Genotype-resolved NS5 stability predicts Japanese encephalitis virus fitness.

Japanese encephalitis virus (JEV) remains a major health threat across Asia, yet the contribution of genotype-specific variation in the multifunctional NS5 protein to viral fitness is not fully resolved. This study evaluated how sequence differences among JEV genotypes G1-G5 shape NS5 stability and, in turn, replication potential. A unified in-silico workflow combined physicochemical profiling, residue-level substitution mapping, and atomistic molecular dynamics to compare structural stability and conformational behavior across genotypes, with a focus on substitutions predicted to modulate enzymatic performance. Analyses revealed that G5 NS5 maintains a balanced electrostatic environment and persistent hydrogen-bonding networks, yielding greater structural stability than other genotypes. In contrast, G4 NS5 presented a charge imbalance and reduced stability. Simulations consistently supported the robustness of G5 dynamics, with specific substitutions, including Y65, M59, E182, and T191, contributing to improved packing, favorable local interactions, and putative gains in catalytic efficiency. These molecular attributes align with heightened replication capacity and provide a mechanistic rationale for the recent prominence of G5 strains relative to G1-G4. Comprising together, our results demonstrate that genotype-linked substitutions in NS5 directly influence protein stability, replication efficiency, and adaptive potential. Translationally, prioritizing G5-informed NS5 features may guide the design of small-molecule inhibitors and vaccine antigens with broader protective value.