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Eight in vitro selection experiments under brincidofovir pressure elicited the known cytomegalovirus DNA polymerase amino acid substitutions N408K and V812L and the novel exonuclease domain substitutions D413Y, E303D, and E303G, which conferred ganciclovir and cidofovir resistance with 6- to 11-fold resistance to brincidofovir or 17-fold when E303G was combined with V812L. The new exonuclease domain I resistance mutations selected under brincidofovir pressure add to the single instance previously reported and show the expected patterns of cross-resistance.

Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance