Construction Of Recombinant Chimeric NDV Expressing HA Gene Of H9 Subtype Avian Influenza Virus And Evaluation Of Its Immune Efficacy

The H9 subtype avian influenza virus(Avian Influenza Virus,AIV)was widespread in our country,and its infection in poultry was more common.H9 subtype AIV infection has led significant economic losses due to a drop of egg production and increased mortality while con-infection with other pathogens.Traditional inactivated vaccines can provide clinical protection for H9 subtype AIV,but they cannot completely prevent the virus shedding from the vaccinated chickens.Traditional inactivated vaccine immunization methods are time-and labor-consuming.With the development of poultry industry in China,new vaccines that are easy to operate and suitable for large-scale immunization have become the trend in future poultry vaccine research and application.In order to develop new and high-efficiency H9 subtype avian influenza vaccine that meets the requirement of intensive breeding,in this study,chimeric Newcastle disease virus AI4-2FHN which can overcome the interference of Newcastle disease maternal antibody,was used as a vector to express the HA gene of the prevalent H9 AIV strain.The new vaccine strains generated in this study were safety,easy production and application,and can induce effective immune efficacy in chickens.1.Construction of recombinant chimeric NDV expressing HA gene of H9 subtype AIVIn order to develop a new live vaccine of H9N2 subtype,five recombinant full-length cDNA clones were constructed by inserting different modified H9N2 HA genes between the P and M genes in the cDNA clone of chimeric NDV(AI4-2FHN strain).The complete HA gene open reading frame was inserted between the P and M genes resulting in pNDV/AI4-2FHN-292HA;the extracellular region of HA gene was inserted resulting in pNDV/AI4-2FHN-HAG;the extracellular region of HA gene fsed with the transmembrane and the intracellular region from the F gene of NDV was inserted resulting in pNDV/AI4-2FHN-HAF;the HA gene with the non-coding region from the M and F genes of NDV was inserted,respectively resulting in pNDV/AI4-2FHN-HAMU and pNDV/AI4-2FHN-HAFU.The 5 recombinant viruses were rescued by co-transfection of full-length cDNA with three helper plasmids in BSR cells,and the inserted genes were confirmed by RT-PCR and sequencing.The biological properties of the five recombinant viruses were tested.The results showed that the titer of the virus-positive allantoic fluid reached 8log2,MDT>120h,and the ICPI were 0,which indicated that the rescued viruses replicated efficiently in SPF embryonated chicken eggs and were all lentogenic.2.Evaluation of genetic stability and immune efficacy of recombinant chimeric NDVsFive recombinant viruses(AI4-2FHN-292HA,AI4-2FHN-HAMU,AI4-2FHN-HAFU,AI4-2FHN-HAG,AI4-2FHN-HAF)generated above were continuously passaged for 10th generation in chicken embryos.The biological properties of the 2th,5th,and 8th passages showed that the rescued viruses could replicated efficiently and were safe to chickens.Results of RT-PCR,Western blot and IFA revealed that the HA protein were expressed stably in recombinant viruses during the passage process.High vaccination dose of recombinant viruses were administrated to SPF chickens,and all the chickens displayed no any clinical symptoms post-infection,which indicated that the recombinant viruses were safety to the chickens.The results of the immune efficacy showed that the five recombinant viruses can effectively induce H9N2 AIV-specific HI antibodies in SPF chickens and significantly prevent the virus shedding post-challenge.Additionally,the immune efficacy of the recombinant viruses in commercial chickens was also evaluated.The commercial chickens were immunized with the recombinant viruses firstly at 2-week-old,and were vaccinated with the H9 AIV inactivated vaccine 3 weeks later.After the boost vaccination,the specific antibodies against H9 AIV increased quickly in all recombinant virus vaccinated groups,especially the chickens vaccinated with AI4-2FHN-HAG.The immunization method of recombinant live virus plus inactivated vaccine can promote the generation of H9 AIV-specific antibody quickly,and can significantly reduce the amount of virus shedding.