Host-specific functional evolution of seal influenza A virus NS1 protein following avian-to-seal transmission.

Marine mammals, particularly seals, are susceptible to both avian and human influenza A viruses (IAVs), making them potential intermediates for zoonotic virus emergence. In recent decades, repeated transmissions of avian influenza viruses (AIVs) from wild aquatic birds, their natural reservoir, have caused significant mortality in seals. Defining the molecular determinants of viral adaptation in marine mammals, and their implications for replication in human cells, is therefore essential. The non-structural protein 1 (NS1) of AIV, a key antagonist of the interferon (IFN) response, plays a central role in host adaptation. Here, we analyzed NS1 proteins from seal influenza viruses (H3, H4, H5, H7, and H10 subtypes) and their closest avian relatives isolated between 1980 and 2023, and evaluated their function in seal, avian, and human cells. Phylogenetic analysis confirmed multiple bird-to-seal transmission events. Seal-derived NS1 proteins generally contained few strain-specific amino acid substitutions and showed comparable expression and IFN antagonism to their avian precursors. A notable exception was the seal H10N7 virus isolated in 2014 in Northeastern Europe, which harbored three previously uncharacterized substitutions at NS1 amino acid residues 94, 104, and 171. These amino acid substitutions markedly altered NS1 properties to enhance protein stability, suppress IFN induction, mediate host transcription shut-off, and increase polymerase activity in human cells, without affecting NS1 expression or reducing virus replication in avian cells. Overall, these results reveal how NS1 undergoes host-specific functional evolution following avian-to-seal transmission and provide mechanistic insight into the adaptation of influenza A viruses to mammalian hosts.IMPORTANCEAvian influenza viruses (AIVs) circulate naturally in wild aquatic birds but occasionally infect mammals, including seals, where they can cause severe outbreaks. Seals are of particular concern because they can harbor both avian and human influenza viruses, creating opportunities for reassortment and the emergence of novel zoonotic strains. Understanding how AIVs adapt to mammalian hosts is therefore critical for anticipating and mitigating future influenza threats. Here, we investigated the role of the NS1 protein, a key viral factor that suppresses host immune responses, in seal-derived AIVs. Overall, NS1 expression and function were conserved across different subtypes and host cells. However, we identified unique amino acid substitutions in the NS1 of a seal H10N7 virus that enhanced protein stability, interferon antagonism, and viral adaptation in human cells. These findings illustrate how minor changes in NS1 protein can drive host adaptation and underscore the need for continued surveillance of AIVs in seals.