Isolation And Identification Of Avian Infectious Bronchitis Nephritis Virus In Shandong Province, And Genetic Variations Of Nucleocapsid Gene

Ten avian infectious bronchitis (IB) nephritis viruses (IBV) were isolated from outbreaks in chickens in Shandong Province between 2006 and 2007, and identified by morphological observation, inoculating virus into chicken embryos, viral interference test, HA test and returning pathogen to host. Nucleocapsid protein gene of the ten strains was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and then was cloned, sequenced and compared with IBV reference strains. The phylogenetic trees were also constructed based on the nucleotide sequences of the Nucleocapsid protein gene in order to trace the source of IBV isolated in Shandong Province, to determine IBV genotypes exist in Shandong Province, and the variation, the sequence identity, the phylogenetic relationships between the strains isolated in Shandong Province and reference strains.Typical signs including dwarfing, stunting, curling and death of embryo were observed in the third to seventh passages when each of the ten isolates was inoculated into 9-day-old to 11-day-old specific-pathogen-free (SPF) chicken embryos. Diagnoses based on electron microscopy examination performed on allantoic fluids of different passages showed all ten isolates had typical coronavirus morphology (Virions enveloped, slightly pleomorphic, spherical, 80nm-120nm in diameter. Surface projections of envelope distinct, club-shaped, spaced widely apart and dispersed evenly over all the surface.). No other agents such as Newcastle disease virus were detected. The replication of NDV was evidently affected in chicken embryos infected by the ten isolates. The allantoic fluid of the ten isolates had no activity of haemagglutination, but when treated with I type of phosphoesterase C, them could agglutinate 10mL/L chicken's red blood cell. The nucleic acid snippets of ten isolates were amplified by RT-PCR using specific primer which can amplify the N gene of IBV. The results of electrophoresis showed that the nucleic acid snippets of ten isolates were composed of about 1600 nucleotides. All the above showed that the viruses we isolated were strains of IBV. The kidneys of chickens were swelling after returning pathogen to host. So we can come to a conclusion that the ten viruses we isolated were strains of infectious bronchitis nephritis virus.Nucleocapsid protein (N protein) genes of ten IBV strains isolated in Shandong Province between 2006 and 2007 were amplified by RT-PCR, and then were cloned, sequenced, analysed and compared with 20 IBV reference strains. The results showed that all of the N genes of the ten isolates were composed of 1230 nucleotides, encoding a polypeptide of 409 amino acid residues. They were the same as the N gene of Beaudette strain. Nucleotide homogeneity of N gene of the ten IBV strains ranged from 84.6%-99.7%, while 86.6%-99.8% identity was found on amino acid level. Nucleotide homogeneity of N gene of the ten IBV strains with N gene of the twenty IBV reference strains ranged from 84.1%-98.9%, while 86.1%-98.5% identity was found on amino acid level. Mutation were found in the N gene sequences of the ten isolates compared with reference strains which maybe one of the reasons lead to variation of genome of IBV isolates in Shandong Province.The sequence identity and the phylogenetic relationships between the genes derived from the 10 isolates and the twenty reference strains indicated that IBV isolates found in Shandong Province were branched into several genetic clusters. Based on the phylogenetic tree of nucleotide sequences of N genes, IBV isolates in Shandong Province were classified into two Genotypes. Two isolates, SD0605 and SD0608, which might be a reisolation of vaccine strains, clustered into genotype I. Massachusetts serotype was present in China in 1990s and was in this genotype and most of IBV vaccines used in China belonged to this genotype. Nucleotide homogeneity of N gene of the SD0605 isolates with N gene of D41 and H120 strains ranged 98.9%, while 98.5% identity was found on amino acid level. Nucleotide homogeneity of N gene of the SD0608 isolates with N gene of GX2 isolates ranged 94.9%, while 95.6% identity was found on amino acid level. Other eight isolates clustered into genotype II which included GDS14, LX4 and QXIBV isolates.Take together, the emergence of new IBV strains might have a relationship to the application of attenuated vaccines. The comparison and phylogenetic analysis of gene sequences indicated that gene recombinations were occurring within Shandong isolates.Those results suggested most of IBV isolates in Shandong Province formed indigenous genotypes and higher nucleotide sequence identity were found among them. Furthermore, low nucleotide sequence identity between the genes of IBV isolates of Shandong Province and vaccine strains used in China also indicated that IBV vaccine strains and field isolates belonged to different genotypes, which may be responsible for frequent outbreaks of IB in vaccinated flocks in China.All the above suggested the ten IBV isolates of Shandong Province had near relation with the IBV isolates of anywhere else in China.The IBV vaccine strains could protect chickens from attacking of some of the IBV isolates. The nucleotide sequence of the N gene of SD0702 strain had much variation, so the vaccine strains could not protect chickens from attacking of it. It is necessary to develop new vaccine and find better measures to protect chickens.