| Infectious bronchitis (IB) is an acute, highly contagious viral disease of the chicken caused by infectious bronchitis virus (IBV). In most parts of China, many Mass-serotype viruses were always isolated though the extensive use of Mass vaccine in poultry farm. In this study, we sequenced thirty-two IBV isolates and constructed phylogenetic tree on the basis of comparative analysis genome and each fragments in order to clarify phylogenetic relationship between IBV isolates and Mass-serotype reference strains. Then we choose five recombinant viruses to proceed with neutralization test in vitro to analyze antigenic differences between these viruses. After that, we use these five viruses to conduct animal experiment to observe the pathogenicity of viruses and the protective efficacy of HI20vaccine.The complete genome sequences of thirty-two IBV isolates were cloned, sequenced and assembled and we constructed phylogenetic tree on the basis of comparative analysis genome which indicated that twenty-five strains belonged to H120-like type and two strains belonged to Mass41-like type. In addition, five strains belonged to recombinant strains. We analyzed sequences of each fragments and found that all isolates have genetic mutation interspersed among the genome. However, ck/CH/LHB/111172, ck/CH/LDL/110931, ck/CH/LHB/130573, ck/CH/LHB/130598, ck/CH/LSD/111219were recombined with H120vaccine and another strain. Isolates ck/CH/LDL/110931originated from recombination between H120vaccine and Connecticut vaccine which switch site located at the3’end of the S1gene; ck/CH/LHB/130573originated from recombination between H120vaccine and tl/CH/LDT3/03which switch site also located at the3’end of the S1gene; ck/CH/LHB/130598originated from recombination between H120vaccine and4/91vaccine which switch site located at the intergenic sequences between M gene and gene5; ck/CH/LSD/111219originated from recombination between H120vaccine and DY07which has two switch sites at nsp14of ORF1and ORF3b, respectively; ck/CH/LHB/111172originated from recombination between H120vaccine and TW2296/95which has two switch sites at E gene and ORF5a, respectively.We choose five recombinant viruses to proceed with neutralization test in vitro. The β VN method with constant virus and diluted serum was employed in SPF chicken embryos for serotyping. According to the results of neutralization test, ck/CH/LHB/111172, ck/CH/LHB/130598and ck/CH/LSD/111219belonged to Mass-serotype while ck/CH/LDL/110931is a new serotype which different from H120vaccine and Connecticut serotype virus and ck/CH/LHB/130573is a new serotype which different from H120vaccine and tl/CH/LDT3/03serotype virus.We choose five recombinant viruses to infect SPF chickens and immune H120vaccine against these viruses. According to observe clinical symptoms, morbidity and mortality of SPF chickens, and do virus detection of oropharyngeal swabs, we can analyze the pathogenicity of recombinant viruses and the protective efficacy of H120vaccine. Results showed that the five recombinant viruses can only cause mild clinical signs with extended neck, nasal discharge, ruffled feathers and wing-droop. It indicated that these viruses had low virulence. According to clinical symptoms and results of virus detection of oropharyngeal swabs, there are high protective efficacy using H120vaccine against ck/CH/LHB/130598, ck/CH/LSD/111219and ck/CH/LHB/111172and low protective efficacy using H120vaccine against ck/CH/LDL/110931and ck/CH/LHB/130573. |
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