Genetic, Structural, and Phenotypic Properties of MS2 Coliphage with Resistance to ClO₂ Disinfection

Common water disinfectants like chlorine have been reported to select for resistant viruses, yet little attention has been devoted to characterizing disinfection resistance. Here, we investigated the resistance of MS2 coliphage to inactivation by chlorine dioxide (ClO₂). ClO₂ inactivates MS2 by degrading its structural proteins, thereby disrupting the ability of MS2 to attach to and infect its host. ClO₂-resistant virus populations emerged not only after repeated cycles of ClO₂ disinfection followed by regrowth but also after dilution-regrowth cycles in the absence of ClO₂. The resistant populations exhibited several fixed mutations which caused the substitution of ClO₂-labile by ClO₂-stable amino acids. On a phenotypic level, these mutations resulted in a more stable host binding during inactivation compared to the wild-type, thus resulting in a greater ability to maintain infectivity. This conclusion was supported by cryo-electron microscopy reconstruction of the virus particle, which demonstrated that most structural modification occurred in the putative A protein, an important binding factor. Resistance was specific to the inactivation mechanism of ClO₂ and did not result in significant cross-resistance to genome-damaging disinfectants. Overall, our data indicate that resistant viruses may emerge even in the absence of ClO₂ pressure but that they can be inactivated by other common disinfectants.