The Genotyping Of IBDV Epidemic Strains And The Genomic Analysis Of Representative Strains

Infectious bursal disease (IBD) is a highly infectious, immunosuppressivedisease of young chickens of three-to-twelve-weeks old. Infectious bursal diseasevirus (IBDV) is the etiological agent of the disease. The immunosuppressioncaused by IBDV could result in the high susceptibility to other disease and lowerlevel of immune response to vaccines, such as new castle disease and avianinfluenza in the flocks. In order to expand our understanding of the molecularepidemiology of IBDV in southern China, and present sufficient information forthe effectively control of IBD, the present study was carried out.Firstly, two pairs of primers was designed according to a reference IBDVfrom GenBank (CEF94) for amplifying the VP1-b (767bp) fragment in B-segmentby RT-PCR from91IBDV field strains isolated between2000-2012in southernChina. The homology analysis and the phylogenetic analysis of the VP1-bsequences of the isolates were carried out. The VP1-b of the isolates were alsocompared to the VP2hypervariable region (vVP2) sequences of the correspondingisolates. The results showed that the homology between all isolates was85.9~100%and89~100%in nucleotides and amino acid level respectively.91isolates showed87.1-99.2%identity to the reference vvIBDV strains and42out of91strains shared higher homology with classical strain (identities of96.1-100%).In the phylogenetic tree, all the IBDVs were grouped into4large clusters, noisolates grouped with vvIBDV, consistent with homology analysis. These resultssuggested that there were genetic diversity in VP1among the IBDV isolates in thepast12years in southern China. When the results from VP1-b were compared tothe phylogenetic tree based on the vVP2, only13/91isolates kept the same geneticrelatives in the segments A-and B-defined consensus tree, whereas the majority ofthe isolates (85.71%,78/91) were identified to be natural reassortment strains. Based on the origin of each segment, at least6types of reassortment IBDVsprevailed in southern China:24strains (including BH11) showed as VP1-b fromclassical strain, vVP2from vvIBDV strain;26strains (including JS7) showed asVP1-b from the002-73similar strain, vVP2from vvIBDV strain;18strains(including NN1172) showed as VP1-b from HLJ-0504similar strain, vVP2fromvvIBDV strain;5strains (including GD10111) showed as VP1-b from classicalstrain, vVP2from the Classical strain;YL052showed as VP1-b from the002-73similar strain, vVP2from the Classical strain;4strains (including NN1005)showed as VP1-b from unidentified origin, vVP2from vvIBDV strain. Based onthe reassortment and the type of reassortant,91isolates were classified into7different genotypes. The results suggested that genomic segment exchangebetween viruses could be the most common means of evolution of IBDV in thefield, and the genotype Ⅱ, Ⅲ and Ⅳ as the main prevalent strains in southernChina in the past12years.Secondly, based on the genotyping results derived from the VP1-b and vVP2sequence analysis,7representative genotype isolates (NN040124, NN1005, BH11,JS7, NN1005, NN1172and YL052) were selected out for whole genome analysis.In this study, A-segment (64bp-3171bp) and B-segment (12bp-2805bp) of the7representative strains and1vaccine strains were successfully obtained. The aminoacid analysis showed that isolates BH11, JS7, NN1005, and NN1172had similarcharacteristic amino acid sites to the reference vvIBDV strains in A-segment;however, isolates NN040124, YL052, and GD10111not only had similarcharacteristic amino acid sites with the reference vvIBDV strains but also similarwith the reference attenuated strains in A-segment, while different strains differedin specific sites to the reference strains. In addition,7isolates had specific aminoacid sequence which didn’t show in reference strains (including classic strains,vvIBDV strains and attenuated strains), but none of these mutations located in thefunction domain of segment A or B. These results suggested that the epidemicIBDV strains remains in constant evolution. The homology and the phylogeneticanalysis of segment A and B of the isolates indicated that7isolates were genomicsegment reassortant, consistent with the results from partial sequence (VP1-b andvVP2) analysis. However, the type of reassortant of the isolates NN040124, YL052and JS7were different: NN040124was more close to vvIBDV strains in segmentA, whose vVP2was belonging to Classical IBDV strains; YL052and JS7strainswere not similar to any reference strains in segment B, but similar to002-73strain based on VP1-b. We proposed that these inconsistent results between wholegenomes and partial sequences analysis might suggested that the homologousrecombination event would have happened in these isolates.This is the first report which analyzed91field strains isolated from southernChina in the latest12years based on the partial sequences (VP1-b and vVP2) andwhole genome segments analysis. The molecular epidemiological data from thisstudy would help to improve IBD prevention, especially new type IBD vaccineresearches.