Genotypic, phenotypic and mechanistic insights into viruses with resistance to common water disinfectants

Infectious diseases caused by waterborne viruses contribute to the global disease burden. An effective barrier to prevent the discharge of waterborne viruses is a disinfection step, yet disinfection is not always efficient at inactivating viruses. This thesis seeks to understand the limits of virus inactivation by disinfection, by investigating the emergence and mechanisms of virus resistance to disinfectants. Firstly, the resistance of MS2 coliphage, a surrogate for human enteric viruses, to inactivation by chlorine dioxide (ClO2) was studied. ClO2-resistant virus populations emerged 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 structural modifications in the assembly protein. This led to a more stable host binding during inactivation compared to the wild-type, which ultimately resulted in a greater ability to maintain infectivity. Finally, ClO2 resistance did not result in significant cross-resistance to other disinfectants. The results obtained for the surrogate virus MS2 were validated for an actual human virus, echovirus 11 (E11). As for MS2, ClO2 resistance emerged in E11 after both inactivation-regrowth and dilution-regrowth passages. Mutations fixed in the genome were linked to modifications in viral protein stability, structure and functions. Specifically, the capacity of E11 to bind to host cell was enhanced, and thus E11 became more resistant through their greater ability to interact with host cells. The enhanced binding coincided with the substitution of ClO2-labile by ClO2-stable amino acids, and a greater affinity toward alternative cell receptors. Interestingly, the resistant E11 populations also exhibited an enhanced fitness, indicated by the fact that they outcompeted susceptible strains during co-infection. To prevent the proliferation of ClO2-resistant E11, alternative control methods must thus be sought. To identify disinfection methods that successfully control resistant E11, their mechanisms of action must be understood. For this purpose, the effect of ClO2, free chlorine (FC), UV254, sunlight, and heat on host binding and genome replication was assessed. ClO2 and FC targeted both host binding and genome replication, whereas UV254 and sunlight caused mainly genome damage, and heat only impaired host binding. A ClO2-resistant and a UV254-resistant E11 population were then exposed to these disinfection methods, which revealed that their resistance was mostly mechanism-specific, and not a general trait. This implies that E11 resistant to one disinfectant can be controlled by another disinfectant with a different mechanism. Overall, this thesis contributes to a better understanding of mechanisms of virus inactivation by, and resistance to, disinfection. It demonstrated the potential emergence and the underlying mechanism of disinfectant-resistance in waterborne viruses. The findings support an improved disinfection design that incorporates two disinfection steps with distinct mechanisms of action, in order to control the proliferation of disinfectant-resistant viruses.