2008年H1N1流感病毒之神經胺酸酶之流行病學研究及 IND-820抗流感病毒作用之探討

Abstract

克流感(Oseltamivir, TamifluTM)為一種治療A型流行性感冒的神經胺酸酶抑制劑，但在世界各地已陸續傳出H1N1抗藥性病毒株的流行。其抗藥性的產生主要為神經胺酸酶第274個胺基酸組胺酸(Histidine)突變為酪胺酸(Tyrosine)。本研究針對台大醫院2008年分離出的18株H1N1 A型流感病毒，並藉由比較第274個胺基酸差異，將病毒分成兩類：克流感敏感株(Oseltamivir-sensitive strain)以及克流感抗藥株(Oseltamivir-resistant strain)。與疫苗株(A/NewCaledomia/20/1999)的胺基酸序列比較發現，除了第274胺基酸位點的差異，克流感敏感株在第34、52、150、262、331、335、353、373等位點也有突變；克流感抗藥株則分別在第18、29、39、79、83、275、353、450、451等位點突變。進一步藉由溶斑減少實驗(plaque reduction assay)來確定胺基酸位點突變與藥物敏感性的相關性，並且於MDCK細胞比較克流感敏感株和抗藥株生長特徵的差異。結果顯示，相較於克流感敏感株，在病毒感染十二小時後，除了第274以及第275個位點皆突變的抗藥株VI08-6867具有明顯偏低的病毒產量，其餘僅在第274胺基酸位點突變的抗藥株的生長趨勢與克流感敏感株並則無明顯差異。接著，針對病毒的釋放量作探討，發現在克流感的存在下，VI08-6867在24小時的病毒量明顯較其它時間點低。隨後利用hemagglutination elution test及神經胺酸酶活性試驗(neuraminidase activity assay, MUNANA method)來探討胺基酸位點改變對於神經胺酸酶活性的影響。結果顯示，VI08-6867病毒株之神經胺酸酶活性較其它抗藥株為低。綜合以上，H1N1流感病毒若同時帶有H274Y及Y275F突變，可能影響神經胺酸酶之正常功能，此假設須進一步利用反轉錄基因技術作更深入的探討。 另一方面，由於近年來世界各地H1N1克流感抗藥株的出現比例已趨近100%，尋找新的藥物標靶及研發新型抗流感藥物乃為當務之急。對於藥物的研發，我們取得一混合物IND-820，並以此進行H1N1流感病毒的藥物敏感性測試。結果顯示，IND-820不僅抑制克流感敏感株(EC50=36.45μg/mL)，對於克流感抗藥株也有良好的抑制的效果(EC50=43.92μg/mL)。而針對2009年爆發大流行的H1N1新型流感以及H3N2 A型流感，IND-820也有明顯的抑制效果(EC50=21.00μg/mL及43.64μg/mL)。接著，利用藥物加入時間點試驗(time of addition assay)來決定IND-820可能影響病毒複製的步驟顯示，IND-820可能影響流感病毒感染細胞的早期階段。進一步以細胞前處理試驗(cell pretreatment assay)及病毒前處理試驗(virus pretreatment)來探討IND-820在病毒感染早期所影響的確切目標。然而，經前處理的細胞，其細胞內的病毒量上升並伴隨細胞型態的改變。細胞毒性試驗結果顯示IND-820對於MDCK細胞的CC50約為58.19μg/mL，換算其針對2008年克流感抗藥株與敏感株H1N1病毒、2009年H1N1新型流感病毒以及季節性H3N2流感病毒之SI(selective index)則僅介於1.3至2.8。由此推測，IND-820此混合物對於細胞毒性過強，可能須純化其有效成分，才能進一步探討其對抗流感病毒作用之主要機制。

Oseltamivir, a neuraminidase inhibitor, is commonly used for treatment of influenza A virus infections; however, circulation of resistant strains has been widely reported throughout the world since 2007. Resistance to oseltamivir was mainly contributed by the H274Y mutation of the neuraminidase. To understand the prevalence and phenotype of resistant strains in Taiwan, a total of 18 H1N1 influenza A virus strains isolated from National Taiwan University Hospital in 2008 were analyzed. These viruses were further divided into two groups, oseltamivir-sensitive and oseltamivir-resistant strains based on the amino acid at residue 274. After alignment of amino acid sequences with the reference strain (A/NewCaledomia/20/1999), besides the H274Y mutation, other amino acid changes were observed at residues 18, 29, 39, 83, 275, 353, 450, and 451 of oseltamivir-resistant strains, and residues 34, 52, 79, 150, 262, 331, 335, 353, and 373 of oseltamivir-sensitive strains. The correlation of genetic mutation and viral susceptibility to oseltamivir was further confirmed by plaque reduction assay. Except one oseltamivir-resistant virus strain VI08-6867 with both H274Y and Y275F mutations, no significant differences in growth kinetics of the other 8 oseltamivir-resistant H1N1 were observed as compared to oseltamivir-sensitive strains. Meanwhile, the virus replication efficiencies of VI08-6867 was not impaired in the presence of oseltamivir, but its releasing ratio was significantly lower than VI08-6706 having only H274Y mutation. Furthermore, the neuraminidase (NA) activity was determined by the hemagglutination elution test and MUNANA. Again, the VI08-6867 strain showed lower NA activity than the other resistant H1N1 strains. In summary, the H274Y mutation combined with Y275F amino acid change might impair the neuraminidase function, such finding requires further confirmation by reverse genetics. The spreading of oseltamivir-resistant virus has raised a concern about development of new anti-influenza compounds. An herbs-mixture, IND-820 was investigated for its anti-influenza activity. IND-820 showed effective inhibition of oseltamivir-sensitive, oseltamivir-resistant H1N1, novel influenza A (H1N1/2009), and H3N2 influenza A virus at EC50 of 36.45 μg/mL, 43.92 μg/mL, 21.00 μg/mL, and 43.64 μg/mL, respectively. The results of time of addition assay showed that IND-820 might interfere the early stage of influenza life cycle. Unfortunately, in the cell and virus pretreatment assay, IND-820 showed cytotoxicity to MDCK cells. The CC50 of IND-820 was 58.19μg/mL, and the selective index (SI) of IND-820 was about 1.3 to 2.8 for H1N1 and H3N2. In brief, due to the cytotoxicity of IND-820 mixture, identification of the effective ingredients of IND-820 is required for future development of IND-820 as a candidate of clinical therapeutic compounds.