Molecular Epidemiology Of The Infectious Bronchitis Virus Isolated In China Between 2005 And 2006

The Biological characteristics of twelve avian infectious bronchitis viruses (IBV) were isolated from outbreaks in chickens in eight provinces of China between 2005 and 2006 were analyzed. In order to trace the source of domestic IBV isolates, to determine IBV genotypes exist in China, and 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. Typical signs including stunting, curling and death of embryo were observed in the second to eighth passages when each of the twelve Chinese isolates was inoculated into 9-day-old to 11-day-old specific-pathogen-free (SPF) chicken embryos. Furthermore, the twelve isolates could not hemagglutinates chicken red blood cells and showed typical coronavirus.The above results elementarily suggested that the twelve viruses isolated in this study were avian infectious bronchitis viruses.S1 and partial N protein gene sequences of twelve IBV strains isolated in china between 2005 and 2006 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 S1 and partial N structural protein genes in order to trace the source of domestic IBV isolates.Phylogenic analysis based on entire S1 gene sequences showed that eight isolates clustered together with LX4-type strains, having 95.3-97.0% nucleotide identity with LX4 strain, and shared no more than 80.1% amino acid sequence identities with H120 vaccine strains. which may be responsible for frequent outbreaks of IB in vaccinated flocks in China;One IBV isolate, CK/CH/LSD/05I, formed a"novel"group. Three isolates CK/CH/LDL/05I,CK/CH/LNM/05I and CK/CH/LGX/06I had close sequence relationship with tl/CH/LDT3/03 strain and belonged to the same cluster based on S1 gene sequences. and three isolates had higher S1 gene nucleotide identity with each other and had unique deletion and insertion sites and similar spike cleavage recognition site, In contrast, phylogenic analysis of more conserved partial N gene sequences revealed different clustering. In addition, CK/CH/LSD/05I had the closest relationship with Mass-type vaccines H120 (93.4%). It demonstrated that isolate CK/CH/LSD/05I formed a"novel"Variant and might emerge by selection pressure, isolate CK/CH/LNM/05I and CK/CH/LGX/06I could happen recombination events. Moreover, The results showed that in the S1 protein gene sequences, nucleotide sequence alignments revealed extensive insertion and deletion in S1 gene hypervariable region 1(HVR1) and HVR2 of isolates in this study, whereas the partial N gene sequences displayed mostly point mutations compared to published sequence of H120 vaccine strain.Thus, taking into account these findings it can be demonstrated that not only point mutations, insertions and deletions but also a recombination events having contributed to the genetic diversity and emergency of IBV variants in China.Phylogenic analysis based on entire S1 gene sequences showed that majority isolates clustered together with the close phylogenetic relationships. Therefore, we researched pathogenicity of the selected isolate IBV CK/CH/LDL/05II in this group, Pathogenic study showed that this virus caused 100% morbidity and 54.5% mortality to 15-day-old specific-pathogen-free (SPF).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.