infection japanese encephalitis virus RNA can be synthesized from in vitroligated cDNA templates.pdf

JOURNAL OF VIROLOGY, Sept. 1992, P. 5425-5431 Vol. 66, No. 9 0022-538X/92/095425-07$02.00/0 Copyright © 1992, American Society for Microbiology Infectious Japanese Encephalitis Virus RNA Can Be Synthesized from In Vitro-Ligated cDNA Templates HIDEO SUMIYOSHI,12* CHARLES H. HOKE, AND DENNIS W. TRENT2 Department of Virus Diseases, Walter ReedArmy Institute ofResearch, Washington, D. C. 20307-5100,1 and Division of Vector-Borne Infectious Diseases, National Centerfor Infectious Diseases, Centers for Disease Control P.O. Box 2087, Fort Collins, Colorado 805222* Received 10 April 1992/Accepted 4 June 1992 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 fIl-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5 (nucleotidepositions 1 to 5576) and 3 (nucleotidepositions 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 mutation 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