Construction And Immunogenicity Of Recombinant Newcastle Disease Virus Expressing Mosaic HA Protein Of H9 Subtype Avian Influenza Virus

Since the end of the 20th century,H9N2 subtype avian influenza virus（AIV）has become the most widely circulating avian influenza subtype in Chinese poultry.Although this subtype of AIV is less pathogenic,it is susceptible to mixed infection with other pathogens,resulting in reduced egg production rates and even death in poultry,causing serious economic losses to the farming industry.Although vaccination of poultry in China has not been interrupted,the H9N2 subtype AIV continues to spread among chickens.Therefore,new H9N2 subtype avian influenza vaccines need to be developed to reduce their losses to the farming industry and the threat to public health security.In this study,the reverse genetic operating system of the Newcastle disease virus（NDV）vaccine strain Mukteswar strain（M strain）was established,and on this basis,the recombinant virus r M-H9M expressing the H9subtype AIV HA mosaic sequence（H9M）and the recombinant virus r M-H9M-3M2e expressing the H9 subtype AIV HA mosaic sequence（H9M）and three M2 protein extracellular tandem（3M2e）were constructed.The biological characteristics and immunogenicity of the study were carried out,and a new H9 subtype avian influenza-Newcastle disease bifurcation vector vaccine was developed to provide technical support for the prevention and control of H9 subtype avian influenza and Newcastle disease.1.Establishment of reverse genetics operating system of M strainIn this experiment,9 pairs of primers were designed based on the Mukteswar genome of the NDV gene type III vaccine strain,9 small fragments of the whole genome were obtained by RT-PCR amplification,and fused into 3 medium fragments H1,H2 and H3 by overlapping PCR,and then the three medium fragments were connected to the vector p ACNR-T7 in turn in an In-fusion seamless way to obtain the recombinant plasmid p AC-Mukteswar（p T7-M）.It is co-transfected with three co-plasmids to BHK-21 cells pre-infected with pox virus for viral rescue to obtain r T7-M recombinant virus.The results showed that the full-length c DNA infectious clone p T7-M of the NDV M strain was successfully obtained,and the recombinant virus r T7-M was obtained,and the biological characteristics were similar to that of the vaccine strain M,indicating that the reverse genetic operating system of the M strain was successfully established.2.Construction of recombinant virus r M-H9M and r M-H9M-3M2eThe HA protein mosaic sequence（H9M）of H9N2 subtype AIV is inserted between the P and M genes of the NDV M strain vector to obtain the recombinant virus r M-H9M.At the same time,3M2e was fused with the C-end of the HN protein of r M-H9M to construct the r M-H9M-3M2e recombinant virus.Indirect immunofluorescence（IFA）and western blot（Western-blot）experimental results show that both recombinant viruses can stably express exogenous proteins.The biological characteristics test results showed that the recombinant virus r M-H9M、r M-H9M-3M2e had similar proliferative characteristics to the parent strain M.The mean time to death（MDT）of the minimum chicken embryo lethal doses of recombinant virus r M-H9M and r M-H9M-3M2e was 64 h and 68 h,respectively,and the brain pathogenic index（ICPI）of 1-day-old SPF chicks was 1.50 and 1.41,respectively,which decreased slightly compared with M strain.The results showed that two strains of recombinant viruses r M-H9M and r M-H9M-3M2e were successfully constructed,which had similar biological characteristics to the parent strain,and the insertion of exogenous genes slightly reduced the virulence of recombinant viruses.3.Immunogenicity evaluation of recombinant virus r M-H9M and r M-H9M-3M2eIn order to evaluate the immunogenicity of recombinant virus r M-H9M and r M-H9M-3M2e,the recombinant virus r M-H9M and r M-H9M-3M2e were immunized to4-week-old SPF chickens by nasal and eye drops,respectively,at an immune dose of10⁶EID₅₀/pc.After 14 days of immunization,the antibody levels of the two groups of experimental chickens were tested.The NDV HI antibody level was above the critical protection value of 4log₂,and the AIV HI antibody level exceeded 7log₂.After 14days of immunization,two H9N2 heterologous strains（DY0602,JM0305）were used to conduct poison tests,and throat swabs and cloaca swabs were collected on the 3th,5th and 7th days after challenged to detect the detoxification of test chickens.The results showed that the positive rates of the 5th day were the largest among the groups,with the positive rates of laryngeal swabs in the DY0602 strain attack group,40%and80%,and the positive rate of laryngeal swabs in the r M-H9M and r M-H9M-3M2e immune groups,and 0%in the laryngeal swabs in the PBS group,while the positivity rate of laryngeal swabs in the PBS group was 100%,and the positivity rate of cloaca swabs was 70%.The positive rates of throat swabs in the JM0305 strain attack group and r M-H9M-3M2e in the two immune groups were 50%and 40%,respectively,while the positive rates of throat swabs in the PBS group were 100%.The above results show that recombinant viruses can effectively reduce the detoxification of experimental chickens.The viral titer measurement results of the positive swab at the same time showed that at the 3 d,the viral titer of the positive swabs in each immune group was significantly lower than that in the PBS group,and in the JM0305 strain challenged group,the viral titers of the positive throat swabs in the r M-H9M and r M-H9M-3M2e immune group were 10^3.25EID₅₀/0.1 m L,10^3.5EID₅₀/0.1 m L,respectively,while the virus titers of the PBS group(10^6.25EID₅₀/0.1m L)were respectively.is 1000times and 562.3 times the titer of the immune group virus,respectively.Therefore,both recombinant virus r M-H9M and r M-H9M-3M2e can induce SPF chickens to produce high levels of antibodies to Newcastle disease and H9 subtype avian influenza,and can significantly reduce the detoxification level.In summary,this study successfully established the reverse genetic operating system platform of the NDV vaccine strain Mukteswar（M）,and based on this,two recombinant viruses r M-H9M and r M-H9M-3M2e were constructed,which can induce the body to produce a higher level of Newcastle disease and H9 subtype avian influenza antibodies after immunization with SPF chickens,and can effectively inhibit the detoxification of AIV,and has a certain cross-protection effect on heterologous H9N2 subtype AIV.Can be used as a candidate for live vaccines for the prevention and control of H9 subtype avian influenza and Newcastle disease.