Inactivation and Tailing during UV₂₅₄ Disinfection of Viruses: Contributions of Viral Aggregation, Light Shielding within Viral Aggregates, and Recombination

UV disinfection of viruses frequently leads to tailing after an initial exponential decay. Aggregation, light shielding, recombination, or resistant virus subpopulations have been proposed as explanations; however, none of these options has been conclusively demonstrated. This study investigates how aggregation affects virus inactivation by UV₂₅₄ in general, and the tailing phenomenon in particular. Bacteriophage MS2 was aggregated by lowering the solution pH before UV₂₅₄ disinfection. Aggregates were redispersed prior to enumeration to obtain the remaining fraction of individual infectious viruses. Results showed that initial inactivation kinetics were similar for viruses incorporated in aggregates (up to 1000 nm in radius) and dispersed viruses; however, aggregated viruses started to tail more readily than dispersed ones. Neither light shielding, nor the presence of resistant subpopulations could account for the tailing. Instead, tailing was consistent with recombination arising from the simultaneous infection of the host by several impaired viruses. We argue that UV₂₅₄ treatment of aggregates permanently fused a fraction of viruses, which increased the likelihood of multiple infection of a host cell and ultimately enabled the production of infective viruses via recombination

Conceptual Model and Experimental Framework to Determine the Contributions of Direct and Indirect Photoreactions to the Solar Disinfection of MS2, phiX174, and Adenovirus

Sunlight inactivates waterborne viruses via direct (absorption of sunlight by the virus) and indirect processes (adsorption of sunlight by external chromophores, which subsequently generate reactive species). While the mechanisms underlying these processes are understood, their relative importance remains unclear. This study establishes an experimental framework to determine the kinetic parameters associated with a virus’ susceptibility to solar disinfection and proposes a model to estimate disinfection rates and to apportion the contributions of different inactivation processes. Quantum yields of direct inactivation were determined for three viruses (MS2, phiX174, and adenovirus), and second-order rate constants associated with indirect inactivation by four reactive species (¹O₂, OH^•, CO₃^•–, and triplet states) were established. PhiX174 exhibited the greatest quantum yield (1.4 × 10^–2), indicating that it is more susceptible to direct inactivation than MS2 (2.9 × 10^–3) or adenovirus (2.5 × 10^–4). Second-order rate constants ranged from 1.7 × 10⁷ to 7.0 × 10⁹ M^–1 s^–1 and followed the sequence MS2 > adenovirus > phiX174. A predictive model based on these parameters accurately estimated solar disinfection of MS2 and phiX174 in a natural water sample and approximated that of adenovirus within a factor of 6. Inactivation mostly occurred by direct processes, though indirect inactivation by ¹O₂ also contributed to the disinfection of MS2 and adenovirus

Kinetics of Inactivation of Waterborne Enteric Viruses by Ozone

Ozone is an effective disinfectant against all types of waterborne pathogens. However, accurate and quantitative kinetic data regarding virus inactivation by ozone are scarce, because of the experimental challenges associated with the high reactivity of ozone toward viruses. Here, we established an experimental batch system that allows tailoring and quantifying of very low ozone exposures and simultaneously measuring virus inactivation. Second-order ozone inactivation rate constants (<i>k</i>_O₃‑virus) of five enteric viruses [laboratory and two environmental strains of coxsackievirus B5 (CVF, CVEnv1, and CVEnv2), human adenovirus (HAdV), and echovirus 11 (EV)] and four bacteriophages (MS2, Qβ, T4, and Φ174) were measured in buffered solutions. The <i>k</i>_O₃‑virus values of all tested viruses ranged from 4.5 × 10⁵ to 3.3 × 10⁶ M^–1 s^–1. For MS2, <i>k</i>_O₃‑MS2 depended only weakly on temperature (2–22 °C; <i>E</i>_a = 22.2 kJ mol^–1) and pH (6.5–8.5), with an increase in <i>k</i>_O₃‑MS2 with increasing pH. The susceptibility of the selected viruses toward ozone decreases in the following order: Qβ > CVEnv2 > EV ≈ MS2 > Φ174 ≈ T4 > HAdV > CVF ≈ CVEnv1. On the basis of the measured <i>k</i>_O₃‑Virus and typical ozone exposures applied in water and wastewater treatment, we conclude that ozone is a highly effective disinfectant for virus control

