| Avian Infectious Bronchitis(IB) is caused by the avian infectious bronchitis virus (IBV), an acute and highly infectious respiratory disease. The disease can threaten poultry husbandry worldwide and result in huge economic loss. IBV is a single-stranded RNA virus, the gene of the virus always mutated owing to it's point-mutation and gene recombination, So the serotype of IBV has more than 30 categories. Different serotypes have lower or no protection. Currently, IB has often been found in China. Even if in the farm where had vaccinate IB vaccine,the incidence is still widely,Which result in serious economic losses to the poultry industry. Therefore, carrying on analysis of IB's epidemiology and pathogen mutation will contribute to synthesis preventing and controlling IB.This research came to carry on the laboratory virus isolation and identification from Jiangsu provincial area doubting for the infectious bronchitis sickness material. The results showed, these four isolates could cause the embrionic body maldevelopment, small, the fingernail joint head, curl and shrink; the allantoic fluid of the isolates had no hemagglutination activity, but after treatment with 2% trypsin,it can agglutinate chickens' red blood cell;The isolates had the interference effect in the chicken embryo to NDV LaSota strain; The SPF chickens which inoculated with the isolates replicated the typical IB clinical signs and pathological changes, which proved that four isolates were IBV strains.S1 gene and N gene sequences of four isolates 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 gene and N gene in order to trace the source of domestic IBV isolates.the S1 gene of four isolates were composed of 1611 bp,1611 bp,1650 bp, 1641 bp, encoding for a polypeptide of 537,537,550,547amino acids, respectively. Nucleotide homogeneity of SI gene of four IBV isolates ranged from 80.0%～97.4%, while 82.9%～95.9%identity was found on amino acid level. Identity of the S1 gene sequence between the JS3 and JS4 strains and IBV reference strains is 72.9%～81.8%, mutations, insertions and deletions were found in S1 gene of JS3 and JS4 strains. A phylogenetic tree based on nucleotide of S1 gene showed that JS1 and JS2 strains have higher homology with respiratory strains, while JS3 and JS4 strains have higher homology with nephritis strains isolated in China. Four isolates in this research have similar spike cleavage sites with normal respiratory strains, the amino acids in cleavage sites is composed of RRFRR.The N gene of four isolates were composed of 1230bp, encoding for a polypeptide of 409 amino acids. Nucleotide homogeneity of N gene of four IBV isolates ranged from 86.7%～96.1%, while 88.0%～98.3%identity was found on amino acid level. The phylogenetic analysis based on nucleotide of N gene indicated that JS1 and JS2 strains and JS3 and JS4 strains were grouped into two different clusters. No insertions and deletions were found in N gene of four isolates,however, a multitude of point- mutation resulted in the animo acid replacements at many places, which also occurred in three conserved alkalescent amino acid areas.Thus, taking into account these findings it can be demonstrated that not only point mutations,insertions and deletions but also a recombition events having contributed to the genetic diversity and emergency of IBV variants. JS1 and JS2 were respiratory strains, while JS3 and JS4 were nephritis strains in this research.The recombinant plasmids pET-N was constructed by inserting the N gene of IBV into the pET-32a vector. And the expression protein were induced by IPTG in E.coli BL21(DE3). SDS-PAGE analysis showed that N was well expressed in E.coli BL21 (DE3) after induced by IPTG. Western blot analysis showed that the protein was specifically recognized by polyclonal antibody IgG against IBV. |
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