| Three infectious bronchitis virus isolates named as Q1, J2 and T3 were isolated from proventricular tissue taken from vaccinated chicken flocks in China. The biological characteristics of the three isolates were observed. After the viruses were inoculated blindly into 9-11 days old chicken embryonated egg through five to six passages, animal artificial infection test and vaccination protection test were done. In addition, the determination of physical and chemical property of the viruses by using tracheal ring culture, virulence measurement and virus neutralization test were applied too. The results indicated that the isolates induced high titers of IBV neutralization antibody in vaccinated SPF chickens,but the viruses could not be neutralized by the antisera specific to IBV serotype M41 and Australia T strain, and that the clinical signs in the tested SPF chickens were similar as those in the infected chickens of outbreaks and the corna virus like virions were reisolated from the damaged tissue, at the same time , expected DNA fragment was amplified from the reisolated viruses by using the primers of amplifying IBV S1 gene. The biological observation proved the pathogenic agent of the chicken infectious disease characterized by the swollen proventriculus to be IBV.For understanding the molecular basis of these isolates, the hypervariable S1 glycoprotein was analyzed. The complete S1 genes of 1.7Kb of these isolates wereamplified by using RT-PCR. The amplified DNA were then cloned and sequenced. The analysis of the sequence data further confirmed that 1BV was the pathogenic agent of the infectious disease associated with proventricular lesion.The deduced SI amino acid sequences of the isolates Ql, J2 and T3 were compared with 47 published sequences by the Clusteral V method of DNAstar software package. The results showed that the difference among the three isolates was 16.5% and 17 amino acid replacement and an unique cleavage site occurred in the sequences of these isolates, which implied the isolates might belong to an unique genomic type .The difference of SI amino acids (25.9%)among isolate T3 and vaccine strain H120 may explain why the infection of the isolates can result in serious disease in the chicken flocks vaccinated with HI20 vaccine.The amino acid sequence of the 99-127 residures in the SI glycoprotein may play a significant role in the pathogenicity. The 5' hypervariable regions of SI glycoprotein (56-69; 117-131) are closely related to neutralization epitope and serotype. The three isolates have four specific replacements in 87 (E), 88 (L), 95 (G), and 119 (N) in SI hypervariable region, and a unique cleavage site R-X-R-T-G-R in 3' terminal region. These data may be valuable for investigation of molecular basis of the serotyping, tissue tropism and pathogenicity of these virus isolates.In order trace the origin and evolution of avian infectious bronchitis virus (IBV) isolates in China and Southeast Asia, genomic sequencing was used for molecular characterization of 24 IBV isolates and two reference strains in comparison with the published sequences. The 5'region of the SI genes, containing hypervariable regions I and II, and 3' region of the nuceocapsid genes, containing cytotoxic T lymphocyte epitopes, were used to construct phlogenetic trees for analysis. The results showed that the 24 isolates could be divided into three distinct groups, that is, American, Asian, and European. Some isolates formed a distinct Asian phylogenetic group, suggesting that IBV has existed for some time in Asia. Our results also showed that invivo recombination of IBV may have occurred at a rather high frequency, contributing to the diversity of these IBV isolates. Importantly, recombination events have probably occurred between vaccine strains and field strains in the natural condition.
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