Article

Propidium Monoazide Reverse Transcriptase PCR and RT-qPCR for Detecting Infectious Enterovirus and Norovirus

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Abstract

Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. Copyright © 2015. Published by Elsevier B.V.

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... In addition, such techniques have reportedly been used for the detection of infectious viruses related to human diseases, such as bacteriophage T4 (Fittipaldi et al., 2010), enteric viruses (Parshionikar et al., 2010;Karim et al., 2015;Leifels et al., 2015;Fongaro et al., 2016;Quijada et al., 2016), hepatitis A viruses (Sánchez et al., 2012;Leifels et al., 2015), rotaviruses (Leifels et al., 2015), adenoviruses (Leifels et al., 2015), norovirus (Karim et al., 2015), and murine norovirus (Lee et al., 2015). However, very few studies have focused on viable cell detection techniques based on PMA and molecular biology in animal disease-related viruses. ...
... In addition, such techniques have reportedly been used for the detection of infectious viruses related to human diseases, such as bacteriophage T4 (Fittipaldi et al., 2010), enteric viruses (Parshionikar et al., 2010;Karim et al., 2015;Leifels et al., 2015;Fongaro et al., 2016;Quijada et al., 2016), hepatitis A viruses (Sánchez et al., 2012;Leifels et al., 2015), rotaviruses (Leifels et al., 2015), adenoviruses (Leifels et al., 2015), norovirus (Karim et al., 2015), and murine norovirus (Lee et al., 2015). However, very few studies have focused on viable cell detection techniques based on PMA and molecular biology in animal disease-related viruses. ...
... We consider the combination of PMA pretreatment and molecular biology detection methods as a great enhancement of the diagnostic efficiency and prevention and control capabilities of ASF. Currently, in the field of virus detection, the established rapid detection methods for infectious viruses based on PMA pretreatment are mainly PMA-PCR (Parshionikar et al., 2010;Karim et al., 2015;Fongaro et al., 2016) and PMA-qPCR (Fittipaldi et al., 2010;Sánchez et al., 2012;Quijada et al., 2016). For the prevention, control, and detection of ASFV, accuracy, convenience, low-cost, and suitability for field detection are all of great significance in addition to rapid detection. ...
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Article
African swine fever (ASF) is a hemorrhagic and often fatal disease occurring in domestic pigs and wild boars. ASF can potentially greatly impact the global trade of pigs and pork products and threaten global food security. Outbreaks of ASF must be notified to the World Organization for Animal Health. In this study, we analyzed the feasibility of applying propidium monoazide (PMA) pretreatment-based infectious virus detection technology to ASF prevention and control and investigated the prospects of applying this technology for epidemic monitoring, disinfection effect evaluation, and drug development. PMA as a nucleic acid dye can enter damaged cells and undergo irreversible covalent crosslinking with nucleic acid under halogen light to prevent its amplification. Although this technology has been widely used for the rapid detection of viable bacteria, its application in viruses is rare. Therefore, we analyzed the theoretical feasibility of applying this technology to the African swine fever virus (ASFV) in terms of gene and cell composition. Rapid infectious ASFV detection technology based on PMA pretreatment would greatly enhance all aspects of ASF prevention and control, such as epidemic monitoring, disinfection treatment, and drug development. The introduction of this technology will also greatly improve the ability to prevent and control ASF.
... The optimal PMA concentration was 120 µmol·L −1 . PMA-RT-qPCR has been applied to detect active human and environmental viruses in several previous studies [34][35][36][37][38][39][40]50]. Among these studies, there is little consensus on optimal PMA concentrations, which range from 5 to 100 µmol·L −1 . ...
... For the detection of infectious SARS-CoV-2, the optimal PMA concentration was 50 µmol·L −1 coupled with 0.005% SDS [40]. To detect infectious enterovirus and norovirus, e.g., poliovirus 1, MNV-1 and Norwalk virus, the optimal concentration of PMA was 348 µmol·L −1 [37]. Although dark incubation time and light exposure time may affect the efficiency of the PMA-RT-qPCR assay, recent studies have shown that these conditions have little effect [37,39,41,50]. ...
... To detect infectious enterovirus and norovirus, e.g., poliovirus 1, MNV-1 and Norwalk virus, the optimal concentration of PMA was 348 µmol·L −1 [37]. Although dark incubation time and light exposure time may affect the efficiency of the PMA-RT-qPCR assay, recent studies have shown that these conditions have little effect [37,39,41,50]. In this study, the dark incubation time and light exposure time were set at 15 min and 30 min, respectively. ...
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Article
Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is an important quarantine virus of cucurbit crops. Seedborne transmission is one of the principal modes for CGMMV spread, and effective early detection is helpful to prevent the occurrence of the disease. Quantitative real-time reverse-transcription PCR (RT-qPCR) is a sensitive and rapid method for detecting CGMMV nucleic acids, but it cannot distinguish between infectious and noninfectious viruses. In the present work, a propidium monoazide (PMA) assisted RT-qPCR method (PMA-RT-qPCR) was developed to rapidly distinguish infectious and inactive CGMMV. PMA is a photoactive dye that can selectively react with viral RNA released or inside inactive CGMMV virions but not viral RNA inside active virions. The formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be amplified. The primer pair cp3-1F/cp3-1R was designed based on the coat protein (cp) gene for specific amplification of CGMMV RNA by RT-qPCR. The detection limit of the RT-qPCR assay was 1.57 × 102 copies·μL−1. PMA at 120 μmol·L−1 was suitable for the selective quantification of infectious CGMMV virions. Under optimal conditions, RT-qPCR detection of heat-inactivated CGMMV resulted in Ct value differences larger than 16 between PMA-treated and non-PMA-treated groups, while Ct differences less than 0.23 were observed in the detection of infectious CGMMV. For naturally contaminated watermelon leaf, fruit and seedlot samples, infectious CGMMV were quantified in 13 out of the 22 samples, with infestation levels of 102~105 copies·g−1. Application of this assay enabled the selective detection of infectious CGMMV and facilitated the monitoring of the viral pathogen in watermelon seeds and tissues, which could be useful for avoiding the potential risks of primary inoculum sources.
... The results of capsid integrity (RT-)qPCR (using PMA, PMAxx, or PtCl 4 ) were close to or equivalent to those of the infectivity-based assay when the temperature and contact time were increased from 72°C to 82°C within 10 min (Fraisse et al., 2018;Kim and Ko, 2012;Oristo et al., 2018). The capsid integrity (RT-) PCR (using PMA) seemed not practical for assessing viral infectivity after chlorination at a dose of <0.06 mg-min/L (Karim et al., 2015); however, it was possibly comparable to an infectivity-based assay at a dose of >1 mg-min/L (Karim et al., 2015;Leifels et al., 2015;Prevost et al., 2016;Sánchez et al., 2012). For the ozone disinfection, the ozone dose (0.25 mg/L, 5.5 s) should also be sufficiently high for successful capsid integrity (RT-)qPCR (using EMA) (Sangsanont et al., 2020). ...
... The results of capsid integrity (RT-)qPCR (using PMA, PMAxx, or PtCl 4 ) were close to or equivalent to those of the infectivity-based assay when the temperature and contact time were increased from 72°C to 82°C within 10 min (Fraisse et al., 2018;Kim and Ko, 2012;Oristo et al., 2018). The capsid integrity (RT-) PCR (using PMA) seemed not practical for assessing viral infectivity after chlorination at a dose of <0.06 mg-min/L (Karim et al., 2015); however, it was possibly comparable to an infectivity-based assay at a dose of >1 mg-min/L (Karim et al., 2015;Leifels et al., 2015;Prevost et al., 2016;Sánchez et al., 2012). For the ozone disinfection, the ozone dose (0.25 mg/L, 5.5 s) should also be sufficiently high for successful capsid integrity (RT-)qPCR (using EMA) (Sangsanont et al., 2020). ...
... The loss of viral infectivity (determined by cell culture methods) was found to be generally higher than the reduction of viruses determined by capsid integrity (RT-)qPCR, suggesting that capsid integrity (RT-)qPCR cannot fully discriminate between infectious and inactivated viruses after inactivation treatments. Moreover, the performance of capsid integrity (RT-)qPCR was closer to the performance of the cell culture method when viruses were inactivated at higher damaging intensity (e.g., heat treatment >72°C or chlorine treatment >1.0 mg-Cl 2 /L) than at lower damaging intensity (e.g., <50°C or 0.06 mg-min/L) (as we have discussed) (Canh et al., 2019b;Fraisse et al., 2018;Karim et al., 2015;Kim and Ko, 2012;Leifels et al., 2015;Oristo et al., 2018;Prevost et al., 2016;Sánchez et al., 2012). A possibility is that the viruses were inactivated, but their capsids remained intact under low damage intensity. ...
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Article
Waterborne diseases caused by pathogenic human viruses are a major public health concern. To control the potential risk of viral infection through contaminated waters, a rapid, reliable tool to assess the infectivity of pathogenic viruses is required. Recently, an advanced approach (i.e., capsid integrity (RT-)qPCR) was developed to discriminate intact viruses (potentially infectious) from inactivated viruses. In this approach, samples were pretreated with capsid integrity reagents (e.g., monoazide dyes or metal compounds) before (RT −)qPCR. These reagents can only penetrate inactivated viruses with compromised capsids to bind to viral genomes and prevent their amplification, but they cannot enter viruses with intact capsids. Therefore, only viral genomes of intact viruses were amplified or detected by (RT-)qPCR after capsid integrity treatment. In this study, we reviewed recent progress in the development and application of capsid integrity (RT-)qPCR to assess the potential infectivity of viruses (including non-enveloped and enveloped viruses with different genome structures [RNA and DNA]) in water. The efficiency of capsid integrity (RT-)qPCR has been shown to depend on various factors, such as conditions of integrity reagent treatment, types of viruses, environmental matrices, and the capsid structure of viruses after disinfection treatments (e.g., UV, heat, and chlorine). For the application of capsid integrity (RT-)qPCR in real-world samples, the use of suitable virus concentration methods and process controls is important to control the efficiency of capsid integrity (RT-)qPCR. In addition, potential future applications of capsid integrity (RT-)qPCR for determining the mechanism of disinfection treatment on viral structure (e.g., capsid or genome) and a combination of capsid integrity treatment and next-generation sequencing (NGS) (capsid integrity NGS) for monitoring the community of intact pathogenic viruses in water are also discussed. This review provides essential information on the application of capsid integrity (RT-)qPCR as an efficient tool for monitoring the presence of pathogenic viruses with intact capsids in water.
... The RNA-PMA complex cannot be amplified during the subsequent RT-PCR assay. PMA has been shown to reduce detection of norovirus genome copies by approximately a 1 log 10 reduction after five-minute heat treatments of 72 °C (Karim et al. 2015), by a log 10 reduction factor of 3.6 after one-minute treatments of 90 °C (Lee et al. 2015), and by a log 10 reduction factor of 0.67 after exposure to 65 °C for ten minutes (Leifels et al. 2015). Differences in PMA treatment efficacy and poor repeatability of assays are probably due to the fact that different subsets of primers (differing amplicon lengths and positions on the viral genome) amplify fragments with varying complexities of RNA secondary structures that present differently potent targets to the intercalating agent (Manuel et al. 2018). ...
... Pre-treatments with intercalating agents PMA/PMAxx™, tested in conjunction with different PCR kits, different primer sets, and varying concentrations of intercalating dye, have previously rendered significantly deviant estimations of norovirus genome copy numbers (Karim et al. 2015;Lee et al. 2015;Leifels et al. 2015;Randazzo et al. 2018). To investigate the impact of the different assay conditions and to develop a standardised method of norovirus genome copy estimation, we tested three different primer sets (selected from pertinent literature) in conjunction with two RT-qPCR processes (one-step versus two-step with long-range RT), and three PMAxx™ concentrations to detect MNV in PBS. ...
... However, the RT-qPCR process following PMAxx™ can influence genome reduction. Differing genome copy reductions reported in the current literature are consequently probably not attributable to fluctuations in intercalating dye concentrations (a range of PMA concentrations of 100-348 µM has previously been tested Karim et al. 2015;Kim and Ko 2012;Lee et al. 2015;Leifels et al. 2015), but are due to the use of differing primer sets and RT-qPCR assay conditions. ...
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Article
Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.
