Avian influenza A virus polymerase gene leads to temperature-resistant genome replication

Influenza A viruses (IAV) pose a persistent pandemic threat. Successful cross-species transmission from waterfowl to mammals requires host adaptation for efficient replication and spread. Avian IAVs are adapted to the temperature of the avian gut, approximately 41°C, while human IAVs replicate at 33°C in the upper respiratory tract. PB1, the catalytic core of the viral heterotrimeric RNA-dependent RNA polymerase (RdRp), is essential for viral RNA synthesis. Three pandemic IAV strains (1918, 1957, and 1968) carried an avian-origin PB1 gene, suggesting a selective advantage. Fever, a critical antiviral response, elevates body temperature to 38°C or higher and can restrict viral replication. We hypothesized that avian PB1 may allow IAV replication at febrile temperatures. Using a mini-replicon system, we demonstrate that a polymerase of a seasonal human H1N1 IAV fails to replicate viral RNA efficiently at 40°C, whereas substitution of the PB1 with that from the 1918, 1957, and 1968 pandemic viruses, or a low-pathogenic avian H2N1 virus, restores RNA synthesis. Chimeric seasonal human H1N1 viruses expressing avian or 1918 PB1 replicate at 40°C in MDCK cells, unlike wild-type H1N1. Activity assays with purified chimeric IAV polymerases further suggest that avian-origin PB1 may intrinsically enhance polymerase activity at elevated temperatures. Together, these data show that avian-origin PB1 enables IAV replication at febrile temperatures, highlighting its role in zoonotic emergence and host adaptation. Surveillance of avian-origin PB1 genes in IAVs circulating in mammals may help identify strains with pandemic potential and inform the development of countermeasures.IMPORTANCEInfluenza A viruses of avian origin remain a persistent threat to human health. The 1918, 1957, and 1968 pandemic influenza viruses each acquired an avian-origin PB1 gene, but the adaptive advantage conferred by these genes remains incompletely understood. Here, we show that avian-origin PB1 genes enhance viral RNA replication by a seasonal human influenza virus polymerase at elevated temperature and enable replication of a human influenza virus at 40°C, a temperature relevant to both avian hosts and human febrile illness. PB1-dependent effects were observed in cell-based polymerase assays, with purified polymerase, and during authentic virus infection. These findings identify temperature-resistant genome replication as a potential adaptive property of avian-origin PB1 and suggest that acquisition of an avian PB1 may help influenza viruses overcome temperature barriers encountered during adaptation to mammalian hosts.