Multiple pathogen contamination of water, hands, and fomites in rural Nepal and the effect of WaSH interventions.

Water, Sanitation, and Hygiene (WaSH) interventions are the most effective in reducing diarrheal disease severity and prevalence. However, very few studies have investigated the effectiveness of WaSH intervention in reducing pathogen presence and concentration. In this study, we employed a microfluidic PCR approach to quantify twenty bacterial pathogens in water (n = 360), hands (n = 180), and fomite (n = 540) samples collected in rural households of Nepal to assess the pathogen exposures and the effect of WaSH intervention on contamination and exposure rates. The pathogen load and the exposure pathways for each pathogen in intervention and control villages were compared to understand the effects of WaSH intervention. Pathogens were detected in higher frequency and concentration from fomites samples, toilet handle (21.42%; 5.4,0 95%CI: mean log10 of 4.69, 5.96), utensils (23.5%; 5.47, 95%CI: mean log10 of 4.77, 6.77), and water vessels (22.42%; 5.53, 95%CI: mean log10 of 4.79, 6.60) as compared to cleaning water (14.36%; 5.05, 95%CI: mean log10 of 4.36, 5.89), drinking water (14.26%; 4.37, 85%CI: mean log10 of 4.37, 5.87), and hand rinse samples (16.92%; 5.49, 95%CI: mean log10 of 4.77, 6.39). There was no clear evidence that WaSH intervention reduced overall pathogen contamination in any tested pathway. However, we observed a significant reduction (p < 0.05) in the prevalence, but not concentration, of some target pathogens, including Enterococcus spp. in the intervention village compared to the control village for water and hands rinse samples. Conversely, no significant reduction in target pathogen concentration was observed for water and hand rinse samples. In swab samples, there was a reduction mostly in pathogen concentration rather than pathogen prevalence, highlighting that a reduction in pathogen prevalence was not always accompanied by a reduction in pathogen concentration. This study provides an understanding of WaSH intervention on microbe concentrations. Such data could help with better planning of intervention activities in the future.

Building Environmental and Sociological Predictive Intelligence to Understand the Seasonal Threat of SARS-CoV-2 in Human Populations.

Current modeling practices for environmental and sociological modulated infectious diseases remain inadequate to forecast the risk of outbreak(s) in human populations, partly due to a lack of integration of disciplinary knowledge, limited availability of disease surveillance datasets, and overreliance on compartmental epidemiological modeling methods. Harvesting data knowledge from virus transmission (aerosols) and detection (wastewater) of SARS-CoV-2, a heuristic score-based environmental predictive intelligence system was developed that calculates the risk of COVID-19 in the human population. Seasonal validation of the algorithm was uniquely associated with wastewater surveillance of the virus, providing a lead time of 7-14 days before a county-level outbreak. Using county-scale disease prevalence data from the United States, the algorithm could predict COVID-19 risk with an overall accuracy ranging between 81% and 98%. Similarly, using wastewater surveillance data from Illinois and Maryland, the SARS-CoV-2 detection rate was greater than 80% for 75% of the locations during the same time the risk was predicted to be high. Results suggest the importance of a holistic approach across disciplinary boundaries that can potentially allow anticipatory decision-making policies of saving lives and maximizing the use of available capacity and resources.

Efficacy of Messenger RNA-1273 Against Severe Acute Respiratory Syndrome Coronavirus 2 Acquisition in Young Adults From March to December 2021.

