Article

Degradation of the Poliovirus 1 genome by chlorine dioxide

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Abstract

This study was undertaken to gain an understanding of the factors that influence viral RNA degradation in the presence of chlorine dioxide (ClO(2)), which will be very useful in helping to define the significance of the presence of the viral genome in disinfected water. We focused our investigation on the influence of ClO(2) on extracted RNA on the one hand, and on the infectious virus on the other. Our first results show that RNA degradation, like viral inactivation, is dose dependent. The influence of the spatial organization of the targeted genomic sequence, as well as that of its size and location (and/or sequence) on degradation of the Poliovirus 1 genome by ClO(2), was studied using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that the preferential sites of action of ClO(2) appear to be located in the untranslated regions, 5'- and 3'-UTR, a phenomenon influenced by both the presence of secondary structures and the genomic sequence in these regions. Our results also reveal a rapid decrease of infectious particles quantified by the cell culture for the applied dose. Comparison between cell culture and real-time PCR for viral detection reveals disagreement following disinfection treatment, even for the largest targeted fragment (a 6,989-base fragment representing the quasi-whole viral genome). The detection of genome fragments is insufficient to confirm the presence of the infectious virus, as each targeted fragment shows a different sensitivity. Hence, the smallest targeted fragment (76 bp) persisted throughout the analysis period, while the longest targeted fragment (6,989 bp) disappeared very rapidly. Highly sensitive regions (i.e. 5'- and 3'-UTR) should be targeted to avoid an overestimation of the risk of viral infection using molecular biology methods in water following disinfection. Further studies in this area are needed. To date, it has not been possible to routinely apply virological controls to drinking water because of the time-consuming nature of the gold standard technique (cell culture) and its inability to detect all serotypes (e.g. Norovirus). Molecular techniques (e.g. real-time RT-PCR) constitute a solution to the rapid and specific detection of all the serotypes. However, ignorance of the mechanisms of viral degradation prevents the validation of PCR for the measurement of the risk of infection to humans following disinfection treatment.

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... Nevertheless, the mechanism of microbial inactivation by ClO2 has not been clearly clarified as lethal incidences are usually associated with complicated processes. Some authors have attempted to elucidate the biocidal mechanisms of ClO2 and have concluded that ClO2 could cause series of damage, such as content leakage (Zhang et al., 2007;Wei et al., 2008), protein and nucleic acid denaturation Li et al., 2004;Cho et al., 2010;Simonet and Gantzer, 2006), and morphological alteration (Chen et al., 2002;Wang et al., 2010). However, there is still a lack of understanding on the major target site of ClO2 interaction with microorganisms. ...
... Nevertheless, the mechanism of microbial inactivation by ClO2 has not been clearly clarified as lethal incidences are usually associated with complicated processes. Some authors have attempted to elucidate the biocidal mechanisms of ClO2 and have concluded that ClO2 could cause series of damage, such as content leakage (Zhang et al., 2007;Wei et al., 2008), protein and nucleic acid denaturation Li et al., 2004;Cho et al., 2010;Simonet and Gantzer, 2006), and morphological alteration (Chen et al., 2002;Wang et al., 2010). However, there is still a lack of understanding on the major target site of ClO2 interaction with microorganisms. ...
... Afterwards, although ClO2 concentration and treatment time continued to increase, no significant increase in metal ion leakage was detected. Similarly, Wang et al. (2010) also found that the content of K + , Ca 2+ , and Mg 2+ in Nosema bombycis spore was lost immediately after the 50 mg/L ClO2 treatment. Moreover, they also indicated that a large amount of protein and DNA of the spores leaked out in a short time after the 50 mg/L ClO2 treatment. ...
Article
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The mechanism of Fusarium tricinctum (Corda) Sacc. spore inactivation by chlorine dioxide (ClO2) was investigated. During F. tricinctum spore inactivation by ClO2, protein, DNA, and metal ion leakage, enzyme activity, and cell ultrastructure were examined. Protein and DNA leakages were not detected, while there were metal ion leakages of K+, Ca2+, and Mg2+, which were well-correlated with the inactivation rate. The enzyme activities of glucose-6-phosphate dehydrogenase, citrate synthase, and phosphofructokinase were inhibited and were also well-correlated with the inactivation rate. Electron micrographs showed the ultrastructural modifications of spores and demonstrated that spores were heavily distorted and collapsed from their regular structure. Spore surface damage and disruption in inner components was also severe. The metal ion leakage, the inhibition of enzyme activities, and the damage of spore structure were significant in F. tricinctum spore inactivation by ClO2.
... La RT-PCR sur fragments courts n'a par contre pas mis en évidence de diminution de la quantité de génome viral. Simonet et Gantzer (2006a) ont étudié l'action du dioxyde de chlore à une concentration initiale de 5 mg/L sur de l'ARN extrait de poliovirus infectieux. La RT-PCR a été utilisée pour mesurer la dégradation de fragments d'ARN de différentes tailles et sur différentes régions du génome. ...
... Pour le virus de l'hépatite A, cette région 5' non codante a été décrite comme la région la plus facilement dégradée après une exposition au chlore et au dioxyde de chlore (Bhattacharya et al., 2004 ;Li et al., 2002). Il en est de même pour le poliovirus, la dégradation de son génome par le dioxyde de chlore se fait préférentiellement sur les régions 5' et 3' non codantes (Simonet et Gantzer, 2006a). ...
Thesis
Les virus entériques abondent dans le milieu hydrique et sont responsables de gastro-entérites aigües,d’hépatites, mais aussi d’autres pathologies comme la poliomyélite. La contamination de l’homme pardes virus entériques pathogènes peut notamment être due à la consommation d’eau ou d’alimentscontaminés. A l’heure actuelle, la détermination de la qualité microbiologique des eaux destinées à laconsommation est uniquement basée sur des indicateurs bactériens qui ne permettent pas uneévaluation satisfaisante du danger viral.Si la culture cellulaire permet de connaitre le statut infectieux des virus détectés, elle n’est pasapplicable à tous les virus entériques. Ainsi, les norovirus qui sont des virus entériques fortementimpliqués dans les cas de gastro-entérites ne sont pas cultivables. Les techniques de biologiemoléculaire comme la RT-PCR permettent quant à elles une détection rapide et spécifique du génomeviral. Cependant plusieurs études ont montré que le génome viral ne peut être utilisé comme marqueurde la présence de virus infectieux dans l’environnement. Devant ce constat, il apparait nécessaire dedévelopper de nouvelles méthodes de détection. Si les méthodes couplant la culture cellulaire à unedétection par biologie moléculaire fournissent des résultats rapides et permettent de conclure surl’infectiosité du virus, elles ne résolvent pas le problème lié à la nécessité de disposer d’une lignéecellulaire permissive du virus recherché. D’autres études se sont intéressées à l’intégrité de la capsidevirale ou du génome de façon indépendante. Cependant, le fait de ne pas prendre en compte la totalitéde la particule virale ne permet pas, avec les méthodes actuelles, d’évaluer son infectiosité. Pour ledéveloppement de nouvelles méthodes de détection des particules virales infectieuses, des pistespourraient être explorées concernant les modifications des interactions entre la capside et le génomelors de l’inactivation virale.
... PCR of a long target region has been proposed to investigate viral infectivity. This approach is based on the fact that an intact viral genome is necessary for the virus to remain infectious, and a long PCR region would be expected to be more representative of an intact viral genome (Allain et al., 2006;Li et al., 2002b;Simonet and Gantzer, 2006a;Wolf et al., 2009). HAV infectivity was previously assessed using this protocol after chlorine treatment; results showed that the HAV genome has different degrees of sensitivity to chlorine, depending on the position of chlorine action -5`NTR and 3`NTR were more sensitive to chlorine (Li et al., 2002a). ...
... HAV infectivity was previously assessed using this protocol after chlorine treatment; results showed that the HAV genome has different degrees of sensitivity to chlorine, depending on the position of chlorine action -5`NTR and 3`NTR were more sensitive to chlorine (Li et al., 2002a). LTR-RT-PCR targets were used to estimate the effect of chlorine dioxide on PV (Simonet and Gantzer, 2006a). The results revealed no linear relationship between the degradation of the viral genome and the size of the target gene. ...
Article
Full-text available
Waterborne diseases have significant public health and socioeconomic implications worldwide. Many viral pathogens are commonly associated with water-related diseases, namely enteric viruses. Also, novel recently discovered human-associated viruses have been shown to be a causative agent of gastroenteritis or other clinical symptoms. A wide range of analytical methods is available for virus detection in environmental water samples. Viral isolation is historically carried out via propagation on permissive cell lines; however, some enteric viruses are difficult or not able to propagate on existing cell lines. Real-time polymerase chain reaction (qPCR) screening of viral nucleic acid is routinely used to investigate virus contamination in water due to the high sensitivity and specificity. Additionally, the introduction of metagenomic approaches into environmental virology has facilitated the discovery of viruses that cannot be grown in cell culture. This review (i) highlights the applications of molecular techniques in environmental virology such as PCR and its modifications to overcome the critical issues associated with the inability to discriminate between infectious viruses and nonviable, (ii) outlines the strengths and weaknesses of Nucleic Acid Sequence Based Amplification (NASBA) and microarray, (iii) discusses the role of digital PCR as an emerging water quality monitoring assay and its advantages over qPCR, (iv) addresses the viral metagenomics in terms of detecting emerging viral pathogens and diversity in aquatic environment. Indeed, there are many challenges for selecting methods to detect classic and emerging viruses in environmental samples. While the existing techniques have revealed the importance and diversity of viruses in the water environment, further developments are necessary to enable more rapid and accurate methodologies for viral water quality monitoring and regulation.
