| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 101 === 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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