Evaluation Of Newcastle Disease Prevention Efficiency Of Simultaneous Expression Of Chicken Granulocyte Monocyte Colony-stimulating Factor And The Hemagglutinin-Neuraminidase Epitope Of Newcastle Disease Virus In A Recombinant Adenovirus

Newcastle disease(ND),caused by the Newcastle disease virus(NDV),is a contagious,life-threatening,transboundary viral disease that affects a wide range of birds,posing a serious threat to poultry production and a technical barrier to trade worldwide.When it comes to the prevention of clinical signs and mortality associated with infection by NDV,vaccination has been very effective.However,recent evidence has proven that more highly virulent NDV genotype VII strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry.Therefore,boosting chickens’ immunological genetic resistance and,therefore,improving the immunological effectiveness of the new-generation genetically matched NDV vaccine to better prevent and control virus infection is urgent.In this study,we engineered human adenovirus type 5 to express chicken granulocyte monocyte colony stimulating factor(Ch GM-CSF)and hemagglutination-neuraminidase(HN)of the NDV genotype VII C22 strain and then evaluated its immunogenicity and ability as a candidate vaccine to prevent and control the NDV infection in vivo and in vitro.1.Packaging of the recombinant adenoviruses that express the Ch GM-CSF and/or HN proteins.Human adenovirus type 5 recombinant vectors that contain either the Ch GM-CSF,HN,or both Ch GM-CSF and HN genes were constructed using polymerase chain reactionbased amplification,enzyme-digestion ligation,homologous recombination,and other related molecular biology techniques.These recombinant adenoviral-constructed plasmids were then transfected into human embryonic kidney(HEK-293A)packaging cells(ATCCCRL1573),and the related recombinant adenoviruses,namely r Adv-Ch GM-CSF,r Adv-HN,and r AdvCh GM-CSF-HN,were successfully rescued and characterized.Also,recovered viruses were purified using a sucrose discontinuous gradient ultracentrifugation technique,and then in vitro genetic stability and safety were verified by infecting HEK-293 A cells and chicken embryo fibroblast(DF-1)cells,respectively,and the results showed that the related adenoviruses remain genetically stable through passage and are safe.2.Determination of biological characteristics of recombinant adenoviruses expressing Ch GM-CSF and HN proteins.The ability of recombinant adenovirus to express related genes was determined by fluorescent quantitative PCR,western blot,indirect immunofluorescent antibody assay,and other related experiments.At the relative m RNA gene expression and protein levels,a significant amount of Ch GM-CSF expression was detected in r Adv-Ch GMCSF-infected cells.A significant amount of NDV HN protein expression was detected in r AdvHN infected cells,and a significant amount of Ch GM-CSF and HN protein expression were detected in r Adv-Ch GM-CSF-HN infected cells at the same time.The related adenoviruses were treated at different temperatures,and their thermostability was determined.It was found that,compared with wt Adv,the recombinant adenoviruses expressing Ch GM-CSF and/or HN proteins did not change the thermostability of the recovered viruses.Further,after the DF-1cells were infected with the proposed recombinant adenoviruses,the RNA of the infected cells was extracted,and the expression levels of interferon,interleukin,and other related cytokines were detected by fluorescent quantitative PCR.It was discovered that r Adv-Ch GM-CSF-HN was able to stimulate the infected cells to express a significant amount of alpha interferon(IFN-α),IFN-β,IFN-γ,interleukin-1β(IL-1β),IL-2,IL-16,IL-18,and IL-22,which were dramatically increased in vitro.3.Determination of the ability of the recombinant adenoviruses expressing Ch GM-CSF and/or HN proteins to prevent and control NDV infection in chickens.The healthy chickens without antibodies to Newcastle disease were immunized with the recombinant adenoviruses obtained above by intramuscular injection,and the antibodies and cytokines of the immunized chickens were regularly measured.It was found that r Adv-Ch GM-CSF-HN and r Adv-HN immunized groups could produce antibodies against NDV,while r Adv-Ch GM-CSF and wt Adv inoculated groups did not produce specific antibodies against NDV,and compared with r Adv-HN and Newcastle disease La Sota in the vaccine-immunized control group,the antibody level produced by the r Adv-Ch GM-CSF-HN-immunized group was the highest.Moreover,the cytokines of immunized chickens were measured by fluorescent quantitative PCR,and it was found that,similarly to the above in vitro infection,r Adv-Ch GM-CSF-HN immunized chickens expressed a significant amount of alpha interferon(IFN-α),IFN-β,IFN-γ,interleukin-1β(IL-1β),IL-2,IL-16,IL-18,and IL-22,as well as other cytokines.After the booster immunization,the chickens were challenged with the virulent NDV genotype VII C22 strain.Following the challenge,clinical symptoms,survival rates,viral shedding,tissue viral loads detection,and pathological tissue section observation were used to evaluate the effectiveness of the vaccinations in preventing and controlling the disease.It was found that the r Adv-Ch GM-CSF-CSF and wt Adv immunized chickens that did not express the HN protein showed obvious clinical symptoms and 100% died,and the r Adv-Ch GM-CSF-HN and r Adv-HN immunized chickens that expressed the HN protein all survived 100%.Compared to the r Adv-HN vaccinated group,the r Adv-Ch GM-CSF-HN immunization group is able to effectively prevent and control viral shedding,tissue viral loads,and histopathological changes.The above experimental findings show that Ch GM-CSF has potential for application as a biological immune adjuvant with the objective of improving the immunogenicity of the Newcastle disease vaccine and providing better prevention and control of NDV infection.So,the antiviral activity of Ch GM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly.Our advance innovation in the recombinant NDV vaccine concepts concluded that poultry health can be improved by boosting chickens’ immunological genetic resistance by combining Ch GM-CSF with a field-circulating strain epitope via live adenovirus type 5 expression,which could lead to the development of a safe,genotype-matched,promising universal transgenic vaccine that could eradicate the NDV disease globally,thereby reducing poverty and food insecurity.