Background: The efficacy of messenger RNA (mRNA)-1273 against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well defined, particularly among young adults. Methods: Adults aged 18-29 years with no known history of SARS-CoV-2 infection or prior vaccination for coronavirus disease 2019 (COVID-19) were recruited from 44 US sites from 24 March to 13 September 2021 and randomized 1:1 to immediate vaccination (receipt of 2 doses of mRNA-1273 vaccine at months 0 and 1) or the standard of care (receipt of COVID-19 vaccine). Randomized participants were followed up for SARS-CoV-2 infection measured by nasal swab testing and symptomatic COVID-19 measured by nasal swab testing plus symptom assessment and assessed for the primary efficacy outcome. A vaccine-declined observational group was also recruited from 16 June to 8 November 2021 and followed up for SARS-CoV-2 infection as specified for the randomized participants. Results: The study enrolled 1149 in the randomized arms and 311 in the vaccine-declined group and collected >122 000 nasal swab samples. Based on randomized participants, the efficacy of 2 doses of mRNA-1273 vaccine against SARS-CoV-2 infection was 52.6% (95% confidence interval, -14.1% to 80.3%), with the majority of infections due to the Delta variant. Vaccine efficacy against symptomatic COVID-19 was 71.0% (95% confidence interval, -9.5% to 92.3%). Precision was limited owing to curtailed study enrollment and off-study vaccination censoring. The incidence of SARS-CoV-2 infection in the vaccine-declined group was 1.8 times higher than in the standard-of-care group. Conclusions: mRNA-1273 vaccination reduced the incidence of SARS-CoV-2 infection from March to September 2021, but vaccination was only one factor influencing risk. Clinical Trials Registration: NCT04811664.

Local and Environmental Reservoirs of Salmonella enterica After Hurricane Florence Flooding.

In many regions of the world, including the United States, human and animal fecal genetic markers have been found in flood waters. In this study, we use high-resolution whole genomic sequencing to examine the origin and distribution of Salmonella enterica after the 2018 Hurricane Florence flooding. We specifically asked whether S. enterica isolated from water samples collected near swine farms in North Carolina shortly after Hurricane Florence had evidence of swine origin. To investigate this, we isolated and fully sequenced 18 independent S. enterica strains from 10 locations (five flooded and five unflooded). We found that all strains have extremely similar chromosomes with only five single nucleotide polymorphisms (SNPs) and possessed two plasmids assigned bioinformatically to the incompatibility groups IncFIB and IncFII. The chromosomal core genome and the IncFIB plasmid are most closely related to environmental Salmonella strains isolated previously from the southeastern US. In contrast, the IncFII plasmid was found in environmental S. enterica strains whose genomes were more divergent, suggesting the IncFII plasmid is more promiscuous than the IncFIB type. We identified 65 antibiotic resistance genes (ARGs) in each of our 18 S. enterica isolates. All ARGs were located on the Salmonella chromosome, similar to other previously characterized environmental isolates. All isolates with different SNPs were resistant to a panel of commonly used antibiotics. These results highlight the importance of environmental sources of antibiotic-resistant S. enterica after extreme flood events.

Improved performance of nucleic acid-based assays for genetically diverse norovirus surveillance.

Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of their genetic diversity. Here, we calculated the variability of nucleotides within the genomes of 10 human viral species in silico and found that endemic viruses exhibit a high percentage of variable nucleotides (e.g., 51.4% for norovirus genogroup II). This genetic diversity led to the variable probability of detection of PCR assays (the proportion of viral sequences that contain the assay's target sequences divided by the total number of viral sequences). We then experimentally confirmed that the probability of the target sequence detection is indicative of the number of mismatches between PCR assays and norovirus genomes. Next, we developed a degenerate PCR assay that detects 97% of known norovirus genogroup II genome sequences and recognized norovirus in eight clinical samples. By contrast, previously developed assays with 31% and 16% probability of detection had 1.1 and 2.5 mismatches on average, respectively, which negatively impacted RNA quantification. In addition, the two PCR assays with a lower probability of detection also resulted in false negatives for wastewater-based epidemiology. Our findings suggest that the probability of detection serves as a simple metric for evaluating nucleic acid-based assays for genetically diverse virus surveillance.IMPORTANCENucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are employed widely as a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of the rapid evolution and genetic variation of viruses. The study analyzed clinical and wastewater samples using multiple PCR assays and found significant performance variation among the PCR assays for genetically diverse norovirus surveillance. This finding suggests that some PCR assays may miss detecting certain virus strains, leading to a compromise in detection sensitivity. To address this issue, we propose a metric called the probability of detection, which can be simply calculated in silico using a code developed in this study, to evaluate nucleic acid-based assays for genetically diverse virus surveillance. This new approach can help improve the sensitivity and accuracy of virus detection, which is crucial for effective infectious disease surveillance and control.

Inactivation Kinetics and Replication Cycle Inhibition of Coxsackievirus B5 by Free Chlorine.