... Incubation des particules avec de la RNase, ou un monoazide photoréactif (PMA, EMA) couplée à une quantification par (RT)qPCR (Nuanualsuwan et al., 2002;Prevost et al., 2016) Antigénicité Capture des virus par des anticorps monoclonaux et titration (Nuanualsuwan and Cliver, 2003) ALTERATION DES CONSTITUANTS Dégradation du génome (RT)qPCR de longs fragments (≈ 1000 nucléotides) (Simonet and Gantzer, 2006b) ...
... C'est un oxydant relativement sélectif qui réagit principalement avec la cystéine, la tyrosine et le tryptophane. L'inhibition de la capacité de liaison par le dioxyde de chlore du fait de l'altération des protéines de capside a été rapportée pour d'autres phages (Noss et al., 1986 (Simonet and Gantzer, 2006b). Le dioxyde de chlore cible notamment le segment comprenant les nucléotides 40 à 80 de la région 5' non codante du PV1 nécessaire à l'initiation de la réplication (Jin et al., 2012). ...
Thesis
Les virus entériques sont l’une des premières causes de gastro-entérites d’origine hydrique. Ils sont excrétés en grand nombre dans les selles et ne sont pas éliminés par les stations de traitement des eaux usées, dont les effluents sont la principale source de contamination des ressources hydriques, qui sont parfois utilisées pour la production d’eau potable. Ces virus sont particulièrement résistants aux traitements de désinfection, et leur génome est parfois détecté dans l’eau potable produite. Il a récemment été démontré que des interactions avec le microbiote intestinal au sein de l’hôte favorisent l’infectivité, pathogenèse et la stabilité de certains virus entériques (entérovirus, norovirus). Dans les milieux hydriques, ils peuvent se retrouver au voisinage d’une grande diversité d’éléments biotiques ou abiotiques, particulaires ou dissous, qui pourraient avoir des conséquences sur leur survie. Ces travaux apportent de nouveaux éléments de réflexion concernant les conséquences des interactions entre la matière organique et les virus entériques sur leur persistance dans les environnements hydriques et l’efficacité de traitements d’inactivation. L’impact de plusieurs composants microbiens sur l’inactivation de quatre sérotypes d’entérovirus a été analysé. D’importants effets protecteurs, ont été mis en évidence dans le cas des traitements d’inactivation ciblant la capside virale (chaleur, chlore). Un effet sérotype-dépendant a de plus été démontré. Dans un deuxième temps, il a été montré que la matière organique dissoute hydrophobe des eaux de surface confère au Coxsackievirus B5 une protection vis-à-vis de la chaleur en stabilisant la capside. La persistance des interactions avec la matière organique dissoute s’est révélée être liée à son hydrophobicité. Enfin, une expérience d’évolution virale sous pression de sélection thermique a révélé que les interactions des virus avec leur environnement participent à la dynamique d’évolution des espèces virales en favorisant leur stabilité génomique. Ainsi l’interaction avec le lipopolysaccharide entraine une levée de la pression de sélection exercée par la température. L’ensemble des résultats indique que la capacité des entérovirus à interagir avec certains types de matières organiques est susceptible d’augmenter leur persistance dans les milieux hydriques et au cours des traitements de désinfection, leur conférant dans certaines conditions un avantage sélectif. Les connaissances acquises sur l’inactivation virale en milieu hydrique pourraient donc surestimer les abattements viraux réels, et nécessiteraient peut-être d’être revisitées en prenant en compte l’existence de telles interactions.
... While the impact of exogenous oxidants on the infectivity of enteric viral particles, including MS2 phages, has been extensively studied [8,[11][12][13][14][15][16][24][25][26][27][28][29], little is known about the potential impact of endogenous ROS produced by host cells on the replication and infectivity of enteric viral particles. It is reasonable to suggest that endogenous oxidants can inactivate a viral particle using the same mechanism(s) as exogenous oxidants. ...
... It is reasonable to suggest that endogenous oxidants can inactivate a viral particle using the same mechanism(s) as exogenous oxidants. For instance, it is well known that exogenous oxidants can alter the viral capsid or genome, thus preventing the first step in the virus's replication cycle (recognition of the host cell receptor; release of the viral genome into the host cell; replication of the viral genome) [10][11][12]16,26,27,[30][31][32][33]. Interestingly, even when it has not been able to successfully complete at least one of the early steps of its replication cycle, a MS2 phage particle that has been inactivated by oxidation has not necessarily been completely disrupted, as it remains visible under electron microscopy [2,33]. ...
Article
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Most of the defective/non-infectious enteric phages and viruses that end up in wastewater originate in human feces. Some of the causes of this high level of inactivity at the host stage are unknown. There is a significant gap between how enteric phages are environmentally transmitted and how we might design molecular tools that would only detect infectious ones. Thus, there is a need to explain the low proportion of infectious viral particles once replicated. By analyzing lysis plaque content, we were able to confirm that, under aerobic conditions, Escherichia coli produce low numbers of infectious MS2 phages (I) than the total number of phages indicated by the genome copies (G) with an I/G ratio of around 2%. Anaerobic conditions of replication and ROS inhibition increase the I/G ratio to 8 and 25%, respectively. These data cannot only be explained by variations in the total numbers of MS2 phages produced or in the metabolism of E. coli. We therefore suggest that oxidative damage impacts the molecular replication and assembly of MS2 phages.
... The latter two were at first neglected because since RNA was considered as not stable; the detected genomes were assumed to be linked to infectious viruses protected by a capsid. Today, such an assumption is no longer acceptable because it has been largely demonstrated that the persistence of the genome is much greater than that of the corresponding infectious virus in most cases (Gassilloud et al. 2003;Ogorzaly et al. 2010;Prevost et al. 2016;Seitz et al. 2011;Simonet and Gantzer 2006). Gaining knowledge about the characteristics of non-infectious viral particles, or more broadly about the molecular mechanisms of viral inactivation, has become a major objective for food and environmental virologists. ...
Article
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The differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60 °C. When exposed to a temperature higher than their critical temperature (72 °C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60 °C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection.
... Olivieri et al. (1985) demonstrated that the genomes of inactivated poliovirus were still infectious, thus suggesting that the genome was not the main target of ClO 2 . In contrast, Simonet and Gantzer (2006), who worked with high ClO 2 exposures (5 mg/L during 120 min), reported that viral RNA did degrade, but did not fully account for inactivation. Genome damage, specifically damage to the 5 ′ non-coding region, was found to be the main target for the treatment of enterovirus 71 and Hepatitis A virus at ClO 2 exposures of 13.5 mg/L * min or higher (Li et al., 2004;Jin et al., 2013). ...
Article
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The emergence of waterborne viruses with resistance to disinfection has been demonstrated in the laboratory and in the environment. Yet, the implications of such resistance for virus control remain obscure. In this study we investigate if viruses with resistance to a given disinfection method exhibit cross-resistance to other disinfectants. Chlorine dioxide (ClO2)- or UV-resistant populations of echovirus 11 were exposed to five inactivating treatments (free chlorine, ClO2, UV radiation, sunlight, and heat), and the extent of cross-resistance was determined. The ClO2-resistant population exhibited cross-resistance to free chlorine, but to none of the other inactivating treatments tested. We furthermore demonstrated that ClO2 and free chlorine act by a similar mechanism, in that they mainly inhibit the binding of echovirus 11 to its host cell. As such, viruses with host binding mechanisms that can withstand ClO2 treatment were also better able to withstand oxidation by free chlorine. Conversely, the UV-resistant population was not significantly cross-resistant to any other disinfection treatment. Overall, our results indicate that viruses with resistance to multiple disinfectants exist, but that they can be controlled by inactivating methods that operate by a distinctly different mechanism. We therefore suggest to utilize two disinfection barriers that act by different mechanisms in order to control disinfection-resistant viruses.
... 40 Similarly, Simonet and Gantzer observed a discrepancy between poliovirus infectivity loss obtained by cell culture and genome decay measured by real-time PCR, suggesting an important role of protein damage in inactivation. 42 In contrast, Alvarez and O'Brien also observed reaction of ClO 2 with the capsid of poliovirus, but reported that the critical target was viral RNA. 44 Given the range of viral components and functions targeted by ClO 2 , ClO 2 -resistance in echovirus may thus result from modification to any of the major viral functions (host binding, host entry or genome replication). ...
