Genetic diversity of murine norovirus populations less susceptible to chlorine.

High genetic diversity in RNA viruses contributes to their rapid adaptation to environmental stresses, including disinfection. Insufficient disinfection can occur because of the emergence of viruses that are less susceptible to disinfection. However, understanding regarding the mechanisms underlying the alteration of viral susceptibility to disinfectants is limited. Here, we performed an experimental adaptation of murine norovirus (MNV) using chlorine to understand the genetic characteristics of virus populations adapted to chlorine disinfection. Several MNV populations exposed to an initial free chlorine concentration of 50 ppm exhibited reduced susceptibility, particularly after the fifth and tenth passages. A dominant mutation identified using whole-genome sequencing did not explain the reduced susceptibility of the MNV populations to chlorine. Conversely, MNV populations with less susceptibility to chlorine, which appeared under higher chlorine stress, were accompanied by significantly lower synonymous nucleotide diversity (πS) in the major capsid protein (VP1). The nonsynonymous nucleotide diversity (πN) in VP1 in the less-susceptible populations was higher than that in the susceptible populations, although the difference was not significant. Therefore, the ability of MNV populations to adapt to chlorine was associated with the change in nucleotide diversity in VP1, which may lead to viral aggregate formation and reduction in chlorine exposure. Moreover, the appearance of some nonsynonymous mutations can also contribute to the alteration in chlorine susceptibility by influencing the efficiency of viral replication. This study highlights the importance of understanding the genetic characteristics of virus populations under disinfection, which can contribute to the development of effective disinfection strategies and prevent the development of virus populations less susceptible to disinfectants.

PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.

Recent Update on UV Disinfection to Fulfill the Disinfection Credit Value for Enteric Viruses in Water.

Ultraviolet (UV) radiation alone or in combination with other oxidation processes is increasingly being considered for water disinfection because of stringent regulatory requirements for pathogen inactivation. To fulfill this requirement, an appropriate UV dose or fluence (mJ/cm2) is applied to combat enteric viruses in surface or treated water. There is a need for a cumulative review on the effectiveness of current and emerging UV technologies against various types of human enteric viruses. We extracted the kinetics data from 52 selected experimental studies on enteric virus inactivation using low pressure (LP-UV), medium pressure (MP-UV), UV-LED, and advanced oxidation processes (AOPs) and applied a simple linear regression analysis to calculate the range of UV fluence (mJ/cm2) needed for 4-log10 inactivation. The inactivation of adenoviruses with LP-UV, MP-UV, and UV/H2O2 (10 mg/L) required the highest fluence, which ranged from 159 to 337, 45, and 115 mJ/cm2, respectively. By contrast, when using LP-UV, the inactivation of other enteric viruses, such as the Caliciviridae and Picornaviridae family and rotavirus, required fluence that ranged from 19 to 69, 18 to 43, and 38 mJ/cm2, respectively. ssRNA viruses exhibit higher sensitivity to UV radiation than dsRNA and DNA viruses. In general, as an upgrade to LP-UV, MP-UV is a more promising strategy for eliminating enteric viruses compared to AOP involving LP-UV with added H2O2 or TiO2. The UV-LED technology showed potential because a lower UV fluence (at 260 and/or 280 nm wavelength) was required for 4-log10 inactivation compared to that of LP-UV for most strains examined in this critical review. However, more studies evaluating the inactivation of enteric viruses by means of UV-LEDs and UV-AOP are needed to ascertain these observations.

A Robust, Safe, and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Wastewater Samples.

Molecular diagnosis and surveillance of pathogens such as SARS-CoV-2 depend on nucleic acid isolation. Pandemics at the scale of COVID-19 can cause a global shortage of proprietary commercial reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open-source method, magnetic-nanoparticle-aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real-world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID-19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field-deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens.

Estimating the minimum number of SARS-CoV-2 infected cases needed to detect viral RNA in wastewater: To what extent of the outbreak can surveillance of wastewater tell us?

There is increasing interest in wastewater-based epidemiology (WBE) of SARS-CoV-2 RNA to serve as an early warning system for a community. Despite successful detection of SARS-CoV-2 RNA in wastewaters sampled from multiple locations, there is still no clear idea on the minimal number of cases in a community that are associated with a positive detection of the virus in wastewater. To address this knowledge gap, we sampled wastewaters from a septic tank (n = 57) and biological activated sludge tank (n = 52) located on-site of a hospital. The hospital is providing treatment for SARS-CoV-2 infected patients, with the number of hospitalized patients per day known. It was observed that depending on which nucleocapsid gene is targeted by means of RT-qPCR, a range of 253-409 positive cases out of 10,000 persons are required prior to detecting RNA SARS-CoV-2 in wastewater. There was a weak correlation between N1 and N2 gene abundances in wastewater with the number of hospitalized cases. This correlation was however not observed for N3 gene. The frequency of detecting N1 and N2 gene in wastewater was also higher than that for N3 gene. Furthermore, nucleocapsid genes of SARS-CoV-2 were detected at lower frequency in the partially treated wastewater than in the septic tank. In particular, N1 gene abundance was associated with water quality parameters such as total organic carbon and pH. In instances of positive detection, the average abundance of N1 and N3 genes in the activated sludge tank were reduced by 50 and 70% of the levels detected in septic tank, suggesting degradation of the SARS-CoV-2 gene fragments already occurring in the early stages of the wastewater treatment process.

