| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 103 === Influenza A virus (IAV) is the major type of influenza virus in the world. In 1997, the first case of H5N1 infection was found in Hong Kong. Since then, there are more than 600 human cases of H5N1 infection, and the fatality rate is also more than 60%. The main receptor of H5N1 is sialic acid, but other studies showed that IAV can infect cell without sialic acid.
DC-SIGN (Dendritic cell specific ICAM-3 grabbing non-integrin) is a C-type lectin on dendritic cell surface. Many studies show that DC-SIGN could be a receptor of various viruses, including HIV type-1, Ebola virus, cytomegalovirus, HCV, HHV8, dengue virus, West Nile virus, Marburg virus, SARS.
From the previous research in our laboratory, we know that H5N1 infection can be enhanced by binding the cells which highly express DC-SIGN and monocyte-derived immature dendritic cell. And DC-SIGN could be an important role of cytokine storm. Since DC-SIGN binds to the virus directly via interaction with high-mannose N-glycan on the viral glycoproteins, and helps the virus infection. After using the software prediction, we found that there are 7 potential N-glycosylation sites on the Hemagglutinin. We want to study whether the any individual N-glycosylation site on hemagglutinin is crucial in DC-SIGN binding. Therefore, we used site directed mutagenesis to mutate these sites form Asn to Gln on the hemagglutinin of A/Vietnam/1203/04 virus and produced the virus by using reverse genetic technique followed by virus preparation in embryonated eggs. In this study, RG-N27Q mutant virus shows reduced virus infection efficiency in both DC-SIGN expressing cell and immature dendritic cells. Next, we found out that RG-N27Q mutant virus caused the less cell death than H5N1 in both DC-SIGN expressing cell and immature dendritic cells, suggesting that 27N on HA is important for DC-SIGN binding. In the previous studies, we know that H5N1 induced the cytokine storm and caused the damage of lung. Next, we compared the cytokine levels induced by the mutant viruses and found that RG-N27Q induced the lowest cytokine expression among all mutants. For virus secretion, we found that the secreted virus particles were lower in RG-N27Q virus compared with wild-type H5N1 at 12, 18, 24hr post infection. In conclusion, our data suggest that 27N glycosylation may play an important role in the interaction between the hemagglutinin of H5N1 virus and DC-SIGN.
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