Article
Waterborne viruses can exhibit resistance to common water disinfectants, yet the mechanisms that allow them to tolerate disinfection are poorly understood. Here, we generated echovirus 11 (E11) with resistance to chlorine dioxide (ClO2) by experimental evolution, and we assessed the associated genotypic and phenotypic traits. ClO2 resistance emerged after E11 populations were repeatedly reduced (either by ClO2-exposure or by dilution) and then regrown in cell culture. The resistance was linked to an improved capacity of E11 to bind to its host cells, which was further attributed to two potential causes: first, the resistant E11 populations possessed mutations that caused amino acid substitutions from ClO2-labile to ClO2-stable residues in the viral proteins, which likely increased the chemical stability of the capsid toward ClO2. Second, resistant E11 mutants exhibited the capacity to utilize alternative cell receptors for host binding. Interestingly, the emergence of ClO2 resistance resulted in an enhanced replicative fitness compared to the less resistant starting population. Overall this study contributes to a better understanding of the mechanism underlying disinfection resistance in waterborne viruses, and processes that drive resistance development.
... In comparison, chlorine dioxide (ClO2) is a water-soluble and yellow gas with a strong oxidizing activity (5,6). Earlier studies have observed that ClO2 has a potent antimicrobial activity against bacteria, fungi, protozoa and viruses (7)(8)(9)(10)(11). This chemical agent has been also utilized for disinfection of supplied water in European countries (maximum 0.5 ppm) and the United States (maximum 0.8 ppm) because of its low production of trihalomethane bodies (12). ...
Article
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In this study, we evaluated the antibacterial activity of chlorine dioxide (ClO2) compared with sodium hypochlorite (NaClO) on various multidrug-resistant strains in the presence of bovine serum albumin and sheep erythrocytes to mimic the frequent blood contamination in clinical environment. The 3 most important species causing nosocomial infections, i.e., methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MDRP) and multidrug-resistant Acinetobacter baumannii (MDRA) were evaluated, with 3 representative strains from each. At a 10 ppm-concentration, ClO2 drastically reduced the number of all MDRP and MDRA, and 2 out of 3 MRSA strains, but NaClO was unable to cause any remarkable attenuation for any of the 9 strains tested in 60 seconds. Increased concentration of 100 ppm enabled ClO2 to completely kill MRSA strains, whereas NaClO failed to significantly lower the number of 2 MRSA and 1 MDRA strains. A time-course experiment demonstrated that, within 15 seconds, 100 ppm of ClO2 could kill completely all tested strains but NaClO at this concentration failed to do so. Together, these data suggest that ClO2 is more effective than NaClO against MRSA, MDRP and MDRA, and 100 ppm could be a practical concentration of ClO2 against these multidrug-resistant strains, which may cause fatal nosocomial infections.
... Cromeans et al. systematically compared the performance of chlorine on the inactivation of EV2, EV40, EV41, CVB3, CVB5, ekovirus 1, ekovirus 11 and murine NoV, and found CVB5 to show the strongest resistance to chlorine, whereas murine NoV exhibited the least resistance [162]. Similarly, the resistance of typical viruses to chlorine dioxide declined as follows: HRoV > coxsackie virus > echovirus > PV > f2 phages > monkey RoV [15,[163][164][165]. Compared to chlorine dioxide, chlorine exhibited a much higher inactivation efficiency for SARS-CoV and SARS-CoV-2, which could be proven by the observation of complete inactivation of SARS-CoV by 20 mg⋅L − 1 chlorine after a 1 min reaction [58,166]. ...
Article
The ongoing coronavirus pandemic (COVID-19) throughout the world has severely threatened the global economy and public health. Due to receiving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a wide variety of sources (e.g., households, hospitals, slaughterhouses), urban sewage treatment systems are regarded as an important path for the transmission of waterborne viruses. This review presents a quantitative profile of the concentration distribution of typical viruses within wastewater collection systems and evaluates the influence of different characteristics of sewer systems on virus species and concentration. Then, the efficiencies and mechanisms of virus removal in the units of wastewater treatment plants (WWTPs) are summarized and compared, among which the inactivation efficiencies of typical viruses by typical disinfection approaches under varied operational conditions are elucidated. Subsequently, the occurrence and removal of viruses in treated effluent reuse and desalination, as well as that in sewage sludge treatment, are discussed. Potential dissemination of viruses is emphasized by occurrence via aerosolization from toilets, the collection system and WWTP aeration, which might have a vital role in the transmission and spread of viruses. Finally, the frequency and concentration of viruses in reclaimed water, the probability of infection are also reviewed for discussing the potential health risks.
... In addition, the specific target sequence for amplification is also important, because the stability of nucleic acids is dependent on the sequence, namely, the stability is increased for sequences with a high G(C) content due to stronger hydrogen bonds and the formation of stable quadruplex structures (Chen and others 2007;Joachimi and others 2009). Sequence stability can be exploited in specific applications, such as detection of the most unstable genomic region of the hepatitis A virus and the poliovirus, namely the 5 NTR, correlated well with that of infectious viruses after inactivation with chlorine dioxide (Li and others 2002;Simonet and Gantzer 2006). Under certain conditions, the food matrix may exert protective effects against degradation, for example, during boiling of soymilk the DNA remained stable (Kharazmi and others 2003a). ...
Article
Full-text available
Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the associated public health risk. Discrepancies between results obtained by molecular and conventional culture methods stem from the difference in target, namely nucleic acids instead of actively growing microorganisms. Nucleic acids constitute 5% to 15% of the dry weight of all living cells and are relatively stable, even after cell death, so they may be present in a food matrix after the foodborne microorganisms have been inactivated. Therefore, interpretation of the public health significance of positive results generated by nucleic acid detection methods warrants some additional consideration. This review discusses the stability of nucleic acids in general and highlights the persistence of microbial nucleic acids after diverse food-processing techniques based on data from the scientific literature. Considerable amounts of DNA and RNA (intact or fragmented) persist after inactivation of bacteria and viruses by most of the commonly applied treatments in the food industry. An overview of the existing adaptations for molecular assays to cope with these problems is provided, including large fragment amplification, flotation, (enzymatic) pretreatment, and various binding assays. Finally, the negligible risks of ingesting free microbial nucleic acids are discussed and this review ends with the future perspectives of molecular methods such as next-generation sequencing in diagnostic and source attribution food microbiology.
... The immunofluorescence tests and the comparison of TCID 50 data, for pellets and supernatants of co-cultures with both adsorbed and un-adsorbed virus demonstrated that the disinfectant killed only the viruses in suspension, while viruses internalized in amoebae (in pellet of co-cultures with adsorbed virus) were able to survive. With respect to our results using qPCR, the positivity of all samples tested may be explained by the detection of viral DNA belonging to non-infectious viruses (inactivated by disinfection), as previously described (Bertrand et al., 2012;Gassilloud et al., 2003;Simonet and Gantzer, 2006). However, comparing quantitative data between internalized and suspended virus a statistical significance difference was observed. ...
Article
Human adenoviruses are responsible for a wide range of clinical infections and are present in aquatic environments, including river, seawater, drinking-water and sewage. Free-living amoebae (Acanthamoeba) in the same environments may internalize them and other microorganisms can act as a reservoir for the internalized viruses. In this study, we studied the interaction between Acanthamoeba polyphaga and Human Adenovirus type 5 (HAdV 5) to determine whether the amoeba played a role in protecting the internalized viruses from chemical disinfection. The efficacy of sodium hypochlorite disinfection against A. polyphaga and HAdV5 either singly or in combination was assessed at three different concentrations. Individually, the amoeba were more resistant to chemical disinfection than HAdV5 and remained alive after exposure to 5 mg/l of sodium hypochlorite. In contrast, HAdV5 lost infectivity following exposure to 2.5 mg/l of sodium hypochlorite. When the amoeba and HAdV5 were co-cultured, infectious virus was found in the cytoplasm of the amoeba at 5 mg/l disinfectant concentration. These findings suggest that the A. polyphaga is providing protection for the HAdV5.
... Genome alterations can also be induced. Some authors have suggested for chlorine dioxide that certain areas of the genome are more sensitive than others and are degraded differently (Simonet and Gantzer 2006;Jin et al. 2012). These mechanisms could partially explain the underestimation induced by RT-qPCR approaches targeting short genome sequences compared to infectivity tests on bacteria or cell cultures for determining the inactivation of bacteriophages or viruses after exposure to disinfectants Park et al. 2014). ...
