There is a plethora of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) serological tests based either on nucleocapsid phosphoprotein (N), S1-subunit of spike glycoprotein (S1) or receptor binding domain (RBD). Although these single-antigen based tests demonstrate high clinical performance, there is growing evidence regarding their limitations in epidemiological serosurveys. To address this, we developed a Luminex-based multiplex immunoassay that detects total antibodies (IgG/IgM/IgA) against the N, S1 and RBD antigens and used it to compare antibody responses in 1225 blood donors across Greece. Seroprevalence based on single-antigen readouts was strongly influenced by both the antigen type and cut-off value and ranged widely [0.8% (95% CI 0.4–1.5%)–7.5% (95% CI 6.0–8.9%)]. A multi-antigen approach requiring partial agreement between RBD and N or S1 readouts (RBD&N|S1 rule) was less affected by cut-off selection, resulting in robust seroprevalence estimation [0.6% (95% CI 0.3–1.1%)–1.2% (95% CI 0.7–2.0%)] and accurate identification of seroconverted individuals.

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Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs.IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.