| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 89 === Many RNA viruses are known to exist as populations of closely related sequences, the so-called quasispecies, probably due to the error-prone nature of the vial reverse transcriptase or the RNA-dependent RNA polymerase. The extent of sequence diversity of dengue virus, a member of Flavirus in the family Flaviridae, within infected individuals and its relationship to disease severity has not been investigated.
The overall objective of this study is to investigate the quasispecies of dengue virus in vivo and the relation between the extent of quasispecies and disease severity. The 18 study participants, 10 patients with dengue fever and 8 patients with dengue hemorrhagic fever, were from a DEN-3 outbreak in southern Taiwan in 1998. In the first specific aim, we used the approach of RT/PCR and clonal sequencing to analyze a fragment of the envelope (E) gene (from residue 254 to 397 of E protein) of plasma dengue viruses. The mean diversity of nucleotide sequence ranges from 0.09% to 0.84%, indicating that dengue viruses are present as quasispecies in vivo. In addition, defective viruses are found in 7% of clones sequenced among 50% of patients. The roles of defective viruses in the pathogenesis of dengue remain to be investigated in the future. Analysis of two sequential samples revealed that quasispecies fluctuated during the course of infection. There is no correlation between the extent of quasispecies and disease severity.
In the second aim, we established a heteroduplex mobility assay (HMA) to examine the extent of dengue sequence diversity. The extent of sequence diversity revealed by HMA was generally correlated with that revealed by clonal sequencing analysis. These findings validated the results of clonal sequencing analysis in our study.
In the third aim, we examined the sequence of 5' NTR, 3' NTR and structural genes (C, prM and E) of plasma dengue viruses from one DF and one DHF patients by direct sequencing of the RT/PCR products. We found one nucleotide difference in the M gene, resulting in amino acid change at residue 13 of M protein. Another nucleotide substitution in the NS1 gene, leading to amino acid change at residue 7 of NS1 protein, was also identified. These findings would provide the basis for future analysis of more cases to find sequence differences between dengue viruses from DF and DHF patients.
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