Article
The phagicidal activity of peroxy products against the virulent bacteriophage P001 infecting lactic acid bacteria (LAB) and bacteriophage MS2 used as a surrogate of enteric viruses was evaluated and compared to sodium hypochlorite using the EN 13610 European suspension test and a surface test developed in our laboratories. Infectivity tests were adapted and/or developed to determine the activity of disinfectants against reference P001 phage of Lactoccocus lactis and F-specific RNA phage MS2 of Escherichia coli in conditions simulating practical use. Similar concentrations of sodium hypochlorite were phagicidal against both bacteriophages, either at 0.05 to 0.125% of active chlorine using the suspension test or at 0.12 to 0.5% using the surface test. For Potassium monopersulphate (MPS), phagicidal concentrations varied from 0.006 to 0.012% whatever the type of test and phages. However, for peracetic acid products (PAP) used in suspension, concentrations 55 times higher were necessary against MS2 (0.271%) than against P001 (0.005%). With the surface test, 0.089 to 0.178% concentrations of PAP were effective against MS2, but these concentrations were 16 to 32 times greater than needed against P001. Sodium hypochlorite and MPS had similar phagicidal activities against P001 and MS2, but PAP did not. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
... ClO 2 is a fast-acting biocide, which is effective against a broad spectrum of micro-organisms, such as bacteria, fungi, spores, mold and viruses [18]. Previous study developed a polymer micro-encapsulating liquid ClO 2 coating that could provide long-term surface disinfection through the sustained release of gaseous ClO 2 from encapsulated liquid ClO 2 [19]. ...
Article
Objective: This study aims to explore the effects of encapsulation of chlorine dioxide in a hydrophilic biodegradable polymer gelatin to reduce its release rate. Methods: An emulsification-coacervation method was adopted. The characterizations of chlorine dioxide-gelatin microspheres were described. Using UV-vis spectrophotometer the λmax of chlorine dioxide was observed at 358 nm. The particle size and distribution of chlorine oxide-gelatin microspheres was measured by a dynamic light scattering (DLS) method, the diameter was (1400~1900) nm. The entrapment of chlorine dioxide-gelatin microspheres was confirmed by IR. The surface morphology, size, and shape of chlorine dioxide-gelatin microspheres were analyzed using Scanning electron microscope (SEM). Results: It showed that the encapsulated microspheres size was around 2000 nm with uniform distribution. The percentage entrapment of chlorine dioxide in the encapsulated samples was about 80~85%. A slow release study of chlorine dioxide from the encapsulated biopolymer (gelatin) in air was also carried out, which showed continuous release up to ten days. Conclusions: It can be concluded that it is possible to make a slow release formulation of ClO2 by entrapped in a hydrophilic biodegradable polymer gelatin. ClO2-gelatin microspheres can stable release low concentration ClO2 gas over an extended period.
... Hauchman and others (61) found that naked viral RNA was more susceptible to degradation than RNA extracted from treated virus particles, suggesting that genomic RNA inside intact viral particles was partially protected from ClO 2 treatment. Similarly, Simonet and Gantzer (62) found that poliovirus genomic RNA was still detectable, even though the virus was completely inactivated. Hence, this evidence suggests that the primary mechanism of MNV-1 inactivation by ClO 2 is the degradation of the viral capsid protein and the disruption of the viral capsid structure, which lead to the leakage of genomic RNA and the subsequent degradation of RNA by ClO 2 . ...
Article
Acute gastroenteritis caused by human norovirus is a significant public health issue. Fresh produce and seafood are examples of high-risk foods associated with norovirus outbreaks. Food contact surfaces also have the potential to harbor noroviruses if exposed to fecal contamination, aerosolized vomitus, or infected food handlers. Currently, there is no effective measure to decontaminate norovirus on food contact surfaces. Chlorine dioxide (ClO2) gas is a strong oxidizer and is used as a decontaminating agent in food processing plants. The objective of this study was to determine the kinetics and mechanism of ClO2 gas inactivation of a norovirus surrogate, murine norovirus 1 (MNV-1), on stainless steel (SS) coupons. MNV-1 was inoculated on SS coupons at the concentration of 107 PFU/coupon. The samples were treated with ClO2 gas at 1, 1.5, 2, 2.5, and 4 mg/liter for up to 5 min at 25°C and a relative humidity of 85%, and virus survival was determined by plaque assay. Treatment of the SS coupons with ClO2 gas at 2 mg/liter for 5 min and 2.5 mg/liter for 2 min resulted in at least a 3-log reduction in MNV-1, while no infectious virus was recovered at a concentration of 4 mg/liter even within 1 min of treatment. Furthermore, it was found that the mechanism of ClO2 gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO2 gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces.
... Scanning electron microscopy (SEM, Hitachi 4300-S) was used for a morphology study of mg/L. The results of simulated air disinfection test were shown inTable 2. Discussions ClO 2 is a fast-acting biocide, which is effective against a broad spectrum of micro-organisms, such as bacteria, fungi, spores, mold and virus- es [18]. Previous study developed a polymer micro-encapsulating liquid ClO 2 coating that could provide long-term surface disinfection through the sustained release of gaseous ClO 2 from encapsulated liquid ClO 2 [19]. ...
... Gene specific primers will bind only to the targeted region of the mRNA and transcribe only the required sequence [ Figure 5]. [75] The RT step is not necessary for viruses whose genome is composed of DNA. ...
Article
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Viral myocarditis is now acknowledged as a leading cause of morbidity as well as mortality in cardiovascular diseases. Its treatment is highly dependent on its proper diagnosis as its clinical features overlap with or mimic many other cardiovascular conditions. Histology by endomyocardial biopsy (EMB) confirms its diagnosis but given its own limitations and complications, the noninvasive imaging methods such as echocardiogram and magnetic resonance imaging as well as the molecular techniques like polymerase chain reaction (PCR) have redefined the entire scenario. Of these, PCR can detect the viral epitopes in peripheral blood samples, heart biopsy tissues samples, or urine/stool sample. Moreover, the best use of PCR is exemplified in the EMB samples where scarcity of the sample is not a limiting factor unlike histopathological examination. Detecting the subclinical infections, identifying different strains, and detecting pathogens which are otherwise difficult to grow gives PCR an edge. As it is said “time is money,” thus rapid detection of specific nucleic acid sequences from minute samples, and the overall cost-effectiveness makes PCR a technique of choice in the diagnostic armamentarium.
... ( 2002 ) also found that the fi rst 600 bases of the HAV genome containing the 5' NTR are more sensitive to chlorine degradation than the rest of the genome. Similar conclusions were reached for poliovirus genome degradation during exposureto chlorine dioxide using a q-PCR approach (Simonet and Gantzer, 2006a ). Similarly, analyses of longer regions of the genome by RT-PCR can screen for damage in the genome that will eventually reduce its infectivity, as an intact genome is necessary for the virus In the second approach, an enzymatic treatment (ET) with proteases (mostly proteinase K) and nucleases (RNase or DNase) before PCR is used: if protein capsid integrity is affected, it is more susceptible to degradation by proteinases. ...
Chapter
Monitoring the presence of enteric viruses in food is a challenging task, and molecular-based methods have become the reference detection methodology. This chapter describes in detail the main steps of the analytical process of detection of foodborne viruses by molecular methods, paying special attention to key aspects such as the interpretation of test results, the use of controls, and the implication for public health of the results obtained by molecular methods.
... Recently, molecular assays such as RT-qPCR have been used to detect NoV from food matrixes and environmental samples; however, because RT-qPCR can detect non-infectious viruses as long as a nucleic acid template exists, it appears to significantly underestimate virus inactivation by chlorination (Lee et al., 2008;Simonet and Gantzer, 2006). These studies are consistent with our results showing underestimation of virus inactivation by RT-qPCR. ...
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.
... The latter two were at first neglected because since RNA was considered as not stable, the detected genomes were assumed to be linked to infectious viruses protected by a capsid. Today, such an assumption is no longer acceptable because it has been largely demonstrated that the persistence of the genome is much greater than that of the corresponding infectious virus in most cases Ogorzaly et al. 2010;Simonet and Gantzer 2006). Gaining knowledge about the characteristics of non-infectious viral particles, or more broadly about the molecular mechanisms of viral inactivation, has become a major objective for food and environmental virologists. ...
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
... The ISO 15216 standard (ISO, 2017) is based on a final detection of the viral genome using realtime reverse transcriptase PCR (RT-qPCR). In food virology, the use of RT-qPCR has been shown to overestimate the quantity of infectious virus or to highly underestimate the effect of the treatment on virus inactivation (Simonet and Gantzer, 2006;de Roda Husman et al., 2009;Fraisse et al., 2011). Therefore, finding an effective method for detecting infectious viral particles is currently crucial for improving the assessment of viral risk. ...
Article
Full-text available
Hepatitis A virus (HAV) is one of the most common agents causing acute liver disease worldwide. HAV has been increasingly reported as the cause of foodborne disease outbreaks. The standard method currently available for detection of the genome of HAV in vulnerable foodstuffs is by RT-qPCR (ISO 15216). Despite its usefulness in the investigation of foodborne viruses, the use of RT-qPCR in food virology has been shown to overestimate the quantity of infectious virus or to highly underestimate the effect of the treatment on virus inactivation. The gold standard methods currently used for evaluating the efficacy of inactivation treatments on the adapted strain of HAV (HM175/18f) are either the plaque assay or the end-point dilution assay (TCID50). However, both assays are labor-intensive and time-consuming. The aim of this study was to evaluate the use of the xCELLigence real-time cell analysis (RTCA) system for detecting the infectivity of the adapted strain of HAV. Kinetics of cell impedance showed that HAV induced a decrease in cell index (CI) correlated with the onset of HAV-induced cell death. In addition, the time to which the HAV-induced CI drop occurred was dependent on the viral concentration. An inverse linear relation could be established over a range of 5 log10 between the concentration of HAV and the time to reach 50% of CI decrease (TCI50), showing that the RTCA assay could be used as a titration method for HAV. In addition, the RTCA-based assay could be performed in less than 6 days instead of 12 to 14 days with the gold standard methods. Therefore, the RTCA-based titration method is a powerful and suitable tool for high-throughput screening of anti-viral treatments. Its usefulness in HAV inactivation studies will improve the assessment of viral risk in food virology, as controlling transmission of viruses through their removal from foodstuffs is also an important challenge in reducing the burden of viral foodborne illnesses.
