| The chicken flocks were infected by infectious bronchitis virus (IBV), which brought greatly economic losses. There are differences among the epidemic strains in different countries and regions. Therefore, grasping the genetic and antigenic changes of variants is very important to the prevention and control of IBV as well as vaccine development. In order to further understand the genetic variation of IBV epidemic strains and provide the basis for research and development of new vaccines in Guangxi in recent years, the gene sequence analysis and serological identification of IBV isolates during 2012-2013 were carried out.Firstly, S-N gene regions (including SI, S2,3a,3b, E, M,5a,5b and N genes) in the 3'terminal of genome of 15 IBV isolates, which were isolated from chickens with immunoprophylaxis defeat in Guangxi, were amplified by RT-PCR and sequenced, Similarity comparison, phylogenetic tree, recombination, O-glycosylation site and positively selected sites analysis were conducted. The results showed that the length of S-N gene regions were 6767 bp?6939 bp. Compared with H120 and reference strains at home and abroad, S1 genes of most isolates occurred extensively amino acid deletion, substitution and insertion, but the other eight genes just had amino acid substitution. The amino acid phylogenetic tree showed that not all genes were parallel. Recombination event was detected in S1 gene, which was a recombinant between vaccine strain 4/91 and isolate LX4-like. O-glycosylation site analysis revealed that N genes of all 15 IBV strains had 21 to 28 glycosylation sites and were more than other genes. In addttion, positive pressure testing result showed that S1 protein had a positive site in aa308. The above results indicated that nine genes of 15 IBV isolates existed diversity variations.Secondly, the TOC-ID50 of 15 IBV strains during the years of 2012-2013 was measured. The 15 isolates were used to run virus neutralization tests on tracheal organ culture with the 7 monovalent antisera which previously identified by our research group. The results showed that 15 IBV strains were divided into seven different serotypes. GX-NN130048, GX-YL13080630, GX-YL130806200 and GX-NN1300-59 belonged to serotype 1; GX-NN 120091 and GX-QZ131126 belonged to serotype 2; GX-YL 130025 belonged to serotype 3; GX-NN 120079 belonged to serotype 4; GX-NN 120084, GX-NN 120089 and GX-QZ 130065 belonged to serotype 5; GX-NN130003, GX-QZ130064 and GX-NN 130067 belonged to serotype 6; GX-NN130021 belonged to serotype 7. And the serotype analysis revealed the serotype of recombinant strain GX-NN 130048 was different from vaccine strains H120 and 4/91. The results demonstrate that multiple serotypes still exist in Guangxi.What's more, the relationship between the four different genes'(S1, E, M and N) genotyping and serotyping of 15 IBV isolates during the years of 2012 to 2013 was analyzed in the study. We found that genotyping based on the sequence of S1 gene matched the serotyping in 66.7% (10/15), but the others were all 46.7%(7/15). Therefore, genotyping based on the sequence of S1 gene had a relatively better correlationship with serotyping than other genes.The results of the present study demonstrated that not only genotype but also serotype of IBV isolates had distinct variation in the epidemic strains. What's more, recombination event existed between vaccine strain and predominant epidemic strains. These may be the main causes of vaccine break in the field. Our study provides a good basic for understanding of molecular mechanisms of IBV variation and development of new vaccines. |
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