Phenotypic characterization of two naturally occurring human Cytomegalovirus sequence polymorphisms located in a distinct region of ORF UL56 known to be involved in in vitro resistance to letermovir.

•Letermovir is a new anti HCMV drug in Phase 3 clinical development.•Letermovir’s novel mode of action targets the UL56 subunit of the viral terminase complex.•Letermovir resistance has been mapped in vitro to a distinct region of ORF UL56 (AA 230–370).•Natural UL56 sequence variants D242G and A327V are not associated with letermovir resistance.•Our data are important to clinicians involved in letermovir resistance testing.