... ClO 2 damages the 5' non-coding region in the viral genome that is necessary for formation of new virus particles within the host cells (Li et al., 2004;Jin et al., 2013). Furthermore, RNA damage, in addition to protein damage, has been attributed to the inactivation of poliovirus (Simonet and Gantzer, 2006). ...
Article
Full-text available
Maintaining microbial safety and quality of fresh fruits and vegetables are a global concern. Harmful microbes can contaminate fresh produce at any stage from farm to fork. Microbial contamination can affect the quality and shelf-life of fresh produce, and the consumption of contaminated food can cause foodborne illnesses. Additionally, there has been an increased emphasis on the freshness and appearance of fresh produce by modern consumers. Hence, disinfection methods that not only reduce microbial load but also preserve the quality of fresh produce are required. Chlorine dioxide (ClO2) has emerged as a better alternative to chlorine-based disinfectants. In this review, we discuss the efficacy of gaseous and aqueous ClO2 in inhibiting microbial growth immediately after treatment (short-term effect) versus regulating microbial growth during storage of fresh produce (long-term effect). We further elaborate upon the effects of ClO2 application on retaining or enhancing the quality of fresh produce and discuss the current understanding of the mode of action of ClO2 against microbes affecting fresh produce.
... An alternative molecular approach to assess virus infectivity is the analysis of long target viral genome regions. The rationale behind this "long template" RT-PCR strategy is that the integrity of viral genome may correlate with virus infectivity (Allain et al. 2006;Li et al. 2004;Simonet and Gantzer 2006;Wolf et al. 2009). The fact that viral inactivation does not necessarily compromise genome integrity, however (e.g., when inactivation occurs through the degradation of surface proteins), limits the applicability of this strategy. ...
Chapter
Enteric viruses are the causes of many sporadic cases and outbreaks originating from contaminated water. Valid and reproducible methods for the detection of waterborne viral pathogens are crucial in order to determine the extent of contamination, the types of pathogens involved and the correlation between viral contamination and environmental factors. Strategies have been developed to overcome the difficulties associated with virological analysis of water samples. Various assays are available for the detection of the major pathogenic viruses potentially present in urban sewage, drinking or recreational waters. Monitoring of sewerage systems provides valuable epidemiological information regarding serotypes circulating in the community.
... Therefore, this compound is commonly used as a strong disinfectant. Few studies have shown that some viruses can be successfully deactivated using ClO 2 , such as human rotavirus (Chen and Vaughn, 1990), HuNoV (Lim et al., 2010;Montazeri et al., 2017), FCV (Montazeri et al., 2017), enterovirus71 (Jin et al., 2013), poliovirus 1 (Simonet and Gantzer, 2006), and echovirus 11 (Zhong et al., 2017). Kingsley et al. (2014) reported a 2.8 log 10 reduction in HuNoV after 1 h of treatment with 350 ppm ClO 2 . ...
Article
Full-text available
A carrier (stainless steel disc as a default carrier) testing method is very needed for use in the actual food-processing fields by following the standard guideline. Here, we aimed to compare the virucidal efficacy of four commercial liquid disinfectants, including sodium hypochlorite (NaOCl), chlorine dioxide (ClO2), and peracetic acid (PAA) against hepatitis A virus (HAV) following the OECD guideline protocol based on the quantitative carrier testing method and compared carrier testing results with the suspension testing results. The OECD method specifies a test for establishing whether a chemical disinfectant or a microbicide has a virucidal activity on hard non-porous surfaces. The antiviral efficacy was evaluated by plaque assays, and disinfectants were considered effective if the virus reduction was greater than or equal to 3 log10 (99.9% decrease) for carrier or 4 log10 (99.99% decrease) for suspension tests. Results indicated that ClO2 above 500 ppm and 50% ethanol were effective in the carrier test method. In contrast, more than 200 ppm NaOCl and 50 ppm ClO2 for all exposure times and 70% ethanol with contact for more than 5 min were effective in suspension tests. Treatment with PAA (80–2500 ppm) were not effective in carrier or suspension tests. Therefore, we recommend the use of more than 500 ppm ClO2 or 50% ethanol with exposure for 10 min to disinfect surfaces that may be contaminated with HAV. Thus, these results could be effective in establishing official antiviral efficacy testing methods and basic data.
... It is well known that viral genomes are more resistant to inactivation factors in the environment or processing technologies than infectious viruses. This has led to an overestimation of the HuNoV hazard in vulnerable foodstuffs, but also an underestimation of the virucidal impact of food processing and water treatments (Espinosa et al., 2008;Ogorzaly et al., 2010;Simonet and Gantzer, 2006b). The impact of different processing technologies on the reduction of HuNoV genomes and the discrepancy between the inactivation of infectious culturable HuNoV surrogates and the reduction of the corresponding viral genomes have been well reviewed (Bosch et al., 2018;DiCaprio et al., 2013;Hirneisen et al., 2010;Knight et al., 2016;. ...
Thesis
Les norovirus (NoV) sont l’une des principales causes de gastro-entérites dans le monde. La recherche des NoV dans les aliments et l’eau est réalisée à l’aide des techniques de biologie moléculaire mais elles présentent l’inconvénient de ne pas apporter d’information sur le caractère infectieux des virus. La détection du génome d’un virus capable de reconnaître préalablement son récepteur cellulaire pourrait limiter la surestimation du danger viral en excluant les particules non infectieuses n’ayant plus de capside intègre. Plusieurs études ont montré que l’interaction spécifique de la capside avec des antigènes tissulaires de groupes sanguins (HBGA) favorisait l’infection des NoV chez l’Homme. Ces sucres complexes, retrouvés dans la salive et à la surface des cellules intestinales, sont considérés comme des facteurs d’attachement mais il n’est pas exclu qu’ils puissent jouer un rôle de (co)-récepteur cellulaire ou un rôle de protection des NoV vis-à-vis des stress imposés par le système digestif de l’Homme avant d’atteindre sa cellule hôte. Dans un premier temps, des facteurs d’inactivation que les virus sont susceptibles de rencontrer avant leur arrivée dans l’intestin de l’Homme (i.e. pH acide, enzymes protéolytiques) ont été testés pour évaluer le rôle de protection des NoV par les HBGA. Dans un second temps, des traitements d’inactivation (i.e. vieillissement naturel, chaleur, oxydants) ont été testés pour évaluer le maintien de la fixation des HBGA par les capsides de NoV GII.4, et donc de l’intégrité de la capside virale. Par des approches méthodologiques complémentaires, il a été montré que l’interaction avec les HBGA ne protégeait pas les NoV GII.4 vis-à-vis du pH acide et des enzymes protéolytiques. Ensuite, nos résultats ont mis en évidence que la fixation spécifique des NoV aux HBGA permettait de sélectionner les virus possédant encore des capsides structurées. Cependant, cette approche montre certaines limites lorsque les traitements induisent des modifications mineures au niveau de la capside, suggérant que la perte de la fixation des HBGA par les capsides ne puisse pas toujours être corrélée à la perte du caractère infectieux des NoV.
... Li et al. revealed that ClO 2 reduced the HAV infectivity through 5 -NCR damage [29]. Simonet et al. reported similar findings as that of Li et al. for Poliovirus-1 (PV-1), whereby the 5 -NCR and 3 -NCR of the PV1 genome appeared to be the most sensitive to the ClO 2 treatment [30]. ...
Article
Full-text available
A viral spread occurrence such as the SARS-CoV-2 pandemic has prompted the evaluation of different disinfectants suitable for a wide range of environmental matrices. Chlorine dioxide (ClO2) represents one of the most-used virucidal agents in different settings effective against both enveloped and nonenveloped viruses. This narrative synthesis is focused on the effectiveness of ClO2 applied in healthcare and community settings in order to eliminate respiratory transmitted, enteric, and bloodborne viruses. Influenza viruses were reduced by 99.9% by 0.5–1.0 mg/L of ClO2 in less than 5 min. Higher concentration (20 mg/L) eliminated SARS-CoV-2 from sewage. ClO2 concentrations from 0.2 to 1.0 mg/L ensured at least a 99% viral reduction of AD40, HAV, Coxsackie B5 virus, and other enteric viruses in less than 30 min. Considering bloodborne viruses, 30 mg/L of ClO2 can eliminate them in 5 min. Bloodborne viruses (HIV-1, HCV, and HBV) may be completely eliminated from medical devices and human fluids after a treatment with 30 mg/L of ClO2 for 30 min. In conclusion, ClO2 is a versatile virucidal agent suitable for different environmental matrices.
