| Infectious bronchitis(IB)is an acute,highly infectious and contagious viral respiratory disease of chickens.The genome of infectious bronchitis virus(IBV)is prone to mutation and recombination,which can lead to diversified genotypes and complicated serotypes.The cross-protection between serotypes is poor,which bring serious economic losses to poultry industry.The predominant genotypes and serotypes of the IBV epidemic strains have regional differences.Therefore,it is of great practical significance to prevent and control the disease to understand the genetic variants and antigenic changes of the IBV epidemic strains in this region in time.Based on the research of our group,this study continued to follow the study on genotypes and serotypes of Guangxi IBV epidemic strains during 2015-2017,with the purpose of understanding the genetic variation of prevalent strains of IBV in Guangxi in recent years,and providing evidence for the selection and development of IBV new vaccines.Firstly,19 IBV isolates were isolated and identified from diseased chickens suspected of suffering from IB by RT-PCR and chicken embryo inoculation,and the four structural genes S1,E,M and N of the isolates were amplified and sequenced.The similarity,phylogenetic tree,recombination,O-glycosylation sites and selection pressure of 19 IBV isolates were analyzed using the molecular biology software MegAlign,MEGA5.0,RDP4,NetOGlyc 4.0 Server,and Datamonkey,respectively.The sequencing results showed that the lengths of the S1,N,M,and E genes were 1611?1638 bp,1230 bp,675?681 bp,and 327?333 bp,respectively.Compared with the vaccine strain H120,the amino acids of the S1 gene were widely inserted,deleted and replaced,and the mutations were concentrated in the three hypervariable regions of the S1 gene.The insertion and deletion of amino acids were present only in a few positions in the M and E genes,whereas the amino acid substitution phenomenon was present in the N genes.The similarity analysis results showed that the similarities of S1,N,E and M genes was 69.0?100.0%,90.5?100.0%,79.0 ?100.0%,and 90.2 ?100.0%,respectively.The phylogenetic tree of S1 gene showed that 19 IBV isolates were divided into six distinct genotypes,of which 4/91-type and Mass-type were dominant genotypes.Phylogenetic tree analysis of N,M and E genes showed that 19 IBV isolates were divided into four,four and three distinct groups,and the dominant genotypes were LX4-type.and their variations were not completely parallel to that of the S1 gene.The results of recombinant analysis showed that none of the four structural genes of the 19 IBV isolates occured recombination.O-glycosylation analysis site were showed that 19 IBV isolates had no glycosylation sites in the S1 and E genes,In the N gene,only strain GX-NN170829 had a glycosylation site at 415Y.In the M gene,only strain GX-YL150727 had a glycosylation site at 4T.Positive selection pressure analysis results showed that there are multiple positive selection sites in the four structural genes,but there were no same amino acid variation sites for the three analysis models.In sunmmary,the four structural genes of 19 isolates have undergone varying degrees of variation,and the evolution of genes is not completely parallel.Secondly,the serotypes of 19 isolates were identified by neutralization test using seven monovalent antisera representing seven different serotypes identified by this group.The results showed that 19 isolates belonged to six serotypes.Seven isolates(GX-NN150019,GX-YL161022,GX-YL170717,GX-QZ170728,GX-YL170805,GX-NN170825,GX-QZ171023)belonged to serotype 2.Two isolates(GX-YL150028,GX-NN171108)belonged to serum type 3;three isolates(GX-YL150619,GX-YL161015,GX-YL170808)belonged to serum type 4;4 isolates(GX-QZ150024,GX-NN170829,GX-NN170901,GX-LZ160322)belonged to serotype 5.Two isolates(GX-YL150727,GX-LZ170609)belonged to serum type 6,and one isolate GX-NN171125 belonged to serum type 7.Among them,serum type 2 was the dominant epidemic serotype in Guangxi IBV from 2015 to 2017,and it was heterologous to serotypes of the commonly used vaccine strains H120 and 4/91.The results showed that there were still mutiple serotypes of strains in Guangxi,and the predominant serotype was different from the vaccine strains,which may be the reason for the failure of the chicken flock in the field.In addition,the correlation between the serotypes and genotypes based on the S1,E,M,and N genes of 19 IBV isolates was analyzed.The results showed that the coincidence rate between serotype and genotype based on S1 gene was 57.89%(11/19),and the coincidence rates based on N,E and M genes were 42.11%(8/19),26.30%(5/19)and 31.6%(6/19),respectively.Therefore,the genotyping based on the S1 gene was most consistent with the results of serotyping.In summary,the IBVs in Guangxi continued to mutated from 2015 to 2017,and there were mutiple genotypes and serotypes.The dominant genotypes of IBV were 4/91-type and Mass-type,and the dominant serotypes 2 was different from those of the vaccine strains.The results illustrated the reasons for immume failure of chicken flock in the field.This study showed the variation and prevalence of IBV in Guangxi from 2015 to 2017,enriching the epidemiological data of the IBV,and providing a scientific basis for the selection and development of IBV vaccines. |
PDF Full Text Request