Immunogenicity And Immune Efficacy Of Recombinant Chimeric NDV Expressing The HA Gene Of H9N2 Subtype AIV

Newcastle disease(ND)caused by Newcastle disease virus(NDV)is a highly contagious and infectious disease.The ND vaccine currently used in China is constructed with the genotype IILaSota strain of NDV,however,the NDV currently circulating in China is the genotype VII strain.The mismatch of genotypes between the prevalent strain and the vaccine strain results in low protection efficacy of the current vaccines.The fusion protein(F)and the hemagglutinin-neuraminidase(HN)of NDV are the primary immunogenic proteins and the determinants for virulence of the virus.In previous studies,we substituted the F and HN genes of the Lasota strain with those of the NDV genotype VII strain to construct a novel chimeric virus,rLaSota-7F/HN.The substituted F gene contains amino acid mutations at the protease cleavage site.Avian influenza caused by avian influenza virus(AIV)subtype H9N2 has caused significant economic losses to the poultry industry of China.AIV subtype H9N2 is a severe threat to public health as it also infects mammals and human.The traditional AIV inactivated vaccines and subunit vaccines mainly induce humoral immune responses,but not mucosal immunity which is critical for AIV prevention.Hemagglutinin(HA)is the primary surface glycoprotein and immunogenic protein of AIV.In the present study,the HA gene of AIV subtype H9N2 was inserted into the genome of rLaSota-7F/HN to construct a recombinant virus rLaSota7-HA.The immunogenicity and protection efficacy of the newly generated virus against AIV subtype H9N2 and NDV were assessed.The thesis contains the following contents:1.The construction and identification of the recombinant chimeric NDV expressing the HA gene of H9N2 subtype avian influenza virus epidemic strainIn order to develop a novel NDV vaccine for the control and prevention of both AIV subtype H9N2 and NDV,the HA gene of AIV subtype H9N2 was inserted into the chimeric NDV rLaSota-7F/HN.The NDV gene-end-like sequence in the HA gene(AGAAAAAA)was modified by nonsense mutation,and the NCR of HN gene of NDV was added to the 5’-end of HA gene.The NCR-HA sequence was inserted between the P and M genes of theLaSota genome to construct a recombinant infectious clone p NDFL-7F/HN-HA.The recombinant NDV rLaSota7-HA was successfully rescued in BHK-21 cells.The results of genome sequencing,western blot and indirect immunofluorescence assay indicated that the HA gene was correctly inserted at the targeting site,and the HA protein stably expressed.The experiment results indicated that the replication kinetics and viral titer of rLaSota7-HA in 10-day-old SPF eggs were similar to the parental LaSota strain.The 50% egg infective dose(EID50)of the 28 th passage of rLaSota7-HA reached 10-8.78/100 μL.The mean death time(MDT)was 132 h,and the intracerebral pathogenicity index(ICPI)and intravenous pathogenicity index(IVPI)were 0.The recombinant chimeric virus rLaSota7-HA constructed in the present study provided a potential vaccine candidate strain for control and prevention of both AIV subtype H9N2 and NDV genotype VII.2.The immunogenicity and protection efficacy evaluation of rLaSota7-HATo evaluate the immunogenicity and protection efficacy of the chimeric virus rLaSota7-HA,a live-attenuated vaccine and an oil-emulsion inactivated vaccine were prepared using the constructed virus.One-week-old SPF chickens were immunized with the prepared vaccines via oculonasal and neck subcutaneous routes.The antibodies against NDV genotype VII and AIV subtype H9N2 were examined by hemagglutination inhibition test.The results indicated that both of the live-attenuated vaccine and oil-emulsion inactivated vaccine could induce specific antibodies against NDV genotype VII and AIV subtype H9N2.Viral challenge tests and examination of viral shedding indicated that both vaccines provided full protection against NDV genotype VII and AIV subtype H9N2.Therefore,the recombinant chimeric virus rLaSota7-HA could be used as a cost-effective and highly efficient vaccine strain for control and prevention of AIV subtype H9N2 and NDV genotype Ⅶ.