Molecular Epidemiology Of The Infectious Bronchitis Virus Isolated In Northeast Of China Between 2006 And 2007

The Biological characteristics of nine avian infectious bronchitis viruses (IBV) were isolated from outbreaks in chickens in the northeast provinces of China between 2006 and 2007 were analyzed. Structural protein genes were cloned, sequenced and compared with IBV reference strains. In order to trace the source of northeast provinces IBV isolates, to determine IBV genotypes exist in northeast provinces and the phylogenetic relationships between northeast provinces isolates and reference strains.Typical signs including stunting, curling and death of embryo were observed in the sixth to tenth passages when each of the nine isolates was inoculated into 9-day-old to 11-day-old specific-pathogen-free (SPF) chicken embryos. Furthermore, the nine isolates could not hemagglutinates chicken red blood cells. Diagnoses based on electron microscopy examination performed on allantoic fluids of different passages showed all nine 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.). Acoording to the NDV interference test all the nine isolates had obvious inhibitory effect to the NDV La Sota strain The above results elementarily suggested that the nine viruses isolated in this study were avian infectious bronchitis viruses.The Structural protein genes which were the most various in IBV genome of the nine isolates were amplified by reverese transcriptase-polymerase chain reaction and they were proved to be IBV by cloning, sequencing and analysis with comparison of other strains such as the H120 which is a reference vaccine strain, all the results showed that all the nine strains isolated from the northeast provinces were IBV.Phylogenic analysis based on S1 gene sequences showed that nine isolates have 76.1% to 99.4% nucleotide identity from each other. Among HLJ0605,HLJ0708,JL0712,LN071001 and TL0608 the S1 gene nucleotide identity were from 96.9% to 97.9%; and HLJ0751, HLJ0752, LN071002, MDJ0610 had S1 gene nucleotide identity from 96.9% to 99.5%. HLJ0752, MDJ0610 and LN071002 had high S1 gene nucleotide identity: between HLJ0752 and MDJ0610 identity were 99.4%; HLJ0752 and LN071002 had 99.5% nucleotide identiy. Between MDJ0610 and LN071002 the identity was about 98.8%. But among HLJ0605, HLJ0708, JL0712, LN071001, TL0608 and HLJ0751, HLJ0752, LN071002, MDJ0610 the nucleotide identity was low from 76.1 % to 79.3%.Acoording to the the results of the serum neutralization test and the phylogenetic tree, the conclusion was as follows: the sequence identity and the phylogenetic relationships between the genes derived from the 9 isolates and other 38 reference strains indicated that IBV isolates of China and world wide were branched into eight genetic clusters. IBV isolates in northeast provinces were classified into 1,4, 8 genetic clusters.Thus, it can be demonstrated that not only point mutations, insertions and deletions but also a recombination events having contributed to the genetic diversity and emergency of IBV variants in northeast provinces in China, that are the main reason causing the variation of the IBV strains.