Isolation And Identification Of Infectious Bronchitis Virus In Henan And The Full Genome Sequence Analysis Of Isolated Strains HN104 And HN091

Avian infectious bronchitis virus(IBV)is a highly infectious and contagious pathogen of domestic fowl, replicating in the respiratory tract and some epithelial tissues of the gut, kidney, and oviduct. IBV infection is commonly followed by secondary bacterial infection, such as with Escherichia coli, resulting in complicated morbidity and increased mortality, causing clinical disease and production problems in vaccinated focks. Therefore,study the phylogeny and variation on the isolates of IBV from the commercial Chickens in Henan is the key for effective control of the disease.Seven viruses were identificated from the chicken flocks with typical clinical symptoms of IB from Kaifeng, Xinmi, Xuchang, Xinzheng, Xingyang of Henan during 2008~2010, and were named HN081, HN082, HN091, HN092, HN101, HN102, HN104. The allantoic fluid that was negative in hemagglutination (HA) and bacterial growth test was amplificated S1 gene and N gene in RT-PCR. The hemagglutination titer of HN104 was 28 after treatment by 1% fresh trypsin; EID50 was10-5.5/0.1ml; It could significantly interfere with Newcastle Disease Virus La Sota strain proliferation in chick embryo; SPF chickens infection experiments showed that the virus could cause swelled and spotted kidneyS1 gene of four IBV isolated strains were sequenced, identity were 77.4~96.9%. Comparative analysis of the S1 gene sequences of the all 43 strains showed that the similarity of the nucleotide sequences were 76~100%. Phylogenetic analysis of the S1 gene sequences of the 4 isolated strains together with 39 strains published in Genbank revealed that all IBV strains were clustered into seven distinct genotypes I~VII. HN104 and HN091 strains were clustered into genotype I which also were belong to predominant LX4 genotype; HN091 was clustered into genotype III which also was belong to Taiwan specific genotype TW2296/95. Genotype III IBV were isolated in the last two years in China. HN092 which was a strain variation of M41 was clustered into genotype II. Compared to Mass, T and 4/91strains show that HN104, HN091 and HN092 isolated strains nucleotide sequences in homology were low 78%. So vaccines have not been effective.N gene of five of six isolated strains in Henan were belong to predominant LX4 genotype. Compared to Mass vaccine strain show that homology was far away.Full genome sequence of IBV HN104 and HN091 isolated strains were obtained successfully. Not only full genome sequence but also all genes show higher identity with other IBV isolated strains. Compared to twelve full genome sequence with HN104 and HN091 in homology show that Non-structural gene 3a is the most conservative; 3b is most variable; 5'UTR is much conservative 92.0~99.8% , which plays an important role in initiation replication. 3'UTR is much unconservative 59.1~99.5%.HN091 isolated strain is presumed to come from an natural recombinant strain. recombined with TW2575/98 genotype strain; 3a and 3b genes of HN091 recombined with GX-YL5 ;The HN091 strain was the result of gene recombinant and gene point mutation. HN104 isolated strain show high homology 95% with DY07 that was isolated in Sichuan on 2007. 5'UTR, 3'UTR and 11 genes of HN104 were gone to serch in BLAST and constructed phylogenetic tree , which show that HN104 was evolved from DY07.Full genome, eleven genes and 5'UTR, 3'UTR of fourteen IBV strains were constructed phylogenetic tree. Few genes of full genome of one strain show different relationship inevolution, which is resumed gene point mutation gene recombinant.Henan located in the Central Plains has large poultry magnate and developed transportation. It provided preconditions for the spread of epidemic IB. Study show that many genotypes strains were survival in Henan. Most of IBV isolates were clustered into predominant LX4 genotype. Amplification of full genome HN104 and HN091 was useful to study molecular mechanism of mutation, and construct reverse genetics of IBV.