Reassessment of PRRSV-2 ORF5 sequence diversity in Japan identifies a previously unrecognized candidate lineage-level clade.

Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) exhibits extensive genetic diversity, and ORF5-based classification provides a standardized framework for global molecular surveillance. In a previous nationwide study in Japan, lineage 4 (L4) was identified as a dominant lineage exhibiting unexpectedly high genetic diversity. This observation motivated a focused reassessment of L4-assigned viruses to determine whether this heterogeneity reflected within-lineage diversification or deeper phylogenetic separation. In this study, we re-evaluated PRRSV-2 ORF5 sequences previously assigned to L4 in Japan using maximum-likelihood phylogenetic reconstruction and pairwise nucleotide distance analyses consistent with the global ORF5 classification framework. Following sequence de-duplication, 321 L4-assigned ORF5 sequences were analyzed together with global reference datasets. Phylogenetic analysis revealed two divergent clades within the L4-assigned population. One clade contained the global L4 reference sequences, whereas the other formed a distinct monophyletic group composed predominantly of Japanese sequences and supported by bootstrap values ≥ 85% at its defining node. Pairwise uncorrected nucleotide p-distance analysis showed that divergence between the two clades exceeded the 11% ORF5 lineage demarcation threshold (median between-clade p-distance = 0.133). Based on combined phylogenetic and genetic distance evidence, we report this distinct clade, provisionally designated JPL-A, as a candidate lineage-level clade separate from the major L4 clade. These findings indicate that contemporary PRRSV-2 diversity in Japan includes a lineage-level group not fully represented in historical global references and highlight the value of regional surveillance data in refining standardized ORF5-based classification resources.