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Virus inactivation mechanisms can be elucidated by methods which measure the loss of specific virus functionality (e.g., host attachment, genome internalization, genome replication). Genome functionality is frequently assessed by PCR-based methods, which are indirect and potentially inaccurate; genome damage that affects detection by high-fidelity PCR enzymes may not adversely affect the ability of actual cellular enzymes to produce functional virus. Therefore, we here develop a transfection-based assay to quantitatively determine viral genome functionality by inserting viral RNA into host cells directly to measure their ability to produce new functional viruses from damaged viral genomes. Echovirus 11 was treated with ozone, free chlorine (FC), UV254 or heat and reductions in genome functionality and infectivity were compared. Ozone reduced genome functionality proportionally to infectivity, indicating that genome damage is the main mechanism of virus inactivation. In contrast, FC caused little or no loss of genome functionality compared to infectivity, indicating a larger role for protein damage. For UV254, genome functionality loss accounted for approximately 60% of virus inactivation, with the remainder presumably due to protein damage. Heat treatment resulted in no reduction in genome functionality, in agreement with the understanding that heat inactivation results from capsid damage. Our results indicate that there is a fundamental difference between genome integrity reductions measured by PCR enzymes in previous studies and actual genome functionality (whether the genome can produce virus) post disinfection. Compared to PCR, quantitative transfection assays provide a more realistic picture of actual viral genome functionality and overall inactivation mechanisms during disinfection. [ABSTRACT FROM AUTHOR]

Treatment of human excreta and animal manure (HEAM) is key in controlling the spread of persistent enteric pathogens, such as viruses. The extent of virus inactivation during HEAM storage and treatment appears to vary with virus genome type, although the reasons for this variability are not clear. Here, we investigated the inactivation of viruses of different genome types under conditions representative of HEAM storage or mesophilic digestion. The goals were to characterize the influence of HEAM solution conditions on inactivation and to determine the potential mechanisms involved. Specifically, eight viruses representing the four viral genome types (single-stranded RNA [ssRNA], double-stranded RNA [dsRNA], single-stranded DNA [ssDNA], and double-stranded DNA [dsDNA]) were exposed to synthetic solutions with well-controlled temperature (20 to 35℃), pH (8 to 9), and ammonia (NH3) concentrations (0 to 40 mmol liter-1). DNA and dsRNA viruses were considerably more resistant than ssRNA viruses, resulting in up to 1,000-fold-longer treatment times to reach a 4-log inactivation. The apparently slower inactivation of DNA viruses was rationalized by the higher stability of DNA than that of ssRNA in HEAM. Pushing the system toward harsher pH (>9) and temperature (>35℃) conditions, such as those encountered in thermophilic digestion and alkaline treatments, led to more consistent inactivation kinetics among ssRNA and other viruses. This suggests that the dependence of inactivation on genome type disappeared in favor of protein-mediated inactivation mechanisms common to all viruses. Finally, we recommend the use of MS2 as a conservative indicator to assess the inactivation of ssRNA viruses and the stable ΦX174 or dsDNA phages as indicators for persistent viruses. [ABSTRACT FROM AUTHOR]

Health risks posed by waterborne viruses are difficult to assess because it is tedious or impossible to determine the infectivity of many viruses. Recent studies hypothesized that quantitative PCR (qPCR) could selectively quantify infective viruses if preceded by an enzymatic treatment (ET) to reduce confounding false-positive signals. The goal of this study was to determine if ET with qPCR (ET-qPCR) can be used to accurately quantify the infectivity of the human viral surrogate bacteriophage MS2 upon partial inactivation by three treatments (heating at 72°C, singlet oxygen, and UV radiation). Viruses were inactivated in buffered solutions and a lake water sample and assayed with culturing, qPCR, and ET-qPCR. To ensure that inactivating genome damage was fully captured, primer sets that covered the entire coding region were used. The susceptibility of different genome regions and the maximum genomic damage after each inactivating treatment were compared. We found that (i) qPCR alone caused false-positive results for all treatments, (ii) ET-qPCR significantly reduced (up to >5.2 log units) but did not eliminate the false-positive signals, and (iii) the elimination of false-positive signals differed between inactivating treatments. By assaying the whole coding region, we demonstrated that genome damage only partially accounts for virus inactivation. The possibility of achieving complete accordance between culture- and PCR-based assays is therefore called into doubt. Despite these differences, we postulate that ET-qPCR can track infectivity, given that decreases in infectivity were always accompanied by dose-dependent decreases in ET-qPCR signal. By decreasing false-positive signals, ET-qPCR improved the detection of infectivity loss relative to qPCR. [ABSTRACT FROM AUTHOR]