... Molecular changes in the capsid protein conformation and composition, which would result in such phenomena, are known for almost three decades and have been observed for both enteric viruses and phages by Minor et al. (1989) and Galisteo et al. (1995). In addition to these general capsid characteristics, it is assumed that the efficiency of dye pretreatment is highly dependent on the virus type: even though proposed a ci-qPCR protocol applicable for both EV and HAdV, highly specific and finely tuned treatment conditions for enteric viruses such as NoV have been published and compared with varying results (Karim et al., 2015;Randazzo et al., 2016). Similar findings have been published by after investigating native and heat inactivated 70 Norwalk virus: Two different real time primer probe sets and an increase of PMA concentration by 100 % failed in distinguishing between infectious and inactive viruses. ...
... Similar findings have been published by after investigating native and heat inactivated 70 Norwalk virus: Two different real time primer probe sets and an increase of PMA concentration by 100 % failed in distinguishing between infectious and inactive viruses. Karim et al. (2015) further investigated the role of capsid integrity for the application of PMA by inactivating both MNV and infectious PV with similar agents as in and reached a comparable conclusion: while the reduction of naked nucleic acids of both viruses under investigation could be observed using RT-qPCR with PMA pretreatment, only comparably high chlorine concentrations resulted in signal suppression for infectious PV and MNV. Therefore, differences in the effect of PMA and EMA on signal reduction for different enteric viruses and phages after heat inactivation at relatively moderate temperatures had to be expected and could be observed. ...
... and MNV) which parallels the findings of and Karim et al. (2015). Both studies applied PMA in concentrations of up to 100 mmol to project the loss of EV infectivity by hypochlorite. ...
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Thesis
Application of molecular methods for the detection and evaluation of enteric human‐pathogenic viruses in complex water matrices
... L'efficacité de ce traitement chimique dépend de l'accessibilité du génome viral, soit par la faculté de la molécule à pénétrer au sein de la capside soit par un accès direct de la molécule au génome viral. Comme observé avec les traitements enzymatiques, des températures supérieures à 72°C permettent une diminution de la quantité de génome détecté après traitement au PMA ou à l'EMA pour différents virus entériques (poliovirus, VHA, rotavirus, astrovirus) et pour le bactériophage MS2 (Karim et al., 2015;Kim et Ko, 2012;Moreno et al., 2015;Parshionikar et al., 2010;Sánchez et al., 2012;Wei et al., 2014). ...
... Nuanualsuwan et Cliver (2003a) Le PMA ou l'EMA ont également été utilisés après exposition au chlore. Une très bonne efficacité de ces molécules est rapportée pour des virus entériques pathogènes et des modèles de ces virus pathogènes (Karim et al., 2015;Leifels et al., 2015;Parshionikar et al., 2010;. Ces molécules amélioreraient la détection des virus infectieux après exposition au chlore pour le poliovirus, l'echovirus, le coxsackievirus, le rotavirus, l'adénovirus ou encore le MNV. ...
... virales infectieuses par millilitre, mais Nuanualsuwan et Cliver (2003a), 10 3 , ce qui pourrait expliquer l'absence d'impact de l'hypochlorite de sodium sur l'accessibilité du génome dans leur étude. Au contraire, l'utilisation du PMA ou de l'EMA, après chloration semble prometteuse pour différents virus comme le MNV, l'adénovirus, le poliovirus ou le rotavirus (Karim et al., 2015;Leifels et al., 2015;Parshionikar et al., 2010;. L'oxydation de la capside par l'hypochlorite de sodium pourrait être la cause d'une modification de la perméabilité de la capside virale. ...
Thesis
Même si les traitements thermiques ou la désinfection par les oxydants ont démontré leur efficacité virucide, les mécanismes liés à la perte du caractère infectieux ne sont pas connus. Ceci pose un réel problème d’interprétation de la présence de génome viral en matière de risque infectieux dans les aliments. Ce travail de thèse a pour objectif d’étudier l’évolution des propriétés de surface (charge et hydrophobie) de virus modèles, bactériophage MS2 et norovirus murin, au cours de l’inactivation par la chaleur, l’hypochlorite de sodium et l’ozone. Pour nos deux virus, nous démontrons l’existence d’une température critique au-delà de laquelle la particule virale se déstructure en libérant son génome. Un simple traitement à la RNase permettrait alors de ne détecter que des virus infectieux par biologie moléculaire. Le traitement thermique implique aussi une augmentation de l’hydrophobie soulignant des modifications conformationnelles de la capside. L’hypochlorite de sodium ne modifie que peu les propriétés de surface mais des phénomènes d’oxydation ont lieu au niveau de la capside puisque la charge du bactériophage MS2 est légèrement modifiée. Ces modifications diminuent la résistance thermique du virus. Nous démontrons un effet synergique de l’hypochlorite de sodium et la chaleur sur le bactériophage MS2 (inactivation, RNase et hydrophobie). Quant à l’ozone gazeux, nous soulignons son intérêt pour le traitement virucide des aliments fragiles. Ainsi, ce travail précise les mécanismes d’inactivation des virus et ouvre de nouvelles perspectives tant pour discriminer les virus infectieux et non-infectieux que pour proposer l’exploration de nouveaux traitements technologiques
... Waterborne transmission of NoV is also suspected to be an important cause of endemic gastroenteritis through fecal contamination of drinking water (Payment et al., 1991). The Safe Drinking Water Act requires the U.S. Environmental Protection Agency to identify and publish a drinking water Contaminant Candidate List (CCL) periodically, which consists of unregulated and emerging contaminants (Karim et al., 2015). Because of the frequent outbreaks caused by NoVs in contaminated water and their potential public health risks, NoVs classified in the Caliciviridae family were recently nominated on CCL 4 (Federal Register, 2016). ...
... The MBS technique has been used successfully to replace enrichment in selective media (Coleman et al., 1995) and, more recently, has been combined with RT-qPCR (MBS-RT-qPCR) to concentrate enter(ic viruses and minimize inhibitors from food (Tian et al., 2008). To discriminate viral integrity, several reports suggested that samples be treated with an intercalating dye, such as propidium monoazide (PMA) or ethidium monoazide (EMA), before nucleic acid extraction and amplification (Coudray-Meunier et al., 2013;Karim et al., 2015). The monoazide agents can penetrate damaged viral capsids and then covalently link to viral nucleic acids under visible-light exposure, thereby inhibiting RT-qPCR (Coudray-Meunier et al., 2013;Escudero-Abarca et al., 2014;Jeong et al., 2017;Leifels et al., 2015;Moreno et al., 2015;Parshionikar et al., 2010;Randazzo et al., 2016;S anchez et al., 2012;Sangsanont et al., 2014). ...
... Most previous studies did not use chlorination, but physical treatments, such as ultraviolet light or heat, for inactivation. Karim et al. (2015) reported that PMA-RT-PCR was able to differentiate between infectious and noninfectious MNV only when inactivation was done by chlorine, not by heat or UV. The differential efficiency of PMA treatment to discriminate non-infectious from infectious viral particles might be owing to the degree of RNA or capsid damage induced by each inactivation method. ...
Article
RT-qPCR allows sensitive detection of viral particles of both infectious and noninfectious viruses in water environments, but cannot discriminate non-infectious from infectious viruses. In this study, we aimed to optimize RT-qPCR-based detection of chlorine-inactivated human norovirus (NoV) and pepper mild mottle virus (PMMoV) in suspension by pretreatment with an optimal combination of a monoazide and a detergent that can efficiently penetrate damaged viral capsids. Four methods were compared to determine the efficacy of chlorine disinfection (at 1, 3, and 5 min mg/L): (A) RT-qPCR alone, (B) RT-qPCR assay preceded by magnetic bead separation for enrichment of viral particles (MBS-RT-qPCR), (C) MBS-RT-qPCR assay with pretreatment with propidium monoazide (PMA-MBS-RT-qPCR), and (D) PMA-MBS-RT-qPCR assay with pretreatment with sodium lauroyl sarcosinate (INCI-PMA-MBS-RT-qPCR). On the basis of a PMA optimization assay, 200 and 300 μM PMA were used in subsequent experiments for NoV GII.4 and PMMoV, respectively. Optimal INCI concentrations, having minimal influence on NoV GII.4 and PMMoV, were found to be 0.5% and 0.2% INCI, respectively. For NoV GII.4, there were significant differences (P < 0.05) in log10 genome copies between the PMA-treated and the INCI + PMA-treated samples (log10 genome copies differed by 1.11 and 0.59 log10 for 3 and 5 min mg/L of chlorine, respectively). For PMMoV, INCI induced differences in log10 genome copies of 0.92, 1.18, and 1.86, for 1, 3, and 5 min mg/L of chlorine, respectively. Overall, the results of this study indicate that an optimal combination of PMA and INCI could be very useful for evaluating disinfection methods in water treatment strategies.
... For quantitative analysis, we performed molecular biology assays, unlike the cell culture technology used by other researchers. The overestimation of infectious viruses after disinfection treatment and the inability to differentiate between inactivated and infectious viruses is considered a main disadvantage of RT-qPCR (Karim et al., 2015). Therefore, the developed method, such as the MBS/PMA/RT-qPCR assay, could allow for more precise quantification of potentially infectious viral particles in environmental samples after disinfection (Lee et al., 2018). ...
... Therefore, the developed method, such as the MBS/PMA/RT-qPCR assay, could allow for more precise quantification of potentially infectious viral particles in environmental samples after disinfection (Lee et al., 2018). In particular, Karim et al. (2015) demonstrated that pretreatment with the intercalating dye PMA combined with RT-qPCR assay could potentially be used with all non-culturable or fastidious viruses, although specific conditions of inactivation must be investigated. ...
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Article
Processes in the food industry that use large amounts of water have been an important cause of waterborne disease outbreaks, as they expose individuals to risks for waterborne disease transmission. Developing technologies to ensure the hygiene and safety of food-processing steps is an urgent concern from an economic perspective. Furthermore, economic benefits can be derived if the processed water can be reused under microbiologically safe conditions. Among the major manufacturing processes in the kimchi industry, the brining process for salted kimchi cabbages requires a considerable amount of brine (approximately 2,000–2,500 l/1,000 kg of raw cabbage). The aim of this study was to establish virucidal conditions with ultraviolet-C light-emitting diodes (UVC LEDs) that can ensure the microbiological safety of brine water samples with various turbidities for reuse after disinfection. For quantitative analysis, first of all, magnetic bead separation (MBS) technique was used to capture and recover the human norovirus (HuNoV) virus particles; propidium monoazide (PMA) combined with RT-qPCR (PMA-RT-qPCR) was subsequently used to selectively detect infectious norovirus. Overall, as the turbidity of the brine water samples increased, the reduction in the HuNoV genogroup II genotype 4 (HuNoV GII.4) levels by UVC LED disinfection decreased. The derived inactivation rate constant (kinac) and inactivation curves (calculated using the log-linear model) were studied as a function of turbidity based on the exponential one-phase inactivation kinetics of HuNoV. Using an impeller system set at 100 rotations/min (rpm) with an eight-nephelometric turbidity unit (NTU) sample (the lowest turbidity studied), the kinact based on the levels of viral genomic RNA concentrations was approximately 2.15-fold higher than that observed without rotation (0 rpm). Moreover, the kinact increased 1.69-fold with a 56-NTU sample (the highest turbidity studied) when the impeller system was set at 100 rpm. UVC LED treatment decreased the HuNoV GII.4 population more effectively in conjunction with the impeller system (100 rpm) than without the impeller system. Our novel findings and model provide fundamental and scientific data that may help reuse brine water and ensure its microbiological safety through disinfection. Our study highlights the benefits of UVC LED treatment in successfully eliminating waterborne viruses in a prompt, resistance-reducing, and energy-efficient approach at the laboratory scale, which lays the foundation for future plant-scale studies of UVC LED-disinfection systems.
... Methods to evaluate the correlation between genomic copies and infective norovirus particles are under investigation. Amongst these, the binding long-range PCR [259] has been proposed to assess genome integrity and both the use of a ligand binding step prior to RT-qPCR [260,261] or viability PCR assays which include pretreatment with intercalating dyes are increasingly utilised to investigate capsid integrity [262,263]. Comparison of RT-qPCR results with newly developed HuNoV infectivity assays (further discussed below) may help determine cycle threshold cut-offs for clinical diagnostic RT-qPCRs, allowing estimation of infectious virus burdens to help guide infection control [264,265]. ...
... Important milestones include new insights into cell tropism [40,44], host-and microbial attachment factors and receptors [43, [59][60][61][71][72][73]79,360], interactions with the cellular translational apparatus [80,83,84], and viral egress from cells [110]. Noroviruses have been detected in previously unrecognised host species [18,19]; detection itself is facilitated by the novel analytical techniques that increasingly supplement established molecular methods [259,[261][262][263]265]. New potential HuNoV transmission routes and/or viral reservoirs have been postulated [223,230,233]. ...
