Development and application of a reverse genetics system for Japanese encephalitis virus.
Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children. Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone. We describe here the development of such a clone. A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome. This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5' and 3' ends. The synthetic RNAs thus produced were highly infectious in susceptible cells (>10(6) PFU/ micro g), and these cells rapidly generated a high titer of synthetic viruses (>5 x 10(6) PFU/ml). The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity. Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli. A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated. Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types. This novel reverse genetics system for JEV will greatly facilitate research into JEV biology. It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.
Department of Internal Medicine, Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea.
Sang-Im
SI
Yun
Seok-Yong
SY
Kim
Charles M
CM
Rice
Young-Min
YM
Lee
D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D018076:DNA, Complementary; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000472:pathogenicity*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D010948:Viral Plaque Assay
D018076:DNA, Complementary; D008164:Luminescent Proteins; D012367:RNA, Viral; D049452:Green Fluorescent Proteins