Ammonia as an In Situ Sanitizer: Inactivation Kinetics and Mechanisms of the ssRNA Virus MS2 by NH₃

Sanitizing human and animal waste (e.g., urine, fecal sludge, or grey water) is a critical step in reducing the spread of disease and ensuring microbially safe reuse of waste materials. Viruses are particularly persistent pathogens and can be transmitted through inadequately sanitized waste. However, adequate storage or digestion of waste can strongly reduce the number of viruses due to increases in pH and uncharged aqueous ammonia (NH₃), a known biocide. In this study we investigated the kinetics and mechanisms of inactivation of the single-stranded RNA virus MS2 under temperature, pH and NH₃ conditions representative of waste storage. MS2 inactivation was mainly controlled by the activity of NH₃ over a pH range of 7.0–9.5 and temperatures lower than 40 °C. Other bases (e.g., hydroxide, carbonate) additionally contributed to the observed reduction of infective MS2. The loss in MS2 infectivity could be rationalized by a loss in genome integrity, which was attributed to genome cleavage via alkaline transesterification. The contribution of each base to genome transesterification, and hence inactivation, could be related to the base p<i>K</i>_a by means of a Bronsted relationship. The Bronsted relationship in conjunction with the activity of bases in solution enabled an accurate prediction of MS2 inactivation rates

Variability in Disinfection Resistance between Currently Circulating <i>Enterovirus B</i> Serotypes and Strains

The susceptibility of waterborne viruses to disinfection is known to vary between viruses and even between closely related strains, yet the extent of this variation is not known. Here, different enteroviruses (six strains of coxsackievirus B5, two strains of coxsackievirus B4 and one strain of coxackievirus B1) were isolated from wastewater and inactivated by UV₂₅₄, sunlight, free chlorine (FC), chlorine dioxide (ClO₂), and heat. Inactivation kinetics of these isolates were compared with those of laboratory enterovirus strains (CVB5 Faulkner and echovirus 11 Gregory) and MS2 bacteriophage. FC exhibited the greatest (10-fold) variability in inactivation kinetics between different strains, whereas inactivation by UV₂₅₄ differed only subtly. The variability in inactivation kinetics was greater between serotypes than it was among the seven strains of the CVB5 serotype. MS2 was a conservative surrogate of enterovirus inactivation by UV₂₅₄, sunlight, or heat but frequently underestimated the disinfection requirements for FC and ClO₂. Similarly, laboratory strains did not always reflect the inactivation behavior of the environmental isolates. Overall, there was considerable variability in inactivation kinetics among and within enteroviruses serotypes, as well as between laboratory and environmental isolates. We therefore recommend that future disinfection studies include a variety of serotypes and environmental isolates

Influence of Amino Acid Substitutions in Capsid Proteins of Coxsackievirus B5 on Free Chlorine and Thermal Inactivation

The sensitivity of enteroviruses to disinfectants varies among genetically similar variants and coincides with amino acid changes in capsid proteins, although the effect of individual substitutions remains unknown. Here, we employed reverse genetics to investigate how amino acid substitutions in coxsackievirus B5 (CVB5) capsid proteins affect the virus’ sensitivity to free chlorine and heat treatment. Of ten amino acid changes observed in CVB5 variants with free chlorine resistance, none significantly reduced the chlorine sensitivity, indicating a minor role of the capsid composition in chlorine sensitivity of CVB5. Conversely, a subset of these amino acid changes located at the C-terminal region of viral protein 1 led to reduced heat sensitivity. Cryo-electron microscopy revealed that these changes affect the assembly of intermediate viral states (altered and empty particles), suggesting that the mechanism for reduced heat sensitivity could be related to improved molecular packing of CVB5, resulting in greater stability or altered dynamics of virus uncoating during infection

Virus Inactivation Mechanisms: Impact of Disinfectants on Virus Function and Structural Integrity