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Article
Human noroviruses are recognised as the major global cause of viral gastroenteritis. Here, we provide an overview of notable advances in norovirus research and provide a short recap of the novel model systems to which much of the recent progress is owed. Significant advances include an updated classification system, the description of alternative virus-like protein morphologies and capsid dynamics, and the further elucidation of the functions and roles of various viral proteins. Important milestones include new insights into cell tropism, host and microbial attachment factors and receptors, interactions with the cellular translational apparatus, and viral egress from cells. No-roviruses have been detected in previously unrecognised hosts and detection itself is facilitated by improved analytical techniques. New potential transmission routes and/or viral reservoirs have been proposed. Recent in vivo and in vitro findings have added to the understanding of host immunity in response to norovirus infection, and vaccine development has progressed to preclinical and even clinical trial testing. Ongoing development of therapeutics includes promising direct-acting small molecules and host-factor drugs.
... It has been found that PMA, a photo-reactive dye, can enter into dead bacteria or viruses characterized by compromised membrane and form PMA-DNA conjugates that prevent DNA-mediated PCR amplification (Karim et al., 2015;Telli and Dogruer, 2019), resulting in PCR being triggered only by the DNA of live microbes. Therefore, we tested if the PMA treatment could block the detection of RNA from heat-inactivated SARS-CoV-2, an enveloped, positive-sense single-stranded RNA virus. ...
... It has been reported that PMA-coupled RT-qPCR can distinguish dead microorganisms from live ones, including bacteria (Telli and Dogruer, 2019), fungi (Vesper et al., 2008), phytoplankton cells (Joo et al., 2018), and viruses (Karim et al., 2015). However, in this study, the PMA-coupled RT-qPCR failed to differentiate live and heatinactivated SARS-CoV-2 without the assistance of SDS or other surfactants. ...
Article
The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.
... More than half of the articles under investigation reported either 50 mM or 100 mM (Table 1), which is in accordance with the recommendations by the manufacturer for bacterial cultures (Biotium Inc., 2019). However, some works indicated optimal reduction rates of presumably false positive signals (as shown in the cell culture) with dye concentrations between 4 mM and 10 mM (Karim et al., 2015;Lee et al., 2016;Leifels et al., 2016Leifels et al., , 2019Prevost et al., 2016). The effect of high azo dye concentration appeared to vary with virus type; for example, two-log reduction in viability of phage MS2 has been reported with >125 mM PMA (Kim and Ko, 2012), while 250 mM PMA successfully removed signals of inactivated viruses for MNV and NoV GII.4 without causing an adverse inactivation effect (Jeong et al., 2017;Lee et al., 2015). ...
... Light in the ultraviolet spectrum, on the other hand, affects the hydrogen bonds between nucleic acids, resulting in the inability to reproduce inside the host cell. Capsid integrity qPCR failed to capture the loss of virus infectivity in most UV studies (Karim et al., 2015;Kim et al., 2017;Leifels et al., 2016Leifels et al., , 2019; it could detect capsid damage caused by medium-pressure UV lamps, Fig. 4. Design of a capsid integrity qPCR assay. Depending on the sample origin (especially the complexity of the matrix), the virus genus and strain as well as the molecular detection method used, several factors like dilution of the sample, concentration of the azo dye, incubation conditions and photo activation can be modified and optimized during assay development. ...
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Article
Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-two recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009 to 2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μ M; incubation on ice or at room temperature (20 - 25ºC) for 5 to 120 min; and dye activation using LED or high light (500 – 800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks 35 such as the current COVID-19 pandemic or endemic diseases like dengue fever.
... Methods to evaluate the correlation between genomic copies and actually infective NoV particles have been developed for a number of years. Amongst these, the binding long-range PCR (Li, Keuckelaere, & Uyttendaele, 2014) and viability PCR, incorporating pre-treatment of extracted particles with intercalating DNA-binding dyes (Karim, Fout, Johnson, White, & Parshionikar, 2015), have recently gained attention. ...
... Such a risk reduction is achievable by a number of different methods, amongst which the observance of minimum distances and dilution effects from sewage discharge points to shellfish harvest areas (Campos et al., 2017) and the use of ultrafiltration membranes in tertiary sewage treatment which has been shown to efficiently reduce HuNoV loads (Sima et al., 2011;Simmons, Kuo, & Xagoraraki, 2011). The prohibition of (accidental) discharge of non-treated overboard human waste from boats is recommended and this practice should be replaced, as is already customary within the European Union, by the use of tank-connected toilet crafts facilitating waste delivery and proper treatment ashore (European Communities, 2013 Quang et al., 2018) and should be compatible with novel molecular testing methods that take into account the infectivity of NoV particles (Karim et al., 2015). Although correct cooking (90°C core temperature for at least 90 s) of contaminated bivalve molluscs reduces viral load, this review provides plentiful evidence of HuNoV disease outbreaks associated with uncooked und undercooked bivalve molluscs on a worldwide scale. ...
Article
Human noroviruses are recognised as the leading worldwide cause of sporadic and epidemic viral gastroenteritis, causing morbidity and mortality in impoverished developing countries and engendering enormous economic losses in developed countries. Transmitted faecal-orally, either via person-to-person contact, or by consumption of contaminated foods or water, norovirus outbreaks are often reported in institutional settings or in the context of communal dining. Bivalve molluscs, which accumulate noroviruses via filter feeding and are often eaten raw or insufficiently cooked, are a common food vehicle implicated in gastroenteritis outbreaks. The involvement of bivalve molluscs in norovirus outbreaks and epidemiology over the past two decades are reviewed. The authors describe how their physiology of filter feeding can render them concentrated vehicles of norovirus contamination in polluted environments and how high viral loads persist in molluscs even after application of depuration practices and typical food preparation steps. The global prevalence of noroviruses in bivalve molluscs as detected by different monitoring efforts is determined and the various methods currently utilised for norovirus extraction and detection from bivalve matrices described. An overview of gastroenteritis outbreaks affirmatively associated with norovirus-contaminated bivalve molluscs as reported in the past 18 years is also provided. Strategies for risk reduction of shellfish contamination and subsequent human infection are discussed.
... Photoactivatable dyes such as propidium monoazide (PMA) and ethidium monoazide (EMA) (Biotium, Inc., Fremont, CA, USA) have been used with RT-qPCR with some success for the selective detection of infectious norovirus using RT-qPCR [8][9][10][11][12][13]. Theoretically, these dyes penetrate a damaged capsid and, in the presence of light, a covalent bond forms with the nucleic acid, resulting in a reduction in nucleic acid extraction efficiency and in the RT-qPCR signal [14]. ...
... The photoactivatable dyes PMA and EMA have been used individually for the selective detection of infectious murine norovirus from heat-inactivated samples [8,13,25,26]. In those studies, PMA was less effective (0.14 to 1.38 log 10 reduction) in discriminating between non-infectious and infectious murine norovirus [13,25] than shown by our results using PEMAX TM (~2.0 log 10 reduction). ...
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Article
Rapid detection of infectious noroviruses from environmental samples is essential to minimize the risk of norovirus outbreaks associated with environmental transmission. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) methods are rapid and sensitive, but cannot differentiate between infectious and non-infectious noroviruses. In this study, a PEMAX TM treatment followed by RT-qPCR (PEMAX TM-RT-qPCR) method was developed for murine norovirus and norovirus GI/GII, and evaluated for the selective detection of infectious viruses following heat inactivation. The norovirus PEMAX TM-RT-qPCR method was then evaluated for the selective detection of infectious viruses from environmental samples. Following heat-treatment (90 • C for 3 min), the murine norovirus PEMAX TM-RT-qPCR showed at least a 2.04 log 10 reduction in detectable virus, compared to a 0.43 log 10 reduction for RT-qPCR alone. Under the same conditions, the norovirus PEMAX TM-RT-qPCR showed a 0.34 to 0.98 log 10 (GI.3) and 0.63 to 2.06 log 10 (GII.4) reduction in detectable viruses, compared to 0.05 to 0.18 log 10 (GI.3) and 0.06 to 0.25 log 10 (GII.4) for RT-qPCR alone. Evaluation of the norovirus PEMAX TM-RT-qPCR on norovirus-contaminated influent and effluent wastewater, and seawater indicated a high proportion of non-infectious norovirus GI and GII (i.e., 56 to 100% in seawater, 32 to 76% in effluent, and 11 to 79% in influent) was present in samples. While potentially overestimating the amount of infectious noroviruses, this approach has potential to provide better information on viral infectivity than RT-qPCR alone.
... However, the efficacy of viability dyes depends on the target virus, the matrix tested, and the inactivation procedure. The application of viability dyes combined with RT-qPCR to NoV yielded mixed results for the discrimination between native and heat-treated virus: PMA and EMA were inefficient in three studies out of five and one study out of two, respectively, and PMAxx was efficient in one study (Escudero-Abarca et al., 2014;Jeong et al., 2017;Karim et al., 2015;Parshionikar et al., 2010;Randazzo et al., 2016). This lack of consistency may be due to the use of different virus strains and viability dye protocols. ...
... The behavior of purified NoV strains in response to heat treatment was highly variable, with no evidence of common behavior among genogroups or genotypes. These results are consistent with the discrepancies found among previous studies: either no effect on NoV genomic titer after heat treatment (Escudero-Abarca et al., 2014;Karim et al., 2015;Randazzo et al., 2016Randazzo et al., , 2018aTuladhar et al., 2012), reduction from 0.2 log 10 to 1 log 10 (Hewitt et al., 2009;Li et al., 2011), or reduction > 1 log 10 (Jeong et al., 2017). Genomic titer reduction of heat-treated NoV treated with PtCl 4 was highly variable, depending on the viral strain but independently of the genogroup (mean reduction of 2.5 log 10 (ranging from 0.2 log 10 to 3.8 log 10 ) for NoV GI strains and 2.2 log 10 (ranging from 1.1 log 10 to 2.7 log 10 ) for NoV GII strains). ...
Article
Human noroviruses (NoV) are major agents of foodborne outbreaks. Because of the lack of a standardized cell culture method, real-time reverse transcriptase PCR is now commonly used for the detection of NoV in foodstuffs and environmental samples. However, this approach detects the viral nucleic acids of both infectious and non-infectious viruses and needs to be optimized to predict infectivity for public health risk assessment. The aim of this study was to develop a viability PCR method to discriminate between native and heat-treated virus, for both NoV and its surrogate, murine norovirus (MNV). To this end, screening of viability markers (monoazide dyes, platinum and palladium compounds) was performed on viral RNA, native virus or heat-treated virus, and incubation conditions were optimized with PtCl4, the most efficient viability marker. Multiple MNV molecular models were designed: no impact of amplicon length was observed on inactivated MNV genomic titer; but the 5'NTR, ORF1 and 3'UTR regions resulted in higher reductions than central genomic regions. The optimal viability PCR conditions developed (incubation with 2.5 mM PtCl4 in PBS for 10 min at 5 °C) were finally applied to MNV by performing heat inactivation studies and to native and heat-treated NoV clinical strains. The viability PCR discriminated efficiently between native and heat-inactivated MNV at 72 °C and 80 °C, and efficiently reduced the genomic titer of heat-treated NoV strains. This viability PCR method could be useful to study heat inactivation kinetics of NoV and MNV. It could also be evaluated for the identification of infectious enteric viruses in foodstuffs and environmental samples.
... With regard to the concentration of PMA to be used, in the present study, we found that a concentration of 50-100 µM is effective (15,16). Karim et al. (16) reported that PMA was able to differentiate between infectious and noninfectious murine norovirus (MNV) only when inactivation was done by chlorine. ...
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Article
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.
... Studies using PMAxx evaluated infectious HAdV particles (serotypes 2, 5, 12, and 40) in samples of phosphatebuffered saline, food (mollusc and sausage), and environmental (soil, sewage, and freshwater from the urban river), thus demonstrating a reduction ranging from 0.42 to 4 log [19,20,22,24,27,45]. PMA or PMAxx were also efficient in detecting another viruses, enteric or not, including SARS-CoV-2 with intact capsids in clinical, food, and environmental matrices [16,31,32,43,[46][47][48][49][50][51][52][53][54] providing valuable data for environmental surveillance. ...
