Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain)

Mini Kapoor, Luwen Zhang, P. Maruthi Mohan, R. Padmanabhan

Research output: Contribution to journalArticle

65 Scopus citations

Abstract

Dengue virus type 2 (DEN-2), a member of the Flaviviridae family, has a positive-strand RNA genome, 10 723 nucleotides (nt) in length and encoding a single polyprotein precursor consisting of 3391 amino acids (aa). In order to construct a full-length cDNA clone, the viral genome was cloned into 5′ (nt 1-2203 under the control of the T7 promoter (pT7)) and 3′ (nt 2203-10723) constructs. A full-length DEN-2 cDNA under pT7 control was assembled in vitro after excising the two cDNA inserts from the 5′ and 3′ constructs, and joining them with T4 DNA ligase. The RNA produced by in vitro transcription of the cDNA using T7 RNA polymerase was infectious, as shown by transfection of permissive BHK-21 and Vero cells, and propagation of the virus particles released into the culture media. The virus particles stably maintained the conservative mutation introduced into the 5' construct, and the cells infected with the infectious RNA-derived virus synthesized virus-specific DEN-2 antigens, as shown by immunofluorescence and immunoprecipitations. The full-length infectious clone for DEN-2 should be useful for the study of molecular mechanisms involved in viral RNA replication and virus assembly.

Original languageEnglish (US)
Pages (from-to)175-180
Number of pages6
JournalGene
Volume162
Issue number2
DOIs
StatePublished - Sep 11 1995

Keywords

  • BHK
  • C
  • DEN
  • DMEM
  • DNA polymerase isolated from Thermus aquaticus YT1
  • Dulbecco's modified Eagle's medium
  • E
  • ELISA
  • ER
  • HMAF
  • IC
  • Klenow (large) fragment ofE. coli DNA polymerase I
  • NS
  • ORF
  • PAGE
  • PBS
  • PCR
  • PMSF
  • PolIk
  • RNA transfection
  • RT
  • Recombinant DNA
  • SDS
  • T7 RNA polymerase
  • T7 promoter
  • Taq polymerase
  • UTR
  • YF
  • aa
  • amino acid(s)
  • baby hamster kidney
  • base pair(s)
  • bp
  • capsid protein
  • dengue virus
  • endoplasmic reticulum
  • envelope protein
  • enzyme-linked immunosorbent assay
  • flavivirus
  • hyperimmune mouse ascetic fluid against a mixture of DEN-2 antigens
  • in vitro transcription
  • infectious clone
  • kb
  • kilobase(s) or 1000 bp
  • non-structural protein(s)
  • nt
  • nucleotide(s)
  • open reading frame
  • p
  • pfu
  • phenylmethylsulfonyl fluoride
  • phosphate-buffered saline (10 mM Na·phosphate pH 7.2/150 mM NaCl)
  • plaque-forming unit(s)
  • polyacrylamide-gel electrophoresis
  • polymerase chain reaction
  • positive-strand RNA virus
  • recombinant vaccinia virus expressing T7 RNA polymerase
  • reverse transcriptase-based PCR
  • reverse transcription
  • site-directed mutagenesis
  • sodium dodecyl sulfate
  • untranslated region(s)
  • vTF 7-3
  • wild type
  • wt
  • yellow fever virus

ASJC Scopus subject areas

  • Genetics

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