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Highly efficient rescue of dengue virus using a co-culture system with mosquito/mammalian cells.

Abstract：

:The production rate of dengue viruses (DENVs), especially low-passage virus isolates, is low, and, therefore, the isolates are generally used only after several passages. However, in vitro passages could induce mutation(s). In this study, we established a system for the characterization of low-passage viral isolates using an infectious cDNA clone. We used R05-624, a plaque derived from type 2 (DENV-2) Thai strain, for the construction of the cDNA clone, named pmMW/R05-624. We found that transfection of both of mammalian Vero cells and mosquito C6/36 cells with viral RNA derived from the cDNA clone produced a significant amount of progeny virus: 3.2x10(6) focus-forming units (FFU) production per ml of cultured fluid only 3days after transfection with 2 microg RNA. Conversely, no detectable level of viruses was produced by conventional methods using a single cell line, Vero or C6/36. When this system was applied for the characterization of eight low-passage clinical viral isolates by placing their 5'-half or 3'-half in the above cDNA clone, we found that all the isolates, except for L04-225, produced similar levels of progeny virus. Among a total of eight cDNA clones reconstructed with the NS4A-3'NCR region derived from L04-225, one clone carried an insertion and produced a low level of progeny virus. Thus, our system to efficiently rescue clinical samples or low-passage viral isolates could be useful for assessing the virological and molecular characteristics of DENV that could be related to disease pathogenesis.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Kurosu T,Khamlert C,Phanthanawiboon S,Ikuta K,Anantapreecha S

doi

10.1016/j.bbrc.2010.02.181

subject

Has Abstract

pub_date

2010-04-02 00:00:00

pages

398-404

issue

2

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(10)00472-9

journal_volume

394

pub_type

杂志文章

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