Background: Influenza A viruses (IAVs) initially infect a few host cells before spreading to neighboring cells. However, the molecular mechanisms underlying this dissemination remain unclear. We have previously demonstrated that intracellular Ca2+ plays a crucial role in facilitating IAV infection. This study aims to clarify the connections between intracellular Ca2+ dynamics and spread of IAV infection.

Methods: Madin-Darby canine kidney (MDCK) cells stably expressing a Ca2+ indicator for optical imaging were established. Cells were cultured in Matrigel to form monolayers, and cell-to-cell Ca2+ dynamics within IAV-infected cells were analyzed using fluorescence microscopy.

Results: IAV infection upregulated the frequency of intercellular calcium wave propagations (iCWPs), facilitating viral spread. ADP released from initially infected cells mediated iCWPs via the P2Y1 receptor. P2Y1 antagonist suppressed both the generation of iCWPs and spread of viral infection. Enhanced endocytosis by the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection. Conversely, an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs.

Conclusions: Intercellular calcium signaling plays a crucial role in the early expansion and establishment of IAV infection, presenting a potential target for IAV prophylaxis.