登革二型病毒PL046感染性質體之建構

胡旻秀

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登革二型病毒PL046感染性質體之建構

Construction of the infectious cDNA clone of dengue virus type 2 PL046 strain

胡旻秀 , Masters　　Advisor：楊昀良　　

繁體中文

登革病毒；感染性質體； dengue virus ； infectious cDNA clone

Abstract │ Reference (44) │ Times Cited (1) │ Altmetrics

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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.

Reference ( 44 ) 〈TOP〉

Alvarez, D.E., De Lella Ezcurra, A.L., Fucito, S. and Gamarnik, A.V., (2005). 'Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication'. Virology, 339 (2):200-212.
連結：
Alvarez, D.E., Filomatori, C.V. and Gamarnik, A.V., (2008). 'Functional analysis of dengue virus cyclization sequences located at the 5' and 3'UTRs'. Virology, 375 (1):223-235.
連結：
Cahour, A., Pletnev, A., Vazielle-Falcoz, M., Rosen, L. and Lai, C.J., (1995). 'Growth-restricted dengue virus mutants containing deletions in the 5' noncoding region of the RNA genome'. Virology, 207 (1):68-76.
連結：
Cech, T.R., Zaug, A.J. and Grabowski, P.J., (1981). 'In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence'. Cell, 27 (3 Pt 2):487-496.
連結：
Clyde, K., Kyle, J. L., and Harris, E., (2006). 'Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis'. J Virol, 80(23): 11418-31.
連結：

Times Cited (1) 〈TOP〉

余幸璇(2011)。登革熱病毒於神經細胞內感染與複製能力之研究。交通大學分子醫學與生物工程研究所學位論文。2011。1-89。

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