The Construction Of PR8/PB2-M2e-CD40L Recombinant Influenza Strain And Evaluation Of Its Immune Effect

Avian Influenza Virus,as a low-pathogenic virus,is widely spread worldwide.To the best of our knowledge,Avian Influenza virus infection will cause chicken flocks appear relatively mild clinical symptoms and lead to the secondary infection which has bring huge economic losses to farmers.To date,influenza virus have 18 haemagglutinin(HA)and 11 neuraminidase(NA)subtypes,most subtypes have been detected in wild birds,except for the recently discovered H17N10 and H18N11 viruses,which found only in bats.It is known that the internal genes of H7N9 and H5N1 viruses are from the same genetic lineage,which all originated from the H9N2 virus.On the other hand,there is no deny that the public health and safety issues caused by H9N2 AIV cannot be underestimated.In this study,H9N2 AIV sequence isolated in 2010-2019 was compared with the representative H9N2 AIV sequence uploaded in NCBI,and a phylogenetic tree was constructed;The research has focused on analysising the genetic evolution rules and key amino acid positions of five H9N2 AIV strain isolated in 2018-2019,and defining the evolutionary trend of H9N2 AIV in South China;Two strains were selected as parent strains,and reverse genetic technology was used to rescue the recombinant virus;animal experiments were conducted to evaluate the immunogenicity of the strains;Using the lentiviral system to construct a cell line that stably expresses foreign proteins;Then,an optimization of rescue virus has been carried out on this cell line;Finally,recombinant influenza virus was obtained that can only be propagated on the target cell line,and conduct animal experiments to evaluate the optimization results of candidate vaccine strains.The research results are as follows:1.The mainstream branch of H9 is H9.4.2.5,and the NA gene belongs to the CK/Bei-like lineage,which clarifies the evolutionary trend of H9N2 AIV in southern China.2.Two influenza viruses(rHA/NA-GD37 and rHA/NA-GD38)were rescued by the eight-plasmid system.The HA and NA genes were derived from the parental strains,and the remaining six endogenous fragments were derived from the H1N1 subtype influenza virus;r HA/NA-GD37 was finally selected for animal experiments based on the virus proliferation and physical and chemical properties.3.The animal test results: rHA/NA-GD37 has stimulated chickens to produce higher antibody products in comparison with wild inactivated vaccine GD37.What’s more,lung tissue HE section and detoxification data showed that r HA/NA-GD37 can provide better protection for animals.4.Constructing a cell line MDCK/Flag-PB2 which can stably expressing the foreign fusion protein Flag-PB2 and a p HW2000-PB2(120nt)-M2e-CD40 L plasmid replacing PCAGGS-PB2.5.MDCK/Flag-PB2 cell line was used to rescue the recombinant influenza virus PR8/PB2-M2e-CD40 L.The growth curve of recombinant influenza virus was measured and found that PR8/PB2-M2e-CD40 L could not proliferate on MDCK cells.6.Animals immunized with PR8/PB2-M2e-CD40 L have higher CD40 L antibody levels.After the immune challenge,the detoxification tests were performed on the throat and cloaca swabs.PR8/PB2-M2e-CD40L+ r HA/NA-GD37 mixed immunized animals could not detect the detoxification of H9N2 subtype avian influenza virus from the third day;HE staining of lung tissue also showed that the PR8/PB2-M2e-CD40 L group provides protection to the chicken.This study clarified the evolutionary rules of H9N2 AIV from 2010 to 2019;The parental strains were selected by reverse genetic system construction,and we successfully obtained a recombinant influenza virus PR8/PB2-M2e-CD40 L that only propagated on MDCK/Flag-PB2 cells,which has produced better protective efficacy by nasal immunization.In conclusion,this study has explored a new way to deal with a possible influenza pandemic.