Adaptation of waterborne echovirus to warm habitats enhances disinfection resistance

Because of their high mutation rate, enteric virus populations excreted from a host are not clonal, but rather consist of a mutant cloud with many different genetic variants. Among these variants, the susceptibility to environmental stressors (e.g., temperature, sunlight, microbial grazing) is expected to differ, and the variants most resistant to environmental stressors are the most likely to persist and encounter a new host. Interestingly, several studies have isolated viruses from the environment with a significantly higher resistance to disinfection compared to the corresponding lab strain. This indicates that environmental persistence and disinfection resistance may be linked. The goal of this study was to determine if exposure to, and persistence under, environmental stress leads to virus resistance to disinfection. Using echovirus 11, we first conducted a series of experimental evolution experiments under different combinations of solar irradiance and temperature, representative of those encountered in temperate and tropical climates. We then determined the persistence of the adapted echovirus 11 populations with respect to thermal inactivation, solar inactivation and microbial grazing, as well as the susceptibility to inactivation by chlorine. None of the adapted populations exhibited enhanced persistence to sunlight or microbial grazing. However, the populations adapted to high temperatures (30 °C) were less prone to thermal inactivation and exhibited a higher capsid stability compared to those adapted to lower temperatures. In addition, the 30 °C-adapted populations were also more resistant to inactivation by chlorine. Interestingly, the mutations associated with thermal stability are commonly found in circulating echovirus 11, indicating a widespread distribution of not only of high environmental persistence, but also disinfection resistance. Overall, our data indicate that viruses that persist in warm surface waters may have enhanced disinfection resistance. As such, disinfection requirements may differ between climate zones, and may change as a function of global warming.

Multi-resistance of human enterovirus to UV-C disinfection and mutagenic nucleoside analogs

Virginie Bachmann, Anna Carratala Ripolles, Tamar Kohn, Qingxia Zhong

Disinfection is an important strategy to control the environmental transmission of human viruses. Specifically, UV254 is increasingly used to disinfect from clinical items or surfaces to drinking water. RNA virus populations have high mutation rates and large population numbers that allow them to rapidly adapt to stressors. It is therefore likely that variants with enhanced resistance to disinfection may eventually emerge in a virus population. The genetic basis and mechanisms that underpin the emergence of virus resistance to disinfection remain, however, unknown. The goal of this study was to investigate the emergence of echoviruses resistant to UV254 disinfection in vitro, and to characterize the genotypic and phenotypic changes in the resistant populations as compared to the wild-type. Resistant virus populations emerged in mutation-accumulation laboratory experiments. Each experiment consisted in exposing echovirus suspensions to a UV254 fluence such that a 4.5 log10 reduction in infective viruses was achieved, and then regrowing the survivors by infecting BGM cells at a very low multiplicity of infection (MOI>0.001). Unexposed control experiments were conducted in parallel by diluting the virus suspensions instead of exposing them to UV254 prior to regrowth. We demonstrated that echovirus populations became increasingly resistant to UV254. Interestingly, a similar extent of resistance was observed in the unexposed control experiments. Fixed mutations were repeatedly identified by next generation sequencing in the 2C and 3D protein region, both involved in the genome replication. The wild-type and resistant echovirus populations exhibited an equivalent replicative fitness under standard culturing conditions. However, the UV254-resistant mutants were more resistant to ribavirin and guanidine hydrochloride. Finally, the exposed populations were able to maintain the production of infective progeny despite the effect of such chemical mutagens. Combined, these findings suggest that multi-resistance of echovirus to UV254 disinfection and treatment with mutagenic nucleotide analogs may arise in virus populations that, subjected to repeated dramatic bottlenecks, evolve towards an increased replication fidelity. The outcomes of this work show that enteric human pathogens may exhibit multi-resistance to disinfection and clinical antiviral therapies. Ultimately, the results from this work may impact future strategies to ensure adequate control of virus environmental transmission.

2016

Adaptation of Human Enterovirus to Warm Environments Leads to Resistance against Chlorine Disinfection

Virginie Bachmann, Anna Carratala Ripolles, Timothy R. Julian, Tamar Kohn

Sunlight, temperature, and microbial grazing are among the environmental factors promoting the inactivation of viral pathogens in surface waters. Globally, these factors vary across time and space. The persistence of viral pathogens, and ultimately their ecology and dispersion, hinges on their ability to withstand the environmental conditions encountered. To understand how virus populations evolve under changing environmental conditions, we experimentally adapted echovirus 11 (E11) to four climate regimes. Specifically, we incubated E11 in lake water at 10 and 30 °C and in the presence and absence of sunlight. Temperature was the main driver of adaptation, resulting in an increased thermotolerance of the 30 °C adapted populations, whereas the 10 °C adapted strains were rapidly inactivated at higher temperatures. This finding is consistent with a source–sink model in which strains emerging in warm climates can persist in temperate regions, but not vice versa. A microbial risk assessment revealed that the enhanced thermotolerance increases the length of time in which there is an elevated probability of illness associated with swimming in contaminated water. Notably, 30 °C-adapted viruses also exhibited an increased tolerance toward disinfection by free chlorine. Viruses adapting to warm environments may thus become harder to eliminate by common disinfection strategies.

2020

Echovirus 11 mutants with increased resistance to common water disinfectants

Virginie Bachmann, Anna Carratala Ripolles, Tamar Kohn, Qingxia Zhong

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 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. 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. This modification also led to change in viral entry pathways. Finally, the resistant echoviruses could outcompete the wild-type in a couple of cell lines tested.

2016

Multi-resistance of human enterovirus to UV-C disinfection and mutagenic nucleoside analogs

Virginie Bachmann, Anna Carratala Ripolles, Tamar Kohn, Qingxia Zhong

2016