Isolation And Identification Of Infectious Bursal Disease Virus And Biological Characterization Of The Isolates

Infectious bursal disease (IBD) is a highly immuno-suppressive disease of young chickens of three-to-twelve-weeks old. Infectious bursal disease virus (IBDV) is the etiological agent of the disease. Lymphoid cells, especially B cells, are primary target cell and the lymphoid tissue of the cloacal bursa is most severely affected. The economic importance of this disease is mainly manifested by a severe prolonged immunosuppresssion of chickens infected at an early age. At present, there are different pathotype of IBDV, namely Classical IBDV(cIBDV), variant IBDV(vIBDV) and vvIBDV co-existed in intensively growing poultry in China. So although both live and inactivated vaccines have been developed to control IBD, difficulties may occur in immplementing these vaccines in combination with efficient sanitary measures under field conditions. In order to control IBD more effectively, IBDV from field samples would be isolated and reseasched in time to learned more about the immunogenicity and pathogenicity of the isolates in molecular level.In this study, tissue samples from chickens that were clinically IBD suspected in broiler and layer chicks of 3–12 weeks of age were collected from the provinces of Guangxi, Jiangsu and Hunan in southern China during the years of 2006~2010 were detected for infectious bursal disease virus (IBDV) by using the developed reverse transcriptase polymerase chain reaction (RT-PCR) technique. Viral isolation was performed on the RT-PCR positive samples by using chicken embryo inoculation via chorio-allantoic membrane (CAM) and totally 30 isolates of IBDV were obtained. A set of primers were designed to amplify the vVP2 of the isolates and the PCR products were analyzed by restriction enzyme analysis (REA) with SspI or SacI. The results showed that PCR products of totally 26 isolates could be digested into two fragments of 201bp and 273bp with Ssp I which suggested that they could be vvIBDV. While two isolates, BB0901 could be digested by SacI which suggested that they could be cIBDV. PCR products of sample JS0822 and JS0806 could be digested neither by SacI nor SspI, while HUN0804 could be digested both by SacI and SspI . The results of the study demonstrated that vvIBDV(about 87.6% of the total) were still epidemic in Guangxi, Hunan and Jiangsu at present.In order to assess the molecular characterization of IBDV epidemic in present, nucleotide sequences of the VP2 hypervariable region (HVR) of 30 infectious bursal disease viruses (IBDVs) isolated in Guangxi, Jiangsu and Hunan from 2006 to 2010 were determined. All sequences obtained were aligned and compared with the sequences of reference viruses included vvIBDV strains, cIBDV strains, intermediate- plus virulent IBDV strains, attenuated vaccine strains, and vIBDV(virulent infectious bursal disease virus,vIBDV) strains. The phylogenetic tree was made based on the nucleotide sequences. The results indicated that 30 isolates were classified into two groups according to their characteristic amino acids at critical sites of vVP2: 28 isolates including QX0604, WZ0608, JS0804, HUN0803 (about 93.3%) had conserved putative virulence marker amino acids (aa) at positions 222(A), 249(Q), 254(G), 256(I), 279(D), 284(A), 294(I) and 299(S), the same as vvIBDV, while the strains HUN0804 and BB0901 had conserved putative virulence marker amino acids (aa) at positions 222(P), 249(Q), 254(G), 256(V), 279(N), 284(T), 294(L) and 299(N), the same as attenuated strains. Sequence comparison of the highly variable region (HVR) of the VP2 proteins showed that these vv isolates had 93%～100% identities to European and Asian vv strains at amino acid level, but there are only 91.1%～96.8%, 88.0%～93.7% identities between the 30 isolates and classic strains (CJ801, F52/70, Cu-1wt, Edgar )and attenuated caccine strains(B87(in), FW2512) respectively. For the vv strains NN0603, NN0704, NN07122, QX0601, QX0604, and HP1001, all had some amino acids substitution at the major hydrophilic domains, indicating that new vv stains are evolving. The results from the study demonstrated that the viruses prevailing in Guangxi, Hunan and Jiangsu in the recently 5 years were vvIBDV and their origins were complex.The antigenicity of some isolates might have been drifted.In order to identify the sub-serotype of the IBDV isolates from Anhui, Guangxi, Hunan, Jiangsu, Hainan and Zhejiang provinces between 2000-2010, virus neutralization(VN) tests were performed between 60 strains (58 isolates, two commonly used vacaine stains (B87, FW2512)) and six rabit anti-IBDV serum(including anti-YL052, anti-B87, anti-FW2512, anti-TSC-2(9), anti-040124 and anti-BH11). The sub-serotype of the 58 isolates could be preliminary determined by the nutrolization titers in vero cells, and the virus (JS7, TSC-1(3), HUN0801) were selected out to prepare for rabbit anti-serum since their VN titer were lowest. Then a cross-VN test was carried out on vero cells between hyper-immuned rabit antuserum against the three isoltes respectively, the six anti-serum previously researched and the anti-serum's virus(YL052, B87, FW2512, BH11, TSC-2(9) ,040124, JS7, TSC-1(3), HUN0801) to determined the sub-serotype of the 9 virus. The results showed that HUN0804, YL052, JS0802, JS0822 ect, altogether 56 isolates belong to a subtype, classified as sub-serotypeⅠ, BH11 belongs to another subtype, classified as sub-serotypeⅡ, JS7 belongs to the third separated subtype, classified as sub-serotypeⅢ, while the two commonly used vacaine stains (B87, FW2512) belong to sub-serotypeⅠ.