Isolation Of Infectious Bursal Disease Virus And Development Of Detection Methods

Infectious bursal disease is a poultry disease caused by the infectious bursal disease virus(IBDV).Chickens infected with IBDV exhibit bursal atrophy and eventually die,causing a substantial economic loss for the poultry industry.So we need to study the molecular epidemiology and develop rapid and specific detection methods for the prevention of IBD.In this study,we isolated and identified infectious bursal disease viruses from eastern China in recent years and the biological properties of them were analyzed.Then we preparated the monoclonal antibodies(mAbs)against IBDV.Based on the mAbs,sandwich ELISA and colloidal gold immunochromatographic assay for detecting IBDV were established.All of these will give help to the integrative prevention of IBD.Part I:Isolation and characterization of biological properties of infectious bursal disease viruses from eastern China in recent years(2013-2015)To characterize genetic variations of infectious bursal disease viruses(IBDV)in eastern China in recent years,we isolated 7 IBDV strains from IBD outbreaks in vaccinated flocks between 2013 and 2015.The IBDV isolates were named J3,A4,S8,S9,A11,S14,and J17.All the isolates were directly cultured in embryonated chicken eggs.However,there were significant differences in adaptability and virus titers when the isolates were propagated on CEF,DF-1 and DF-EGFP-miR-9 cell cultures.All the isolates were relatively easy to grow on DF-EGFP-miR-9 cell cultures and the titer of A11 reached as high as 1011 TCID50/0.1ml.Clinical signs were observed in six-week-old SPF chickens that were inoculated with each of the isolates,which were passaged 3 times in embryonated chicken eggs.The morbidity was as high as 100%and the mortality was 50%-70%.In comparison,no clinical signs and mortality were observed when SPF chickens were inoculated with each of the isolates that were passaged 20 times on cell cultures.Homology analysis of the complete VP2 sequences of the 7 isolates revealed that the nucleotide and amino acid sequence identities were 97.4%-99.6%,and 98.7%-99.8%,respectively.Phylogenetic analysis showed that the 7 isolates all belonged to serotype I IBDV,and distributed in 3 relatively distant vvIBDV clades.Analysis of critical amino acid sites of VP2 showed that they were same with characteristic amino acids of vvIBDV strains.The isolate J17 had a unique 212N residue and the isolate S14 had a unique 222L residue in the first hydrophilic domain of the highly variable region(HVR),which is different from other attenuated or vvIBDV strains.Our results indicate that vvIBDV is prevalent in vaccinated flocks in eastern China.The strains have different biological characteristics and complex origins,which poses new challenges in the control of IBDV.Part II:Establishment of the hybridomas secreting monoclonal antibodies against infectious bursal disease virusThe splenocyte of BALB/c mice immunized with purified IBDV and recombinant VP2 protein expressed in insect cells were fused with SP2/0 cells.Two hybridomas secreting monoclonal antibodies(mAb)against IBDV,designated as 4G8 and 2C9,were selected with inderect ELISA and cloned by the method of limiting dilution.Monoclonal antibodies secreted from hybridomas 4G8 and 2C9 were identified to be subclass antibodies IgG2a and IgM.ELISA titers of the acsites were 107 and 108,respectively.The specificity test proved that all the monocloal antibodies were specific to IBDV,whereas they did not react with other viruses.Indirect immunoffuorescence assay(IFA)showed that the two mAbs could combine with the natural IBDV specifically.In western blotting,mAb 4G8 could react with IBDV and recombinant VP2 protein expressed in insect cells.Neutralization test indicated that mAb 4G8 had neutralizing ability to IBDV,with the neutralizing titres of 107 in ascites.Part ?:Establishment of the monoclonal antibodies-based sandwich ELISA for detecting infectious bursal disease virusThe purified(mAb)4G8 was labeled with horseradish peroxidase as HRP antibody and the purified(mAb)2C9 was used to capture IBDV antigen,then the double antibody sandwich ELISA was established for detecting IBDV.The sandwich ELISA had no cross-reaction with NDV,IBV,EDSV,AIV H9N2,CEF and normal tissue.The minimum detectable level of the sandwich ELISA was 102 8TCID50.The variation coefficients of the intra-assay and inter-assay were less 10%.The sandwich ELISA was specific,sensitive and stable for the diagnosis and epidemiological survey of IBD.Part IV:Development of the monoclonal antibodies-based colloidal gold immunochromatography strip for the detection of infectious bursal disease virusThe monoclonal antibodies-based colloidal gold immunochromatography strip was developed for the detection of infectious bursal disease virus(IBDV).The(mAb)4G8 conjugated with colloidal gold was used to capture IBDV antigen and(mAb)2C9 to bind colloidal-gold mAb-IBDV complexes at a test(T)line on the nitrocellulose strip.A downstream control(C)line of goat anti-mouse immunoglobulin G(IgG)antibody was used to capture excess free colloidal-gold conjugated 4G8 to validate test performance.The detection results indicated that the strip was specific to IBDV and the detection limits of IBDV was 103 9 TCID50.Repeatability and stability of this method were good.To test clinical samples,the assay had 100%agreement with the sandwich ELISA established by using the same monoclonal antibodies.The monoclonal antibodies-based colloidal gold immunochromatography strip apply to the field diagnosis ofIBD.