Article
Molecular methodologies providing data on viral concentration and infectivity have been successfully used in environmental virology, supporting quantitative risk assessment studies. The present study aimed to assess human mastadenovirus (HAdV) intact particles using a derivative of propidium monoazide associated with qPCR (PMAxx-qPCR) in aquatic matrices. Initially, different concentrations of PMAxx were evaluated to establish an optimal protocol for treating different naturally contaminated matrices, using 10 min incubation in the dark at 200 rpm at room temperature and 15 min of photoactivation in the PMA-Lite™ LED photolysis device. There was no significant reduction in the quantification of infectious HAdV with increasing concentration of PMAxx used (20 μM, 50 μM, and 100 μM), except for sewage samples. In this matrix, a reduction of 5.01 log of genomic copies (GC)/L was observed from the concentration of 50 μM and revealed 100% HAdV particles with damaged capsids. On the other hand, the mean reduction of 0.51 log in stool samples using the same concentration mentioned above demonstrated 83% of damaged particles eliminated in the stool. Following, 50 μM PMAxx-qPCR protocol revealed a log reduction of 0.91, 0.67, and 1.05 in other samples of raw sewage, brackish, and seawater where HAdV concentration reached 1.47 × 104, 6.81 × 102, and 2.33 × 102 GC/L, respectively. Fifty micrometers of PMAxx protocol helped screen intact viruses from different matrices, including sea and brackish water.
... Concentrates were incubated with monoazide before DNA extraction to ensure bacterial integrity (Karim et al., 2015;Coudray-Meunier et al., 2013;Prevost et al., 2016). Ethidium monoazide was used in 2018 and was replaced by the propidium monoazide due to better performances. ...
Article
Leptospirosis is a neglected zoonotic disease with a worldwide distribution caused by bacterial pathogenic Leptospira. Rodents are considered as the main reservoir of Leptospira and transmission usually occurs through exposure to urine-contaminated environment. However, interactions between environment, rodent reservoir and human leptospirosis remain poorly studied. Here, we evaluated the concentration of Leptospira in surface water and captured rats in the city of Paris (France) from 2018 to 2020 using an integrity qPCR (Quantitative Polymerase Chain Reaction). All environmental samples (n = 1031) were positive for saprophytic Leptospira but pathogenic Leptospira P1 group were only found in 40% (n = 363; 2018) to 0% (n = 264; 2020) of samples. In the same time, analysis of 200 brown rat corpses trapped in the city, showed about 15% of positivity for Leptospira but the different method used for rats conservation (based on presence or absence of conservative agent) showed important variations in the Leptospira prevalence. Metagenomic analysis, based on rrs gene sequencing, was also carried out to evaluate the distribution of Leptospira in samples. Results could indicate that some species of Leptospira are found in surface waters as well as rats, but further study is needed to accurately describe the nature of the link between these two reservoirs. Quantification of Leptospira and pathogenic species description circulating inside animal reservoir living in the vicinity of freshwater in urban areas, will be helpful to understand the eco-epidemiology of leptospirosis and to establish prevention and intervention strategies, especially in the context of organization of recreative activity events in these urban areas.
... To be able to work, viability dyes must first enter the cell, and therefore it is necessary that damage to the envelope occur. Many publications have already shown, for non-enveloped viruses, that viability dyes are not effective at removing the signal of UV-inactivated viruses (Karim et al., 2015;Leifels et al., 2015). ...
Article
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.
... Before DNA extraction, samples were incubated with propidium monoazide (PMAxx) to ensure bacterial integrity [41][42][43]. The PMAxx solution was diluted in molecular grade water to obtain a final concentration of 10 mM and aliquots were stored at -20˚C. ...
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Article
Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira . Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira , allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.
... The detection of the viral genome using a polymerase chain reaction, for example, can allow for verifying the virus integrity only if photo-reactive DNA intercalating dyes, such as ethidio monoazide or propidium monoazide, are used. These dyes prevent the amplification of genomes outside the viral structure or of genomic fragments inside damaged viruses by binding the double helix of the genome and inhibiting the polymerase reaction [29]. This possibility is clearly not applicable to single-stranded viruses. ...
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Article
Microbiological surveillance carried out in order to verify the effectiveness of endoscope reprocessing does not include the research of viruses, although endoscopes may be associated with the transmission of viral infections. This paper reports the experience of the University Hospital of Pisa in managing the risk from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during an endoscopy. A review of the reprocessing procedure was conducted to assess whether improvement actions were needed. To verify the reprocessing efficacy, a virological analysis was conducted both before and after the procedure. Five bronchoscopes and 11 digestive endoscopes (6 gastroscopes and 5 colonoscopes) were sampled. The liquid samples were subjected to concentration through the use of the Macrosep Advance Centrifugal Devices (PALL Life Sciences, Port Washington, NY, USA) and subsequently analyzed using the cobas® SARS-CoV-2 Test (Roche Diagnostics, Basel, Switzerland), together with eSwab 490 CE COPAN swabs (COPAN, Brescia, Italy), which were used to sample surfaces. In accordance with the first ordinance regarding the coronavirus disease 2019 (COVID-19) emergency issued by the Tuscany Region in March 2020, a procedure dedicated to the management of the COVID-19 emergency in endoscopic practices was prepared, including the reprocessing of endoscopes. The virological analysis carried out on samples collected from endoscopes after reprocessing gave negative results, as well as on samples collected on the endoscopy column surfaces and the two washer-disinfectors that were dedicated to COVID-19 patients. The improvement in endoscope reprocessing implemented during the COVID-19 emergency was effective in ensuring the absence of SARS-CoV-2, thus reducing the risk of infections after an endoscopy on COVID-19 patients.
... Alternatively, culture-independent methods for investigating the infectivity of viruses have been used to evaluate the infectivity of enteric viruses (Sano et al. 2015). The culture-independent methods are based on RT-qPCR, and RNase or photo-reactive intercalators like PMAxx to distinguish undamaged (likely infectious) viral particles from damaged ones (Fraisse et al. 2018;Karim et al. 2015;Park et al. 2016;Randazzo et al. 2016). In considering the results of the culture-independent methods, the correlation with the results of culture-dependent methods is very important. ...
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Article
Human noroviruses are the major cause of non-bacterial acute gastroenteritis worldwide. Since no therapeutic agent has been proven to prevent human norovirus infection yet, preventive healthcare interventions to block the infection routes play an important role in infection control. One of the possible infection routes of human noroviruses are through contaminated hands, but no hand antiseptics have been proven effective. Olanexidine gluconate is a new biguanide compound that has already been approved for sale as an antiseptic for the surgical field in Japan. A new hand antiseptic was developed using olanexidine gluconate in this study, and its virucidal efficacy against human noroviruses was evaluated using modified RT-qPCR that can account for genome derived from intact viruses using RNase A and photo-reactive intercalators. We tested the virucidal efficacy of five materials; two olanexidine gluconate antiseptics (hand rub formulation and surgical field formulation), two kinds of ethanol solutions at different pH (approx. 3 or 7), and a base component of olanexidine gluconate hand rub formulation against 11 human norovirus genotypes by culture-independent methods. The infectivity of murine norovirus (MNV), a surrogate for human norovirus, was significantly reduced after use of the antiseptics. The olanexidine gluconate hand rub demonstrated the strongest virucidal efficacy against human norovirus among the five tested materials. This study showed that olanexidine gluconate has the potential to become a strong tool for the prevention of human norovirus infection.
... The combination of PMA with qPCR has been widely used to distinguish viable pathogenic fungi and bacteria from dead cells, such as Aspergillus fumigatus (Vesper et al., 2008), Escherichia coli O157: H7 (Zhong & Zhao, 2018), Salmonella (Han et al., 2020), Vibrio parahaemolyticus (Ling et al., 2020), Emetic Bacillus cereus (Zhou et al., 2019) and Listeria monocytogenes (Kragh, Thykier, & Truelstrup Hansen, 2020). The method was also adapted for detecting infectious human enteric viruses such as norovirus (Chen, Wu, Sánchez, & Randazzo, 2020;Fraisse et al., 2018), poliovirus (Karim, Fout, Johnson, White, & Parshionikar, 2015) and hepatitis A virus (Randazzo, Piqueras, Rodríguez-Díaz, Aznar, & Sánchez, 2017). ...
Article
Salmonella is a foodborne pathogen that may cause serious neonatal disease. In this study, an isothermal real-time recombinase polymerase amplification (RPA) was established to detect Salmonella at 37 °C within 20 min, with the detection sensitivity of 10³ cfu mL⁻¹ in pure culture. In food applications using powdered infant formula (PIF), the detection limit of Salmonella using this assay was 2 × 10³ cfu mL⁻¹ without a pre-enrichment procedure. When PIF was spiked with Salmonella at 0.1, 1 and 10 cfu mL⁻¹ and enriched at 37 °C, results showed that this assay can detect Salmonella at initial inoculation level of 0.1 cfu mL⁻¹ in PIF after 8 h pre-enrichment. This real-time RPA assay was considerably faster than qPCR and exhibited no losses in detection sensitivity and specificity, therefore it was suitable for on-site detection, especially in resource-poor environments.
... Intercalating dyes, such as propidium monoazide (PMA) and ethidium monoazide (EMA) in conjunction with qPCR (PMA-RT-qPCR and PMA-qPCR for RNA or DNA viruses, respectively), have been used to determine the potential infectivity of enteric viruses in water [11,70,71]. Treatment of virus suspensions with platinum (IV) chloride (PtCl4) has also been applied to discriminate between potentially infectious and thermally inactivated enteric hepatitis viruses in environmental samples [12,72,73]. ...
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Article
Detection of waterborne enteric viruses is an essential tool in assessing the risk of waterborne transmission. Cell culture is considered a gold standard for detection of these viruses. However, it is important to recognize the uncertainty and limitations of enteric virus detection in cell culture. Cell culture cannot support replication of all virus types and strains, and numerous factors control the efficacy of specific virus detection assays, including chemical additives, cell culture passage number, and sequential passage of a sample in cell culture. These factors can result in a 2- to 100-fold underestimation of virus infectivity. Molecular methods reduce the time for detection of viruses and are useful for detection of those that do not produce cytopathogenic effects. The usefulness of polymerase chain reaction (PCR) to access virus infectivity has been demonstrated for only a limited number of enteric viruses and is limited by an understanding of the mechanism of virus inactivation. All of these issues are important to consider when assessing waterborne infectious viruses and expected goals on virus reductions needed for recycled water. The use of safety factors to account for this may be useful to ensure that the risks in drinking water and recycled water for potable reuse are minimized.
... The failure of both dyes to estimate infectious HAdV after UV disinfection is consistent with studies with PMA/EMA-qPCR for UV-treated bacterial assays [35,57,58] and most likely due to the different mode of virus inactivation (UV breaking down di-hydrogen bonds between nucleotides rather than affecting the virus capsid as described by Beck et al. [10]). As for bacteria, similar observations have been described for a variety of enteric viruses (such as MNV, Poliovirus, Norwalk virus, Rotavirus, HAdV, and Coxsackievirus B1) and bacteriophages (like MS2 and PhiX174) after exposure to UV light by [38,[59][60][61]. While culture methods indicated a loss of virus and phage infectivity, the addition of PMA and/or EMA failed to represent this. ...
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Article
Recreational, reclaimed and drinking source waters worldwide are under increasing anthropogenic pressure, and often contain waterborne enteric bacterial, protozoan, and viral pathogens originating from non-point source fecal contamination. Recently, the capsid integrity (ci)-qPCR, utilizing the azo-dyes propidium monoazide (PMA) or ethidium monoazide (EMA), has been shown to reduce false-positive signals under laboratory conditions as well as in food safety applications, thus improving the qPCR estimation of virions of public health significance. The compatibility of two widely used human adenovirus (HAdV) qPCR protocols was evaluated with the addition of a PMA/EMA pretreatment using a range of spiked and environmental samples. Stock suspensions of HAdV were inactivated using heat, UV, and chlorine before being quantified by cell culture, qPCR, and ci-qPCR. Apparent inactivation of virions was detected for heat and chlorine treated HAdV while there was no significant difference between ci-qPCR and qPCR protocols after disinfection by UV. In a follow-up comparative analysis under more complex matrix conditions, 51 surface and 24 wastewater samples pre/post UV treatment were assessed for enteric waterborne HAdV to evaluate the ability of ci-qPCR to reduce the number of false-positive results when compared to conventional qPCR and cell culture. Azo-dye pretreatment of non-UV inactivated samples was shown to improve the ability of molecular HAdV quantification by reducing signals from virions with an accessible genome, thereby increasing the relevance of qPCR results for public health purposes, particularly suited to resource-limited low and middle-income settings.
... Despite being rapid, sensitive and specific, molecular methods are unable to predict the infectivity of norovirus present in the shellfish. In recent years, RT-qPCR methods have been combined with a pretreatment such as enzymatic (RNase) [47][48][49][50][51], photoactivatable dyes (EMA, PMA, PMAxx and PEMAX) [52][53][54][55][56] and a platinum compound [57] for the selective detection of infectious norovirus. Other methods, such as porcine gastric mucin-binding [58][59][60][61] and in situ capture [62][63][64][65][66][67], have also been combined with RT-qPCR for this purpose. ...
