Highly efficient rescue of dengue virus using a co-culture system with mosquito/mammalian cells.
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.
Kurosu
Takeshi
T
Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Research Institute for Microbial Diseases (RIMD), Osaka University, Tiwanon Road, Muang, Nonthaburi 11000, Thailand. tkurosu@biken.osaka-u.ac.jp
Khamlert
Chidchanok
C
Phanthanawiboon
Supranee
S
Ikuta
Kazuyoshi
K
Anantapreecha
Surapee
S
D000818:Animals; D002522:Chlorocebus aethiops; D018920:Coculture Techniques; D009033:Culicidae / Q000166:cytology / Q000821:virology; D018076:DNA, Complementary / Q000235:genetics; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic; D014709:Vero Cells
D018076:DNA, Complementary; D012367:RNA, Viral