Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

Abstract

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/g), and these cells rapidly generated a high titer of synthetic viruses (>5 ؋ 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.

Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus

“…The 39 non-coding region (NCR) was sequenced by using a previously reported method (Kolykhalov et al, 1996;Yun et al, 2003). Briefly, ddATP was incorporated into the 59-phosphorylated primer (T: 59-CCAGTGTTGTGGCCTGCAGGGCGAATT-39) with terminal deoxynucleotidyltransferase (TdT) to prevent intramolecular and intermolecular ligation of the primers.…”

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

“…There is a common difficulty in the construction of full-length clones of flaviviruses, because the plasmids containing a full-length cDNA of these viruses are often unstable during propagation in E. coli. Therefore, by using low-copy-number plasmids and specific bacterial hosts, stable full-length infectious clones have been developed for several flaviviruses (Gritsun and Gould, 1998;Hayasaka et al, 2004;Kinney et al, 1997;Shi et al, 2002;Yamshchikov et al, 2001;Yun et al, 2003). In this study, the low-copy-number plasmid pACNR was used for the construction of the full-length OHFV cDNA.…”

Characterization of an infectious clone of the wild-type yellow fever virus Asibi strain that is able to infect and disseminate in mosquitoes

“…However, many of these clones were unstable. Therefore, through the use of low-copy-number vector plasmids and specific bacterial host cells, a number of stable full-length flavivirus infectious clones have been developed and reported, including those for DENV (Lai et al, 1991;Kinney et al, 1997), TBEV (Gritsun & Gould, 1998;Hayasaka et al, 2004;Mandl et al, 1997), WNV (Shi et al, 2002;Yamshchikov et al, 2001) and JEV (Yun et al, 2003). With the exception of differences in cell culture replication kinetics reported for a TBEV infectious clone (Hayasaka et al, 2004), the resulting infectious clones were stable, and virus produced from these infectious clones retained the biological and phenotypic characteristics of their parental viruses.…”

“…For virus production and measurement of specific infectivity, 10 mg RNA was electroporated into BHK-21 cells in L-15 as described previously (Higgs et al, 1997) using the Gene Pulser Xcell electroporation system (Bio-Rad). The specific infectivity of RNA from YFV-As ic, measured by infectious centre assay in Vero cells following a protocol reported previously by Yun et al (2003), was 1?6610 4 f.f.u. (mg RNA) 21 .…”