The kinetics of coxsackievirus serotype B5 (CVB5) inactivation with free chlorine is characterized over a range of pH and temperature relevant to drinking water treatment with the primary goal of selecting experimental conditions used for assessing inactivation mechanisms. The inactivation kinetics identified in our study is similar to or slower than experimental data reported in the literature and thus provides a conservative representation of the kinetics of CVB5 inactivation for free chlorine that could be useful in developing future regulations for waterborne viral pathogens including adequate disinfection treatment for CVB5. Untreated and free chlorine-treated viruses, and host cells synchronized-infected with these viruses, are analyzed by a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method with the goal of quantitatively investigating the effect of free chlorine exposure on viral genome integrity, attachment to host cell, and viral genome replication. The inactivation kinetics observed results from a combination of hindering virus attachment to the host cell, inhibition of one or more subsequent steps of the replication cycle, and possibly genome damage.

Application of neighborhood-scale wastewater-based epidemiology in low COVID-19 incidence situations.

Wastewater-based epidemiology (WBE), an emerging approach for community-wide COVID-19 surveillance, was primarily characterized at large sewersheds such as wastewater treatment plants serving a large population. Although informed public health measures can be better implemented for a small population, WBE for neighborhood-scale sewersheds is less studied and not fully understood. This study applied WBE to seven neighborhood-scale sewersheds (average population of 1471) from January to November 2021. Community testing data showed an average of 0.004 % incidence rate in these sewersheds (97 % of monitoring periods reported two or fewer daily infections). In 92 % of sewage samples, SARS-CoV-2 N gene fragments were below the limit of quantification. We statistically determined 10-2.6 as the threshold of the SARS-CoV-2 N gene concentration normalized to pepper mild mottle virus (N/PMMOV) to alert high COVID-19 incidence rate in the studied sewershed. This threshold of N/PMMOV identified neighborhood-scale outbreaks (COVID-19 incidence rate higher than 0.2 %) with 82 % sensitivity and 51 % specificity. Importantly, neighborhood-scale WBE can discern local outbreaks that would not otherwise be identified by city-scale WBE. Our findings suggest that neighborhood-scale WBE is an effective community-wide disease surveillance tool when COVID-19 incidence is maintained at a low level.

Inactivation Mechanism and Efficacy of Grape Seed Extract for Human Norovirus Surrogate.

Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess antimicrobial and antifungal properties, but antiviral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, a process that correlated with a decrease in virus titers. This aggregation and disinfection were not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food. IMPORTANCE Human noroviruses are major food- and waterborne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies presented here, plant-derived polyphenols outperformed chemical disinfectants (i.e., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

Peracetic Acid Sanitation on Arugula Microgreens Contaminated with Surface-Attached and Internalized Tulane Virus and Rotavirus.

Hydroponic production of vegetables is becoming more common, especially in regions with unfavorable climate for year-round crop production. However, if viruses are present in the hydroponics feed water, then there is a chance that infectious viruses will be internalized into the tissues of hydroponically grown vegetables. When this happens, surface sanitization of postharvest vegetables may not be effective because the sanitizer cannot disinfect the internalized viruses. In this study, we determined if the effectiveness of peracetic acid (PAA), a sanitizer used in the vegetable industry, is affected by the location of viruses (produce surface or interior tissue) in microgreen arugula. Either internally or externally contaminated hydroponically grown microgreen arugula was then treated with PAA at either 30 or 80 ppm for up to 3 min. The PAA disinfection efficacy was higher when the RV was on the arugula surface (approximately 5-log10 in PFU after 3 min of exposure), instead of the arugula interior (1.5-log10 in PFU after 3 min of exposure). However, PAA disinfection efficacy of TV was not dependent on the virus location in arugula. For both internalized TV and RV, the disinfection efficacy was less than 2-log10 in PFU using all the tested PAA concentrations and exposure times examined here. Thus, both the type and location of virus in fresh vegetables may influence the virus disinfection of postharvest vegetables. Therefore, the optimization of sanitation for postharvest fresh vegetables is needed to reduce foodborne viral infection risks.

Distribution and Antibiotic Resistance Profiles of Salmonella enterica in Rural Areas of North Carolina After Hurricane Florence in 2018.

