Preliminary Study On The Related Gene Loci Of Influenza Virus Vero Cell Adaptation

The outbreak of influenza virus poses a huge threat to human health and economy.In recent years,the death toll caused by influenza virus infection in the United States has exceeded 14,000.At present,the treatment of influenza virus has limited efficacy,mainly including amantadine,zanamivir and oseltamivir.The increasing number of drug-resistant influenza virus strains has made vaccines the most effective and economical means of controlling influenza outbreaks.At present,the production base of influenza vaccine is mainly chicken embryo,and there are various defects in the production of influenza vaccine from chicken embryo.First of all,it is difficult to supply a large number of chicken embryos in a timely manner during the influenza outbreak.Furthermore,the residual ovalbumin in the flu vaccine produced by chicken embryos is difficult to apply to those who are allergic to ovalbumin.In summary,the production matrix for influenza vaccines is more cell-oriented.At present,the cells that develop influenza vaccine production mainly include MDCK and Vero cells.Although MDCK cells are more sensitive to influenza viruses,they are tumor cells,and their application is still controversial.Vero cells are recognized as a matrix for vaccine production with a high safety factor,and its use as a matrix for influenza vaccine production has become a trend.However,the influenza virus is not sensitive to Vero cells,and it is necessary to use the reassortment of the epidemic strain and the Vero cell high-yield adaptive strain to prepare the production virus.At present,Vero cell adaptation strains are very rare,and research on the genetic locus of Vero cell adaptation of influenza virus is very important for the preparation of Vero cell influenza virus vaccine by reverse genetics.In this study,the H3N2 influenza A virus Vero cell adaptive strain selected in this laboratory was taken as the research object.Gene sequencing was used to find the differential sites of the genes within the four segments of virus PB1,PB2,NP,and PA before and after the virus adapted to Vero cells.The bioinformatics software DNASTAR predicts the effect of gene locus changes on protein secondary structure.After analysis,the gene loci to be studied are selected,including PB1 49,PB1 1069,PB2 407,and PA 1277.For NP fragments,because it is shorter than the previous three gene fragments,NP 301,NP 451,NP 593,NP 647 and NP 850 loci were selected for exploration.Gene fusion is carried out by fusion PCR technology to obtain gene fragments containing mutation sites,and then the target gene fragments are constructed on pHW2000 plasmid based on homologous recombination method.The plasmid containing the mutation site was co-transfected with the other seven plasmids into 293T cells.The 72-hour-transfected supernatant was inoculated into 9-11-day-old chicken embryos,cultured for 72 hours,and positive chicken embryo allantoic fluid was harvested.Under the condition that the rescued virus was confirmed by nucleic acid extraction and sequencing without introducing other mutations,the difference in the proliferation of the rescued virus containing the mutation site and the unmutated virus on Vero cells was compared.The changes of nucleotides PB1 49,NP 593,NP 301,NP 451,NP 850,PA 1277,PB2 407 in the coding frame had no effect on the proliferation of the virus on Vero cells.Influenza Vero cell adaptation may involve more single gene loci or gene mutations.The specific mutation loci and mechanism still need further study.