An emerging PB2-627 polymorphism increases the pandemic potential of avian influenza virus by breaking through ANP32 host restriction in mammalian and avian hosts

Alterations in the PB2-627 domain could substantially increase the risk of an avian influenza virus (AIV) pandemic. So far, a well-known mammalian mutation PB2-E627K has not been maintained in AIV in poultry, which limits the spread of AIVs from avian to humans. Here, we discovered a variant, PB2-627V, which combines the properties of avian-like PB2-627E and human-like PB2-627K, overcoming host restrictions and posing a risk for human pandemics. Specifically, by screening the global PB2 sequences, we discovered a new independent cluster with PB2-627V emerged in the 2010s, which is prevalent in various avian, mammalian, and human isolates of AIVs, including H9N2, H7N9, H3N8, 2.3.4.4b H5N1, and other subtypes. And, the increasing prevalence of PB2-627V in poultry is accompanied by a rise in human infection cases with this variant. Then we systematically assessed its host adaptation, fitness, and transmissibility across three subtypes of AIVs (H9N2, H7N9, and H3N8) in different host models, including avian and human cells, chickens, mice, and ferrets where infections naturally occur. We found that PB2-627V facilitates AIVs to efficiently infect and replicate in chickens and mice by utilizing both avian- and human-origin ANP32A proteins. Importantly, and like PB2-627K, PB2-627V promotes efficient transmission between ferrets through respiratory droplets. Deep sequencing in passaged chicken samples and transmitted ferret samples indicates that PB2-627V remains stable across the two distinct hosts and has a high potential for long-term prevalence in avian species. Therefore, the mutation has the ability to continue spreading among poultry and can also overcome the barrier between birds and humans, greatly enhancing the likelihood of AIVs infecting humans. Given the escalating global spread of AIVs, it is crucial to closely monitor influenza viruses carrying PB2-627V to prevent a pandemic. Author summary Avian influenza viruses (AIVs) play a critical role in the evolution of pandemic influenza viruses. Adaptive changes in AIVs enable them to infect mammalian species by altering viral interactions with host-specific factors, and one well-known example is the PB2-E627K mutation. Fortunately, this mutation is sporadic and rare in AIVs, usually occurring after infecting humans, and AIVs with PB2-627E, which is prevalent in poultry, cannot easily infect humans. However, if an AIV manages to overcome this species barrier, the risk of a pandemic originating from an AIV will significantly increase. In our study, we identified a dual adaptive variant called PB2-627V that combines avian-like properties of PB2-627E and human-like properties of PB2-627K. This variant not only maintains viral fitness in chickens but also facilitates aerosol transmission of AIVs between ferrets. Mechanistically, PB2-627V utilizes both avian and human host protein ANP32A to support viral polymerase function. The mutation exhibited good fitness in both birds and humans, thereby greatly increasing the likelihood of AIVs infecting humans. The PB2-627V polymorphism has been found in birds, humans, and mammals worldwide across more than ten AIV subtypes. Given the escalating global spread of AIVs, we recommend considering PB2-627V as a new molecular marker to assess the pandemic potential of AIVs. ### Competing Interest Statement The authors have declared no competing interest.