BGMK cells, Escherichia coli, E11 and bacteriophage MS2 were cultured and maintained as described previously (Zhong et al., 2016, 2017). Infective E11 concentrations were enumerated as most probable number of cytopathic units per mL (MPNCU/mL), and infective MS2 concentrations were determined as plaque forming units per mL (PFU/mL) (Suess, 1982).

ClO₂-resistant and UV₂₅₄-resistant E11 populations were obtained by experimental evolution (Figure 1). The production of ClO₂-resistant populations was described in detail in Zhong et al. (2017). Briefly, the E11 laboratory strain, here denoted as “wild-type” (WT), was subjected to 20 passages of directed evolution. During each passage, the virus population was exposed to ClO₂ up to an exposure of 6 mg/L^*min, resulting in an inactivation of at least 3 log₁₀, before the inactivation was halted and the remaining virus population was regrown on BGMK cells. The resulting ClO₂-resistant population is henceforth referred to as E_ClO₂ (“exposed to ClO₂”). A similar approach was used to obtain UV₂₅₄-resistant E11, except that a different ancestral population of E11 was used. Specifically, prior to any exposure to UV₂₅₄, the E11 lab strain (WT) was first subjected to three cell culture adaptation passages in the presence of the mutagen ribavirin (Fluorochem), to enhance the genetic diversity of the starting population (Crotty et al., 2000). This population (WT_Rib+) was then subjected to 20 passages of titer reduction by UV₂₅₄ followed by regrowth. The details of the UV₂₅₄ setup are given in the following section. The resulting UV₂₅₄-resistant population is named E_UV hereafter. Both evolved populations at their last (20th) passage as well as the corresponding wild-types were sequenced as described below and the mutations of the evolved populations are listed in Table 1.

All inactivation experiments were performed in phosphate-buffered saline [PBS; 5 mM Na₂HPO₄ (99%, Acros), 10 mM NaCl (99.5%, Acros), pH 7.4], at a starting E11 concentration of 10⁶–10⁷ MPNCU/mL. The specific inactivation assays associated with the different methods tested are summarized below. Kinetic analyses were performed by monitoring the loss of infective E11 as a function of disinfectant exposure (oxidants), dose (UV₂₅₄ and solar radiation), or time (heat).

In order to identify the main viral functions affected during inactivation, we quantified the effect of the different disinfectants on genome replication and on host binding. To this end, E11 WT was inactivated by several orders of magnitude by ClO₂, FC, heat, UV₂₅₄, or sunlight. Isothermal conditions were applied for heat inactivation where viruses were incubated at 56°C in a water bath for 5 min. Samples were collected and divided into two aliquots. The first aliquot was diluted and infectious units of the sample were determined by infectivity assay. The other aliquot was subjected to the genome replication or host binding assays described below.

The genomes of virus populations of interest were extracted, and sequencing libraries were prepared as described previously (Zhong et al., 2017). The whole genomes were then sequenced by Next Generation Sequencing (NGS) at the Lausanne Genomics Technologies Core Facility. Briefly, 300 bp PCR amplicons were purified and pooled to obtain 100 ng of nucleic acids per sample. Libraries of 100 bp double-stranded cDNA were constructed using the TruSeq Stranded mRNA Library Prep kit (Illumina). The library nucleic acid concentrations were measured by Nanodrop 2000 (Thermal Fisher Scientific Inc.) and the cDNA quality was checked by Fragment AnalyzerTM (Advanced Analytical). An Illumina HiSeq 2500 platform was used to sequence up to 6 independent, barcoded and pooled libraries. Reads of 100 nucleotides were trimmed and cleaned for further bioinformatics analysis. Only the genomic positions for which the number of reads exceeded 100 were included in downstream analysis. Single-end reads were aligned to E11 Gregory strain using HTS station (David et al., 2014) to call the nucleotide/base at each position The sequence of WT was used as reference to identify the fixed mutations in all evolved populations.

To determine if the evolved (E_ClO₂ and E_UV) populations were more resistant than their corresponding wild-types, their inactivation rate constants were compared first by paired t-test analysis. The comparison of ClO₂ inactivation kinetics was done by Likelihood Ratio (LR) test (Haas et al., 2014), where the test statistics were determined by Chi-squared distribution table. For all tests the threshold p-value for statistical significance was 0.05. The goodness-of-fit was evaluated based on the coefficient of determination (R²), which was determined by GraphPad Prism (Version 6.01, 2012). Unpaired t-test or regular one-way ANOVA was applied to compare all other parameters between populations or treatments.