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Article
Reports of norovirus infections associated with the consumption of contaminated bivalve molluscan shellfish negatively impact both consumers and commercial shellfish operators. Current virus recovery and PCR detection methods can be expensive and time consuming. Due to the lack of rapid, user-friendly and onsite/infield methods, it has been difficult to establish an effective virus monitoring regime that is able to identify contamination points across the production line (i.e. farm-to-plate) to ensure shellfish quality. The focus of this review is to evaluate current detection methods and discuss emerging approaches. Recent advances in omics-based detection approaches have the potential to identify novel biomarkers that can be incorporated into rapid detection kits for onsite use. Furthermore, some omics techniques have the potential to simultaneously detect multiple enteric viruses that cause human disease. Other emerging technologies include microfluidic, aptamer and biosensor-based detection methods developed to detect norovirus with high sensitivity from a simple matrix. Many of these approaches have the potential to be developed as user-friendly onsite detection kits with minimal costs. However, more collaborative efforts on research and development will be required to commercialize products. Once developed, such technologies provide a way forward to minimize public health risks associated with the shellfish consumption.
... This technique was also used for evaluating the effects of high hydrostatic pressure on MNV and Tulane virus . The combination of pig mucin binding and RNAse treatment reduced detection of damaged particles after different inactivation treatments (Karim et al., 2015;Afolayan et al., 2016). ...
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Article
In a recent report by risk assessment experts on the identification of food safety priorities using the Delphi technique, foodborne viruses were recognized among the top rated food safety priorities and have become a greater concern to the food industry over the past few years. Food safety experts agreed that control measures for viruses throughout the food chain are required. However, much still needs to be understood with regard to the effectiveness of these controls and how to properly validate their performance, whether it is personal hygiene of food handlers or the effects of processing of at risk foods or the interpretation and action required on positive virus test result. This manuscript provides a description of foodborne viruses and their characteristics, their responses to stress and technologies developed for viral detection and control. In addition, the gaps in knowledge and understanding, and future perspectives on the application of viral detection and control strategies for the food industry, along with suggestions on how the food industry could implement effective control strategies for viruses in foods. The current state of the science on epidemiology, public health burden, risk assessment and management options for viruses in food processing environments will be highlighted in this review.
... To overcome the limitations encountered with cell culture methods, intercalating dyes such as propidium monoazide (PMA) in conjunction with real time PCR (RT-qPCR or qPCR for RNA or DNA viruses, respectively (PMA-RT-qPCR/qPCR) have been used to determine the potential infectivity of enteric RNA and DNA viruses in water and other environmental matrices (Parshionikar et al., 2010;Karim et al., 2015;Leifels et al., 2015;Fongaro et al., 2016). However, current methods are still limited in this assessment (Rodriguez et al., 2009). ...
Article
Treatment of wastewater for potable reuse requires the reduction of enteric viruses to levels that pose no significant risk to human health. Advanced water treatment trains (e.g., chemical clarification, reverse osmosis, ultrafiltration, advanced oxidation) have been developed to provide reductions of viruses to differing levels of regulatory control depending upon the levels of human exposure and associated health risks. Importance in any assessment is information on the concentration and types of viruses in the untreated wastewater, as well as the degree of removal by each treatment process. However, it is critical that the uncertainty associated with virus concentration and removal or inactivation by wastewater treatment be understood to improve these estimates and identifying research needs. We reviewed the critically literature to assess to identify uncertainty in these estimates. Biological diversity within families and genera of viruses (e.g. enteroviruses, rotaviruses, adenoviruses, reoviruses, noroviruses) and specific virus types (e.g. serotypes or genotypes) creates the greatest uncertainty. These aspects affect the methods for detection and quantification of viruses and anticipated removal efficiency by treatment processes. Approaches to reduce uncertainty may include; 1) inclusion of a virus indicator for assessing efficiency of virus concentration and detection by molecular methods for each sample, 2) use of viruses most resistant to individual treatment processes (e.g. adenoviruses for UV light disinfection and reoviruses for chlorination), 3) data on ratio of virion or genome copies to infectivity in untreated wastewater, and 4) assessment of virus removal at field scale treatment systems to verify laboratory and pilot plant data for virus removal.
... In response, researchers have explored various methods to distinguish between RT-qPCR signals originating from infectious versus noninfectious virions (51). Two sample preparation methods that have been proposed are pretreatment with RNase (52-54) and pretreatment with dyes such as propidium monoazide (PMA) and ethidium monoazide (EMA) (55)(56)(57). The objective of both methods is to align RT-qPCR signals more closely with infectivity measurements by neutralizing nonencapsulated viral RNA and RNA within a damaged capsid prior to amplification. ...
Article
Human noroviruses (hNoVs) are a known public health concern associated with the consumption of leafy green vegetables. While a number of studies have investigated pathogen reduction on the surfaces of leafy greens during the postharvest washing process, there remains a paucity of data on the level of treatment needed to inactivate viruses in the wash water, which is critical for preventing cross-contamination. The objective of this study was to quantify the susceptibility of hNoV genotype I (GI), hNoV GII, murine norovirus (MNV), and bacteriophage MS2 to free chlorine in whole leaf, chopped romaine, and shredded iceberg lettuce industrial leafy green wash waters, each sampled three times over a 4-month period. A suite of kinetic inactivation models was fit to the viral reduction data to aid in quantification of concentration-time (CT) values. Results indicate that 3-log10 infectivity reduction was achieved at CT values of less than 0.2 mg · min/liter for MNV and 2.5 mg · min/liter for MS2 in all wash water types. CT values for 2-log10 molecular reduction of hNoV GI in whole leaf and chopped romaine wash waters were 1.5 and 0.9 mg · min/liter, respectively. For hNoV GII, CT values were 13.0 and 7.5 mg · min/liter, respectively. In shredded iceberg wash water, 3-log10 molecular reduction was not observed for any virus over the time course of experiments. These findings demonstrate that noroviruses may exhibit genogroup-dependent resistance to free chlorine and emphasize the importance of distinguishing between genogroups in hNoV persistence studies. IMPORTANCE Postharvest washing of millions of pounds of leafy greens is performed daily in industrial processing facilities with the intention of removing dirt, debris, and pathogenic microorganisms prior to packaging. Modest inactivation of pathogenic microorganisms (less than 2 log10) is known to occur on the surfaces of leafy greens during washing. Therefore, the primary purpose of the sanitizing agent is to maintain microbial quality of postharvest processing water in order to limit cross-contamination. This study modeled viral inactivation data and quantified the free-chlorine CT values that processing facilities must meet in order to achieve the desired level of hNoV GI and GII reduction. Disinfection experiments were conducted in industrial leafy green wash water collected from a full-scale fresh produce processing facility in the United States, and hNoV GI and GII results were compared with surrogate molecular and infectivity data.
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At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50–100 μM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.
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Cellular responses to stress generally lead to the activation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Several lines of study support that ERAD may be playing a proviral role during flaviviral infection. A key host factor in ERAD is the valosin-containing protein (VCP), an ATPase which ushers ubiquitin-tagged proteins to degradation by the proteasome. VCP exhibits different proviral activities, such as engaging in the biogenesis of viral replication organelles and facilitating flavivirus genome uncoating after the viral particle entry. To investigate the possible antiviral value of drugs targeting VCP, we tested two inhibitors: eeyarestatin I (EEY) and xanthohumol (XAN). Both compounds were highly effective in suppressing Zika virus (ZIKV) and Usutu virus (USUV) replication during infection in cell culture. Further analysis revealed an unexpected virucidal activity for EEY, but not for XAN. Preincubation of ZIKV or USUV with EEY before inoculation to cells resulted in significant decreases in infectivity in a dose- and time-dependent manner. Viral genomes in samples previously treated with EEY were more sensitive to propidium monoazide, an intercalating agent, with 10- to 100-fold decreases observed in viral RNA levels, supporting that EEY affects viral particle integrity. Altogether, these results support that EEY is a strong virucide against two unrelated flaviviruses, encouraging further studies to investigate its potential use as a broad-acting drug or the development of improved derivatives in the treatment of flaviviral infection.
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The increased sensitivity of advanced molecular techniques greatly exceeds the sensitivities of traditional detection methods for infectious agents. This sensitivity causes difficulty in interpreting the biological significance of such detections in fish (and shellfish), especially when the agent(s) cannot be cultured in the laboratory. In the Pacific Northwest, including Canada and Alaska, molecular detections of “new” (unknown or known but discovered in a different geographic location or fish host) potentially infectious agents in fish have received extensive media attention and misinterpretation that call for resource agencies to change current fish health surveillance practices or policies to include these agents. Fish health specialists from several of these agencies and organizations (see Acknowledgments) advise that any policy changes should be made only after further investigations to avoid wasting resources to conduct surveillance for organisms that are not significant to fish health or for non‐infectious genetic material that does not represent a viable agent. Molecular detection is not proof of agent viability within or on host tissues and requires further investigation regarding the agent ability to replicate and evidence that the agent causes substantial risk of disease to exposed fish populations. This document provides examples of molecularly detected agents causing public concern that were accompanied by little or no data to provide context and assessment of biological significance, highlights important questions to be answered regarding these detections and provides a suggested pathway of investigative criteria to determine viability and pathogenicity of such agents that are necessary for consideration of any changes to aquatic animal health practices and policies.
Article
The effective food processing technology is a key step in eliminating human noroviruses in foods mainly due to their stability in diverse environmental conditions. The aim of this study was to evaluate the effect of rising temperatures for inactivation of norovirus genogroup (G) II and murine norovirus 1 in samples of tomato sauce (72-74 °C for 1 min) and ground meat (100 °C for 30 min). Spiking experiments were carried out in triplicate using TRIzol® reagent method associated with quantitative polymerase chain reaction (qPCR) TaqMan™ system combined with previous free RNA digestion. Success rate and efficiency recoveries of both viruses as well limit of detection of a method for each matrix were also conducted. The heat treatment applied here proved to be efficient to reduce the burden of norovirus GII in a range of 1-4 log10 genomic copies per gram (percentage ranging from 0.45 to 104.54%) in both matrices. The experiments in this study showed that the results of norovirus GII and murine norovirus 1 in tomato sauce and ground meat tested during thermal treatments cannot be generalized to other food matrices, since there may be food-specific protective effects, as the presence of different components, that can interfere in virus inactivation. Studies using different food matrices reinforce the importance to investigate viruses' inactivation thermal processes in foods due to the resistance of these viruses to adverse conditions, contributing to food security in food virology.
Thesis
Les leptospires pathogènes sont responsables d’une zoonose de répartition mondiale, la leptospirose, où l’homme peut être un hôte occasionnel. Le réservoir animal, principalement les rongeurs, excrète les leptospires dans ses urines et contamine ainsi l’environnement hydrique. Depuis 2015, près de 600 cas de leptospirose ont été recensés en Métropole. L’augmentation du nombre de cas pourrait être due à plusieurs facteurs dont l'augmentation des comportements à risques (ex : sports aquatiques). L’étude de l’écologie des leptospires en milieu aquatique se développe ces dernières années, mais ces bactéries ne sont toujours pas intégrées dans le contrôle sanitaire des eaux. Les activités nautiques en milieu urbain sont de plus en plus fréquentes et pourraient augmenter l’exposition des usagers à ce pathogène.Dans ces travaux nous avons tenté d’obtenir un premier aperçu de la présence des leptospires dans les eaux de surface parisiennes. Pour ce faire, une qPCR d’intégrité fonctionnant en multiplex a été mise au point. Basée sur deux gènes spécifiques et éprouvée lors de tests d’inactivation (UV, chlore, etc.), elle permet la détection de l’ensemble des leptospires (gène rrs) mais également la distinction de la part de pathogènes (gène lipL32). De juin 2018 à septembre 2020, cette méthode a été appliquée sur la recherche des leptospires pathogènes chez les rats (plus de 190 reins analysés) et sur plusieurs sites d’eau douce (plus de 1000 prélèvements), susceptibles d’accueillir des lieux de baignades. Dans un second temps, une comparaison de la diversité des souches circulantes dans les eaux et celles présentes chez le rat (réservoir principal potentiel) a également pu être étudiée par séquençage NGS. L’analyse des reins de rats a révélé la présence d’une seule OTU, regroupant des espèces pathogènes. Du fait de la grande diversité de leptospires et de bactéries dans les eaux de surface, la diversité au sein des échantillons environnementaux est plus grande mais comprend parfois des leptospires pathogènes. La concentration de ces espèces semble diminuer ces dernières années.Ces résultats permettent pour la première fois de connaitre la contamination environnementale dans les zones de baignade urbaine, au sein d’une grande agglomération (sous climat tempéré), en utilisant Paris comme région « témoin » dans un contexte d’ouverture programmée d’espaces de baignade dans des eaux de surface. Inscrit dans l’approche One Health, ce projet a pour objectif de sensibiliser la communauté médicale, la population et les services de contrôles sanitaires de l’eau sur le risque potentiel lié à ces bactéries ; et si cela est justifié, de mettre en place des mesures de prévention.
