Infection And Inflammatory Cytokine Response Induced By Recombinant RSD01 H9N2 AIV On Cells

Avian influenza?AI?is a kind of infectious disease caused by influenza A virus,which seriously endangering poultry health.According to its pathogenicity,it can be divided into highly pathogenic avian influenza and low pathogenic avian influenza.The H9N2 subtype avian influenza virus?AIV?is one of the subtypes of the influenza A virus and belongs to the low pathogenic AIV,but when it is mixed with other pathogens,it can cause high morbidity and mortality in poultry.In addition,H9N2 subtype AIV can be transmitted across species to pigs and humans,and at the same time the?2009?H1N1 new influenza A virus has become widespread in humans and mammals and has been present in pigs.As a genetic"mixer"for influenza virus,pigs can infect both bird flu virus and human influenza virus,which provides favorable conditions for the reorganization of influenza virus.Therefore,in nature,these two subtypes of viruses have a great chance of recombination to produce new viruses and pose potential threats to human health.Studies have shown that the H1N1 influenza virus is highly compatible with the H9N2subtype AIV gene.The two viruses are easily recombined.The recombinant virus is susceptible to infect mice and some of the recombinant virus are significantly more pathogenic than the two parental viruses.Through gene analysis of virulent viruses,the PA gene was derived from the H1N1 influenza virus.After the outbreak of 2009 H1N1 influenza,a genome analysis of the virus revealed that its PA gene was derived from avian influenza virus in North America.And studies have shown that the introduction of PA gene of the human H1N1 influenza virus into the avian polymerase gene can overcome the restriction of avian polymerase in human cells.Therefore,the polymerase PA gene plays an important role in host adaptation and virulence of the virus.Based on this,this study used reverse genetic technology to recombines PA genes of H1N1 subtype swine influenza virus.and H9N2 subtype avian influenza virus to construct a recombinant virus rSD01-PA,whose PA gene was derived from the H1N1 influenza virus.Using human lung adenocarcinoma epithelial cells?A549?and chicken embryo fibroblasts?DF-1?as models,determined the replication of H9N2 parent strain and recombinant virus on A549and DF-1 cells and the expression of related pattern recognition receptors and cytokines by mapping virus growth curves,indirect immunofluorescence,and fluorescent quantitative PCR techniques.This part helps to understand the replication of H9N2 subtype AIV and its recombinant strains on different host cells and the difference of host immune response at different time points.Experiments are mainly divided into the following sections:First,Rescue of Recombinant VirusesBased on the principle of homologous recombination,eight plasmids of SD01 strain and SD07 strain of PA plasmid of SD07 strain were constructed using the primers containing the homologous sequence of the target fragment and the bidirectional transcription expression vector pHW2000,and verified by sequencing.Using the reverse genetic technique,293T cells were co-transfected with 7 plasmids of SD01 and PA plasmid of SD07 to rescue the new recombinant virus named rSD01-PA.The TCID₅₀ of the SD01 and rSD01-PA strains was determined.The results showed that the TCID₅₀ of the SD01 strain was 10^-6.67/0.1mL,while the TCID₅₀ titer of the recombinant virus rSD01-PA strain was 10^-7.39/0.1mL,which was higher than that of the parent strain.The results showed that the infectivity of recombinant strains increased.Second,detection of virus replication ability on A549 cells and DF-1 cellsThe A549 cells and DF-1 cells were infected with the H9N2 parent strain SD01 and the recombinant strain rSD01-PA,respectively,virus-infected cells were collected at 6h,12h,18h,24h,36h,and 48h,and the growth curves of the two viruses on A549 cells and DF-1 cells were plotted by detecting their TCID₅₀.Indirect immunofluorescence was performed to detect the replication of the virus on cells at 12 h and 24 h after virus infection.The results showed that both SD01 and recombinant virus rSD01-PA could effectively replicate on A549 cells.The recombinant virus rSD01-PA had higher virus titer at all time points and was more obvious at12 h,and at the same time point,rSD01-PA also emitted more fluorescence after infecting A549cells.In addition,both strains were also able to replicate effectively on DF-1 cells.With the extension of cultivation time,the virus titer continued to rise and tended to balance after 24 h.The SD01 strain had a higher virus titer than the rSD01-PA strain between 12 h and 24 h after infection.The results in this section showed that both the parental strain and the recombinant virus can effectively replicate on both cells,and the recombinant strain rSD01-PA has a stronger ability to replicate on A549 cells,indicating that the introduction of porcine H1N1 PA gene enhances the replication ability of avian influenza virus on A549 cells.Third,detection of virus induced pattern recognition receptors and cytokines expression.After the A549 cells and DF-1 cells were infected with the H9N2 parent strain SD01 and the recombinant strain rSD01-PA,the pattern recognition receptors and related cytokine expression levels were detected at different time points by qRT-PCR.The results showed that the expression of four pattern recognition receptors and related cytokines could be significantly up-regulated after both strains infected A549 cells within 48h.The expression levels of the pattern recognition receptor TLR-3,TLR-7 and inflammatory cytokines such as IL,IFN and MX induced by rSD01-PA on A549 cells were higher.And the expression level of pattern recognition receptor TLR-3,TLR-7 and IFN-?,MX,OAS and TNF-?induced by rSD01-PA on DF-1 cells was higher.In the process of viral infection of both cells,the antiviral proteins MX and OAS were significantly upregulated,indicating that they play an important role in cell resistance to viral infection.