| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 89 === Dengue virus is a positive, single-stranded RNA enveloped flavivirus that is transmitted by mosquitoes, Aedes aegypti and Aedes albopictus. The virus is divided into four serotypes, DEN-1, DEN-2, DEN-3 and DEN-4.
In the process of virus entry, the envelope protein, E protein, plays an important role. It is a glycoprotein of 495 amino acids, with a transmembrane domain at the C-terminal. It is believed to form a stable, non-covalently linked homodimer. The extracellular domains of E glycoprotein are thought to interact with the host cell receptor.
The overall objective of this study is to investigate the dengue virus entry by establishing an E protein-mediated cell fusion assay.
In the first aim, we examined the expression of five DEN-2 E protein constructs, including pD2ME, pD2ME His, E His, pCB8D2J2 and pCB8D2J2VSV, in the mammalian cell line, 293 cells, to identify the ideal dengue E protein expression construct.
In the second aim, we developed a dengue cell fusion assay by cotransfecting NPCTW04 cells with pCB8D2J2VSV (from DEN-2, 16681 strain) and a reporter construct, pET-21a-GFP, followed by coculturing with target cells which were infected with recombinant vaccinia virus. Examination by fluorescent microscope revealed that NPCTW04 cell can fuse with BHK and K562 cells, two known dengue target cells, but not with H9 or Hut78 cells. The infectivity assay in different target cells revealed that DEN-2 (16681 strain) virus can replicate in BHK and K562 cells efficiently, but not in H9 or Hut78 cells. This finding indicated that our cell fusion assay correlated with cellular tropism of dengue virus.
In the third aim, we developed a real-time RT-PCR assay for quantification of DEN-2 and DEN-3 viruses, using the probe and primers targeting a highly conserved region in the capsid gene. This assay was used to study replication kinetics, in comparison with traditional plaque assay and immunofluorescence assay. It is a convenient, sensitive and accurate method of quantification, and has potential for future application.
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