台灣北部地區2001、2006-2007年人類流行性感冒病毒(A/H1N1)表面蛋白質基因及其大小溶斑病毒生物學性質之研究

Abstract

A型流行性感冒病毒屬於正黏液病毒科(Orthomyxoviridae)，為單股負鏈RNA病毒，共有八段基因，可以產生十種不同的病毒蛋白質；A型流感病毒根據十六種血球凝集素(H1~H16)及九種神經胺酸酶(N1~N9)再分類為不同亞型，感染人類的A型流感病毒主要以H1N1及H3N2為主。根據台灣疾病管制局之文獻資料，A型流感病毒H1N1在2001/2002年及2005/2006年有流行之高峰；且A/H1N1病毒株為2001/2002年及2005/2006年主要之流感病毒分離株，對此些病毒之基因及生物特性之瞭解甚少，因此本研究針對台灣北部2001及2006、2007年之A/H1N1病毒株進行相關探討分析。研究主要包括兩部份，第一部份為A/H1N1病毒表面醣蛋白質血球凝集素、神經胺酸酶基因分子流行病學之研究；第二部份為對具有大小溶斑病毒形態之A/H1N1病毒之生物學性質進行研究。藉由這些實驗來進一步瞭解NA蛋白質(N1)在流感病毒所扮演之角色，並進一步利用NA蛋白質結構模擬，來討論重要位置之發現對於病毒NA蛋白質和抗流感藥物之結合是否會有影響。 研究結果發現2001年病毒株屬於A/New Caledonia/20/99-like strain，而2006及2007年病毒株則是接近A/Solomon Islands/3/06-like strain，透過A/H1N1臨床病毒株HA及NA基因種系圖分析，可以知道2001、2006及2007年的病毒株分為兩群；而胺基酸序列分析結果也發現2001年病毒株、2006及2007年病毒株的胺基酸序列有一些改變。在神經胺酸酶活性測定的實驗中也發現小溶斑病毒並不一定會完全失去神經胺酸酶的活性，有些小溶斑病毒還是具有感染細胞的能力。接著再針對溶斑實驗所純化出來的大小溶斑病毒進行研究，結果發現6593S 小溶斑病毒神經胺酸酶的第443個胺基酸殘基由Serine改變為Asparagine；此改變的確會造成其神經胺酸酶活性下降，而此一小溶斑病毒透過血球凝集釋出實驗，證明其無法由血球凝集中釋出，實驗結果證明6593S 小溶斑病毒神經胺酸酶第443個胺基酸殘基的改變會影響神經胺酸酶的活性與功能。而進一步透過蛋白質結構模擬分析，可以知道6593S小溶斑病毒神經胺酸酶第443個胺基酸殘基的改變，使得小溶斑病毒神經胺酸酶酵素催化位置和唾液酸及神經胺酸酶抑制藥物之間結合的鍵結有所改變，此結果可和實驗結果相互印證。神經胺酸酶第443個胺基酸殘基的改變會影響神經胺酸酶的活性與功能的發現，仍有待進一步實驗確認。

The influenza A virus is a member of the Orthomyxoviridae family. It is a single-stranded RNA virus with envelope. The genome consists of eight RNA fragments and can encode 10 viral proteins. There are 16 H (H1~H16) and 9 N (N1~N9) subtypes. Currently, the H1N1 and H3N2 subtypes are circulating among humans. According to the Center for Disease Control, Taiwan, the A/H1N1 strain reached peak prevalence in the years 2001/2002 and 2005/2006, and was the major strain isolated in the influenza seasons of these years. In order to understand the variation of genetic sequences and biological characteristics of these viruses, this study was focused on the A/H1N1 virus strains isolated in northern Taiwan in the years 2001, 2006, and 2007. The study consists of two parts. The first part includes molecular epidemiological studies, which focus on the surface glycoprotein hemagglutinin (HA) and neuraminidase (NA) genes. In second part, the biological function of A/H1N1 isolates with large or small viral plaques and the role of NA protein (N1) in the virulence of the influenza virus were examined. The structure modeling and the key positions of NA protein were discussed. The results revealed that the dominant strains isolated in 2001 was an A/New Caledonia/20/99-like strain, whereas the strains isolated in 2006 and 2007 was similar to the A/Solomon Islands/3/06 strain. Through phylogenetic analysis of the HA and NA of clinical isolates of the A/H1N1 virus, we found that these strains were clustered into two groups. The correlation between plaque size and neuraminidase activity was not existed. The inconsistent results were also found between the plaque size and the virus growth activity. However, by using plaque purification, there was one unique strain (6593) showed different biological characteristics between small plaques and large plaques. The results showed that the amino acid residue at position 443 of neuraminidase enzyme in the 6593S virus (small plaque) was asparagine different from the serine residue of the large plaque derived from the same parental strain. This change resulted in a decrease in neuraminidase activity. Further, a hemagglutination elution test revealed that the 6593S virus was unable to release from hemagglutinated RBC. Through the protein structure modeling, it was suggested that the change in amino acid residue 443 of neuraminidase might alter the neuraminidase catalytic sites. The variation in neuraminidase structure may interfere the interactions among sialic acid, neuraminidase, and enzyme inhibitor. However, this finding needs to be elucidated by further studies.