| Avian influnenza virus(AIV) is type A influenza virus which isolated from avian hosts, including many subtypes. Currently, AIV was isolated routinely and propagated in specific pathogen free embryonated chicken eggs(ECEs). The use of ECEs tends to be inefficient, high energy consumption and exogenous virus pollution inevitably. Thus, application of cell culture technology in large scale bioreactor will be an alternative road which avoid above disadvantages and further upgrading vaccine manufacturing technology.DF-1 cell line is a chicken-origin fibroblast cell and it was assumed to be a better substitute of ECEs. However, the instablility in virus proliferation and lower antigen production of this cell line compared to ECEs limitted its application in industrial production. Consequently, this study was conducted primarily to reform DF-1 cell line using genetic modification technique in order to establish a stable cell strain which can be passaged persistently and could promote the replication of AIV.In this study, the kinetics of replication of highly pathogenic(H5N1) compared with low pathogenic(H9N2) avian influenza viruses were measured and compared. Besides, 10 innate immune-related genes including IFN-α, IFN-β, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST and KHSRP were analyzed and compared at different time points using real-time PCR method in immortal DF-1 cells following infection by both AIVs. Potential antiviral function of these selected genes were also analysed. Based on above results, mi RNA-mediated and levirus- mediated RNAi were used respectively to knockdown or silence expression of IFNAR1 and IFNAR2 genes and further analysis the influence of AIV replication following knockdown or silencing of both genes. The cell strains were screened accompanied by silencing of target genes. These cell strains could improve replication titer of avain influenza virus.The results were as follow: 1. DF-1 supported the replication of both H5N1 and H9N2 viruses and presented different replication ability. H5N1 virus replicated more efficiently than H9N2 virus. Moreover, cells infected by either virus overall exhibited a similar expression profile for three ISGs(Mx1, OASL, and ISG12) and SST gene which up-reglutaed at 6 hpi or 9 hpi and steadily rising until 15 hours post infection. In terms of the differential expression of IFN-α and IFN-β genes, we found that both gene expressions were not induced significantly at early stage(3, 6, and 9 hpi) in virus-infected cells. However, a significant upregulation of IFN-α and IFN-β gene expression was observed in cells infected by both viruses at 12 hpi or 15 hpi. This result indicated a strong ability of both viruses in suppression of IFN genes expression at the early stage of virus replication. IFIT5 and USP18 gene expression presented different gene expression pattern from other genes which could distinguish the two different viruses infection in DF-1 cells. There was a continuous upward increase for IFIT5 and USP18 genes until 12 hpi followed by a significant decline at 15 hpi in H5N1 virus infection. In contrast, a steady rise for both gene expressions at various time points was observed in DF-1 cells infected by H9N2 virus. However, an exception to above gene expression pattern, two immune-regulatory genes(IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly upregulated in DF-1 cells infected with low pathogenic H9N2 infection with a dynamic up-regulation at 3 hpi or 6 hpi in DF-1 cells. The host response observed in this study was subtype-dependent. Further comparison between the two viruses demonstrated that KHSRP and IRF7 genes in H9N2 virus infected DF-1 cells displayed significantly high expression than their expression levels in H5N1-infected cells. This study confirmed immune-related genes were responsive to AIV infection and futher analysed their potential antiviral function, which offers new insights to the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtype or strains of avian influenza A virus.2. The most effective plasmids including MR-A3 for IFNAR1 knockdown, MR-B2 and MR-B3 for IFNAR2 knockdown were selected using real-time PCR method. Transfection of effective miRNA knockdowned mRNA expression of both IFN-α and IFN-β genes. However, significant upregulation of gene expressions including Mx1, OASL and IRF7 were induced in DF-1 cells transfected by MR-B2 plasmid. And the transfected cells improved AIV replication which suggested activation ofⅠIFNs siginal pathway(changes of Mx1, OASL and IRF7 gene expression) may play important roles during AIV replication. While, the replication of AIV was inhibited significantly in DF-1 cells tranfected by MR-A3 plasmid, which demonstrated nonspectific regulation of miRNA during the process of gene silence. Though stable cell strain was not established in this study, it provided experimental reference for research of antiviral mechanism and construction of immunodeficient cell line for AIV.3. shRNA sequences were designed and synthetized targeted for IFNAR1 and IFNAR2 genes. And recombinant plasmids were constructed according to shRNA and p LKO.1 vector. Based on above, most effective plasmids including pR1-sh1 and pR1-sh3 for IFNAR1 knockdown, pR2-sh3 for IFNAR2 knockdown were selected in tranfected DF-1 cells using real-time PCR method. shRNA sequences of effective plasmids were carried to lentivirus packaging by JiKai gene company of Shanghai. Then, DF-1 cells were infected by lentivirus at most suitable ratio(MOI 0.5) and monoclonal cell strains were selected and purified using puromucin(4 μg/m L). Furhtermore, seven cell strains and one negative control cell strain were selected for analysis. Among them, five cell strains were selected targeted for IFNAR1 genes and two other strains targeted for IFNAR2 gene. Cell morphological observation under fluorescence microscope demonstrated homogeneous cell morphology and better purified condition. Among five cell strains for IFNAR1 gene silencing, three cell strains(DF1-R1-sh1-3, DF1-R1-sh3-1 and DF1-R1-sh3-2) reduced mRNA expression of IFNAR1 genes compared with two other cell strains and controls. However, only two cell strains(DF1-R1-sh1-3 and DF1-R1-sh3-1) among above three cell strains with IFNAR1 konwdown increased their virus titer 4.6 folds and 4 folds respectively compared to parental cells at 36 hours post infection. In terms of IFNAR2 gene, both cell strains(DF1-R2-sh3-1 and DF1-R2-sh3-2) downregulated expression of silenced genes significantly, but changes of virus titer were not detected in both cell strains. Two transformed cell strains of DF-1 cells screened in this experiment which promoted virus replication meet the needs of this experiment.In conclusion, this study confirmed IFN and ISGs presented different expression pattern responsed to AIVs infection, and two immune-regulatory genes(IRF7 and KHSRP) were identified which could distinguish the two different viruses infection in DF-1 cells. Further study affirmed knockdown or silencing of IFNAR1 and IFNAR2 genes improved AIV replication and established two cell strains with IFNAR1 gene silencing. The two cell strains increased virus titer four to five folds compared to parental cells. This study possessed some theoretical significance and practical value for further research of AIV pathogenesis and promoting the improvement of vaccine production system. |
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