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

Influenza B virus (IBV), though often overshadowed by influenza A viruses (IAVs), remains a significant global public health concern, particularly during seasons when it predominates. However, the molecular mechanisms underlying IBV pathogenicity remain largely unknown. In this study, we analyzed polymerase polymorphisms in an IBV isolated from a single patient’s oropharyngeal swab and identified two plaque colonies with distinct replication and pathogenicity phenotypes, associated with the PB2:N460S and NP:I163T substitutions. Using reverse genetics, we generated recombinant IBV mutants to evaluate the impact of these substitutions. The results showed that while neither mutation alone significantly affected viral replication or pathogenicity, their combination markedly enhanced both. Transcriptomic analysis of infected lung tissues revealed heightened immune activation, including upregulation of antiviral and immune-related genes, contributing to excessive inflammation and disease severity. Mechanistically, both substitutions increased protein expression and strengthened PB2-NP interaction, but only together did they enhance polymerase activity. Structural modeling suggested that PB2:460 is positioned at the PB2-NP interface, whereas NP:163 is not, indicating 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 exacerbating disease outcomes.