Oxidative processes are often harnessed as tools for pathogen disinfection. Although the pathways responsible for bacterial inactivation with various biocides are fairly well understood, virus inactivation mechanisms are often contradictory or equivocal. In this study, we provide a quantitative analysis of the total damage incurred by a model virus (bacteriophage MS2) upon inactivation induced by five common virucidal agents (heat, UV, hypochlorous acid, singlet oxygen, and chlorine dioxide). Each treatment targets one or more virus functions to achieve inactivation: UV, singlet oxygen, and hypochlorous acid treatments generally render the genome nonreplicable, whereas chlorine dioxide and heat inhibit host-cell recognition/binding. Using a combination of quantitative analytical tools, we identified unique patterns of molecular level modifications in the virus proteins or genome that lead to the inhibition of these functions and eventually inactivation. UV and chlorine treatments, for example, cause site-specific capsid protein backbone cleavage that inhibits viral genome injection into the host cell. Combined, these results will aid in developing better methods for combating waterborne and foodborne viral pathogens and further our understanding of the adaptive changes viruses undergo in response to natural and anthropogenic stressors

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

Inactivation of Bacteriophage MS2 with Potassium Ferrate(VI)

Ferrate [Fe­(VI); FeO₄^2–] is an emerging oxidizing agent capable of controlling chemical and microbial water contaminants. Here, inactivation of MS2 coliphage by Fe­(VI) was examined. The inactivation kinetics observed in individual batch experiments was well described by a Chick–Watson model with first-order dependences on disinfectant and infective phage concentrations. The inactivation rate constant <i>k</i>_<i>i</i> at a Fe­(VI) dose of 1.23 mgFe/L (pH 7.0, 25 °C) was 2.27(±0.05) L/(mgFe × min), corresponding to 99.99% inactivation at a <i>Ct</i> of ∼4 (mgFe × min)/L. Measured <i>k</i>_i values were found to increase with increasing applied Fe­(VI) dose (0.56–2.24 mgFe/L), increasing temperature (5–30 °C), and decreasing pH conditions (pH 6–11). The Fe­(VI) dose effect suggested that an unidentified Fe byproduct also contributed to inactivation. Temperature dependence was characterized by an activation energy of 39(±6) kJ mol^–1, and <i>k</i>_<i>i</i> increased >50-fold when pH decreased from 11 to 6. The pH effect was quantitatively described by parallel reactions with HFeO₄^– and FeO₄^2–. Mass spectrometry and qRT-PCR analyses demonstrated that both capsid protein and genome damage increased with the extent of inactivation, suggesting that both may contribute to phage inactivation. Capsid protein damage, localized in the two regions containing oxidant-sensitive cysteine residues, and protein cleavage in one of the two regions may facilitate genome damage by increasing Fe­(VI) access to the interior of the virion

Bacteria Inactivation during the Drying of Struvite Fertilizers Produced from Stored Urine

Human urine can be processed into market-attractive fertilizers like struvite; however, concerns regarding the microbial safety of such products remain. The present study evaluated the inactivation of in situ heterotrophs, total bacteria as observed by flow cytometry, and inoculated <i>Enterococcus</i> spp. and <i>Salmonella typhimurium</i> during the drying of struvite under controlled temperature (from 5 to 35 °C) and relative humidity (approximately 40 and 80%) as well as dynamic field conditions. Bacteria accumulated in the struvite cake during struvite filtration. Despite the use of sublethal temperatures, all bacteria types were subsequently inactivated to some degree during struvite drying, and the inactivation typically increased with increasing drying temperature for a given relative humidity. Heterotrophic bacteria inactivation mirrored the trend in total bacteria during struvite drying. A linear relationship was observed between inactivation and sample moisture content. However, bacteria survivor curves were typically nonlinear when struvite was dried at low relative humidity, indicating bacterial persistence. Weibull model survivor curve fits indicated that a shift in the mechanism of inactivation may occur with changing humidity. For increased efficiency of bacterial inactivation during the production of struvite, initial heating under moist conditions is recommended followed by desiccation