Thesis
As population growth, climate change, and urbanization strain drinking water sources, the increasingly common use of diverse and impacted water supplies necessitates a better understanding of contaminant fate in this setting. Among the human health hazards found in water supplies, viral pathogens are of principal concern, because they can be present in elevated concentrations, are highly infectious, and are difficult to remove due to their small size. Effective viral pathogen removal is of particular importance in direct potable water reuse, in which wastewater is transformed into drinking water. A multibarrier approach to treatment is traditionally used for contaminant removal, where different treatment processes are placed in series and cumulatively reduce virus concentrations to levels that pose no significant public health risk. However, the persistence of several important waterborne viruses (e.g., human norovirus) through treatment processes is not well characterized due to difficulties in virus culturability. This raises questions about whether proposed reuse treatment schemes are sufficient to protect human health. In addition, monitoring strategies used to ensure treatment performance in real-time are not sufficiently sensitive to validate virus reductions, likely resulting in the design of overengineered treatment schemes for virus removal. This dissertation sheds light on alternative molecular and predictive modeling approaches for estimating virus fate through disinfection when traditional methods are not feasible and evaluates flow virometry as a novel approach to accurately validate virus reductions through treatment in real-time. Results demonstrate that alternative methods to accurately determine virus susceptibility to UV254 disinfection treatments can be applied effectively when culture-based approaches are not possible. Specifically, the UV254 sensitivity of human norovirus was established with these alternative approaches and confirmed through use of a novel culture system. The findings show that commonly used approaches to estimate infectious human norovirus levels overestimate norovirus survival through UV254 disinfection. Further, flow virometry, a high-throughput method for detecting and enumerating virus particles, was explored as a sensitive method to ensure virus reductions through treatment in real-time. Work revealed that flow virometry could effectively detect large dsDNA virus populations, while smaller RNA and DNA viruses were not reliably measured. Proof-of-concept experiments evaluating virus removal through ultrafiltration indicated that while flow virometry could detect particles in the same size range as viruses, little improvement over currently used monitoring approaches was observed due to limitations in the detection capabilities of current flow cytometers. Taken together, this dissertation research improves our understanding of human norovirus fate through treatment and provides novel methods that can be applied to monitor virus behavior through treatment. Ultimately, this research aids in the development of a regulatory framework that will make direct potable reuse more feasible, economical, and environmentally sustainable while still guaranteeing public health protection.
Article
Human norovirus (hNoV) is an important etiology of gastrointestinal illness and can be transmitted via ingestion of contaminated water. Currently impractical to culture, hNoV detection is reliant on real-time polymerase chain reaction (RT-PCR)-based methods. This approach cannot distinguish between infective and inactivated viruses because intact regions of the RNA genome can amplify even if the damage is present in other regions of the genome or because intact genetic material is not contained within an infectious virion. Herein, we employ a multiple long-amplicon RT-qPCR extrapolation approach to assay genome-wide damage and an enzymatic pretreatment to study the impact of simulated sunlight on the infectivity of hNoV in clear, sensitizer-free water. Using MS2 coliphage as an internal control, the genome-wide damage extrapolation approach, previously successfully applied for UV-254 inactivation, vastly overestimated sunlight inactivation, suggesting key differences in photoinactivation under different spectral conditions. hNoV genomic RNA was more susceptible to simulated sunlight degradation per base compared to MS2 genomic RNA, while enzymatic pretreatment indicated that hNoV experienced more capsid damage than MS2. This work provides practical and mechanistic insight into the endogenous sunlight inactivation of single-stranded RNA bacteriophage MS2, a widely used surrogate, and hNoV GII.4 Sydney, an important health-relevant virus, in clear sensitizer-free water.
Article
Cronobacter sakazakii is a common foodborne pathogen that may cause serious neonatal disease. The quantification of the viable bacteria is a major concern during food processing. The traditional culture-based method is time-consuming and laborious. In this work, the improved propidium monoazide (PMAxx) was successfully combined with real-time recombinase polymerase amplification (qRPA) and real-time polymerase chain reaction (qPCR) to quantify the viable C. sakazakii in spiked powdered infant formula (PIF). Primers specific for C. sakazakii were designed to amplify different sized fragments of the alpha-glucosidase (gluA) gene. The impact of amplicon size on differentiation of viable and heat-killed cells (90°C, 15 min) was determined following PMAxx treatment. Results demonstrated that the developed PMAxx-qRPA and PMAxx-qPCR assay can distinguish viable and dead bacterial cells. The long amplicon (256 bp and 338 bp) significantly reduced the signal from dead cells compared to the short amplicon (110 bp and 128 bp, P < 0.05). The PMAxx-qRPA assay was completed within 20 min at 37°C compared to the PMAxx-qPCR assay which required a longer reaction time and multiple temperature gradients to complete the amplification. The food matrix in PIF significantly affected the detection of viable cells in PMAxx-qPCR assay whereas not in PMAxx-qRPA assay (P < 0.05). When different number of viable cells (0 - 10⁷ cfu/g) were mixed with dead cells (10⁷ cfu/g) in the PIF matrix, both PMAxx-based assays eliminated the false-positive signals from the dead cells, and the quantitative results of PMAxx-qRPA assays for viable cells were consistent with the traditional culture-based method (P > 0.05). Thus, PMAxx-qRPA with longer amplicons can be used as an effective tool to quantify the viable C. sakazakii in complex food samples such as PIF, which greatly improve detection efficiency and can be a promising technique for risk assessments in food processing.
Article
Evaluating the efficacy of disinfection processes to inactivate human enteric viruses is important for the prevention and control of waterborne diseases caused by exposure to those viruses via drinking water. Here, we evaluated the inactivation of two representative human enteric viruses (adenovirus type 40 [AdV] and coxsackievirus B5 [CV]) by thermal or free-chlorine disinfection. In addition, we compared the infectivity reduction ratio of a plant virus (pepper mild mottle virus [PMMoV], a recently proposed novel surrogate for human enteric viruses for the assessment of virus removal by coagulation‒rapid sand filtration and membrane filtration) with that of the two human enteric viruses to assess the suitability of PMMoV as a human enteric virus surrogate for use in thermal and free-chlorine disinfection processes. Finally, we examined whether conventional or enhanced viability polymerase chain reaction (PCR) analysis using propidium monoazide (PMA) or improved PMA (PMAxx) with or without an enhancer could be used as alternatives to infectivity assays (i.e., plaque-forming unit method for AdV and CV; local lesion count assay for PMMoV) for evaluating virus inactivation by disinfection processes. We found that PMMoV was more resistant to heat treatment than AdV and CV, suggesting that PMMoV is a potential surrogate for these two enteric viruses with regard to thermal disinfection processes. However, PMMoV was much more resistant to chlorine treatment compared with AdV and CV (which is chlorine-resistant) (CT value for 4-log10 inactivation: PMMoV, 84.5 mg-Cl2·min/L; CV, 1.15-1.19 mg-Cl2·min/L), suggesting that PMMoV is not useful as a surrogate for these enteric viruses with regard to free-chlorine disinfection processes. For thermal disinfection, the magnitude of the signal reduction observed with PMAxx-Enhancer-PCR was comparable with the magnitude of reduction in infectivity, indicating that PMAxx-Enhancer-PCR is a potential alternative to infectivity assay. However, for free-chlorine disinfection, the magnitude of the signal reduction observed with PMAxx-Enhancer-PCR was smaller than the magnitude of the reduction in infectivity, indicating that PMAxx-Enhancer-PCR underestimated the efficacy of virus inactivation (i.e., overestimated the infectious virus concentration) by chlorine treatment. Nevertheless, among the PCR approaches examined in the present study (PCR alone, PMA-PCR or PMAxx-PCR either with or without enhancer), PMAxx-Enhancer-PCR provided the most accurate assessment of the efficacy of virus inactivation by thermal or free chlorine disinfection processes.
Article
Metal-doped TiO2 photocatalysis are recognized as effective materials for eliminating human norovirus (HuNoVs). In recent years, the airborne transmission of viral particles of HuNoVs has been a cause for concern. In this study, we evaluated the virucidal effects of a Cu/TiO2 non-woven fabric (NWF) on viral particles of HuNoV genogroup II genotype 4 (HuNoV GII.4) under an ultraviolet A light-emitting diode (UVA-LED) source. For the optimized parameters, a multivariate statistical analysis using the Box-Behnken design (BBD) technique combined with the response surface methodology (RSM) was applied. The experimental results showed that the Cu/TiO2-based NWF degraded HuNoV viral particles in the air samples. The BBD-based RSM indicated that the optimum treatment conditions for inactivating the HuNoV GII.4 droplets with the Cu/TiO2 NWF were a 1:7.7 ratio of Cu:TiO2 and the use of a 373-nm UVA-LED source for 48.08 min. The optimal conditions for the photocatalytic efficacy in HuNoV GII.4 of Cu/TiO2 NWF were verified experimentally, giving a value of 2.89 ± 0.11 log10 genomic copies, which was the same as the predicted value (2.91611) within experimental uncertainty. This result adequately validated the predicted model and confirmed that viral particles of HuNoVs could efficiently be disinfected using Cu/TiO2 NWF stimulated by UVA-LED light.
Article
Noroviruses are the most common cause of gastroenteritis outbreaks in humans and bivalve shellfish consumption is a recognized route of infection. Our aim was to detect and characterize norovirus in bivalves from a coastal city of Brazil. Nucleic acid was extracted from the bivalve's digestive tissue concentrates using magnetic beads. From March 2018 to June 2019, 77 samples were screened using quantitative RT-PCR. Noroviruses were detected in 41.5%, with the GII being the most prevalent (37.7%). The highest viral load was 3.5 × 106 and 2.5 × 105 GC/g in oysters and mussels, respectively. PMA-treatment demonstrated that a large fraction of the detected norovirus corresponded to non-infectious particles. Genetic characterization showed the circulation of the GII.2[P16] and GII.4[P4] genotypes. Norovirus detection in bivalves reflects the anthropogenic impact on marine environment and serves as an early warning for the food-borne disease outbreaks resulting from the consumption of contaminated molluscs.
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Preprint
Capsid-integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo-dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-two recent peer-reviewed studies employing capsid-integrity qPCR for viruses in the last decade (2009 to 2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 µM; incubation on ice or at room temperature (20 - 25°C) for 5 to 120 min; and dye activation using LED or high light (500 – 800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever. Graphical abstract
Article
Introduction. The results of experimental studies on the comparative assessment of the effects of various doses of UV radiation on the survival of poliovirus type I LSc2ab, phage MS-2, hepatitis A viruses and their RNA in tap water are presented. Material and methods. Poliomyelitis viruses of type I strain LSc2ab (PV), viruses of hepatitis A, strain HAS-15 (HAV), phages MS-2, free RNA isolated from hepatitis viruses and poliomyelitis were introduced into model reservoirs with dechlorinated Moscow tap water. Water samples were taken from each tank and subjected to ultraviolet irradiation (UVR) with a wavelength of 254 nm with doses of 25, 40, 60, 80 and 100 mJ/cm2. PV titration was performed on a BGM monkey kidney cell transplant line; MS-2 phages were determined by the agar layer method using the E. coli K12F + Str. detector; determination of PV RNA and HAV was carried out on the Rotor GeneTM 6000 amplifier in RT-PCR reaction in real-time using appropriate test systems. Extraction and isolation of RNA from samples of PV and HAV were also performed using reagent kits of domestic and foreign production. Results. Ultraviolet irradiation in doses from 25 to 100 mJ/cm2 was shown to have a pronounced inhibitory effect on phages MS-2 and PV, determined by traditional methods in accordance with the methodological guidelines MUK 4.2.1018-01 and MUK 4.2.2029-05. At UVR doses of 80 and 100 mJ/cm2, complete inactivation of MS-2 and PV phages in water was noted. At the same time, these same doses of UVR had a less inhibitory effect on PVA, HAV RNA, as well as on isolated free PVA RNA/X and HAV, which were more stable and continued to be determined by RT-PCR in water at all doses of UVR, including 80 and 100 mJ/cm2. Conclusion. If only RNA viruses are detected in the treated drinking water and there are no other direct or indirect indices of viral contamination, it is impossible to unambiguously judge the extent of the potential epidemic hazard of the water body. This requires the development of reliable additional tests confirming the infectivity of viruses, determined only by RNA or DNA markers.