... Specifically, the inactivation by ClO 2 is caused by damage in the 5′ noncoding region within the genome, which is necessary for the formation of new virus particles within the host cell (Jin et al. 2013(Jin et al. , 2012Li et al. 2004). Moreover, it has been reported that although protein damage plays an important role in inactivation of poliovirus, inactivation is ultimately attributed to viral RNA damage (Alvarez and O'Brien 1982;Simonet and Gantzer 2006). Disinfection resistance of viruses is closely related to these two kinds of inactivation mechanisms by ClO 2 and the details are provided in Text S1. ...
Article
Full-text available
Chlorine dioxide (ClO2), an alternative disinfectant to chlorine, has been widely applied in water and wastewater disinfection. This paper aims at presenting an overview of the inactivation kinetics and mechanisms of ClO2 with viruses. The inactivation efficiencies vary greatly among different virus species. The inactivation rates for different serotypes within a family of viruses can differ by over 284%. Generally, to achieve a 4-log removal, the exposure doses, also being referred to as Ct values (mutiplying the concentration of ClO2 and contact time) vary in the range of 0.06–10 mg L−1 min. Inactivation kinetics of viruses show two phases: an initial rapid inactivation phase followed by a tailing phase. Inactivation rates of viruses increase as pH or temperature increases, but show different trends with increasing concentrations of dissolved organic matter (DOM). Both damages in viral proteins and in the 5′ noncoding region within the genome contribute to virus inactivation upon ClO2 disinfection.
... There is evidence of virucidal activity of ClO2 against echovirus [15], enterovirus [16], poliovirus [17], rotavirus [18], norovirus [19,20], calicivirus [19], and coronavirus [21]. However, there are few publications that explore antiviral effects in vitro and in vivo. ...
Preprint
Full-text available
Background The need for safe and effective antiviral treatments is pressing given the number of viral infections that are prevalent in animal and human populations, often causing devastating economic losses and mortality. Informal accounts of anecdotal use of chlorine dioxide (ClO 2 ), a well-known disinfectant and antiseptic, in COVID-19 patients has raised concern about potential toxicity, but also raises the question that ClO 2 might elicit antiviral effects, a possibility that has never been examined in vivo in any animal model. Here, we challenged the hypothesis that ClO 2 decreases the viral load and virus-induced mortality in a vertebrate model. For this, we determined viral load, virus-induced lesions and mortality in 10-day old chick embryos inoculated with 10 ⁴ mean EID 50 /mL of attenuated Massachusetts and Connecticut avian coronavirus (IBV) strains. Results The ClO 2 treatment had a marked impact on IBV infection. Namely, viral titres were 2.4-fold lower and mortality was reduced by half in infected embryos that were treated with ClO 2 . Infection led to developmental abnormalities regardless of treatment. Lesions typical of IBV infections were observed in all inoculated embryos, but severity tended to be significantly lower in ClO 2 -treated embryos. We found no gross or microscopic evidence of toxicity caused by ClO 2 at the doses used herein. Conclusions Our study shows that ClO 2 could be a safe and viable way of treating and mitigating the effects of avian coronavirus infections, and raises the possibility that similar effects could be observed in other organisms. Graphical abstract
... 11,81 Chlorine dioxide is a disinfectant widely used in veterinary medicine; however, in one report, disinfection did not degrade all genome fragments of a nonenveloped RNA virus. 64 More studies are needed to determine the efficacy of chlorine dioxide as a nucleic acid decontamination chemical. The use of UV lights contained in biological safety cabinets, when allowed by local regulations, may assist in decontamination 82 provided that the UV output is monitored during annual biological cabinet calibration and the bulbs are cleaned weekly to maintain efficacy. ...
Article
The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.
... more resistant to inactivation factors in the environment or processing technologies than infectious viruses. This has led to an overestimation of the HuNoV hazard in vulnerable foodstuffs, but also an underestimation of the virucidal impact of food processing and water treatments (Espinosa et al., 2008;Ogorzaly et al., 2010;Simonet and Gantzer, 2006b). The impact of different processing technologies on the reduction of HuNoV genomes and the discrepancy between the inactivation of infectious culturable HuNoV surrogates and the reduction of the corresponding viral genomes have been well reviewed (Bosch et al., 2018;DiCaprio et al., 2013;Hirneisen et al., 2010;Knight, 2016;. ...
Article
Human noroviruses (HuNoVs) are a main cause of acute gastroenteritis worldwide. They are frequently involved in foodborne and waterborne outbreaks. Environmental transmission of the virus depends on two main factors: the ability of viral particles to remain infectious and their adhesion capacity onto different surfaces. Until recently, adhesion of viral particles to food matrices was mainly investigated by considering non-specific interactions (e.g. electrostatic, hydrophobic) and there was only limited information about infectious HuNoVs because of the absence of a reliable in vitro HuNoV cultivation system. Many HuNoV strains have now been described as having specific binding interactions with human Histo-Blood Group Antigens (HBGAs) and non-HBGA ligands found in food and the environment. Relevant approaches to the in vitro replication of HuNoVs were also proposed recently. On the basis of the available literature data, this review discusses the opportunities to use this new knowledge to obtain a better understanding of HuNoV transmission to human populations and better evaluate the hazard posed by HuNoVs in foodstuffs and the environment.
... ClO 2 was found to oxidize the mercapto groups of enzymes to develop disulfide analogs, damaging the normal physiological function of those enzymes (Huang et al. 1997). In addition, ClO 2 may also damage protein by oxidation, causing genotoxicity (Buschini et al. 2004), degradation of viral RNA (Simonet and Gantzer 2006), disruption of important compounds in biochemical reactions (Sharma and Sohn 2012), and thereby mediating microbial death . Guntiyaa et al. (2016) reported that electrolyte leakage, the activation of the pathogen cell wall damaging enzymes chitinase (CHI) and glucanase (GLU), and the inhibition of mycelial growth and spore germination were vital events in controlling Cladosporium spp., Fusarium spp., and Lasiodiplodia spp. on harvested 'Daw' logan fruit with ClO 2 gaseous treatment. ...
Article
Full-text available
Agricultural products are perishable and easily contaminated by spoilage microorganisms or foodborne pathogens, causing decay of food and illness in consumers. Chlorine dioxide (ClO2) has many advantages as a fungicide in comparison to chlorine, NaClO, and ozone, and it can be used for sterilization and preservation due to its strong oxidizing properties. ClO2 gas is more widely used in food safety and food preservation than its aqueous solutions. However, the ClO2 gas produced on a large scale from a chemical reactor cannot be applied to food in small packages. Several recent reports have discussed the use of ClO2 self-releasing packaging. This article highlights the systematic evaluation of the performance of ClO2 alone and in combination with other treatments, such as 1-MCP, chitosan, and UV irradiation, in maintaining food safety and freshness of agricultural products. This article also focuses on the biological functions and mechanisms of ClO2-mediated preservation of plant or animal food, including damage to the cell wall and the membrane of pathogens, inhibition of ethylene biosynthesis, restoration of redox balance, and change of the compound structure of toxins, such as pesticides, insecticides, and mycotoxins.
... ClO 2 is regarded as a potent and suitable disinfectant widely used in recent years. Many studies have certified that several viruses were inactivated effectively by the chlorination of ClO 2 , including human rotavirus (HRV) (Chen and Vaughn, 1990), human norovirus (HNoV) (Lim et al., 2010;Montazeri et al., 2017), feline calicivirus (FCV) (Montazeri et al., 2017), enterovirus71 (EV71) (Jin et al., 2013), poliovirus 1 (PV1) (Simonet and Gantzer, 2006) and echovirus 11 (E11) (Zhong et al., 2017). However, the mechanism that the inactivation of PRRSV by ClO 2 has not been reported. ...
Article
Porcine reproductive and respiratory syndrome virus (PRRSV) causes a great economic loss to the swine industry globally. Current prevention and treatment measures are not effective to control the outbreak and spread of porcine reproductive and respiratory syndrome (PRRS). In other words, new antiviral strategies are urgently needed. Chlorine dioxide (ClO2) is regarded as a broad-spectrum disinfectant with strong inhibitory effects on microbes and parasites. The purpose of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of ClO2 against PRRSV infection in vitro. Here, we identified ClO2 (the purity is 99%) could inhibit the infection and replication of PRRSV in both Marc-145 cells and porcine alveolar macrophages (PAMs). ClO2 could block PRRSV binding to cells rather than internalization and release, suggesting that ClO2 blocks the first stage of the virus life cycle. We also demonstrated that the inhibition exerted by ClO2 was attributed to the degradation of PRRSV genome and proteins. Moreover, we confirmed that ClO2 could decrease the expression of inflammatory cytokines induced by PRRSV. In summary, ClO2 is an efficient agent and potently suppressed PRRSV infection in vitro.