Human viruses are ubiquitous contaminants in surface waters, where they can persist over extended periods of time. Among the factors governing their environmental persistence, the control (removal or inactivation) by microorganisms remains poorly understood. Here, we determined the contribution of indigenous bacteria and protists to the decay of human viruses in surface waters. Incubation of echovirus 11 (E11) in freshwater from Lake Geneva and seawater from the Mediterranean Sea led to a 2.5-log10 reduction in the infectious virus concentration within 48 h at 22°C, whereas E11 was stable in sterile controls. The observed virus reduction was attributed to the action of both bacteria and protists in the biologically active matrices. The effect of microorganisms on viruses was temperature dependent, with a complete inhibition of microbial virus control in lake water at temperatures of ≤16°C. Among three protist isolates tested (Paraphysomonas sp., Uronema marinum, and Caecitellus paraparvulus), Caecitellus paraparvulus was particularly efficient at controlling E11 (2.1-log10 reduction over 4 days with an initial protist concentration of 103 cells ml-1). In addition, other viruses (human adenovirus type 2 and bacteriophage H6) exhibited different grazing kinetics than E11, indicating that the efficacy of antiviral action also depended on the type of virus. In conclusion, indigenous bacteria and protists in lake water and seawater can modulate the persistence of E11. These results pave the way for further research to understand how microorganisms control human viral pathogens in aquatic ecosystems and to exploit this process as a treatment solution to enhance microbial water safety. [ABSTRACT FROM AUTHOR]

Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quanti- fied the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin. IMPORTANCE Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-tomouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with waterrelated activities. [ABSTRACT FROM AUTHOR]

Viral disinfection kinetics have been studied in depth, but the molecular-level inactivation mechanisms are not understood. Consequently, it is difficult to predict the disinfection behavior of nonculturable viruses, even when related, culturable viruses are available. The objective of this work was to determine how small differences in the composition of the viral genome and proteins impact disinfection. To this end, we investigated the inactivation of three related bacteriophages (MS2, fr, and GA) by UV254, singlet oxygen (¹O2), free chlorine (FC), and chlorine dioxide (ClO2). Genome damage was quantified by PCR, and protein damage was assessed by quantitative matrix-assisted laser desorption ionization (MALDI) mass spectrometry. ClO2 caused great variability in the inactivation kinetics between viruses and was the only treatment that did not induce genome damage. The inactivation kinetics were similar for all viruses when treated with disinfectants possessing a genome-damaging component (FC, ¹O2, and UV254). On the protein level, UV254 subtly damaged MS2 and fr capsid proteins, whereas GA's capsid remained intact. ¹O2 oxidized a methionine residue in MS2 but did not affect the other two viruses. In contrast, FC and ClO2 rapidly degraded the capsid proteins of all three viruses. Protein composition alone could not explain the observed degradation trends; instead, molecular dynamics simulations indicated that degradation is dictated by the solvent-accessible surface area of individual amino acids. Finally, despite the similarities of the three viruses investigated, their mode of inactivation by a single disinfectant varied. This explains why closely related viruses can exhibit drastically different inactivation kinetics. [ABSTRACT FROM AUTHOR]

Human adenoviruses (HAdV) are important pathogens in both industrialized and developing nations. HAdV has been shown to be relatively resistant to monochromatic UVC light. Polychromatic UVC light, in contrast, is a more effective means of disinfection, presumably due to the involvement of viral proteins in the inactivation mechanism. Solar disinfection of HAdV, finally, is only poorly understood. In this paper, the kinetics and mechanism of HAdY inactivation by UVC light and direct and indirect solar disinfection are elucidated. PCR and mass spectrometry were employed to quantify the extent of genome and protein degradation and to localize the affected regions in the HAdV proteins. For this purpose, we used for the first time an approach in- volving stable isotope labeling by amino acids in cell culture (SILAC) of a human virus. Inactivation by UVC light and the full sunlight spectrum were found to efficiently inactivate HAdY, whereas UVA-visible light only caused inactivation in the presence of external sensitizers (indirect solar disinfection). Genome damage was significant for UVC but was less important for solar disinfection. In contrast, indirect solar disinfection exhibited extensive protein degradation. In particular, the fiber protein and the amino acids responsible for host binding within the fiber protein were shown to degrade. In addition, the central domain of the penton protein was damaged, which may inhibit interactions with the fiber protein and lead to a disruption of the initial stages of infection. Damage to the hexon protein, however, appeared to affect only regions not directly involved in the infectious cycle. [ABSTRACT FROM AUTHOR]