Abstract
Human noroviruses (HuNoVs) are the leading cause of non-bacterial gastroenteritis worldwide, exhibiting extensive genetic diversity driven by recombination and mutation. The norovirus RNA-dependent RNA polymerase (RdRp), encoded by the NS7 gene, is the main driver of virus evolution by determining the mutation and recombination rates, and impacting viral fitness. Herein, we conducted spatiotemporal phylodynamic analysis on 1,094 complete RdRp amino acid sequences from genogroups GI and GII collected between 1972 and 2024 to investigate molecular evolution, amino acid variation, evolutionary rates, and selection pressures. Time-scaled phylogenetic analysis revealed that GI polymerases diverged into three major lineages since the year ~ 1630, while GII polymerases formed four lineages, including a distinct cluster for P16. Genotype distribution was dominated by GII.P16 (43%) and GII.P31 (20%), both historically associated with GII.4 capsids. Amino acid substitutions were rare to non-existent within conserved motifs (A–G), indicating strong structural and functional constraints. Evolutionary rate estimates showed GII polymerases evolve faster (mean 1.5 × 10⁻³ substitutions/site/year) than GI (mean 3.8 × 10⁻⁴ substitutions/site/year), though overall RdRp evolves more slowly than the major capsid protein VP1. Selection analyses demonstrated pervasive purifying selection, with limited evidence of positive selection at codons outside of the conserved motifs. These findings highlight the high conservation of RdRp, its slower evolutionary dynamics compared to capsid gene, and its role in norovirus diversification, supporting its relevance as a target for antiviral development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-40248-5.