Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca²⁺) binding in the low-affinity Ca²⁺-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca²⁺-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca²⁺ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca²⁺ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca²⁺ binding in the low-affinity calcium-binding pocket.