference strains domestic and foreign were 84.5%-99.6%.In addition the homology between GDTS13 with the LX4 type was 87.6%.The S1 sequence of GDTS13 showed68.0%homology to stain LX4.Moreover,the M sequence of GDTS13 showed 87.0%homology to stain LX4.Furthermore,the N sequence of GDTS13 showed 86.5%homology to stain LX4.The chicken embryo neutralization test used a method of fixing virus concentration and diluting serum.The results were showed that none of the five vaccine strains serums?4/91,H120,M41,Holte and Conn46?could neutralize GDTS13.It was suggested that GDTS13 and 5 vaccine strains might not belong to the same serotype.GDTS13 was continuously subcultured on the SPF chicken embryo.The pathogenicity of SPF chickens were tested with different generation of GDTS13.The results were showed that the F40 strain could infect 50%SPF chickens and cause the typical symptoms and pathology.In addition,F60 strain could infect 20%SPF chickens and cause the typical symptoms and pathology.However,F80 and F100 strains did not infect SPF chicks.The results of the S1 gene analysis of F1,F20,F40,F60,F80 and F100 strains was displayed that there were four mutation sites in the nucleotide sequence resulting three in amino acid residues.The homology between F80 and F100 generation strains was 100%,which was expressed that the GDTS13 had good genetic stability after multiple passages.The GDTS13F100 was inoculated with 2-day-old SPF chicks and passed for 5 consecutive passages.The viral virulence was judged by observing clinical symptoms,lesions,and Egg Infectious Dose 50%(EID₅₀).There was no obvious IBV clinical symptoms and pathological changes in selected test.Meanwhile,the virus strain,s EID₅₀ were similar in selected test.These results were mean that the GDTS13 F100 was stable and had attenuated.During vaccination-challenge test,2-day-old chickens were vaccinated with GDTS13F100 at a dose of 10³.5.5 EID₅₀ and challenged with homologous strain GDTS13 and heterogenous strain BJSN17 at a dose of 10⁵ EID₅₀0 after two weeks,respectively.The result showed that chickens vaccinated with F100 had at least 90%protection against virulent attacks.The results mean that GDTS13 F100 is an ideal candidate attenuated vaccine against the new genotype IB.In conclusion,this study investigated the epidemiology of IBV in Guangdong Province to research the epidemic genotypes and trends of IBV strains.It was succeeded in breeding new genotype IB attenuated vaccine candidate strains through passaging in chicken embryos.