| Fifty-six isolates of avian infectious bronchitis virus (IBV) were obtained from 216 flocks in China in 2010 and characterized by electron microscope observation, pathogenicity to chicken embryo, hemagglutination test and RT-PCR. The genes of these strains were emplifyed through RT-PCR, then ligated to pMD18-T vector, transferred into JM109 competent cell, cloned and seqnenced, analysed genes and genomes through biology software.Phylogenetic analysis based on the nucleotide sequences of the S1 gene of 56 IBV isolates in this study and 19 reference strains of IBV showed that 33 isolates in LX4 cluster I and 18 isolates in LX4 cluster II. The results showed that the LX4-type isolates is still the predominant IBVs in China and it could be grouped further into two clusters. Viruses in each cluster had favourite amino acid residues at different positions in S1 subunit of spike protein, implicating viruses in the different clusters were evolving dichotomously.When we conducted the pairwise comparison between the S1 genes of IBV LX4-type and tl/CH/LDT3/03-type isolates in this study, we found that recombination event was observed to be occurred between LX4-type and tl/CH/LDT3/03I-type strains which contributed to the emergency of the new strain ck/CH/LSD/100408. Some findings provided important evidence that ck/CH/LSD/100408 S1 gene sequence is a mosaic which descended from ck/CH/LSD/101223 and ck/CH/LSD/100504-like viruses. To our knowledge, this is the first report of natural recombination among LX4 and tl/CH/LDT3/03-like field strains in China.Though extensive vaccination using Mass-type vaccine, Mass-type strains are frequently isolated from vaccinated chicken flocks. This is the case for the IBV isolate ck/CH/LDL/101212 which had only one nonsynonymous mutation of S1 gene with the vaccine H120 strain. Isolate ck/CH/LHLJ/100902 had the closer relationship with pathogenic M41 in phylogenetic analysis and sequence pairwise comparison as well.The most important in the study is the isolation and identification of Taiwan II-type (TW II-type) IBV strains in the mainland of China in recent years. The genome of TW II-type IBV strains isolated in mainland of China experienced mutations and deletions, as demonstrated by comparison of the entire genome sequence with those of IBV strains isolated in Taiwan. The pathogenicity test and sequence analysis of 3′terminal untranslated region (UTR) revealed that TW II-type IBV strains isolated in mainland of China had closer relationship with the embryo-passaged, attenuated TW2296/95. The results of ck/CH/LHB/100801 virulent study showed that IBV strain ck/CH/LHB/100801 had low virulence to SPF chickens. However, the seroconversion of the birds after inoculation with isolate ck/CH/LHB/100801 was comparable with those of the virulent ck/CH/LDL/091022 strain but not H120 strain. A 49bp deletion was found in the 3′UTR of isolate ck/CH/LHB/100801, which was consistent with that of the embryo-passaged, attenuated TW2595/96. In this study, by comparing the sequence of more IBV strains could result the speculation that the deletion in this region of those IBV strains could be involved in pathogenicity and attenuation.China had large populations of commercial chickens maintained at a high density. The increasing poultry populations and greater use of vaccines will improve the more rapid generation of new types. These conditions implicated the importance of epidemiology and dynamic surveillance of IBV. |
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