人類流行性感冒主要由A及B型流感病毒所引起，其中A型流感病毒常引起高罹患率及死亡率，由於A型流感病毒具有八段負股RNA，常因基因突變或是片段重配而引起大流行。在A型流感病毒所產生的11個蛋白中，非結構蛋白-NS1為一多功能蛋白，可以和宿主內多種形式RNA結合，也可以和多種蛋白作用而抑制第一型干擾素的合成及反應，因此NS1被認為是A型流感病毒中非常重要的毒力因子。在本研究中，我們主要想探討A型流感病毒H1N1亞型NS1基因序列改變是否會影響病毒株對於第一型干擾素的反應。首先我們收集由成大臨床病毒實驗室所分離出的A型流感病毒株並分析1999至2008年間NS1基因的演化，發現NS片段並無不同型間重配的現象，經分析NS1基因位置發生non-synonymous （dN）synonymous（dS）突變的機率（dN/dS）比例則為小於1，表示這十年來A型流感病毒NS1基因在演化上呈現穩定狀態，胺基酸序列並不太改變。接著我們分析各臨床病毒株對第一型干擾素基因表現的影響及對於干擾素的感受性，結果發現有一株流感病毒Taiwan/N39/06可引起較高干擾素基因表現同時對於干擾素較具抗性。因此接下來我們便利用反轉基因系統製造出具有不同NS片段的病毒株，透過病毒對於干擾素的反應找出新的功能性位點。由重組病毒株引起干擾素基因表現的結果中，可以看到NS片段對於第一型干擾素的反應有顯著的影響，其中Taiwan/N39/06的NS片段可引起較高干擾素表現，並且在外加干擾素時呈現較低的生長能力。經由NS序列比對，我們發現Taiwan/N39/06的NS片段只有在NS1上第49位點(RNA結合位區)有著與其他病毒株的threonine不同的isoleucine。因此我們利用單點突變方式製造出含有NS1-I49T突變的重組病毒株，並進一步測試其對於第一型干擾素，IL-8及RANTES基因的表現，結果發現此位點為threonine時可有效抑制這些細胞激素的基因表現，並且利用reporter assay也發現含有NS1-I49T突變的重組病毒株可抑制IFN-beta promoter的活性。由演化分析中，我們發現大部分A型流感病毒株在NS1第49個胺基酸均為threonine，而本研究發現此胺基酸對於抑制抗病毒的免疫細胞激素有著顯著的影響。總之，此研究結果找出NS1基因新的功能性位點，且發現此位點在A型流感病毒演化上的重要性。

Human influenza, caused by influenza A and B viruses, is a highly infectious respiratory disease which can result in high morbidity and mortality. New influenza virus strains may originate from mutations and reassortment of viral genes to cause an epidemic or pandemic. Influenza A virus contains 8 segmented negative strand RNA genome that encode 11 proteins. Among them, non-structural protein 1 (NS1) is known to act as a virulence factor which enhances viral pathogenesis by antagonizing the host interferon (IFN) defense mechanism. In this study, we evaluated the effect of NS1 genetic variations on the response to type I interferons. Firstly, genetic analysis of NS1 gene of influenza viruses isolated at NCKU Hospital from 1999 to 2008 suggested no reassortment in NS segment. In addition, the nonsynonymous/ synonymous rate ratio (dN/dS) showed NS1 was negative selection in the evolution. Subsequently, a panel of influenza viruses was selected for further study based on their variations in the functional domains of NS1 gene. The expression of IFN-beta gene in virus infected A549 cells and the susceptibility to interferon-alpha pre-treatment of those selected influenza isolates were investigated. The results showed different isolates varied in their induction of interferon and susceptibility to interferon-alpha. Moreover, we used reverse genetic system to generate recombinant viruses which contained various NS1 genes to evaluate the difference in expression of IFN-beta gene and susceptibility to interferon-alpha. Interestingly, we found the NS gene from one isolate, A/Taiwan/N39/06, induced a strong type I interferon gene expression and was sensitive to anti-interferon response when compared with others. Furthermore, sequence analysis revealed a threonine-to-isoleucine substitution at the position of amino acid 49 in the RNA-binding domain of NS1 gene. Finally, WSN/N39-NSI49T mutant was generated by site directed mutagenesis and reverse genetics. The results showed IFN-beta，IL-8 and RANTES gene expression levels of this single substitution were reduced to the level similar to others. Moreover, the IFN-beta promoter activity was also reduced by WSN/N39-NSI49T. In conclusion, we demonstrated that the type I interferon response is influenza strain specific and the T49 in the NS1 is responsible for the inhibition of type I IFN gene expression.

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