| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 89 === Dengue virus is a single-stranded positive sense RNA flavivirus that is transmitted by mosquito to human host. Probably due to the non-proofreading nature of the RNA-dependent RNA polymerase, RNA viruses are known to exist as a swarm of viruses that have slightly different genome compositions, the so-called quasispecies. Although the relationship between the degree of sequence diversity and disease severity has been reported in other RNA viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), little is known about dengue virus.
The first aim of this study focuses on analyzing the extent of sequence diversity in the capsid gene of DEN-3 viruses from plasma samples of 18 patients, including 10 dengue fever (DF) and 8 dengue hemorrhagic fever (DHF), during an outbreak in southern Taiwan in 1998. Using the approach of RT/PCR and clonal sequencing, the mean diversity of nucleotide sequences of each patient range from 0.05%~0.78%, indicating the existence of dengue viral quasispecies in vivo. In addition, defective viruses were found in 4.6% of total clones sequenced among 3 out of 18 patients（16.7%）. There is no difference between the extent of sequence diversity and disease severity.
The second aim of this study examines the differences in dengue virus envelope gene, which is believed to play an important role in virus-cell interaction, between dengue viruses isolated from three different cells. Based on the results of direct sequencing, there is no difference in the envelope gene between plasma dengue virus and dengue isolates.
In the third aim, we established a real-time RT-PCR assay to quantify Den-3 virus. The primers and probe were designed to target a highly conserved region in the capsid gene, based on the sequence information obtained from our first aim and two sequences available in the Genebank. We evaluated this assay by studying the replication kinetics of dengue virus, in comparison with the traditional plaque assay and immunofluorescence assay. It is a convenient, sensitive and accurate method of quantification, and has great potential in the future research.
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