Article
Depuis une dizaine d'années, les norovirus et le virus de l'hépatite A (VHA) sont les virus entériques les plus fréquemment incriminés dans les épidémies alimentaires. En 2013, la proportion d'épidémies causées en Europe par ces virus entériques a légèrement augmenté par rapport à l'année précédente, passant de 14 % à 18 % (EFSA, 2015). C'est ainsi que le risque viral dans les aliments ou l'eau n'est plus considéré comme un risque émergent, mais plutôt comme un risque avéré au regard des données de surveillance épidémiologiques et des alertes sanitaires diffusées par l'Institut national de veille sanitaire (InVS), le réseau européen Rapid Alert System for Food and Feed (RASFF) ou la revue Eurosurveillance. Par exemple, il est désormais bien établi que les toxi-infections liées à la consommation de coquillages sont majoritairement d'origine virale (par ex. norovirus). Entre 1996 et 2010, le nombre de foyers où les virus entériques étaient impliqués dans les toxi-infections alimentaires collectives (TIAC) à coquillages s'élevait à 251 en France, représentant près de 54 % du total de ces TIAC (Vaillant et al., 2012b). Pour les végétaux, des toxi-infections d'origine virale liées à la consommation de fruits rouges ou de salades sont aussi rapportées. Par exemple en Allemagne, une large épidémie alimentaire à norovirus a eu lieu en 2012 dans des cantines scolaires. Pas moins de 10 950 personnes contaminées, dont 38 cas d'hospitalisation, ont été enregistrées suite à la consommation de fraises congelées importées de Chine (Mäde et al., 2013). Aux États-Unis, 67,7 % des épidémies alimentaires ont été imputées aux norovirus entre 2009 et 2010 (Hall et al., 2013). Ces virus sont des virus nus, constitués d'une capside protéique de vingt à trente nanomètres et d'un génome à acide ribonucléique (ARN) simple brin de polarité positive (environ 7 500 nucléotides). Comme tous les virus entériques humains, ils pénètrent chez l'Homme par voie orale. La réplication des virus s'effectue alors soit au niveau des cellules intestinales (norovirus, VHA), soit au niveau des cellules hépatiques (VHA). La capside virale reconnaît spécifiquement un récepteur de la cellule permissive et le génome, une fois libéré dans le cytoplasme, permet à lui seul de synthétiser de nouvelles particules virales. C'est ainsi que le cycle infectieux peut être à l'origine chez l'hôte des symptômes de gastro-entérite ou d'hépatite. Les virus néo-formés sont rejetés dans l'environnement par l'intermédiaire des selles et des vomissures. Comme pour tout virus, les virus entériques n'ont aucun moyen de se répliquer à l'extérieur de l'hôte. Pour que le pouvoir pathogène s'exprime au sein d'un nouvel hôte, il est indispensable que les virus ingérés possèdent à la fois une capside et un génome viral intègres. Si tel est le cas, les virus sont considérés comme infectieux. Par analogie avec d'autres virus entériques (par ex. rotavirus, enterovirus), la dose infectieuse pour l'Homme évaluée avec un modèle in vivo devrait se situer entre un et cent virus infectieux (Afssa, 2007). Cette dose infectieuse n'a pas encore été quantifiée avec précision par culture cellulaire pour les norovirus, car les seuls systèmes in vitro potentiellement disponibles pour les cultiver n'ont été décrits que récemment (Jones et al., 2014). En utilisant une approche moléculaire par Reverse Transcription – Polymerase Chain Reaction (RT-PCR), la dose infectieuse 50 % (ID 50) a été estimée à dix-huit particules pour les norovirus humains (Teunis et al., 2008). Dans ce cas, la part de virus réellement et strictement infectieux demeurait inconnue.
Article
Depuis une dizaine d'années, les norovirus et le virus de l'hépatite A (VHA) sont les virus entériques les plus fréquemment incriminés dans les épidémies alimentaires. En 2013, la proportion d'épidémies causées en Europe par ces virus entériques a légèrement augmenté par rapport à l'année précédente, passant de 14 % à 18 % (EFSA, 2015). C'est ainsi que le risque viral dans les aliments ou l'eau n'est plus considéré comme un risque émergent, mais plutôt comme un risque avéré au regard des données de surveillance épidémiologiques et des alertes sanitaires diffusées par l'Institut national de veille sanitaire (InVS), le réseau européen Rapid Alert System for Food and Feed (RASFF) ou la revue Eurosurveillance. Par exemple, il est désormais bien établi que les toxi-infections liées à la consommation de coquillages sont majoritairement d'origine virale (par ex. norovirus). Entre 1996 et 2010, le nombre de foyers où les virus entériques étaient impliqués dans les toxi-infections alimentaires collectives (TIAC) à coquillages s'élevait à 251 en France, représentant près de 54 % du total de ces TIAC (Vaillant et al., 2012b). Pour les végétaux, des toxi-infections d'origine virale liées à la consommation de fruits rouges ou de salades sont aussi rapportées. Par exemple en Allemagne, une large épidémie alimentaire à norovirus a eu lieu en 2012 dans des cantines scolaires. Pas moins de 10 950 personnes contaminées, dont 38 cas d'hospitalisation, ont été enregistrées suite à la consommation de fraises congelées importées de Chine (Mäde et al., 2013). Aux États-Unis, 67,7 % des épidémies alimentaires ont été imputées aux norovirus entre 2009 et 2010 (Hall et al., 2013). Ces virus sont des virus nus, constitués d'une capside protéique de vingt à trente nanomètres et d'un génome à acide ribonucléique (ARN) simple brin de polarité positive (environ 7 500 nucléotides). Comme tous les virus entériques humains, ils pénètrent chez l'Homme par voie orale. La réplication des virus s'effectue alors soit au niveau des cellules intestinales (norovirus, VHA), soit au niveau des cellules hépatiques (VHA). La capside virale reconnaît spécifiquement un récepteur de la cellule permissive et le génome, une fois libéré dans le cytoplasme, permet à lui seul de synthétiser de nouvelles particules virales. C'est ainsi que le cycle infectieux peut être à l'origine chez l'hôte des symptômes de gastro-entérite ou d'hépatite. Les virus néo-formés sont rejetés dans l'environnement par l'intermédiaire des selles et des vomissures. Comme pour tout virus, les virus entériques n'ont aucun moyen de se répliquer à l'extérieur de l'hôte. Pour que le pouvoir pathogène s'exprime au sein d'un nouvel hôte, il est indispensable que les virus ingérés possèdent à la fois une capside et un génome viral intègres. Si tel est le cas, les virus sont considérés comme infectieux. Par analogie avec d'autres virus entériques (par ex. rotavirus, enterovirus), la dose infectieuse pour l'Homme évaluée avec un modèle in vivo devrait se situer entre un et cent virus infectieux (Afssa, 2007). Cette dose infectieuse n'a pas encore été quantifiée avec précision par culture cellulaire pour les norovirus, car les seuls systèmes in vitro potentiellement disponibles pour les cultiver n'ont été décrits que récemment (Jones et al., 2014). En utilisant une approche moléculaire par Reverse Transcription – Polymerase Chain Reaction (RT-PCR), la dose infectieuse 50 % (ID 50) a été estimée à dix-huit particules pour les norovirus humains (Teunis et al., 2008). Dans ce cas, la part de virus réellement et strictement infectieux demeurait inconnue.
Article
Depuis une dizaine d'années, les norovirus et le virus de l'hépatite A (VHA) sont les virus entériques les plus fréquemment incriminés dans les épidémies alimentaires. En 2013, la proportion d'épidémies causées en Europe par ces virus entériques a légèrement augmenté par rapport à l'année précédente, passant de 14 % à 18 % (EFSA, 2015). C'est ainsi que le risque viral dans les aliments ou l'eau n'est plus considéré comme un risque émergent, mais plutôt comme un risque avéré au regard des données de surveillance épidémiologiques et des alertes sanitaires diffusées par l'Institut national de veille sanitaire (InVS), le réseau européen Rapid Alert System for Food and Feed (RASFF) ou la revue Eurosurveillance. Par exemple, il est désormais bien établi que les toxi-infections liées à la consommation de coquillages sont majoritairement d'origine virale (par ex. norovirus). Entre 1996 et 2010, le nombre de foyers où les virus entériques étaient impliqués dans les toxi-infections alimentaires collectives (TIAC) à coquillages s'élevait à 251 en France, représentant près de 54 % du total de ces TIAC (Vaillant et al., 2012b). Pour les végétaux, des toxi-infections d'origine virale liées à la consommation de fruits rouges ou de salades sont aussi rapportées. Par exemple en Allemagne, une large épidémie alimentaire à norovirus a eu lieu en 2012 dans des cantines scolaires. Pas moins de 10 950 personnes contaminées, dont 38 cas d'hospitalisation, ont été enregistrées suite à la consommation de fraises congelées importées de Chine (Mäde et al., 2013). Aux États-Unis, 67,7 % des épidémies alimentaires ont été imputées aux norovirus entre 2009 et 2010 (Hall et al., 2013). Ces virus sont des virus nus, constitués d'une capside protéique de vingt à trente nanomètres et d'un génome à acide ribonucléique (ARN) simple brin de polarité positive (environ 7 500 nucléotides). Comme tous les virus entériques humains, ils pénètrent chez l'Homme par voie orale. La réplication des virus s'effectue alors soit au niveau des cellules intestinales (norovirus, VHA), soit au niveau des cellules hépatiques (VHA). La capside virale reconnaît spécifiquement un récepteur de la cellule permissive et le génome, une fois libéré dans le cytoplasme, permet à lui seul de synthétiser de nouvelles particules virales. C'est ainsi que le cycle infectieux peut être à l'origine chez l'hôte des symptômes de gastro-entérite ou d'hépatite. Les virus néo-formés sont rejetés dans l'environnement par l'intermédiaire des selles et des vomissures. Comme pour tout virus, les virus entériques n'ont aucun moyen de se répliquer à l'extérieur de l'hôte. Pour que le pouvoir pathogène s'exprime au sein d'un nouvel hôte, il est indispensable que les virus ingérés possèdent à la fois une capside et un génome viral intègres. Si tel est le cas, les virus sont considérés comme infectieux. Par analogie avec d'autres virus entériques (par ex. rotavirus, enterovirus), la dose infectieuse pour l'Homme évaluée avec un modèle in vivo devrait se situer entre un et cent virus infectieux (Afssa, 2007). Cette dose infectieuse n'a pas encore été quantifiée avec précision par culture cellulaire pour les norovirus, car les seuls systèmes in vitro potentiellement disponibles pour les cultiver n'ont été décrits que récemment (Jones et al., 2014). En utilisant une approche moléculaire par Reverse Transcription – Polymerase Chain Reaction (RT-PCR), la dose infectieuse 50 % (ID 50) a été estimée à dix-huit particules pour les norovirus humains (Teunis et al., 2008). Dans ce cas, la part de virus réellement et strictement infectieux demeurait inconnue.
