Molecular Characterization Of Infectious Bronchitis Virus Isolated In Shandong(China) Between 2006 And 2008

Infectious bronchitis is currently one of the highly contagious respiratory diseases in chickens caused by infectious bronchitis virus (IBV) which is a member of coronavirus. IBV mainly causes damages in respiratory tissue, reproductive system and kidney, and results in decreased both egg production and quality and poor performance. Initially,IB was recognized primarily as a disease of young chicks; however, it was observed to be common in semimature and laying flocks. Infectious bronchitis is of major economic importance because it causes poor weight gain and feed efficiency with often being a component of mixed infections that produce airsacculitis that may result in condemnations at processing of broiler, and being a cause of egg-production and egg-quality declines. The highly transmissible nature of the disease, and the occurrence of multiple serotypes of infectious bronchitis virus(IBV) have sophisticated and increased the cost of attempts to prevent the disease by immunization. 1BV virion contains three major virus-specific proteins: the spike (S) glycoproteins, the membrane (M) glycoproteins and the internal nucleocapsid (N) protein. The S protein comprises two glycopolypeptides, S1 and S2. Hemagglutination-inhibitin(HI) antibodies are induced by Sl.The Biological characteristics of 16 avian infectious bronchitis viruses (IBVs) were isolated from outbreaks in chickens in six cities of Shandong province between 2006 and 2008 were analyzed. In order to trace the source of domestic IBV isolates, to determine IBV genotypes exist in Shandong, and the phylogenetic relationships between Shandong isolates and reference strains.Typical signs including dwarfing, stunting, curling and death of embryo were observed in the second to fifth passages when each of the 16 Shandong 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 16 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 above results elementarily suggested that the 16 viruses isolated in this study were avian infectious bronchitis viruses.These Shandong IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Eleven of fourteen isolates were similar to the LX4 RFLP pattern, which is a common pattern in China. The RFLP patterns for 2 of 16 isolates corresponded to the patterns of IBV Massachusetts strains. One isolate showed variant RFLP pattern.Phylogenic analysis based on entire S1 gene sequences showed that thirteen isolates clustered together with LX4-type strains, having 94.5-99.7% nucleotide identity with LX4 strain, and shared no more than 80.1% amino acid sequence identities with H120 vaccine strains. which may be responsible for frequent outbreaks of IB in vaccinated flocks in Shandong; Two isolates SDTA06111 and SDJY0701 had close sequence relationship with LX4 strain and belonged to the same cluster based on N gene sequences. and two isolates had higher N gene acid identity with each other. In contrast, phylogenic analysis of more conserved S1 gene sequences revealed different clustering. Isolate SDWF0608 is of different cluster in the analysis of N and S1 gene. In addition, SDYT0605 had the closest relationship with Mass-type vaccines H120 in the analysis of N and S1 gene. It demonstrated that isolate SDYT0605 formed a"novel"Variant and might emerge by selection pressure. SDTA06111 ,SDJY0701 and SDWF0608 isolates could happen recombination events. Moreover, The results showed that in the S1 protein gene sequences, nucleotide sequence alignments revealed extensive insertion and deletion in this study, whereas the N gene sequences displayed mostly point mutations compared to published sequence of H120 vaccine strain.Thus, taking into account these findings 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 Shandong.