Waterborne viruses can exhibit resistance to common water disinfectants, yet the mechanisms that allow them to tolerate disinfection are poorly understood. Here, we generated echovirus 11 (E11) with resistance to chlorine dioxide (ClO2) by experimental evolution, and we assessed the associated genotypic and phenotypic traits. ClO2 resistance emerged after E11 populations were repeatedly reduced (either by ClO2-exposure or by dilution) and then regrown in cell culture. The resistance was linked to an improved capacity of E11 to bind to its host cells, which was further attributed to two potential causes: first, the resistant E11 populations possessed mutations that caused amino acid substitutions from ClO2-labile to ClO2-stable residues in the viral proteins, which likely increased the chemical stability of the capsid toward ClO2. Second, resistant E11 mutants exhibited the capacity to utilize alternative cell receptors for host binding. Interestingly, the emergence of ClO2 resistance resulted in an enhanced replicative fitness compared to the less resistant starting population. Overall this study contributes to a better understanding of the mechanism underlying disinfection resistance in waterborne viruses, and processes that drive resistance development.

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 (ClO). ClO inactivates MS2 by degrading its structural proteins, thereby disrupting the ability of MS2 to attach to and infect its host. ClO-resistant virus populations emerged not only after repeated cycles of ClO disinfection followed by regrowth but also after dilution-regrowth cycles in the absence of ClO. The resistant populations exhibited several fixed mutations which caused the substitution of ClO-labile by ClO-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 ClO 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 ClO pressure but that they can be inactivated by other common disinfectants.

The emergence of waterborne viruses with resistance to disinfection has been demonstrated in the laboratory and in the environment. Yet, the implications of such resistance for virus control remain obscure. In this study we investigate if viruses with resistance to a given disinfection method exhibit cross-resistance to other disinfectants. Chlorine dioxide (ClO2)- or UV-resistant populations of echovirus 11 were exposed to five inactivating treatments (free chlorine, ClO2, UV radiation, sunlight, and heat), and the extent of cross-resistance was determined. The ClO2-resistant population exhibited cross-resistance to free chlorine, but to none of the other inactivating treatments tested. We furthermore demonstrated that ClO2 and free chlorine act by a similar mechanism, in that they mainly inhibit the binding of echovirus 11 to its host cell. As such, viruses with host binding mechanisms that can withstand ClO2 treatment were also better able to withstand oxidation by free chlorine. Conversely, the UV-resistant population was not significantly cross-resistant to any other disinfection treatment. Overall, our results indicate that viruses with resistance to multiple disinfectants exist, but that they can be controlled by inactivating methods that operate by a distinctly different mechanism. We therefore suggest to utilize two disinfection barriers that act by different mechanisms in order to control disinfection-resistant viruses.

TailingReversible protection MALDI Resistant subpopulation a b s t r a c tThis study investigates the mechanisms underlying the deviation from ChickeWatson kinetics, namely a tailing curve, during the disinfection of viruses by chlorine dioxide (ClO 2 ). Tailing has been previously reported, but is typically attributed to the decay in disinfectant concentration. Herein, it is shown that tailing occurs even at constant ClO 2 concentrations. Four working hypothesis to explain the cause of tailing were tested, specifically changes in the solution's disinfecting capacity, aggregation of viruses, resistant virus subpopulations, and changes in the virus properties during disinfection. In experiments using MS2 as a model virus, it was possible to rule out the solution's disinfecting capacity, virus aggregation and the resistant subpopulation as reasons for tailing. Instead, the cause for tailing is the deposition of an adduct onto the virus capsid over the course of the experiment, which protects the viruses. This adduct could easily be removed by washing, which restored the susceptibility of the viruses to ClO 2 . This finding highlights an important shortcoming of ClO 2 , namely its self-limiting effect on virus disinfection. It is important to take this effect into account in treatment applications to ensure that the water is sufficiently disinfected before human consumption. ª 2013 Elsevier Ltd. All rights reserved. IntroductionChlorination is among the oldest and most commonly used disinfection process worldwide. However, over the years it has been shown that chlorine produces harmful by-products such as trihalomethanes and other halogenated compounds with potential carcinogenic effects (Xie, 2004). It is therefore of interest to investigate other disinfectants that have a similar disinfection potential but generate fewer problematic byproducts. As a good alternative, chlorine dioxide (ClO 2 ) has shown to efficiently disinfect water for human consumption (Huang et al., 1997;Jin et al., 2013;Zoni et al., 2007).Importantly, it is effective at inactivating Cryptosporidium, whereas free chlorine is not (Chauret et al., 2001). Except from exhibiting a good disinfection capacity, ClO 2 can also oxidize iron and manganese, as well as help controlling taste and odor compounds (Aieta and Berg, 1986;Li et al., 1996). The disadvantage of using chlorine dioxide is that it reacts to chlorite, which may be neurotoxic at high doses (Xie, 2004). In 1908, Chick published the first model for describing bacteria inactivation by disinfecting agents (Chick, 1908) Available online at www.sciencedirect.com journal homepage: www.else vier.com/locate /wa tres w a t e r r e s e a r c h 4 8 ( 2 0 1 4 ) 8 2 e8 90043-1354/$ e see front matter ª

Ultraviolet light in the UVC range is a commonly used disinfectant to control viruses in clinical settings and water treatment. However, it is currently unknown whether human viral pathogens may develop resistance to such stressor. Here, we investigate the adaptation of an enteric pathogen, human echovirus 11, to disinfection by UVC, and characterized the underlying phenotypic and genotypic changes. Repeated exposure to UVC lead to a reduction in the UVC inactivation rate of approximately 15 per cent compared to that of the wild-type and the control populations. Time-series next-generation sequencing data revealed that this adaptation to UVC was accompanied by a decrease in the virus mutation rate. The inactivation efficiency of UVC was additionally compromised by a shift from first-order to biphasic inactivation kinetics, a form of ‘viral persistence’ present in the UVC resistant and control populations. Importantly, populations with biphasic inactivation kinetics also exhibited resistance to ribavirin, an antiviral drug that, as UVC, interferes with the viral replication. Overall, the ability of echovirus 11 to adapt to UVC is limited, but it may have relevant consequences for disinfection in clinical settings and water treatment plants.