In this study, water samples were analyzed from a rural area of North Carolina after Hurricane Florence in 2018 and the distribution of the ttrC virulence gene of Salmonella enterica were investigated. We also examined the distribution of culturable S. enterica and determined their antibiotic resistance profiles. Antibiotic resistance genes (ARGs) in the classes of aminoglycoside, beta-lactam, and macrolide-lincosamide-streptogramin B (MLSB) were targeted in this study. The ttrC gene was detected in 23 out of 25 locations. There was a wider and higher range of the ttrC gene in flooded water versus unflooded water samples (0-2.12 × 105 copies/L vs. 0-4.86 × 104 copies/L). Culturable S. enterica was isolated from 10 of 25 sampling locations, which was less prevalent than the distribution of the ttrC gene. The antibiotic resistance profiles were not distinct among the S. enterica isolates. The aminoglycoside resistance gene aac(6')-Iy had the highest relative abundance (around 0.05 copies/16S rRNA gene copy in all isolates) among all ARGs. These findings suggested that the 2018 flooding event led to higher copy numbers of the ttrC genes of S. enterica in some flooded water bodies compared to those in unflooded water bodies. The high ARG level and similar ARG profiles were observed in all S. enterica isolates from both flooded and unflooded samples, suggesting that the antibiotic resistance was prevalent in S. enterica within this region, regardless of flooding.

The Basis of Peracetic Acid Inactivation Mechanisms for Rotavirus and Tulane Virus under Conditions Relevant for Vegetable Sanitation.

We determined the disinfection efficacy and inactivation mechanisms of peracetic acid (PAA)-based sanitizer using pH values relevant for vegetable sanitation against rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). TV was significantly more resistant to PAA disinfection than RV: for a 2-log10 reduction of virus titer, RV required 1 mg/liter PAA for 3.5 min of exposure, while TV required 10 mg/liter PAA for 30 min. The higher resistance of TV can be explained, in part, by significantly more aggregation of TV in PAA solutions. The PAA mechanisms of virus inactivation were explored by quantifying (i) viral genome integrity and replication using reverse transcription-quantitative PCR (RT-qPCR) and (ii) virus-host receptor interactions using a cell-free binding assay with porcine gastric mucin conjugated with magnetic beads (PGM-MBs). We observed that PAA induced damage to both RV and TV genomes and also decreased virus-receptor interactions, with the latter suggesting that PAA damages viral proteins important for binding its host cell receptors. Importantly, the levels of genome-versus-protein damage induced by PAA were different for each virus. PAA inactivation correlated with higher levels of RV genome damage than of RV-receptor interactions. For PAA-treated TV, the opposite trends were observed. Thus, PAA inactivates each of these viruses via different molecular mechanisms. The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease.IMPORTANCE In this study, we examined the inactivation mechanisms of peracetic acid (PAA), a sanitizer commonly used for postharvest vegetable washing, for two enteric viruses: Tulane virus (TV) as a human norovirus surrogate and rotavirus (RV). PAA disinfection mechanisms for RV were mainly due to genome damage. In contrast, PAA disinfection in TV was due to damage of the proteins important for binding to its host receptor. We also observed that PAA triggered aggregation of TV to a much greater extent than RV. These studies demonstrate that different viruses are inactivated via different PAA mechanisms. This information is important for designing an optimal sanitation practice for postharvest vegetable washing to minimize foodborne viral diseases.

Assessment of microbial risks by characterization of Escherichia coli presence to analyze the public health risks from poor water quality in Nepal.

The combination of natural disasters and inadequate infrastructure introduce unique challenges in providing safe drinking water in low-income countries. For example, Nepal faces several challenges in managing sporadic diarrheal outbreaks across the country, given its complex geographic terrain, and its susceptibility to extreme natural events like earthquakes, floods, and landslides. To assess the risks of diarrheal diseases caused by fecal contamination in several water sources in different geographical regions of Nepal, we conducted a two months cross-sectional study throughout 37 out of 75 districts in Nepal, including the ones affected by Nepal 2015 earthquake. Quantitative Microbial Risk Assessment (QMRA) was applied to estimate the human health risk based on Escherichia coli (E. coli) count for 2,822 water samples collected at source and households. Disease burden calculations suggested that Hilly and Terai (low-land) regions are at the highest risk with 0.27 and 0.16 DALYs per person per year (DALYpppy), respectively, whereas mountain region disease burden was 0.02 DALYpppy. The risk comparison among exposure pathways indicated that the water used in households, including drinking water and water for washing, posed higher risks than from source water, reservoir water or tap water, suggesting deteriorated sanitation conditions in households. These findings suggest that the combination of QMRA and spatial analysis can help to prioritize intervention activities after a major natural disaster.

