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Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone

¹ Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan
² Chengdu Institute of Biological Products, Chengdu 610063, Sichuan Province, PR China

Correspondence
Takaji Wakita
wakita{at}tmin.ac.jp

A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus–cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus–cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.

The GenBank/EMBL/DDBJ accession numbers for the sequences described in this study are given in Table 1.

Supplementary figures and tables are available in JGV Online.

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