Solar disinfection of viruses: role of carbonate radicals

Lack of improved drinking water sources contributes a lot to many waterborne diseases caused by pathogens which include viruses, protozoa, bacteria or worms. It is essential to develop and promote treatment methods in order to provide pathogen-free safe drinking water. Sunlight plays an important role in disinfection of viruses through direct photolysis or indirect photolysis through reactive transient species such carbonate radical. This study demonstrated that carbonate radical can be generated using 4-carboxylbenzophenone (CBBP) as sensitizer to react with carbonate/bicarbonate ions. The steady state concentration of carbonate radical was determined by measuring the degradation of probe compounds using HPLC. 4-nitroaniline proved to be an appropriate probe compound to be used in this method. CBBP concentration was varied in order to generate different carbonate radical concentrations and subsequently vary the inactivation rate. The measured carbonate radical concentration varied from 10-14 to 10-12 M which corresponded to the values obtained from previous studies. The resultant inactivation kinetics showed that the reaction order of carbonate radical was 0.13±0.064 which may be due to the surface reaction or the interference of 4-NA. The pseudo-first order inactivation rate constant of Phi-X174 bacteriophage was 5 times smaller than that of MS2 while bacteriophage GA had similar inactivation rate constant with MS2

The impact of hospital attributes on patient choice for first visit

The underutilization of primary care in urban China threatens the efficiency and effectiveness of the Chinese health system. To guide patient flow to primary care, the Chinese government has rolled out a sequence of health care reforms which improve the affordability, the infrastructure and workforce of the primary care system. However, these measures have not yielded the desired effect on the utilization of primary care, which is lowest in urban areas. It is unclear how the factors identified to influence facility choice in urban China are actually impacting choice behaviour. We conducted a discrete choice experiment to elicit the quantitative impact of facility attributes when choosing a health care facility for first visit and analysed how the stated choice varies with these attributes. We found that the respondents placed different weights on the identified attributes, depending on whether they perceived their condition to be minor or severe. For conditions perceived as minor, the respondents valued visit time, equipment and medical skill most. For conditions perceived as severe, they placed most importance on equipment, travel time and facility size. We found that for conditions perceived as minor, only 14% preferred visiting a facility over opting out, a percentage which would more than double to 37% if community health centres were maximally improved. For conditions perceived as severe, improvements in community health centres may almost double first visits to primary care, mostly from patients who would otherwise choose higher-level facilities. Our findings suggest that for both severity conditions, improvements to medical equipment and medical skill at community health centres in urban China can effectively direct patient flow to primary care and promote the efficiency and effectiveness of the urban health system

Cross-resistance of UV- or chlorine dioxide-resistant echovirus 11 to other 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

On the cause of the tailing phenomenon during virus disinfection by chlorine dioxide

This study investigates the mechanisms underlying the deviation from Chick-Watson kinetics, namely a tailing curve, during the disinfection of viruses by chlorine dioxide (ClO2). 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 ClO2 concentrations. Four working hypothesis to explain the cause of tailing were tested, namely changes in the solution’s disinfecting capacity, aggregation of viruses, resistant virus subpopulations, and changes in the virus properties over the course of reaction. 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 ClO2. This finding highlights an important shortcoming of ClO2, 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

Resistance of echovirus 11 to ClO2 is associated with enhanced host receptor use, altered entry routes and high fitness

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

Genetic, structural and phenotypic properties of MS2 coliphage with resistance to ClO2 disinfection

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 (ClO2). ClO2 inactivates MS2 by degrading its structural proteins, thereby disrupting the ability of MS2 to attach to and infect its host. 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 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 ClO2 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 ClO2 pressure, but that they can be inactivated by other common disinfectants

Beta-Catenin Signaling Plays a Disparate Role in Different Phases of Fracture Repair: Implications for Therapy to Improve Bone Healing

In a study in mice Benjamin Alman and colleagues show that β-catenin functions differently in different stages of fracture repair; moreover, activation of β-catenin by lithium improves fracture healing when used in later phases of repair

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