| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 100 === Influenza A virus is an important pathogen with worldwide prevalence. The major circulating subtypes are H3N2 and H1N1. Influenza A virus contains 8 negative stranded RNA segments that encode 11 or 12 viral proteins. Among them, NS (non-structural) gene encodes NS1 protein which can increase viral pathogenicity and virulence by regulating viral translation and antagonizing host defense immune responses, especially type I interferon (IFN-α/β) production and antiviral activities of IFN-induced proteins. Our phylogenetic analysis of NS genes of H3N2 viruses from 1999 to 2011 showed that most NS genes fall into one group, H3 group, except that NS genes of 2002 isolates (H3 variants, H3v) were separated into another group, H3v group. Two amino acid changes, E71G in the RNA binding domain and V82A in the effector domain, were identified in NS1 proteins of these 2002 isolates. To test whether these amino acid mutations contribute to IFN response, we investigated IFN-β protein production, IFN-β promoter activity and IFN-α susceptibility of isolates in H3 group and H3v group. The result showed that the H3 variants induced higher IFN-β protein production and IFN-β promoter activity. In addition, the H3 variants showed comparable reduction to other isolates in virus productions when pretreated A549 cell with IFN-α. The result indicated that the two substitutions V82A and E71G in NS1 protein of H3 variants can affect the IFN-β protein level and promoter activation. We further constructed recombinant NS1 protein to elucidate the effect of E71G and V82A mutations on IFN-β and ISRE promoter activity. Recombinant NS1 proteins with E71G or double mutation of E71G/V82A substitution showed higher induction of IFN-β promoter than NS1 protein from H3 group. Interestingly, recombinant NS1 proteins with either substitution exhibited stronger inhibitory effects on ISRE promoter activity when compared with H3 NS1. We further investigated the effect of recombinant virus with difference in NS gene sequence on replication curve. It showed that the replication ability of recombinant virus with different NS gene were similar. Our results indicated that NS1 with E71G and V82A mutations may affect type I IFN response in the IFN-β protein secretion, promoter activation and the downstream pathway of IFN-β which may be the disadvantage to virus infection and lead to disappearance in the later season. The substitutions in NS1 can be applied to vaccine strain development. Our study will be helpful to understand the contribution of NS1 genetic variation to the pathogenesis of influenza viruses.
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