| Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV), which results inheavy economic losses to the commercial poultry industry, worldwide. The virus causesrespiratory disease, reduced production performances, nephrosis and irreparable damage tooviduct leading to production of abnormal eggs. Immunization programmes now seem to beineffective due to the extreme genetic variations of IBV. To date information on IBV localepidemic strains has been scare, although the disease is endemic and continues to have aconsiderable impact on China poultry industry. In our study, we have isolated andidentificated four isolates of IBV from Shaanxi province, cloned and sequenced them, andtested the pathogenicity of some strains. Four different parts of results have been obtained asfollows:1. Two IBV isolates were collected from suspected flocks with respiratory infectionsymptoms. The viruses were indentified based on inoculated chick embryo, HA test,molecular biology tests and animal infection. The two IBV isolates showed highnephropahtogencity and infectivity to chicken embryo.2. Three pairs of IBV special primers were designed according to the nucleotidesequences of S1, M and N gene published in GenBank, and the three genes of four isolateswere amplified, cloned, sequenced for analyzing the genetic variation of IBV.In comparingwith W93vaccine strain, many point mutations occurred within the S1gene in all viruses andgene insertions occurred in all of isolates. Amino acid sequence homology of S1gene amongthe four IBV isolates varied from75.8%to99.4%.Mutations occurred in M gene, and genedeletions occurred in isolates of W09and WN12. Amino acid sequence homology of M geneamong the four IBV isolates varied from91.0%to99.6%.Deletion and insertion were notfound in the N genes, but point mutations were found. Amino acid sequence homology of Ngene among the four IBV isolates varied from99.3%to99.5%. In the phylogenetic treesbased on the deduced amino acid sequences of S1, M and N genes, the four isolates werecategorized into different branches, which suggested the genes may be occurredrecombination. Moreover，4isolates had further relationship with W118strain. The results indicated that the epidemic IBV strains in Shaanxi province had been mutated, which wasprobably the leading reason of immunization failure.3. According to IBV strain DY07, twenty-two pairs of primers were designed.Genomeof IBV strain CK/CH/Shaanxi/2009/H09was amplified, sequenced and aligned with otherIBV strains. The sequences analysis showed that the27675bp of H09had the5′cap~Replicase~S~3a~3b~3c~M~5a~5b~N~poly（A）3′gene order, and the typical aviancoronavirus cleavage site of536RRFRR540in spike protein, and the length of the3a,5a andN genes were conserved while other genes, length were varied. The sequence analysis resultsrevealed that homology of the strain with reference strains was85.2%~93.5%, geneticallyclose to ck/CH/LDL/091022, YX10and DY07. Isolate CK/CH/Shaanxi/2009/H09was thefirst IBV strain whose complete genome sequence has been determined in Shaanxi province.Although the strain belonged to epidemic serotype of IBV, it has been mutated at the genelevel.4. Toxicity attack test also shows that the symptoms presented can be appeared on15-day-old non-immune health cockerel after challenged with strain H09of IBV. Chicken hadonset after challenged3days.The kidney of ill chicken was swelling and ureteral was expansiveand had a lot of white urate deposition. Theearly histopathological changes were mainlyglomerulus atrophy, tubular epithelial cells deformation necrosis and tracheal mucosaswelling off.To sum up, the results showed that the four IBV isolates in this paper had highlyvirulence to kidney. Based on animal challenge results, the two IBV isolated showed highnephropahtogencity. These strains belonged to epidemic serotype of IBV, but they have beenmutated at the gene level. |
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