Molecular Epidemiology Of Avian Infectious Bronchitis Virus Isolated In Partial Regions Of China In 2007

Thirty-two avian infectious bronchitis viruses (IBV) were isolated from outbreaks in chickens in some provinces of China in 2007. The biological characteristics of the thirty-two IBV isolates were analyzed. Spike glycoprotein genes were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and then were cloned, sequenced and compared with IBV reference strains. The phylogenetic trees were also constructed based on the nucleotide sequences of the spike glycoprotein genes in order to investigate the source of domestic IBV isolates, to determine IBV genotypes exist in China, and the variation, the sequence identity, the phylogenetic relationships between Chinese isolates and reference strains.Characterization of the virus, such as pathological role to chicken embryo, hemagglutination activity and morphological characterization were studied. The above results elementarily suggested that the thirty-two viruses isolated in this study were avian infectious bronchitis viruses. Serotypic evolution in IBV is associated primarily with the sequences of S glycoprotein and the genetic diversity of IBV is mainly monitored on analysis of S1 gene. Because the S1 glycoprotein of IBV is responsible for inducing neutralizing and serotype-specific antibodies in chickens and mutations in the antigenically important spike glycoprotein S1 subunit leads to the emergence and proliferation of variant serotypes associated with disease outbreaks. Based on the phylogenetic tree of nucleotide sequences of S1 genes, IBV isolates in China were classified into three Genotypes. Genotypes I was LX4-type IBVs which included thirty isolates out of the thirty-two field isolates, isolated from different vaccinated flocks in china. An isolate,CK/CH/LDL/07III, was clustered into CK/CH/LSC/99I-type group and showed more close relationship with the IBV strains isolated in the same region from 2004 to 2005. Finally, isolate CK/CH/LHLJ/07VII was grouped into the serotype of Mass vaccine strains and field isolates from different countries of the world in the phylogenetic tree. Those results suggested most of IBV isolates in China formed indigenous genotypes and indicated that IBV vaccine strains and field isolates belonged to different genotypes, which may be responsible for frequent outbreaks of IB in vaccinated flocks in China. Hence, developing vaccines from local strains is necessary for IBV control in China.Comparison of nucleotide and amino acid in S1 gene of CK/CH/LHLJ/07VII with those of other Mass-type strains identified common unique deletions and insertions in three different regions among IBV types. The three regions were at amino acid positions 72 -79, 82 -85, 127-130. It clearly showed the presence of common unique deletion and insertions in the different types of IBVs, which may become a criterion for typing IBV isolates.In order to study the pathogenicity of the LX4-type IBVs isolate circulating in China in recent years, one of the IBV isolate, CK/CH/LHLJ/07IV, was used for inoculation to forty-two SPF chickens and sixty-six commercial broilers, causing mortality of 10.8%-30%. The kidneys from the dead chickens showed the predominant gross lesions which were pale, mottled, and swollen. The renal tubules and ureters were distended with excess urates. The results indicated that the pathogenicity of the LX4-type IBV isolates didn't change in the recent years in China and they were mainly nephropathologenic IBVs.Although Mass-type vaccines were commonly used in China, Chinese Mass-type strains were also isolated. Phylogenetic relationships indicated that Mass type IBV strain CK/CH/LHLJ/07VII in China formed a genotype together with H120 vaccine strains, therefore, the nucleotide and amino acid sequences of S1 gene of the IBV isolate, CK/CH/LHLJ/07VII, were assembled, aligned and compared with other 12 reference IBV strains. Maximum nucleotide and amino acid identities between ck/CH/LHLJ/07VII and reference strains were 99% and 97.1%, respectively (CK/CH/LHLJ/07VII and SD/97/01). However, 18 unique nucleotide mutations and15 amino acid substitutions were found in S1 protein gene of isolate CK/CH/LHLJ/07VII comparing with those of other Mass-type reference strains which maybe one of the reasons lead to variation of genome of IBV, indicating that it is unlikely to be reisolation of vaccine strain used in the immunization of the flock. Point mutation occurred in these isolates due to immunologic pressures caused by the widespread use of live vaccines. Mass-type strains have been circulating in our courtry, so it's necessary to use Mass-type vaccines for IB control.