Abstract
This study investigates the codon usage characteristics of Japanese encephalitis virus (JEV) genotype 5 (G5). Based on 339 complete JEV genome sequences, we systematically compared the codon usage patterns of G5 with other genotypes (G1-G4) using a multi-faceted approach, including evolutionary analysis, nucleotide composition, Relative Synonymous Codon Usage (RSCU), Principal Component Analysis (PCA), Effective Number of Codons Plot analysis (ENC-Plot), Parity Rule 2 analysis (PR2), Neutrality plot analysis, dinucleotide abundance analysis and Codon Adaptation Index analysis (CAI). The results indicate that G5 forms a distinct evolutionary branch, with both its overall GC content (50%) and GC content at the third codon position (GC3, 53%) being lower than those of other genotypes. RSCU analysis revealed a preferential use of A/U-ended codons in G5, indicating a trend towards reduced GC3 usage. ENC analysis demonstrated a stronger codon usage bias in G5 (mean ENC = 54.2). Furthermore, ENC-plot, PR2, and neutrality plot analyses collectively suggested that G5 is subject to stronger natural selection pressure. Analysis of dinucleotide abundance showed a significant increase in CA values in G5, while CAI analysis indicated higher translational efficiency in human hosts compared to Culex mosquito hosts. Our findings suggest that G5 JEV, potentially through reduced Cytosine-phosphate-Guanine (CpG) usage and optimized codon preference, may enhance its capabilities for immune evasion and host adaptation, and could possess the potential for efficient replication in humans or other mammalian hosts. This research provides crucial theoretical insights into the molecular evolutionary mechanisms of G5 JEV and informs related vaccine development.