台灣地區2009-2011年新型流行性感冒病毒(A/H1N1)非結構蛋白質(NS1)之基因變化

Abstract

流行性感冒病毒屬於正黏液病毒科，具有套膜且含有八條單股RNA，可以轉譯出11種病毒蛋白質。流行性感冒病毒分為A､B､C三型，其中A型流感病毒的抗原較B型易發生突變，所以A型較B型更容易引起流行。每年台灣冬季11月到3月都有小規模流行，約每十年有一次大規模的流行。 根據行政院衛生署疾病管制局統計資料顯示，2009年全球流行的H1N1病毒(2009 pandemic H1N1)在台灣引起兩波大規模的流行，分別是2009年的第31週到2010年的第5週，和2010年第48週到2011年的第10週。這兩波流行的2009 pandemic H1N1病毒是否具有相異之處，是值得探討之課題。 2009 pandemic H1N1病毒在血球凝集素(Hemagglurinin; HA)和神經胺酸酶(Neuraminidase; NA) 之變化已有許多文獻發表；但第八片段NS（nonstructural）基因，則很少分析。由於NS基因可以轉譯出兩個非結構性蛋白質，NS1與NS2。其中NS1為一非結構性蛋白質，與病毒的複製和抑制宿主干擾素反應有關。於是，探討這兩波在台灣廣為流行的流感病毒其NS1基因序列是否有所差異，為本研究主要目標。 共有57株完成了NS基因全長序列之定序，2009年總計15株、2010年總計6株、2011年總計36株。 由定序結果發現2009 pandemic H1N1流感病毒可依NS1序列的不同分為四種基因型，分別為NS1 55E 123I、55E 123V、55Q 123V和55Q 123A，其中包含55及123位點之single mutation和co-mutation的型別。接著就對病毒single mutation和co-mutation的生長情形做觀察，發現在感染24小時後，co-mutation QA的病毒量會小於EV，single mutation EV會和EI差不多，而single mutation QV會小於EV。 進一步分析不同基因型誘發IFN-β mRNA的量發現，在感染24小時後， co-mutation QA誘發IFN-β mRNA的量會大於EI，single mutationEV誘發IFN-β mRNA的量會和EI差不多，而single mutation QV會大於EV。綜合以上的實驗結果可知，若病毒的NS1 55和123位點突變成QA和QV時，病毒的毒力會下降；而在EI和EV這兩株病毒則無太大的差異。 由實驗結果可以得知，NS1 55Q 123A的流行性感冒病毒，會產生大量的IFN-β，而且病毒量也有大幅度的下降，可能為病毒毒力減弱的影響，至於更精確的機制，可能需要更多的研究來探討。

Influenza A virus belongs to the family of Orthomyxoviridae. It is an enveloped virus with eight segment negative sense, single-stranded RNA. These eight segments of encode for the 11 viral proteins. Three types A, B and C of influenza viruses were identified. Seasonal influenza epidemics occurre in winter between Nov and Mar every year in Taiwan. According to the report from Taiwan Centers for Disease Control, pandemic H1N1 induced two large-scale epidemic waves in Taiwan. One was at week 31 of 2009 to week 5 of 2010 and the other was week 48 of 2010 to week 10 of 2011. In order to analyze the variation between viruses of the two large-scale epidemic waves, virus strains isolated at National Taiwan University Hosptial during 2009-2011 were collected for study. Because the variations in hemagglurinin and neuraminidase were studied extensively, the NS gene was chosen in this study. The sequence variation of NS1 gene between the viruses of the two large-scale epidemic waves in Taiwan was explored. NS gene can encode two non-structural proteins, NS1 and NS2. NS1 is one of the non-structural protein and is critical for viral replication and inhibition of host induced interferon response. The whole NS genes of 57 strains were sequenced. 15 strains isolated from 2009, 6 strains isolated from 2010, 36 strains isolated from 2011. Based on the NS1 gene sequence analysis, influenza virus can be divided into four genotypes, NS1-55E/123I、55E/123V、55Q/123V and 55Q/ 123A. In order to know the growth characteristics of these four genotypes, their growth activity in MDCK cell were compared. The viral load of co-mutation, QA, was lower than EV at the hour post infection. The single mutation, EV and EI exhibit comparable growth ability in MDCK. Neverthelss, the single mutation, QV had showen the growth activity than EV at 24 hour post infection. For cytokine analysis, the expression level of IFN-beta mRNA upon infection of the co-mutation, 55Q/123A virus wes greater than the co-mutation, 55E/123I at 24 hour post infection. Whereas, no significant difference of the IFN-beta mRNA was found between the virus with single mutation at 55E/123V and 55E/123I. In contrast, the IFN-beta mRNA level was higher in the single mutation, 55Q/123V virus than 55E/123V virus. It suggestes that viruses with NS1 55 and 123 site mutate into QA and QV are related with a decrease of viral virulence. The real mechanism of decrease of viral virulence needs further investigation.