Example inactivation curves of E11 WT by ClO₂, FC, UV₂₅₄, sunlight, and heat are shown in Figure 2. The inactivation of E11 WT by to FC, UV₂₅₄, and sunlight were first-order with respect to the disinfectant exposure over the inactivation range tested. The inactivation curve of ClO₂, in contrast, tailed off at higher ClO₂ exposures. Despite this tail, a 3 log₁₀ (99.9%; 6.9 ln) reduction in the infective viral load could be readily achieved (within minutes) by ClO₂ at the disinfectant exposures tested. Inactivation by FC and UV₂₅₄ at the conditions employed herein were similarly effective. In contrast, inactivation by simulated sunlight proceeded more slowly, such that a 3 log₁₀ inactivation required several hours of exposure. Finally, inactivation by thermal shift revealed that the decay temperature of E11 was around 41°C. Beyond this temperature, 3 log₁₀ virus decay was achieved within four temperature shifts during which the temperature increased to 50°C.

The susceptibilities of the ClO₂- and UV₂₅₄-tolerant E11 populations, as well as their corresponding wild-type were tested for the five inactivating treatments considered. Figure 3 shows the extent of resistance to the original stressor (ClO₂ for E_ClO₂ and UV₂₅₄ for E_UV), along with the extent of cross-resistance to each additional treatment considered. The data are presented as the inactivation rate constants of the E_ClO₂ and E_UV populations relative to their respective wild-types, such that a value below unity indicates greater resistance than the wild-type. The absolute inactivation rate constants are reported in Supplementary Tables 1–4. For heat inactivation, the difference in decay temperature to the corresponding wild-type is shown, with the original decay temperatures reported in Supplementary Table 5.

E_ClO₂ exhibited a 43% reduction in k_ClO2compared to its wild-type (p < 0.0001; Figure 3A). E_ClO₂ was furthermore cross-resistant to FC, with a 19% reduction in k_FC compared to its wild-type (p < 0.0001). Compared to ClO₂, resistance to FC was thus less pronounced. Finally, no significant resistance was observed toward UV₂₅₄, sunlight or heat.

E_UV exhibited resistance to UV₂₅₄ inactivation, though its extent was low. Specifically, the reduction in k_UV compared to its wild-type was 15% (p = 0.0008; Figure 3B). In addition, E_UV exhibited a 14% reduction in susceptibility to ClO₂ compared to the wild type, though the difference was not significant at the 95% confidence level (p = 0.0977). Finally, this population was more susceptible than its wild-type to heat, yielding a 4°decrease in Td (p = 0.0070), whereas no measurable resistance to FC or sunlight was observed.

Disinfection resistance arises from mutations to the viral genome which confer an advantage with respect to withstanding disinfection. A comparison of the mutations fixed in each of the resistant populations, along with their likely effects on the viral phenotype, may further aid in understanding the occurrence or absence of cross-resistance.

A total of 12 non-synonymous mutations were identified (Table 1). Of these, five were shared by both resistant populations, and may be a result of adaptation to cell culturing. E_ClO₂ furthermore exhibited unique non-synonymous mutations in the structural protein VP1, whereas unique non-synonymous mutations in E_UV were located in the replication-related proteins 2C and 3D, as well as in structural protein VP4. Given that there was no cross-resistance of E_ClO₂ to UV₂₅₄ (Figure 3A) and only minor cross-resistance of E_UV to ClO₂, these unique mutations serve as candidate mutations responsible for the resistance of E_UV to UV₂₅₄ and of E_ClO₂ to ClO₂ respectively.

Based on our findings of reduction in genome integrity (Figures 4A,B) and inhibition of host binding (Figures 4C,D), an overview of the main mechanisms of inactivation is presented in Figure 6. Roughly, the modes of action of the different disinfectants can be categorized into three groups, depending on the major viral function impaired. First, for heat, inactivation can be attributed entirely to a loss in host binding. Accordingly, no other viral functions are implicated in inactivation and all genomes remain as replicable as in the untreated samples. Second, for UV₂₅₄ and sunlight, no or minimal binding loss was detected. Inactivation must thus be due to losses in other viral functions, such as genome internalization, replication or virion assembly. While these functions were not tested individually, both UV₂₅₄ and sunlight resulted in a considerable decrease in the fraction of PCR-replicable genome copies. The extensive genome degradation observed by PCR supports the conclusion that the main mechanism of inactivation by both UV₂₅₄ and sunlight involves genome damage, and hence inhibition of replication. Third, inactivation by the oxidants ClO₂ and FC cannot be attributed to loss in a single virus function. Their mode of action mainly involves a reduction in host binding, yet losses in other functions also contribute significantly. As observed for UV₂₅₄ and sunlight, treatment by FC and ClO₂ also leads to extensive genome degradation, which likely causes a loss of genome replication.