Synergistic Combination of Oncolytic Virotherapy and Immunotherapy for Glioma.

PURPOSE: We hypothesized that the combination of a local stimulus for activating tumor-specific T cells and an anti-immunosuppressant would improve treatment of gliomas. Virally encoded IL15Rα-IL15 as the T-cell activating stimulus and a prostaglandin synthesis inhibitor as the anti-immunosuppressant were combined with adoptive transfer of tumor-specific T cells. EXPERIMENTAL DESIGN: Two oncolytic poxviruses, vvDD vaccinia virus and myxoma virus, were each engineered to express the fusion protein IL15Rα-IL15 and a fluorescent protein. Viral gene expression (YFP or tdTomato Red) was confirmed in the murine glioma GL261 in vitro and in vivo. GL261 tumors in immunocompetent C57BL/6J mice were treated with vvDD-IL15Rα-YFP vaccinia virus or vMyx-IL15Rα-tdTr combined with other treatments, including vaccination with GARC-1 peptide (a neoantigen for GL261), rapamycin, celecoxib, and adoptive T-cell therapy. RESULTS: vvDD-IL15Rα-YFP and vMyx-IL15Rα-tdTr each infected and killed GL261 cells in vitro. In vivo, NK cells and CD8+ T cells were increased in the tumor due to the expression of IL15Rα-IL15. Each component of a combination treatment contributed to prolonging survival: an oncolytic virus, the IL15Rα-IL15 expressed by the virus, a source of T cells (whether by prevaccination or adoptive transfer), and prostaglandin inhibition all synergized to produce elimination of gliomas in a majority of mice. vvDD-IL15Rα-YFP occasionally caused ventriculitis-meningitis, but vMyx-IL15Rα-tdTr was safe and effective, causing a strong infiltration of tumor-specific T cells and eliminating gliomas in 83% of treated mice. CONCLUSIONS: IL15Rα-IL15-armed oncolytic poxviruses provide potent antitumor effects against brain tumors when combined with adoptive T-cell therapy, rapamycin, and celecoxib.

UV Inactivation of Rotavirus and Tulane Virus Targets Different Components of the Virions.

Enteric viruses are shed in fecal material by humans and other animals and are common contaminants in wastewater and surface water. Wastewater treatment plants often disinfect this effluent with low-pressure and medium-pressure UV lamps, which emit 254-nm and 220- to 280-nm irradiation, respectively. It is not known whether this treatment is efficacious against enteric viruses or how such treatments may inactivate these enteric viruses. This study examined UV disinfection for two enteric viruses: rotavirus (RV) (strain OSU with double-stranded RNA and a three-layer capsid) and Tulane virus (TV) (a cultivable surrogate for human norovirus with single-stranded RNA and a single-layer capsid). Viruses were treated with UV irradiation at 220 or 254 nm under conditions relevant to wastewater stabilization ponds, whose water is often used for irrigation. TV was susceptible to 220- or 254-nm UV at similar levels. It appears that UV irradiation inactivated TV by mutagenizing both its genome and capsid binding proteins. RV was more susceptible to UV at 220 nm than to UV at 254 nm. UV irradiation of RV at either 220 or 254 nm resulted in a virus that retained its ability to bind to its host cell receptor. After 220-nm treatment, the VP7 segment of the RV genome could not be amplified by PCR, suggesting that this treatment mutagenized the viral genome. However, this correlation was not observed when UV at 254 nm was used. Thus, RV and TV, with different genome and capsid contents, are targeted by UV irradiation in different ways.IMPORTANCE UV irradiation is becoming common for disinfection in water treatment plants, but little is known about the effectiveness of this treatment for enteric RNA viruses. Here, we observed that 220-nm UV irradiation was efficacious against rotavirus (RV) and Tulane virus (TV). UV irradiation at 254 nm inactivated TV to a greater extent than RV. Additional assays showed that UV irradiation compromised different portions of the RV and TV life cycles. UV irradiation decreased the binding of TV to its host receptor and mutagenized the TV genome. UV irradiation at 220 nm appeared to allow RV-host receptor interaction but halted RV genome replication. These findings provide knowledge about the disinfection of waterborne viruses, information that is important for the safe reuse or release of treated wastewater.

