Q ZHONG¹, A CARRATALÀ ¹, T KOHN<sup>1

1</sup>Environmental Chemistry Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland

Common water disinfectants like chlorine have been reported to select for resistant viruses. Yet, only little attention has been devoted to characterizing disinfection resistance. The goal of this study was to produce disinfectant-resistant viruses and to characterize the underlying resistance mechanisms. Resistant virus populations of echovirus 11 and MS2 coliphage were established by experimental evolution. Specifically, viruses were repeatedly inactivated by ClO2 and subsequently regrown. In parallel, control experiments were conducted in which virus numbers were reduced by dilution rather than ClO2 exposure. Virus inactivation kinetics were quantified to capture the emergence of resistance. Once a resistant population was established, their genomes and structures were studied and the changes were linked to modifications in vital viral functions. Furthermore, we investigated changes in the replicative fitness, and the cross-resistance towards other disinfectants. Both MS2 and echovirus 11 exhibited resistance after repeated disinfection-regrowth passages. Interestingly, resistance also emerged without exposure to ClO2, indicating that cell passaging rather than ClO2-exposure drives resistance development. The resistant populations exhibited several fixed mutations that caused the substitution of ClO2-labile by ClO2-stable amino acid. These mutations imply a greater protein stability toward oxidation by ClO2. We thus propose that one of the mechanisms underlying resistance is the protection of the host attachment site from oxidation. Alternatively, host attachment may be maintained by switching to a different attachment site. Cross-resistance to other disinfectants was shown to depend on the disinfectants' main viral target: the resistant populations also exhibited resistance against free chlorine, which acts on both viral proteins and genome. In contrast, no cross-resistance was observed for UV light, which exclusively targets the viral genome. Finally, the mutants were inactivated more readily by heat, which indicates that the protection of the viral proteins from oxidation came at the cost of lower thermal stability.