The proposed mechanisms agree with previous studies on enterovirus that demonstrated that heat and FC inhibited host binding of poliovirus (Nuanualsuwan and Cliver, 2003a) and that UV₂₅₄'s primary target is the genome (Helentjaris and Ehrenfeld, 1977; Nuanualsuwan and Cliver, 2003b). The proposed mechanisms of inactivation of E11 are also are largely consistent with those previously described for MS2 (Wigginton et al., 2012). Major discrepancies were only found for ClO₂: this disinfectant was previously reported to have no effect on the genome integrity of MS2 (Wigginton et al., 2012), and no effect on host binding for poliovirus (Alvarez and O'Brien, 1982), whereas both these functions were inhibited in E11. The disagreement may be linked to differences in the viral species investigated and their binding motifs, as well as to the disinfectant exposures and solution conditions considered. Furthermore, the inactivation curve of E11 by ClO₂ exhibits a pronounced tail (Figure 2A). This feature has previously been reported for virus inactivation by ClO₂, and has been attributed to multiple causes, including the presence of resistant subpopulations or the gradual accumulation of protein oxidation products that form a protective layer on the viral capsid (Berman and Hoff, 1984; Chen and Vaughn, 1990; Thurston-Enriquez et al., 2005; Lim et al., 2010; Jin et al., 2013; Sigstam et al., 2013). The tailing inactivation curve may cause the extent of genome damage by ClO₂ to not scale linearly with inactivation, but instead to increasingly exceed the extent of inactivation (Figure 5). As such, it is likely that the relative contribution of genome damage to inactivation by ClO₂ depends on the ClO₂ exposure and the extent of inactivation considered.

In the ClO₂-resistant population E_ClO₂, we previously identified that resistance was rooted in the ability to utilize an additional host receptor, which was in turn linked to mutations in VP1 (Zhong et al., 2017). This trait allowed the resistant population to better maintain host binding in the presence of ClO₂, and hence to tolerate higher ClO₂ exposure. Cross-resistance of E_ClO₂ may thus be expected to any disinfectant that inhibits host binding (Figure 6). Consistent with this hypothesis, E_ClO₂ also exhibited resistance to FC. In contrast, no cross-resistance to heat was observed, even though this treatment also affects host binding. This result can be rationalized by considering that ClO₂ and FC both oxidize viral proteins, whereas heat acts by denaturation (Rombaut et al., 1994; Dodd, 2012). While the mutations in E_ClO₂ protected the virus from oxidation by allowing alternative receptor use, and by replacing oxidation-reactive by stable amino acids (Table 1) (Sharma and Sohn, 2012), they may not yield the same benefits for protection from denaturation. Finally, the absence of cross-resistance to UV₂₅₄ and sunlight, which do not act on host binding, supports that resistance to ClO₂ is a mechanism-specific trait.

In population E_UV, resistance to UV₂₅₄ implies a greater ability of the resistant population to deal with mutations accumulated through the action of UV₂₅₄. This ability should also extend to sunlight, since solar UVB (280–315 nm) is also known to have mutagenic action (Pfeifer et al., 2005). Yet, population E_UV did not demonstrate measurable cross-resistance to sunlight. This observation can be explained by considering that the UV₂₅₄-resistance of this population was relatively mild, and that UV at different wavelengths have distinct mutational specificity (Pfeifer et al., 2005).

No cross-resistance of E_UV was observed for heat, which is consistent with its mode of action being entirely protein dependent. Instead, E_UV exhibited enhanced susceptibility to heat. We tentatively attribute this feature to mutation Y33F, which was only found in E_UV, and which is located on the N-terminus of structural protein VP4. The intertwining N-terminus extension of VP1, VP3, and VP4 form a network of protein-protein interactions on the interior of the capsid that is crucial to viral stability (Knipe and Howley, 2007). Therefore, we argue that Y33F on VP4 rendered the E_UV less structurally stable and hence more heat-sensitive. This proposition is supported by the prediction of protein stability changes upon single point mutations using I-Mutant2.0 (Capriotti et al., 2005, 2008). Mutation Y33F was estimated to yield a Gibbs free energy change of −0.76 at 45°C compared to the wild-type. Therefore, Y33F is destabilizing the protein. I-Mutant, however, considers only single proteins, hence free energy calculations that take into consideration inter-protein interactions are needed to validate the result.

