Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates

Hideo Sumiyoshi, Charles H. Hoke, Dennis W. Trent

Research output: Contribution to journalArticle

116 Citations (Scopus)

Abstract

Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae. Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural. A full-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5′ (nucleotide positions 1 to 5576) and 3′ (nucleotide positions 5577 to 10976) halves of the genome. T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells. To identify the recombinant virus, a silent mutations was introduced into the clone encoding the 3′ half of the genome, which abolished an XbaI site at nucleotide position 9131. Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity. Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice. Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful. Synthesis or infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.

Original languageEnglish (US)
Pages (from-to)5425-5431
Number of pages7
JournalJournal of Virology
Volume66
Issue number9
StatePublished - Sep 1992

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Japanese Encephalitis Virus
Japanese encephalitis virus
Complementary DNA
RNA
Genome
genome
Nucleotides
nucleotides
Viruses
viruses
Flaviviridae
mutation
Flavivirus
plasmid vectors
RNA Viruses
cells
transfection
DNA-directed RNA polymerase
Transfection
Ligation

ASJC Scopus subject areas

  • Immunology

Cite this

Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. / Sumiyoshi, Hideo; Hoke, Charles H.; Trent, Dennis W.

In: Journal of Virology, Vol. 66, No. 9, 09.1992, p. 5425-5431.

Research output: Contribution to journalArticle

Sumiyoshi, Hideo ; Hoke, Charles H. ; Trent, Dennis W. / Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. In: Journal of Virology. 1992 ; Vol. 66, No. 9. pp. 5425-5431.
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