| Summary: | 博士 === 國立交通大學 === 生物科技系所 === 97 === Dengue virus (genus Flavivirus, family Flaviviridae) is a single-stranded and positive-sense RNA virus with a 10.7 kb genome. Generally, most of the dengue virus infectious clones (or infectious cDNA clones) are under the control of prokaryotic promoter. To generate the virus RNA, the process requires in vitro transcription and addition of 5’-cap. According to the study of West Nile virus (Flavivirus), the virus could be recovered from the plasmid DNA-transfected cells directly while prokaryotic promoter was replaced by cytomegalovirus (CMV) promoter. Based on this concept, the objective of the present study was to employ this strategy to construct the DNA-launched dengue virus type 2 PL046 strain (Taiwan local isolated) infectious clones.
Previously, the whole genome of dengue virus type 2 PL046 strain was successfully constructed in pcDNA3 plasmid by laboratory predecessors. This infectious clone was named pcDNA3/DV2F and the viral genome was placed under the control of CMV promoter. However, no virus particles were produced in transfected host cells (BHK-21). After full-length sequencing of the clones, several mutations such as nonsense mutation and frame shift mutation were discovered in this study. In addition, there remained certain superfluous nucleotide at the 5’- and 3’- end of viral genome. These non-viral sequence may affect viral replication. Restriction fragment replacement, polymerase chain reaction (PCR), and introduction of ribozyme were applied to correct the mutation and remove excess sequence from the 5’- and 3’- untranslated region (UTR) of viral genome.
The new infectious clone was transfected into host cells for the production of viruses. However, no virus particle was detected, although viral RNA was detected.
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