Abstract
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.
Highlights
Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia and parts of the Western Pacific, with,60% of the world’s population at risk of infection [1]
We have developed a reverse genetics system for SA14-14-2, a live JE vaccine that is most commonly used in JE-endemic areas, by constructing an infectious bacterial artificial chromosome that contains the full-length SA14-142 cDNA
Using this infectious SA14-14-2 cDNA, combined with a mouse model for JEV infection, we have identified a key viral neurovirulence factor, a conserved single amino acid in the ij hairpin adjacent to the fusion loop of the viral E glycoprotein, which regulates viral infectivity into neurons within the central nervous system in vivo and neuronal cells of mouse and human in vitro
Summary
Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia and parts of the Western Pacific, with ,60% of the world’s population at risk of infection [1]. Within the family Flaviviridae (genus Flavivirus), JEV belongs to the JE serological group, which includes medically important human pathogens found on every continent except Antarctica [2,3]: West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus (MVEV). Worldwide, ,50,000–175,000 clinical cases of JE are estimated to occur annually [10]; this incidence is undoubtedly a considerable underestimate because surveillance and reporting are inadequate in most endemic areas, and only ,0.1–4% of JEV-infected people develop clinical disease [11,12]. On average, ,20–30% of patients die, and ,30–50% of survivors suffer from irreversible neurological and/or psychiatric sequelae
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