Article
The removal and inactivation of infectious human norovirus is a major focus in water purification, but its fate through disinfection treatment processes is largely unknown owing to the lack of a readily available infectivity assay. In particular, norovirus behavior through unit processes may be over- or underestimated using current approaches for assessing human norovirus infectivity (e.g., surrogates, molecular methods). Here we fill a critical knowledge gap by estimating inactivation data for human norovirus after exposure to UV254, a commonly used disinfection process in the water industry. Specifically, we used a PCR-based approach that accurately tracks positive-sense single-stranded RNA virus inactivation without relying on culturing methods. We first confirmed that the approach is valid with a culturable positive-sense single-stranded RNA human virus, coxsackievirus B5, by applying both qPCR- and culture-based methods to measure inactivation kinetics with UV254 treatment. We then applied the qPCR-based method to establish a UV254 inactivation curve for human norovirus (inactivation rate constant = 0.27 cm² mJ ⁻¹). Based on a comparison with previously published data, human norovirus exhibited similar UV254 susceptibility compared with other enteric single-stranded RNA viruses (e.g., Echovirus 12, feline calicivirus), but degraded much faster than MS2 (inactivation rate constant = 0.14 cm² mJ⁻¹). In addition to establishing a human norovirus inactivation rate constant, we developed an approach using a single qPCR assay that can be applied to estimate human norovirus inactivation in UV254 disinfection systems.
Article
Objective: To establish a non-coated enzyme-linked immunosorbent assay (ELISA) based on zika virus envelope (E) protein for detecting the expression of E protein in infected cells. Methods: Adherent Vero-143 cells infected with zika virus in a 96-well plate were fixed, and the antibodies against zika virus E protein were added at an optimized concentration to establish the non-coated ELISA method for E protein. The antiviral activities of lignans compound C1 was evaluated using this method. The accuracy of this non-coated ELISA was verified by RT-PCR, and the cross reaction with dengue virus was assessed. Results: After optimization, the background absorbance at 450 nm of uninfected cells was reduced to about 0.20. The antiviral activities of lignans compound C1 detected by this method were basically consistent with the results of RT-PCR. No cross reaction with dengue virus was found in this assay. Conclusions: A non- coated ELISA method based on zika virus E protein was established, which can be used for screening antiviral agents against zika virus.
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Article
Ultraviolet (UV) disinfection is widely used to inactivate microorganisms prior to release of treated municipal wastewater. However, limited data are available for in situ inactivation of infectious enteric viruses by UV treatment at full-scale. In this study, a total of 51 pre-UV and 50 post-UV samples were collected over a two-year period from two wastewater treatment plants (WWTPs) and analyzed for noroviruses, rotavirus, reovirus, sapovirus, astrovirus, enteroviruses, adenoviruses and JC virus. Both pre-UV and post-UV samples had relatively high concentrations of these viruses determined by qPCR. Infectious viruses were also observed in 98% of pre-UV samples and 76% of post-UV samples by cell culture, using either cytopathic effect (CPE) or integrated cell culture with qPCR (ICC-qPCR). Reovirus was the most common virus detected by ICC-qPCR, present in 92% of pre-UV and 48% of post-UV samples. Infectious enterovirus and adenovirus were detected by ICC-qPCR in 33% and 31% of pre-UV samples, 14% and 20% of post-UV samples, respectively. Mean log10 reduction estimates for infectious reovirus was 1.2 and 1.8 log for the two WWTPs as assessed by ICC-qPCR, which was similar to the reduction of total infectious viruses (1.5 and 1.7 log) as assessed by CPE in cells culture. Overall, quantification of infectious reovirus appears to provide a useful index of enteric virus inactivation during wastewater treatment at full-scale. To our knowledge, this is the first comprehensive study to assess UV inactivation of human enteric viruses at full-scale in WWTPs using both molecular and cell culture techniques, providing important information for quantitative microbial risk assessment of UV inactivation of human viruses in municipal wastewater.
Chapter
Human noroviruses (HNoVs) are primarily transmitted by the fecal–oral route, either by person-to-person contact, or by ingestion of contaminated food or water as well as by aerosolization. Moreover, HNoVs significantly contribute to foodborne diseases being the causative agent of one-fifth of acute gastroenteritis worldwide. As a consequence of globalization, transnational outbreaks of foodborne infections are reported with increasing frequency. Therefore, in this review, state-of-the-art information regarding molecular procedures for human norovirus detection in food as well common food processing technologies have been summarized. Besides, the purpose of this chapter is to consolidate basic information on various aspects of HNoVs and to summarize food processing technologies that can potentially be applied in the food industry.
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Les virus entériques sont à l’origine de pathologies liées au péril fécal et dans l’état actuel des connaissances, la recherche des indicateurs de pollution fécale conventionnels (i.e. Escherichia coli, entérocoques) peut s’avérer inefficace pour évaluer le danger viral. La définition d’autres indicateurs pour gérer le danger lié à la présence des virus entériques dans les matrices hydriques et alimentaires est aujourd’hui nécessaire. Parmi eux, les bactériophages ARN F-spécifiques (FRNAPH) présentent plusieurs intérêts. Ces virus d’origine entérique sont présents en quantité importante dans les eaux usées. Très proches des virus entériques en termes de structure, ces microorganismes présentent l’avantage d’être facilement cultivables. Ils sont enfin souvent étudiés pour déterminer l’origine d’une pollution fécale (i.e. humaine ou animale). Certaines limites leur sont cependant fréquemment associées, que ce soit en termes de corrélation avec les pathogènes entériques ou concernant leur potentiel pour discriminer l’origine d’une pollution. Dans ce contexte, l’objectif du travail présenté ici était de préciser l’intérêt des FRNAPH en tant qu’indicateurs de pollution fécale mais aussi en tant qu’indicateurs de pollution virale dans l’environnement et les coquillages. Ces travaux ont permis dans un premier temps d’améliorer la capacité des FRNAPH à identifier les contaminations d’origine humaine. Nos résultats soulignent par ailleurs la plus-value apportée par la recherche des FRNAPH en cas de pollution fécale massive, en particulier si on s’intéresse à la contamination des coquillages. En effet, contrairement aux indicateurs bactériens, l’accumulation des FRNAPH ainsi que leur persistance dans ces aliments est très comparable à celles des virus entériques (i.e. norovirus). Enfin, en utilisant des méthodes de détection comparables, une forte corrélation entre la présence des FRNAPH d’origine humaine et celle des norovirus a été observée dans les coquillages. Compte tenu de ces résultats, une méthode de détection assurant la détection sensible des FRNAPH infectieux d’origine humaine dans différents types de matrices hydriques ou alimentaires (e.g. eaux de surface, fruits de mer, fruits rouges, salades) est proposée pour améliorer la gestion du danger viral
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The water-borne protozoan parasite Cryptosporidium parvum forms oocysts that can persist for long periods of time in the environment, even though the sporozoites inside the oocysts may no longer be viable, making it difficult to assess the associated risk of infection. In this study, we compared the ability of various in vitro methods to discriminate viable from non-viable oocysts, including excystation, DAPI/PI staining, RNA FISH, PMA-qPCR and a novel polymer slide adhesion method. With the notable exception of our in vitro excystation protocol, all methods were found to be useful for identifying viable oocysts.
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Aims: Detection/Quantitation of RNA viruses is mostly done by reverse transcriptase (RT)-(q)PCR, but it does not distinguish between infectious and non-infectious viruses. Our aim was to test, how different pretreatments before RT-qPCR could eliminate positivity originated from external nucleic acids or genomes of damaged particles. Methods and results: Heat-inactivated (80 °C for 10 min) rotavirus Wa strain and fecal samples containing rotavirus or norovirus were treated with PMA/PMAxx, benzonase or crude extract RNase prior to RT-qPCR. PMA/PMAxx pretreatments were not consistently efficient for RV, although they seemed to work to some extent for heat-inactivated norovirus. Benzonase and RNase provided consistently 2.2 - 2.8 log10reductions in the titer of fecal rotavirus. Conclusions: All pretreatments need to be further validated for each virus separately, taking into account sample matrix and inactivation conditions. Although none of the pretreatments could completely render inactivated viruses undetectable, RNase worked most consistently for both rota- and norovirus. Significance and impact of the study: This study sheds light on capacity of the most common pre-RT-qPCR treatments to eliminate damaged, non-infectious rotaviruses and noroviruses after thermal treatment. To our knowledge, this is the first time, when benzonase has been used in this context. This article is protected by copyright. All rights reserved.
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The objective of this study was to characterize human norovirus (hNoV) GI and GII reductions during disinfection by peracetic acid (PAA) and monochloramine in secondary wastewater (WW) and phosphate buffer (PB) as assessed by reverse transcription-qPCR (RT-qPCR). Infectivity and RT-qPCR reductions are also presented for surrogate viruses murine norovirus (MNV) and bacteriophage MS2 under identical experimental conditions to aid in interpretation of hNoV molecular data. In WW, RT-qPCR reductions were less than 0.5 log10 for all viruses at concentration-time (CT) values up to 450 mg-min/L except for hNoV GI, where 1 log10 reduction was observed at CT values of less than 50 mg-min/L for monochloramine and 200 mg-min/L for PAA. In PB, hNoV GI and MNV exhibited comparable resistance to PAA and monochloramine with CT values for 2 log10 RT-qPCR reduction between 300 to 360 mg-min/L. Less than 1 log10 reduction was observed for MS2 and hNoV GII in PB at CT values for both disinfectants up to 450 mg-min/L. Our results indicate that hNoVs exhibit genogroup dependent resistance and that disinfection practices targeting hNoV GII will result in equivalent or greater reductions for hNoV GI. These data provide valuable comparisons between hNoV and surrogate molecular signals that can begin the process of informing regulators and engineers on WW treatment plant design and operational practices necessary to inactivate hNoVs.
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Aim: To assess the potential of a viability dye and an enzymatic RT-qPCR pretreatment to discriminate between infectious and non-infectious enteric viruses. Methods and results: Enterovirus, Norovirus GII.4 and Hepatitis A virus were inactivated at 95 °C for 10 min and four methods were used to compare the efficiency of inactivation: (a) cell culture plaque assay for Hepatitis A virus and Enterovirus, (b) RT-qPCR alone, (c) RT-qPCR assay preceded by RNase treatment, and (d) pretreatment with a viability dye (Reagent D) followed by RT-qPCR. Additionally, heat-inactivated Norovirus were treated with reagent D coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from non-infectious viruses. Reagent D-RT-qPCR reduced more efficiently the detection of non-infectious viruses with little to no removal observed with RNase. Reagent D-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and Reagent D did not show relevant improvements on the removal of inactivated viruses signal compared to viability RT-qPCR, with the exception of Triton X-100. Conclusion: The use of surfactant/Reagent D-RT-qPCR, although not being able to completely remove the signal from non-infectious viral particles, yielded a better estimation of viral infectivity. Significance and impact of the study: Surfactant/Reagent D-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses. This article is protected by copyright. All rights reserved.
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The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual’s histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.
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Significance Human noroviruses are the predominant cause of acute gastroenteritis worldwide, but they remain noncultivatable. A tractable system is needed to understand the host restriction to cultivation. We established a reverse genetics system driven by a mammalian elongation factor-1α promoter without helper virus. This system supports genome replication, particle formation, and particles containing a GFP-marked genomic RNA. RNA from these particles is infectious. The system also produces infectious murine norovirus, confirming its broad applicability to other noroviruses.
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Human enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR. Once the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 muM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 muM) for RV (WA) and PMA (50 muM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37[degree sign]C, 68 C, 72[degree sign]C, 80[degree sign]C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37[degree sign]C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68[degree sign]C to 80[degree sign]C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV. We concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples.
