每年流感造成了全球數百萬的人受到嚴重的影響，而A型流感病毒為主要造成全世界性流感的重要致病原因。在A型流感病毒表面具有神經胺酸酶蛋白 (Neuraminidase, NA)，其功能主要是切除細胞受體上的唾液酸和流感病毒血球凝集素 (Hemagglutinin, HA) 之間的鍵結，以促進流感病毒的釋放及擴散。本研究中，我們分析了1999到2012年由成大醫院分離出的H1N1病毒株的NA序列，在演化樹當中大部分的2009新型流感H1N1都落於同一的演化群 (clade 1)，但是在2010年末至2012年有一新的演化群的出現 (clade 2)。而在clade 1中，2011年的病毒株又自成一群 (clade 1.1)。我們發現所有的2009新型流感H1N1的臨床病毒之NA蛋白和疫苗株 (A/California/07/09) 比較皆具有V106I及N248D的突變，而在clade 2和clade 1.1的病毒株分別帶兩組不同的胺基酸突變: N44S、V241I、N369K及S299A 、I374V。接著，我們進一步分析在NA這幾位點的突變對於NA酵素活性上是否會造成影響，因為臨床病毒株可能會受到流感病毒的其他七段基因影響較不易觀察到這些NA蛋白突變對其酵素活性之影響，所以，我們利用了重組NA蛋白及重組病毒 (reverse genetics viruses) 來分析NA突變對其酵素活性的影響，結果發現NA蛋白或重組病毒同時具有V106I 和N248D的突變時，NA活性會上升；在同時有N44S、V241I和N369K則會造成NA活性下降；而在同時有S299A、I374V則會造成NA活性的上升。另外，由重組病毒的生長曲線顯示，這些NA位點的改變不影響重組病毒的複製。但是，由分析病毒初期的感染性得知，NA同時有V106I 及N248D的突變下，初期感染力會上升，顯示這兩點位點可能對流感病毒進入細胞有幫助。總結，本研究有助於了解NA蛋白位點的改變對於流感病毒進入細胞及釋放的影響。

Influenza is a disease that deeply affects millions of people every year. Influenza A virus is an important pathogen with worldwide prevalence. It encodes the membrane neuraminidase (NA) protein that cleaves sialic acids from cellular receptors to facilitate progeny virus release and to promote the spread of the infection. Our phylogenetic analysis of NA genes of 2009 pandemic and old seasonal H1N1 viruses in Taiwan from 1999 to 2012 revealed that 2009 pandemic H1N1 isolates from 2009 to 2011 fell into one major clade (clade 1), but there were two separate clades in 2011 (clade 1.1 & clade 2). Five amino acids changes, S299A and I374V in clade1.1 and N44S, V241I and N369K in clade 2, were identified in NA proteins of these 2011 isolates. To investigate whether these amino acid mutations influenced their NA activity, selected isolates were tested for NA activity. However, the results showed that the NA activities of the selected isolates were similar which may due to the influence of various genetic backgrounds. To investigate NA activity under same genetic background, reverse genetics 2009 pandemic H1N1viruses in the background of A/PR/8/34 (PR8) (H1N1) with different NA genes were generated. To explore whether these amino acid mutations influenced on the viral fitness, we compared the viral replication of reverse genetics viruses. These reverse genetics viruses showed similar growth kinetics in MDCK cells. When their NA activity was examined, the results showed that the NA double mutants (V106I, N248D) enhanced NA activity, and the NA double mutants (S299A , I374V) also showed enhanced NA activity. In contrast, other NA triple mutants (N44S, V241I, and N369K) showed reduced NA activity. The same results were obtained when examined the cell surface NA activities of eight different NA recombinant proteins. Because the NA protein also facilitates viral entry, we further investigated whether these mutations on NA proteins could influence the viral entry. Faster infection process of the reverse genetics viruses that contained NA double mutants (V106I, N248D) were observed when compared with wild type (A/California/07/09). This study will be helpful to understand contributions of NA genetic variations to influenza virus release and entry.

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