Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza 
virus and mediates the critical "cap-snatching" step of viral RNA transcription, 
which is considered to be a promising anti-influenza target. Here, we describe 
in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the 
active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription 
via selective inhibition of CEN activity in enzymatic assays, and inhibits viral 
replication in infected cells without cytotoxicity in cytopathic effect assays. 
The antiviral activity of BXA is also confirmed in yield reduction assays with 
seasonal type A and B viruses, including neuraminidase inhibitor-resistant 
strains. Furthermore, BXA shows broad potency against various subtypes of 
influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, 
serial passages of the viruses in the presence of BXA result in isolation of 
PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic 
analyses with reverse genetics demonstrate the mechanism of BXA action via CEN 
inhibition in infected cells. These results reveal the in vitro characteristics 
of BXA and support clinical use of BXM to treat influenza.