Genotyping And Serotyping Of Infectious Bronchitis Viruses Isolated In Shandong Province Between 2006 And 2010

Ninteen strains of infectious bronchitis virus in Shandong province were isolated and identified between 2006 and 2010. In order to trace the source of IBV isolates and determine IBV genotypes exist in Shandong province, the S1, N, and M gene of these strains were sequenced and phylogenetically analyzed with the published strains. Vaccine strains H120,MA5,491 and some isolates were prepared sera by use of special free chicken. Cross neutralization tests were also took and antigen relatedness values were calcuLated to identify its serotypes. In order to study the relationship between serotypes and genotypes, S1 gene were also analyzed with the results of cross neutralization tests.Suspected diseased chicken tissues treated by conventional methods and were inoculated into 9-day-old to 11-day-old specific-pathogen-free (SPF) chicken embryos, 96h later, collection of chick embryo allantoic fluid , and observed lesions of the remaining embryo at 18 days. The first generation of inocuLation showed no or a small number of chick embryo growth inhibition. Typical signs including dwarfing, stunting, curling and death of embryo were observed in the second to fifth passages. This isolates also can interfere the formation of NDV hemagglutination. Diagnoses based on electron microscopy examination performed on allantoic fluids showed all 19 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 were detected. The above results elentarily suggested that the 19 viruses isolated in this study were avian infectious bronchitis viruses.With vaccine strain H120 as a reference, point mutations, amino acid substitution and insertion in the S1 protein can be observed at many positions. N gene have no base deletion and insertion, there is only point mutations of nucleotide and amino acid substitution. M gene of the majority isolate strains only have a small number of mutations and amino acid substitution except the isolate CK/CH/SD09/005 has three bases insertion.Phylogenetic analysis indicated that the S1, N and M genes of thirteen isolates were genetically relatively parallel and closely related to strain LX4. However, Genetic recombination may have happened in three isolates SDTA06111, SDWF0608 and CK/CH/SD09/005, as their S1, N and M genes belong to different evolutionary branch. The resuLts showed that point mutation, insertion and recombination may have contributed to the emergence of the variant IBV strains at the immune pressure.The neutralization tests result showed that SDWF0608, SDLY0612, SDLY0701 with the vaccine strains H120 belong to the same subtypes of Mass serotypes, the other five isolates with H120 and 491 do not belong to the same serotype, the antigen relatedness values less than 25% (belonging to different serotypes). The results from the perspective of humoral immunity in chickens immune to explain the clinical H120 or 491 IBV vaccine is still frequent cause of the infection, but also prompted IBV strains present muLtiple serotypes in Shandong Province. Based on the type of serum of these epidemic strains, multivalent vaccine must be produced.S1 gene sequence with antigenic relatedness results show that the H120 and strain SDTA06111were clustered into gene I, and the S1 gene nucleotide sequence homologies were 99.3%, but the R between the two strains only 18% ; with the H120 strain of SDLY0612 highestantigen relatedness value (70.71%), but both S1 gene sequence homology of only 78.2%, indicating that combined with S1 gene sequence analysis, their resuLts can not be completely correlation, IBV can lead to neutralizing epitope changes by a small number of mutations , resuLting in a new serotype.In summary, the IBV gene mutation, insertion, deletion and recombination continue to generate new serotypes, resuLting in the production of vaccine failure, we must strengthen the monitoring of epidemic strains of IBV and take including biosafety, including the comprehensive prevention and control measures can effectively prevent the occurrence of IB.