Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain.
The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides. DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR. These fragments were assembled into a full-length cDNA clone in the yeast-E. coli shuttle vector pRS424, using homologous recombination in yeast. RNA produced by in vitro transcription of this clone was infectious upon electroporation into LLCMK2 cells, as shown by cytopathic effects and detection of viral antigens by indirect immunofluorescence, and by propagation of the virus released into the culture media. Biological properties of the transcript-derived virus, such as the pattern of dengue-specific protein synthesis and growth rate in LLCMK2 or C6/36 cells, resembled those of the parent DEN1 WP virus.
Puri
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Infectious Diseases Department, Naval Medical Research Institute, Bethesda, MD 20889-5607, USA. purib@nmripo.nmri.nnmc.navy.mil
Polo
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Hayes
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CG
Falgout
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B
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral; D003716:Dengue Virus / Q000235:genetics* / Q000472:pathogenicity; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000032:analysis; D015003:Yeasts / Q000235:genetics
D012367:RNA, Viral; D014764:Viral Proteins