N460S in PB2 and I163T in nucleoprotein synergistically enhance the viral replication and pathogenicity of influenza B virus

Influenza B viruses (IBVs), though often overshadowed by influenza A viruses (IAVs), remain a significant global public health concern, particularly during seasons when they predominate. However, the molecular mechanisms underlying IBV pathogenicity remain largely unknown. In this study, we identified two amino acid substitutions, PB2-N460S and NP-I163T, from IBV clinical isolates with distinct replication and pathogenicity profiles. Using reverse genetics, we generated recombinant IBV viruses to evaluate the impact of these substitutions. In vitro and in vivo infections revealed that viral replication and pathogenicity were not significantly affected by either substitution alone but were substantially enhanced when both substitutions occurred together. Lung transcriptomics in mice infected with virus containing PB2-N460S/NP-I163T substitutions showed heightened immune activation. This was characterized by upregulated transcription of antiviral and immune-related genes, contributing to excessive inflammation and severe disease outcomes. Mechanistic investigations demonstrated that each substitution independently increased protein expression and strengthened PB2-NP interactions. However, only the combined presence of PB2-N460S and NP-I163T significantly enhanced polymerase activity. Structural modeling indicated that PB2–460 residue is positioned at the PB2-NP interface, while NP-163 site resides distally, suggesting an indirect functional interplay. These findings provide new insights into the molecular determinants of IBV pathogenesis, highlighting the synergistic effect of PB2-N460S and NP-I163T in enhancing viral fitness and worsening disease outcomes.