Finally, E_UV was slightly cross-resistant to ClO₂, even though these two disinfectants act by drastically different mechanisms. This finding indicates that populations with a more general resistance spectrum can exist. However, the inverse cross-resistance was not found: E_ClO₂ remained susceptible to UV₂₅₄. This supports the notion that the multi-resistance of E_UV is not linked to the resistance to UV₂₅₄ per se, but may be induced by the experimental evolution assay used to produce the evolved populations. Specifically, resistant populations were produced by repeated and drastic reduction of their population numbers by either ClO₂ or UV₂₅₄ exposure, followed by regrowth. This action likely selected for those variants that most efficiently proliferated under the experimental conditions used. Efficient proliferation may be aided by enhanced host binding, which in turn is also beneficial to resistance to ClO₂. Interestingly, both evolved populations shared mutations that are confirmed or probable sites associated with a host receptor switch (VP2-G139R, VP1-M238V, and VP1-K259E; Table 1) (Zhong et al., 2017). This supports the notion that analogous to E_ClO₂, the ClO₂ resistance manifested in E_UV is also associated with a better use of an alternative cell receptor.

Given the significant cross-resistance of ClO₂ and FC observed in E_ClO₂, the presence of cross-resistance of E_UV to ClO₂ but absence of cross-resistance to FC is surprising. However, resistance of E_ClO₂ to FC is less pronounced compared to ClO₂ (Figure 3A), and cross-resistance to ClO₂ in E_UV was only slight (Figure 3B). Combined, these two factors likely rendered any cross-resistance of E_UV to FC too small to be experimentally measured.

Overall, this study supports the hypothesis that cross-resistance is mainly found among disinfectants that act by a similar mechanism. To confirm this result, future studies should include viruses with resistance to less specific stressors, such as ozone or free chlorine, which significantly target both viral proteins and genomes. It is conceivable that viruses evolved under pressure of such non-selective disinfectants evolve more general resistances that extend to both genome- and protein-active disinfectants.

As discussed in the introduction, the presence of disinfection-resistant viruses in the environment and is already well-established, though the origin of their resistance is not always known. A potential new source of resistant viruses may be the increasing practice of direct potable reuse of wastewater. In these systems, waterborne viruses may remain in the “treatment-consumption-excretion-treatment” cycle, where they can become subjected to iterate disinfectant exposures and cause new infections. In such a setting, we should be conscious of the potential emergence of disinfection-resistant viruses, and evaluate the best approaches to control their occurrence.

Our results to date suggest that viruses with resistance to a given disinfectant can be controlled by a disinfectant with a different mode of action. This may be achieved by implementing a double disinfection barrier that uses different disinfectants in sequence. From our study on echovirus, a smart choice of disinfectant to include in a double barrier setup is UV₂₅₄. First, UV₂₅₄ is a rather non-selective disinfectant that acts on all genetic material to a roughly similar extent (Lytle and Sagripanti, 2005). This is a stark contrast to a disinfectant like ClO₂, which only efficiently targets specific amino acids, namely cysteine, tyrosine, tryptophan, histidine, and proline (Tan et al., 1987; Sharma and Sohn, 2012). Compared to ClO₂, it is thus unlikely that any virus will ever fully escape the pressure of UV₂₅₄. Second, the resistance to UV₂₅₄ was slight compared to that to ClO₂ (though we cannot exclude that different experimental approaches to produce the resistant virus result in greater resistance). Even if the resistance is minor, however, it remains necessary to include an additional disinfection step using a different disinfectant, such as free chlorine, to control UV₂₅₄-resistant organisms.

The efficiency of different double disinfection barriers to control resistant viruses remains to be tested in future work. In particular, this approach should be validated for additional viruses, as their inactivation mechanisms by the disinfectants tested may differ from that of E11. Furthermore, research should identify ideal combinations of disinfectants and optimal treatment regimes. Ultimately, such a setup should be able to successfully inactivate resistant viruses while avoiding the emergence of multi-resistant viruses.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.