A Conserved NP Mutation Quartet Drives the Avian-to-Canine Host Jump and Establishes a Molecular Foundation for Zoonotic Adaptation

Understanding the molecular principles of influenza A virus (IAV) host-switching remains a fundamental challenge, as many landmark spillovers occurred before the era of high-resolution genomics. Here, we demonstrate that H3N2 canine influenza virus (CIV) provides a uniquely powerful evolutionary proxy to resolve the real-time molecular logic of host-switching. As a paradigm of rapid avian-to-mammalian adaptation, CIV successfully established a stable lineage and achieved global dominance while consistently lacking the classic PB2 E627K and D701N mutations, which are typically considered prerequisites for mammalian adaptation. We identify a conserved nucleoprotein (NP) mutation quartet (T373K, A428T, R452K, and N473K) as a non-canonical adaptive axis that bypasses the traditional requirement for polymerase-specific mutations. Mechanistically, these mutations optimize the viral RNP’s utilization of a previously unrecognized functional interface: residue 30 within the ANP32A Leucine-Rich Repeat (LRR) domain. While this quartet was primarily selected to exploit the I30 signature—conserved across canines, felines, ferrets, and mice—it concurrently enhances compatibility with the N30 signature shared by humans, swine, equines, and guinea pigs. This dual-track compatibility underscores how adaptation within an initial mammalian reservoir provides a potent molecular foundation for broad host-range expansion. Collectively, our findings establish the NP-ANP32A interface as a critical interspecies barrier and demonstrate that NP-mediated adaptation can serve as a pre-adaptive springboard for zoonotic transmission, offering a new dimension for monitoring the pandemic potential of avian influenza viruses.