Establishment Of Indirect ELISA Based On Newcastle Disease Virus V Protein For Differentiating Infected From Vaccinated Chicken

Newcastle disease,caused by Newcastle disease virus(NDV),is an acute,highly contagious avian disease,which presents worldwide and affects chicken and many other species of birds,causing severe economic losses in the poultry sector.Currently,vaccination is the main method of controlling ND.Differentiation of infected from vaccinated animals(DIVA)is a necessary pathway of achieving the full control of viral diseases.However,the common methods of NDV serological diagnosis cannot be used for DIVA.The non-structural protein V is produced during the replication of NDV and is mainly found in host cells infected with the virus,but it is not involved in the assembly of virus particles.Conventional inactivated NDV vaccine is virus particles that lost their infection ability and cannot replicate in cells,so it fails to produce new V proteins.Thus,this study distinguish between the chicken inoculated with NDV vaccines and the chicken infected with wild-type NDV by detecting V protein antibody response levels.The main research contents are as follows:Firstly,since the V protein of NDV is produced by the RNA editing of P gene,P protein and V protein share the same N-terminal region and vary at the C-terminal region.Therefore,in order to avoid the possible cross reaction between the two proteins,recombinant proteins of the unique C-terminal domain of V protein(Vc)were expressed in E.coli prokaryotic expression system to ensure the specificity of the antigen.Then,the recombinant protein Vc obtained in previous step was purified and used as coating antigen of indirect ELISA method for detecting V protein antibody.After multiple optimizations,the optimal ELISA conditions were determined.The optimal antigen concentration was 1.6 ?g/mL.The optimal serum sample dilution was 1:50.The optimal antigen coating method was 4 ?C overnight.The optimal blocking solution was 7.5% skim milk-PBS,and the optimal blocking time was 2 h at 37 ?C.The optimal reaction time for antigen and serum sample was 60 min at 37 ?C.The optimal Goat anti-Chicken IgG/HRP dilution was 1:8 000,and the optimal reaction time for antibody and IgG/HRP was 60 min at 37 ?C.The optimal reaction time for IgG/HRP and TMB substrate is 10 min at room temperature.The cut-off value of this ELISA was determined by detecting sixty NDV negative sera.When the serum samples with S/P ratios of ? 0.29 were considered as positive,those with S/P ratios ? 0.25 were judged to negative,and the others were suspicious.Crossreactivity assay showed this method was of high specificity and had no cross reaction with the positive sera of other avian viruses.In addition,the CV values of intra-plate and interplate repeating assay were all less than 10%,which showed a nice repeatability.Finally,SPF chicken were used for animal experiment.Sera were collected,and V protein antibodies were tested using the established ELISA method described above.(1)None of sera tested positive in the group of chicken inoculated with PBS and the two groups of chicken inoculated with two different inactivated NDV vaccines.(2)Three weeks after immunization with two different inactivated NDV vaccines,the two groups of chicken subsequently challenged with virulent NDV strain.On the 7th,14 th and 21 st days after challenge,the positive rate of V protein antibody was 60%(6/10),80%(8/10),70%(7/10)and 50%(5 /10),80%(8/10),70%(7/10)respectively.After calculation,all these S/P mean values of these two groups were more than 0.29.(3)Only 20%(2/10)and 10%(1/10)of the sera were positive in the two groups of chicken inoculated with two different live attenuated NDV vaccines on the 21 st day after inoculation,but the antibody levels were relatively low.After calculation,the S/P mean values of these two groups were less than 0.25.Above results indicated that there was a significant difference in the levels of V protein antibody response between the groups inoculated with NDV vaccines and the groups infected with virulent NDV strain.Therefore,this method can distinguish between vaccinated chicken and virulent NDV infected chicken at the population level.In summary,an indirect ELISA method for detecting NDV non-structural protein antibodies was successfully established using the recombinant protein Vc in this study,which could distinguish the chicken infected with virulent NDV strain from those chicken inoculated with NDV vaccines at the population level and provide a new potential detection tool for NDV serological diagnosis,antibody detection and epidemiological surveys.It may have a good prospect.