Isolation and Transcriptomic Characterization of Genotype 5 Japanese Encephalitis Virus E138K Mutant Strain

The recent emergence of the genotype five Japanese encephalitis virus (G5 JEV) has once again drawn public attention to public health concerns. Plaque assay analysis of G5 JEV parental strain XZ0934 revealed two distinct plaque morphologies. To investigate this phenotypic heterogeneity, we performed single-plaque purification and isolated two strains, designated as XZ0934-L (large plaque) and XZ0934-S (small plaque). Subsequently, deep mutational scanning revealed an amino acid mutation at position 138 (E138K) in the E protein of the XZ0934-S strain. Viral titer determination and plaque morphology analysis showed that the titers of XZ0934-L and XZ0934-S in BHK-21 cells were 10^7.06 PFU/mL and 10^7.35 PFU/mL, respectively, with plaque diameters of 0.87 ± 0.12 mm and 0.38 ± 0.08 mm. However, while both strains induced cytopathic effects across six cell lines used in this study, XZ0934-S produced markedly weaker CPE than XZ0934-L in N2a cells. Spatial modeling predicted that the E138K substitution did not significantly alter the overall conformation of the E protein. In contrast, transcriptomic analysis demonstrated that infections with different JEV genotypes induced significantly distinct host gene expression profiles in N2a cells. The XZ0934-S strain caused the mildest transcriptional perturbations, and the perturbation of regulatory pathways was markedly weaker than those of the XZ0934-L strain. Previous studies have suggested that the E138K mutation can attenuate the neurovirulence of JEV. This study provides the first comprehensive in vitro characterization of an E138K mutant in G5 JEV. The XZ0934-S mutant strain, given its small plaque phenotype and reduced transcriptional impact, represents a promising candidate for further vaccine development. Future studies should rigorously evaluate its safety profile, genetic stability, and immunogenicity in animal models.