A Molecularly Cloned, Live-Attenuated Japanese Encephalitis Vaccine SA14-14-2 Virus: A Conserved Single Amino Acid in the ij Hairpin of the Viral E Glycoprotein Determines Neurovirulence in Mice

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.

Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice

“…The location of these mutations in all three E protein structural domains and the stem-anchor region indicates that multiple E protein functions, including cell attachment and entry or virus assembly and release, are probably impaired in neurons of the central nervous system. Reversion of individual or combinations of these JEV SA14-14-2 E protein mutations to the WT amino acid (such as F107L, K138E, G244E, and possibly V315A) is capable of restoring some level of neurovirulence in mice (27)(28)(29), emphasizing that their cumulative effect on neurovirulence attenuation is critical for vaccine safety.…”

“…More recent studies support this conclusion and have demonstrated that passage of JEV SA14-14-2 in mouse brain results in amino acid substitutions in the E protein that produce a neurovirulent phenotype (27,28). However, a recombinant WT JEV Nakayama clone containing prM-E or 5=-UTR-C-prM-E of the JEV SA14-14-2 vaccine strain produced viruses with similar or moderately lower levels of neurovirulence than that of the WT parent virus (29), suggesting that mutations in other regions of the virus genome also contribute to attenuation.…”

A single mutation in the prM protein of Zika virus contributes to fetal microcephaly

“…We used one-day-old neonatal mice, as this stage of development is analogous to the second trimester of pregnancy in humans. Upon intracerebral injection (18,19), all three contemporary strains led to 100% mortality in neonatal mice, with typical neurological manifestations such as inactivity, motor weakness, and bilateral hind limb paralysis. By contrast, CAM/2010 killed only 16.7% animals (Fig.…”

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

“…There again, the molecular basis of JEV-SA 14 -14-2 attenuation is unclear. Two mutations in the E protein were found as important factors: the mutation E138K was involved in viral clearance (81) and interferon antagonism (46), while the mutation E244G was recently linked to neurovirulence attenuation (82). Additionally JEV-SA 14 -14-2 did not appear to produce the NS1= protein (83), an important factor in viral neuroinvasiveness (84).…”