Roles of Vegetable Surface Properties and Sanitizer Type on Annual Disease Burden of Rotavirus Illness by Consumption of Rotavirus-Contaminated Fresh Vegetables: A Quantitative Microbial Risk Assessment.

Enteric viruses are often detected in water used for crop irrigation. One concern is foodborne viral disease via the consumption of fresh produce irrigated with virus-contaminated water. Although the food industry routinely uses chemical sanitizers to disinfect post-harvest fresh produce, it remains unknown how sanitizer and fresh produce properties affect the risk of viral illness through fresh produce consumption. A quantitative microbial risk assessment model was conducted to estimate (i) the health risks associated with consumption of rotavirus (RV)-contaminated fresh produce with different surface properties (endive and kale) and (ii) how risks changed when using peracetic acid (PAA) or a surfactant-based sanitizer. The modeling results showed that the annual disease burden depended on the combination of sanitizer and vegetable type when vegetables were irrigated with RV-contaminated water. Global sensitivity analyses revealed that the most influential factors in the disease burden were RV concentration in irrigation water and postharvest disinfection efficacy. A postharvest disinfection efficacy of higher than 99% (2-log10 ) was needed to decrease the disease burden below the World Health Organization (WHO) threshold, even in scenarios with low RV concentrations in irrigation water (i.e., river water). All scenarios tested here with at least 99.9% (3-log10 ) disinfection efficacy had a disease burden lower than the WHO threshold, except for the endive treated with PAA. The disinfection efficacy for the endive treated with PAA was only about 80%, leading to a disease burden 100 times higher than the WHO threshold. These findings should be considered and incorporated into future models for estimating foodborne viral illness risks.

Free Chlorine Disinfection Mechanisms of Rotaviruses and Human Norovirus Surrogate Tulane Virus Attached to Fresh Produce Surfaces.

To fill the knowledge gap on how effective free chlorine is against viral-contaminated produce, we inoculated the surfaces of outdoor- or greenhouse-grown kale and mustard with Rotavirus (RV) or a human norovirus surrogate (Tulane virus, TV) and then disinfected the leaves with free chlorine. Disinfection efficacies for RV strain OSU and Wa were approximately 1-log10 higher when attached to mustard than to kale. Similar disinfection efficacies were observed for TV attached to mustard or kale. When examining TV and RV OSU in suspension (not attached to leaf surfaces), TV was more resistant to free chlorine than RV OSU. Inactivation efficacies were higher for these viruses in suspension versus viruses attached to produce the surface. We also found that free chlorine damaged viral capsids, allowing free chlorine access to viral RNA to damage viral genomes. Exposure to free chlorine at 1.7 ppm over 1 min caused VP8* of RV OSU to lose its ability to bind to its host receptors. TV lost its ability to bind to its receptor only after exposure to free chlorine at 29 ppm over 1 min. Thus, to reduce foodborne viral infections, it is important to consider the differences in virus' reactivity and inactivation mechanisms with free chlorine.

A cautionary note on the selectivity of oncolytic poxviruses.