Required Chlorination Doses to Fulfill the Credit Value for Disinfection of Enteric Viruses in Water: A Critical Review.

A credit value of virus inactivation has been assigned to the disinfection step in international and domestic guidelines for wastewater reclamation and reuse. To fulfill the credit value for water disinfection, water engineers need to apply an appropriate disinfection strength, expressed as a CT value (mg × min/L), which is a product of disinfectant concentration and contact time, against enteric viruses in wastewater. In the present study, we extracted published experimental data on enteric virus inactivation using free chlorine and monochloramine and applied the Tobit analysis and simple linear regression analysis to calculate the range of CT values (mg × min/L) needed for 4-log10 inactivation. Data were selected from peer-reviewed papers containing kinetics data of virus infectivity and chlorine residual in water. Coxsackie B virus and echovirus require higher CT values (lower susceptibility) for 4-log10 inactivation than adenovirus and a human norovirus surrogate (murine norovirus) with free chlorine. On the other hand, adenovirus has lower susceptibility to monochloramine compared to murine norovirus, coxsackievirus, and echovirus. The factors that influence the required CT value are virus type, pH, water temperature, and water matrix. This systematic review demonstrates that enteroviruses and adenovirus are appropriate representative enteric viruses to evaluate water disinfection using free chlorine and monochloramine, respectively.

Elucidating the Role of Virulence Traits in the Survival of Pathogenic E. coli PI-7 Following Disinfection.

Reuse and discharge of treated wastewater can result in dissemination of microorganisms into the environment. Deployment of disinfection strategies is typically proposed as a last stage remediation effort to further inactivate viable microorganisms. In this study, we hypothesize that virulence traits, including biofilm formation, motility, siderophore, and curli production along with the capability to internalize into mammalian cells play a role in survival against disinfectants. Pathogenic E. coli PI-7 strain was used as a model bacterium that was exposed to diverse disinfection strategies such as chlorination, UV and solar irradiation. To this end, we used a random transposon mutagenesis library screening approach to generate 14 mutants that exhibited varying levels of virulence traits. In these 14 isolated mutants, we observed that an increase in virulence traits such as biofilm formation, motility, curli production, and internalization capability, increased the inactivation half-lives of mutants compared to wild-type E. coli PI-7. In addition, oxidative stress response and EPS production contributed to lengthening the lag phase duration (defined as the time required for exposure to disinfectant prior to decay). However, traits related to siderophore production did not help with survival against the tested disinfection strategies. Taken together, the findings suggested that selected virulence traits facilitate survival of pathogenic E. coli PI-7, which in turn could account for the selective enrichment of pathogens over the non-pathogenic ones after wastewater treatment. Further, the study also reflected on the effectiveness of UV as a more viable disinfection strategy for inactivation of pathogens.

Fecal Source Tracking in A Wastewater Treatment and Reclamation System Using Multiple Waterborne Gastroenteritis Viruses.

Gastroenteritis viruses in wastewater reclamation systems can pose a major threat to public health. In this study, multiple gastroenteritis viruses were detected from wastewater to estimate the viral contamination sources in a wastewater treatment and reclamation system installed in a suburb of Xi'an city, China. Reverse transcription plus nested or semi-nested PCR, followed by sequencing and phylogenetic analysis, were used for detection and genotyping of noroviruses and rotaviruses. As a result, 91.7% (22/24) of raw sewage samples, 70.8% (17/24) of the wastewater samples treated by anaerobic/anoxic/oxic (A2O) process and 62.5% (15/24) of lake water samples were positive for at least one of target gastroenteritis viruses while all samples collected from membrane bioreactor effluent after free chlorine disinfection were negative. Sequence analyses of the PCR products revealed that epidemiologically minor strains of norovirus GI (GI/14) and GII (GII/13) were frequently detected in the system. Considering virus concentration in the disinfected MBR effluent which is used as the source of lake water is below the detection limit, these results indicate that artificial lake may be contaminated from sources other than the wastewater reclamation system, which may include aerosols, and there is a possible norovirus infection risk by exposure through reclaimed water usage and by onshore winds transporting aerosols containing norovirus.

Free-Chlorine Disinfection as a Selection Pressure on Norovirus.

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure.IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.