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Unlabelled: Human noroviruses (HuNoVs) cause significant morbidity and mortality worldwide. However, despite substantial efforts, a small-animal model for HuNoV has not been described to date. Since "humanized" mice have been successfully used to study human-tropic pathogens in the past, we challenged BALB/c mice deficient in recombination activation gene (Rag) 1 or 2 and common gamma chain (γc) (Rag-γc) engrafted with human CD34+ hematopoietic stem cells, nonengrafted siblings, and immunocompetent wild-type controls with pooled stool isolates from patients positive for HuNoV. Surprisingly, both humanized and nonhumanized BALB/c Rag-γc-deficient mice supported replication of a GII.4 strain of HuNoV, as indicated by increased viral loads over input. In contrast, immunocompetent wild-type BALB/c mice were not infected. An intraperitoneal route of infection and the BALB/c genetic background were important for facilitating a subclinical HuNoV infection of Rag-γc-deficient mice. Expression of structural and nonstructural proteins was detected in cells with macrophage-like morphology in the spleens and livers of BALB/c Rag-γc-deficient mice, confirming the ability of HuNoV to replicate in a mouse model. In summary, HuNoV replication in BALB/c Rag-γc-deficient mice is dependent on the immune-deficient status of the host but not on the presence of human immune cells and provides the first genetically manipulable small-animal model for studying HuNoV infection. Importance: Human noroviruses are a significant cause of viral gastroenteritis worldwide, resulting in significant morbidity and mortality. Antivirals and vaccines are currently not available, in part due to the inability to study these viruses in a genetically manipulable, small-animal model. Herein, we report the first mouse model for human noroviruses. This model will accelerate our understanding of human norovirus biology and provide a useful resource for evaluating antiviral therapies.
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Background: Groundwater supplies for drinking water are frequently contaminated with low levels of human enteric virus genomes, yet evidence for waterborne disease transmission is lacking. Objectives: We related quantitative polymerase chain reaction (qPCR)–measured enteric viruses in the tap water of 14 Wisconsin communities supplied by nondisinfected groundwater to acute gastrointestinal illness (AGI) incidence. Methods: AGI incidence was estimated from health diaries completed weekly by households within each study community during four 12-week periods. Water samples were collected monthly from five to eight households per community. Viruses were measured by qPCR, and infectivity assessed by cell culture. AGI incidence was related to virus measures using Poisson regression with random effects. Results: Communities and time periods with the highest virus measures had correspondingly high AGI incidence. This association was particularly strong for norovirus genogroup I (NoV-GI) and between adult AGI and enteroviruses when echovirus serotypes predominated. At mean concentrations of 1 and 0.8 genomic copies/L of NoV-GI and enteroviruses, respectively, the AGI incidence rate ratios (i.e., relative risk) increased by 30%. Adenoviruses were common, but tap-water concentrations were low and not positively associated with AGI. The estimated fraction of AGI attributable to tap-water–borne viruses was between 6% and 22%, depending on the virus exposure–AGI incidence model selected, and could have been as high as 63% among children < 5 years of age during the period when NoV-GI was abundant in drinking water. Conclusions: The majority of groundwater-source public water systems in the United States produce water without disinfection, and our findings suggest that populations served by such systems may be exposed to waterborne viruses and consequent health risks.
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Since 1971, CDC, the Environmental Protection Agency (EPA), and the Council of State and Territorial Epidemiologists have collaborated on the Waterborne Disease and Outbreak Surveillance System (WBDOSS) for collecting and reporting data related to occurrences and causes of waterborne disease outbreaks associated with drinking water. This surveillance system is the primary source of data concerning the scope and health effects of waterborne disease outbreaks in the United States. Data presented summarize 48 outbreaks that occurred during January 2007--December 2008 and 70 previously unreported outbreaks. WBDOSS includes data on outbreaks associated with drinking water, recreational water, water not intended for drinking (WNID) (excluding recreational water), and water use of unknown intent (WUI). Public health agencies in the states, U.S. territories, localities, and Freely Associated States are primarily responsible for detecting and investigating outbreaks and reporting them voluntarily to CDC by a standard form. Only data on outbreaks associated with drinking water, WNID (excluding recreational water), and WUI are summarized in this report. Outbreaks associated with recreational water are reported separately. A total of 24 states and Puerto Rico reported 48 outbreaks that occurred during 2007--2008. Of these 48 outbreaks, 36 were associated with drinking water, eight with WNID, and four with WUI. The 36 drinking water--associated outbreaks caused illness among at least 4,128 persons and were linked to three deaths. Etiologic agents were identified in 32 (88.9%) of the 36 drinking water--associated outbreaks; 21 (58.3%) outbreaks were associated with bacteria, five (13.9%) with viruses, three (8.3%) with parasites, one (2.8%) with a chemical, one (2.8%) with both bacteria and viruses, and one (2.8%) with both bacteria and parasites. Four outbreaks (11.1%) had unidentified etiologies. Of the 36 drinking water--associated outbreaks, 22 (61.1%) were outbreaks of acute gastrointestinal illness (AGI), 12 (33.3%) were outbreaks of acute respiratory illness (ARI), one (2.8%) was an outbreak associated with skin irritation, and one (2.8%) was an outbreak of hepatitis. All outbreaks of ARI were caused by Legionella spp. A total of 37 deficiencies were identified in the 36 outbreaks associated with drinking water. Of the 37 deficiencies, 22 (59.5%) involved contamination at or in the source water, treatment facility, or distribution system; 13 (35.1%) occurred at points not under the jurisdiction of a water utility; and two (5.4%) had unknown/insufficient deficiency information. Among the 21 outbreaks associated with source water, treatment, or distribution system deficiencies, 13 (61.9%) were associated with untreated ground water, six (28.6%) with treatment deficiencies, one (4.8%) with a distribution system deficiency, and one (4.8%) with both a treatment and a distribution system deficiency. No outbreaks were associated with untreated surface water. Of the 21 outbreaks, 16 (76.2%) occurred in public water systems (drinking water systems under the jurisdiction of EPA regulations and water utility management), and five (23.8%) outbreaks occurred in individual systems (all of which were associated with untreated ground water). Among the 13 outbreaks with deficiencies not under the jurisdiction of a water system, 12 (92.3%) were associated with the growth of Legionella spp. in the drinking water system, and one (7.7%) was associated with a plumbing deficiency. In the two outbreaks with unknown deficiencies, one was associated with a public water supply, and the other was associated with commercially bottled water. The 70 previously unreported outbreaks included 69 Legionella outbreaks during 1973--2000 that were not reportable previously to WBDOSS and one previously unreported outbreak from 2002. More than half of the drinking water--associated outbreaks reported during the 2007--2008 surveillance period were associated with untreated or inadequately treated ground water, indicating that contamination of ground water remains a public health problem. The majority of these outbreaks occurred in public water systems that are subject to EPA's new Ground Water Rule (GWR), which requires the majority of community water systems to complete initial sanitary surveys by 2012. The GWR focuses on identification of deficiencies, protection of wells and springs from contamination, and providing disinfection when necessary to protect against bacterial and viral agents. In addition, several drinking water--associated outbreaks that were related to contaminated ground water appeared to occur in systems that were potentially under the influence of surface water. Future efforts to collect data systematically on contributing factors associated with drinking water outbreaks and deficiencies, including identification of ground water under the direct influence of surface water and the criteria used for their classification, would be useful to better assess risks associated with ground water. During 2007--2008, Legionella was the most frequently reported etiology among drinking water--associated outbreaks, following the pattern observed since it was first included in WBDOSS in 2001. However, six (50%) of the 12 drinking water--associated Legionella outbreaks were reported from one state, highlighting the substantial variance in outbreak detection and reporting across states and territories. The addition of published and CDC-investigated legionellosis outbreaks to the WBDOSS database clarifies that Legionella is not a new public health issue. During 2009, Legionella was added to EPA's Contaminant Candidate List for the first time. CDC and EPA use WBDOSS surveillance data to identify the types of etiologic agents, deficiencies, water systems, and sources associated with waterborne disease outbreaks and to evaluate the adequacy of current technologies and practices for providing safe drinking water. Surveillance data also are used to establish research priorities, which can lead to improved water quality regulation development. Approximately two thirds of the outbreaks associated with untreated ground water reported during the 2007--2008 surveillance period occurred in public water systems. When fully implemented, the GWR that was promulgated in 2006 is expected to result in decreases in ground water outbreaks, similar to the decreases observed in surface water outbreaks after enactment of the Surface Water Treatment Rule in 1974 and its subsequent amendments. One third of drinking water--associated outbreaks occurred in building premise plumbing systems outside the jurisdiction of water utility management and EPA regulations; Legionella spp. accounted for >90% of these outbreaks, indicating that greater attention is needed to reduce the risk for legionellosis in building plumbing systems. Finally, a large communitywide drinking water outbreak occurred in 2008 in a public water system associated with a distribution system deficiency, underscoring the importance of maintaining and upgrading drinking water distribution system infrastructure to provide safe water and protect public health.
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Adenoviruses are resistant to monochromatic, low-pressure (LP) UV disinfection—but have been shown to be susceptible to inactivation by polychromatic, medium-pressure (MP) UV—when assayed using cell culture infectivity. One possible explanation for the difference between UV lamp types is that the additional UV wavelengths emitted by MP UV enable it to cause greater damage to viral proteins than LP UV. The objective of this study was to examine protein damage in adenoviruses treated with LP and MP UV. Results show that MP UV is more effective at damaging viral proteins at high UV doses, though LP UV caused some damage as well. To our knowledge, this study is the first to investigate protein damage in UV-treated adenovirus, and the overview presented here is expected to provide a basis for further, more detailed work.
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Tangential-flow ultrafiltration was optimized for the recovery of Escherichia coli, Enterococcus faecalis, Clostridium perfringens spores, bacteriophages MS2 and PRD1, murine norovirus, and poliovirus seeded into 100-liter surface water (SW) and drinking water (DW) samples. SW and DW collected from two drinking water treatment plants were then evaluated for human enteric viruses.
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Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.
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Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1(60) (3.36-MDa) and VP1(80) (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1(180)). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1(180) particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1(60) and VP1(80) species.
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Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41 (HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and E11), and murine norovirus (MNV) are compared in this study. Experiments were performed with 0.2 mg of free chlorine or 1 mg of monochloramine/liter at pH 7 and 8 in buffered reagent-grade water at 5 degrees C. CT values (disinfectant concentration x time) for 2- to 4-log(10) (99 to 99.99%) reductions in virus titers were calculated by using the efficiency factor Hom model. The enteroviruses required the longest times for chlorine inactivation and MNV the least time. CVB5 required the longest exposure time, with CT values of 7.4 and 10 mg x min/liter (pH 7 and 8) for 4-log(10) inactivation. Monochloramine disinfection was most effective for E1 (CT values ranged from 8 to 18 mg x min/liter for 2- and 3-log(10) reductions, respectively). E11 and HAdV2 were the least susceptible to monochloramine disinfection (CT values of 1,300 and 1,600 mg-min/liter for 3-log(10) reductions, respectively). Monochloramine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disinfection. These data will be useful in drinking water risk assessment studies and disinfection system planning.
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Human noroviruses (NoVs) of genogroup II, genotype 4 (GII.4) are the most common strains detected in outbreaks of acute gastroenteritis worldwide. To gain insight into the epidemiology and genetic variation of GII.4 strains, we analyzed 773 NoV outbreaks reported to the CDC from 1994 to 2006. Of these NoV outbreaks, 629 (81.4%) were caused by GII viruses and 342 (44.2%) were caused by GII.4 strains. The proportion of GII.4 outbreaks increased from 5% in 1994 to 85% in 2006, but distinct annual differences were noted, including sharp increases in 1996, 2003, and 2006 each associated with newly emerging GII.4 strains. Sequence analysis of the full-length VP1 gene of GII.4 strains identified in this study and from GenBank segregated these viruses into at least 9 distinct subclusters which had 1.3 to 3.2% amino acid variation between strains in different subclusters. We propose that GII.4 subclusters be defined as having >5% sequence variation between strains. Our data confirm other studies on the rapid emergence and displacement of highly virulent GII.4 strains.
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Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.
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Noroviruses (NoVs) are the most common cause of viral gastroenteritis. Their high incidence and importance in health care facilities result in a great impact on public health. Studies from around the world describing increasing prevalence have been difficult to compare because of differing nomenclatures for variants of the dominant genotype, GII.4. We studied the global patterns of GII.4 epidemiology in relation to its genetic diversity. Data from NoV outbreaks with dates of onset from January 2001 through March 2007 were collected from 15 institutions on 5 continents. Partial genome sequences (n=775) were collected, allowing phylogenetic comparison of data from different countries. The 15 institutions reported 3098 GII.4 outbreaks, 62% of all reported NoV outbreaks. Eight GII.4 variants were identified. Four had a global distribution--the 1996, 2002, 2004, and 2006b variants. The 2003Asia and 2006a variants caused epidemics, but they were geographically limited. Finally, the 2001 Japan and 2001 Henry variants were found across the world but at low frequencies. NoV epidemics resulted from the global spread of GII.4 strains that evolved under the influence of population immunity. Lineages show notable (and currently unexplained) differences in geographic prevalence. Establishing a global NoV network by which data on strains with the potential to cause pandemics can be rapidly exchanged may lead to improved prevention and intervention strategies.
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