BACKGROUND: Oncolytic viruses selectively infect cancer cells while avoiding infection of normal cells. Usually, selectivity is demonstrated by injecting a virus into tumor-bearing mice and observing infection and lysis of tumor cells without infection of other tissues. The general view is that this selectivity is due to tropisms of the virus. However, apparent selectivity could be due to accessibility. For example, intravenously injected virus may not gain access to cells within the central nervous system (CNS) because of the blood-brain barrier. PURPOSE: We tested the CNS safety of two oncolytic poxviruses that have been demonstrated to be safe for treatment of peripheral tumors (vaccinia virus vvDD-IL15-Rα and myxoma virus vMyx-IL15Rα-tdTr). METHODS: Two poxviruses were tested for selectivity in vitro and in vivo. RESULTS: Both viruses infected glioma cells in vitro. In vivo, both viruses infected glioma cells and did not infect neurons when injected into a tumor or into the normal striatum. However, viral gene expression was observed in ependymal cells lining the ventricles, implying that these poxviruses were not as selective as originally predicted. For vvDD-IL15-Rα, some tumor-bearing mice died soon after virus treatment. If the same titer of vvDD-IL15-Rα was injected directly into the lateral cerebral ventricle of nontumor-bearing mice, it was uniformly fatal. Infection of ependymal cells, subventricular cells, and meninges was widespread. On the other hand, vMyx-IL15Rα-tdTr only transiently infected ependymal cells and was safe even when injected directly into the lateral cerebral ventricles. The two poxviruses also differed in their infection of dendritic cells; vvDD-IL15-Rα infected dendritic cells and lysed them but vMyx-IL15Rα-tdTr did not. CONCLUSION: Vaccinia virus vvDD-IL15-Rα is very promising for treating cancer types outside of the brain. However, for cancers located within the brain, myxoma virus vMyx-IL15Rα-tdTr offers a safer alternative.

Influence of algal organic matter on MS2 bacteriophage inactivation by ultraviolet irradiation at 220 nm and 254 nm.

We determined the potential interference of extracellular algal organic matter (EAOM) and intracellular algal organic matter (IAOM) extracted from Microcystis aeruginosa on MS2 bacteriophage inactivation under UV irradiation at two wavelengths (220 and 254 nm). UV irradiation at 220 nm doubled the inactivation rate of MS2 in water containing EAOM than in organic-free phosphate buffered solution. In contrast, EAOM did not change MS2 inactivation by exposure to UV 254 nm. The presence of IAOM did not significantly influence MS2 inactivation by exposure to either UV 254 or UV 220 nm. To achieve 3 log10 inactivation of MS2, UV254 nm required more than double the dose of UV220 nm (45 mJ/cm2 vs. 20 mJ/cm2). Linear correlations between the reduction in infectivity and the reduction in genome copies detected by reverse transcription quantitative polymerase chain reaction suggested that genomic damage is the main mechanism responsible for MS2 inactivation in water containing algal organic matter (AOM) by exposure to UV irradiation. These findings suggest that the presence of AOM did not negatively influence MS2 inactivation by either 220 or 254 nm irradiation, and that a lower UV dose of 220 nm irradiation can be used to achieve the same level of inactivation in water containing AOM.

Inactivation Mechanisms of Human and Animal Rotaviruses by Solar UVA and Visible Light.

Two rotavirus (RV) strains (sialidase-resistant Wa and sialidase-sensitive OSU) were irradiated with simulated solar UVA and visible light in sensitizer-free phosphate buffered solution (PBS) (lacking exogenous reactive oxygen species (ROS)) or secondary effluent wastewater (producing ROS). Although light attenuated for up to 15% through the secondary effluent wastewater (SEW), the inactivation efficacies increased by 0.7 log10 for Wa and 2 log10 for OSU compared to those in sensitizer-free phosphate buffered solution (PBS) after 4 h of irradiation. A binding assay using magnetic beads coated with porcine gastric mucin containing receptors for rotaviruses (PGM-MB) was developed to determine if inactivation influenced RV binding to its receptors. The linear correlation between the reduction in infectivity and the reduction in binding after irradiation in sensitizer-free solution suggests that the main mechanism of RV inactivation in the absence of exogenous ROS was due to damage to VP8*, the RV protein that binds to host cell receptors. For a given reduction in infectivity, greater damage in VP8* was observed with sialidase-resistant Wa compared to sialidase-sensitive OSU. The lack of correlation between the reduction in infectivity and the reduction in binding, in SEW, led us to include RNase treatment before the binding step to quantify virions with intact protein capsids and exclude virions that can bind to the receptors but have their capsid permeable after irradiation. This assay showed a linear correlation between the reduction in RV infectivity and RV-receptor interactions, suggesting that RV inactivation in SEW was due to compromised capsid proteins other than the VP8* protein. Thus, rotavirus inactivation by UVA and visible light irradiation depends on both the formation of ROS and the stability of viral proteins.