Isolation And Identification Of Shannxi Strains Of Avian Infectious Bronchitis Virus And Analysis Of S1 Gene

Recently, infectious bronchitis (IB) are epidemic in Shaanxi province that result in serious economic loss. In order to discover the law of genesis and development of this pestilence, control the epidemic disease, we did some research on isolation and identification of avian infectious bronchitis virus. Four field strains of infectious bronchitis virus (IBV)were isolated from chicken kidney, trachea and lung of the young layer flocks in some regions of Shaanxi province. Biological characterizations of the virus were investigated by virus pathogenicity to chicken embryo and healthy chickens, hemagglutination test(HA), hemagglutination inhibition test(HI), dwarfing embryonated chicken eggs, electron microscopy and reverse transcription polymerase chain reaction(RT-PCR). It demonstrated that allantoic fluid of each passage virus could hemagglutinate chicken red blood cells by treatment with 1% trypsin. The standard positive serum to IBV could inhibited the hemagglutination. The isolates could make respiratory and kidney diseases in chickens. Gross lesions were observed in chicken embryo after passaged for four times. The spherical virons covered with spike like corona about 100 nm in diameter were observed under electron microscope. We named the four strains IBV BJ-04, FF-04, XP-03 and YL-04. The isolates RNA were extracted and used to amplify S1 gene of IBV by reverse transcription polymerase chain reaction(RT-PCR). 1.8 kb fragments were obtained. The PCR production were purified and cloned into pMD18-T vector. The recombinant plasmid was transformed into E. coli DH5a, then the positive clones were obtained by Amp+ selection. The recombinant DNA was purified, digested with BamHâ… and Hind â…¢, and sequenced. To compare the homogenieity of isolated strains with standard strains include M41, T, etc., make phylogenic trees, reveal the genomic relatedness among different strains, the sequence results were blasted on NCBI and the sequence of nucleotide and amino acid were analyzed by DNAStar 5.0. Meanwhile the hydrophilicity plot, antigenic index, glycosylation plot and some restriction endonuclease sites of S1 gene were analyzed. The results demonstrate that gene substitution, insertion and deficiency in S1 of the four isolates cause genotypic variation, and the genetic homologystrain of S1 gene between FF-04 and XP-03 are higher than that of BJ-04 and YL-04. The genetic distance of four isolates and respiratory type and kidney type standard strains is a little far, and the genotypes are all anomaly. The work enriches molecular epidemiologic information of IB, and establishes the foundation for IB diagnosing and curing.