Recovery And Evaluation Of Immune Responses Induced By A Recombinant Newcastle Disease Virus Expressing Fowl Adenoviruses Antigen Fiber2

Since 2015,domestic poultry populations have been increasingly changing their Hepatitis and Hydropericardium Syndrome(HHS)due to the infection of FAdV.HHS mainly occurs in broilers aged 3-5 weeks and occasionally occurs in layers aged 10-20 weeks,causing serious economic losses to domestic poultry industry.Epidemiological investigation showed that the prevalent strain of FAdV in domestic chicken population was mainly highly pathogenic FAdV-4.Although partially commercialized FAdV inactivated vaccine and attenuated live vaccine have been available in foreign markets,they are expensive and difficult to be promoted in China.At present,a number of domestic patent applications related to FAdV-4 vaccine have been successful,including various inactivated vaccine,genetic engineering subunit vaccine and DNA vaccine,but there is no commercial vaccine for FAdV-4.In this study,a recombinant plasmid expressing the FAdV-4 Fiber2 antigen gene was constructed and virus rescue was performed using reverse genetic manipulation with the attenuated genotype ? Newcastle disease virus(NDV)strain(MG7)as the vector(its F protein cleavage site mutated from the original base sequence to the cleavage site of the LaSota vaccine strain F protein).Then chicken embryos through testing the mean death time(MDT),1 day-old SPF chicks Intracerebral pathogenicity index(ICPI)and 6 weeks of SPF chicken Intravennous pathogenicity index(IVPI),chicken embryos batches strains sequencing validation and replicate the level in BHK-21 cells,etc.,for the successful rescue of the virulence of the virus,genetic stability and biological activity,such as growth characteristics were discussed.At the same time,an indirect ELISA method for FAdV antibody detection was established using Fiber2 protein as the antigen.Finally,the successfully saved virus strain,commercial attenuated LaSota vaccine strain and subunit Fiber2 protein were used as vaccines to immunize SPF chickens and evaluate their immune effects.The results showed that a recombinant strain named rNDV-Fiber2 expressing the Fiber2 antigen gene with NDV vector was successfully rescued.MDT of rNDV-Fiber2?120 h,both ICPI and IVPI were o,and no mutation occurred in chicken embryo.The virus replication level on BHK-21 cells was consistent with that of the parent virus rNDV.In the indirect ELISA method established with the expressed Fiber2 protein as the coated antigen,when the S/P value ? 0.20,the serum sample was positive.When the S/P value of serum sample is<0.20,it is a FAdV negative serum sample.After immunization with rNDV-Fiber2 strain,we were able to induce the body to produce NDV-specific HI antibody,neutralizing antibody and successfully stimulate the IgA mucosal immune response,but not FAdV-specific antibody.After immunization,the proportion of CD8+ and CD4+ T lymphocyte subsets in peripheral blood lymphocytes(PBMCs)increased significantly at 3 d and 5 d.After immunization 3 weeks Challenge experiments show that:the genotype ?strong NDV strains NDV97 experiment,rNDV-Fiber2 vaccine group protection ratio reached 100%,detoxification did not happen,and all the organs are not detected;The LaSota vaccine group showed partial detoxification and organ toxicity.In the challenge test with the highly pathogenic FAdV-4 strain,the rNDV-Fiber2 vaccine group failed to protect the attack of FAdV?4.As the control group,all the immunized chickens died and the main organs were dissected and severe lesions were found.Detoxification was observed with pericardial effusion,hepatorenal enlargement,etc.The protective rate of both the 50 ?g Fiber2 and 100 ?g Fiber2 vaccine groups was 100%,and no FAdV detoxification or toxicity was detected,and no obvious lesions were observed in the viscera at necroptomies.The results showed that the rNDV-Fiber2 had similar biological characteristics with MG7 strains,both with weak virulence characteristics,and could be expressed stably in the subsequent passage process.Indirect ELISA method was established with Fiber2 protein as the coated antigen,with good specificity,high sensitivity,stable and reliable response results,providing a reliable method for the subsequent detection of FAdV antibody level after immunization.After immunization with the rNDV-Fiber2 vaccine,it stimulated the body to produce a comprehensive humoral,cellular,and mucosal immune response,providing 100%protection against virulent NDV97 and inhibiting NDV replication in vivo.Although Fiber2 antibody could not be induced and the protection against FAdV-4,both 50?g Fiber2 and 100 ?g Fiber2 proteins obtained 100%protection.Therefore,whether rNDV-Fiber2 can be used as genetic engineering dual vaccine for prevention and control of ND and HHS is urgent to be further tested.