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Avian influenza viruses cause a considerable threat to humans and animals. In this study, we investigated whether alkaline disinfectant solution can inactivate H5N1, H3N2, H6N1, and H9N2 subtypes of avian influenza virus. When H5N1, H3N2, H6N1, and H9N2 avian influenza viruses were treated with alkaline solution diluted with PBS (pH 7.2) prior to infection into MDCK cells, alkaline disinfectant solution (at dilutions up to 10^{-2}) completely inactivated all avian influenza subtypes tested. To confirm the inactivation of avian influenza viruses by alkaline disinfectant solution, we used an immunofluorescence assay with influenza A anti-nucleoprotein antibody and FITC-labeled secondary antibody to stain MDCK cells infected with avian H9N2 influenza viruses. No staining was observed in MDCK rells infected with H9N2 viruses that were pre-treated with a 10^{-2} dilution of alkaline disinfectant solution, while strong staining was observed in MDCK cells infected with H9N2 viruses without pre-treatment. Our results indicate that alkaline solution could help to control avian influenza viruses including the highly pathogenic H5N1 subtype.
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Norovirus (NoV) is considered the major causative agent of food poisoning outbreaks in Korea. Most such outbreaks originate from vegetables or drinking water, but bivalves are known to be an important NoV vector. Environmental contamination of NoV in ground and river water has been reported in Korea, but the NoV contamination status of shellfish-growing areas based on regular surveys is not available. In this study, we investigated the NoV contamination status of oyster-growing areas in the Jinhae Bay of Korea to evaluate the circulation of various NoV genotypes. A total of 39 NoV-positive samples detected from February, 2010 to February, 2011 were sequenced and evaluated. Based on the sequencing results, five genotypes (GI.1, GI.2, GI.5, GI.6, and GI.7) were identified in NoV GI, and seven genotypes (GII.2, GII.3, GII.4, GII.7, GII.12, GII.13, and GII.17) in NoV GII. GI.1 (52.6%; 10 of 19). Both GII.3 and GII.4 (30.0%; 6 of 20, respectively) were identified as the most prevalent GI and GII strains in oyster during the investigation period.
Chapter
Viruses are important human pathogens causing substantial mortality and morbidity as well as economic damage. The lack of vaccination strategy and viral resistance to antiviral chemotherapy reemphasize the need for microbicides to eliminate or control viral outbreak. The activity and interactions of microbicides with the viral particle has been far less studied than their effects on bacteria. In addition, differences in virus size and structure as well as the diversity of efficacy test protocols used add to the fragmentation of information and the general lack of understanding of virucides. This chapter is focusing on the use of microbicides to destroy or control the transmission of viruses. The test methodologies are reviewed together with the general virucidal efficacy of different microbicides. Finally, the mechanisms of virucidal action of a number of microbicides are described as well as virus survival to microbicidal exposure.
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In recent years, a variety of conventional and novel food sanitation technologies have been developed, among which some may adversely affect the organoleptic properties and the nutrients of foods. The increasing demand for fresh-like foods has promoted efforts for developing innovative technologies. The detrimental effects of some technologies on the sensorial and nutritional values of foods could be overcome by using the hurdle technology that has become a promising approach. The interest in using chlorine dioxide for food sanitation has increased due to its many advantages over chlorine such as its powerful antimicrobial activity and less formation of harmful disinfection by-products. However, using chlorine dioxide to achieve a complete pathogen elimination from foods is still hard. In this context, chlorine dioxide has been combined with other technologies to enhance microbial food safety. This review, therefore, aims to present the application of chlorine dioxide-based hurdle technology through sequential or simultaneous treatments to control foodborne pathogens. The antimicrobial effects of chlorine dioxide combined with thermal and non-thermal physical, chemical, and biological technologies on various foodborne pathogens in a wide range of food commodities are critically reviewed.
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The feasibility of substituting a conventional pre-treatment, consisting of dioxi-chlorination, coagulation/flocculation, settling and sand filtration, of a drinking water treatment plant (DWTP) by direct ultrafiltration (UF) has been assessed from a microbiological standpoint. Bacterial indicators, viral indicators and human viruses have been monitored in raw river, ultrafiltered and conventionally pre-treated water samples during two years. Direct UF has proven to remove bacterial indicators quite efficiently and to a greater extent than the conventional process does. Nevertheless, the removal of small viruses such as some small bacteriophages and human viruses (e.g. enteroviruses and noroviruses) is lower than the current conventional pre-treatment. Membrane integrity has been assessed during two years by means of tailored tests based on bacteriophages with different properties (MS-2, GA and PDR-1) and bacterial spores (Bacillus spores). Membrane integrity has not been compromised despite the challenging conditions faced by directly treating raw river water. Bacteriophage PDR-1 appears as a suitable microbe to test membrane integrity, as its size is slightly larger than the considered membrane pore size. However, its implementation at full scale plant is still challenging due to difficulties in obtaining enough phages for its seeding. Copyright © 2015 Elsevier Ltd. All rights reserved.
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This chapter describes viruses that are enterically transmitted and cause systemic disease. These are recognized as important food and waterborne pathogens. The chapter first summarizes the general characteristics of the viruses, then describes their typical epidemiological patterns. The chapter then discusses methods to detect and to inactivate the viruses, with an emphasis on strategies that can be implemented for food safety.
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When ready-to-eat salami was treated in a closed system with ³⁶Cl-labeled ClO2 (5.5 mg/100 g of salami), essentially all radioactivity was deposited onto the salami. Administered ³⁶ClO2 was converted to ³⁶Cl-chloride ion (>97%), trace levels of chlorate (<2%), and detectable levels of chlorite. In residue studies conducted with nonlabeled ClO2, sodium perchlorate residues (LOQ, 4 ng/g) were not formed when reactions were protected from light. Sodium chlorate residues were present in control (39.2 ± 4.8 ng/g) and chlorine dioxide treated (128 ± 31.2 ng/g) salami. If sanitation occurred under conditions of illumination, detectable levels (3.7 ± 1.5 ng/g) of perchlorate were formed along with greater quantities of sodium chlorate (183.6 ± 75.4 ng/g). Collectively, these data suggest that ClO2 is chemically reduced by salami and that slow-release formulations might be appropriate for applications involving the sanitation of ready-to-eat meat products.
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RNA carries the genetic instructions for many viruses to replicate in their host cells. The photochemical reactions that take place in RNA and affect viral infectivity in natural and engineered environments, however, remain poorly understood. We exposed RNA oligomer segments from the genome of bacteriophage MS2 to UV254, simulated sunlight, and singlet oxygen (¹O2), and analyzed the oligomer reaction kinetics with RT-qPCR and quantitative MALDI-TOF mass spectrometry (MS). Following UV254 exposure, quantitative MALDI-TOF-MS detected significantly more RNA modifications than RT-qPCR, suggesting that certain chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In contrast, MALDI-TOF-MS tracked as much ¹O2-induced RNA damage as RT-qPCR. After 5 hours of simulated sunlight exposure (5100 J/m² UVB and 1.2 × 10⁵ J/m² UVA), neither MALDI-TOF-MS nor RT-qPCR detected significant decreases in the oligomer concentrations. High-resolution ESI-Orbitrap MS analyses identified pyrimidine photohydrates as the major UV254 products, which likely contributed to the discrepancy between the MS- and RT-qPCR-based results. Reactions between RNA oligomers and ¹O2 resulted in an unidentified major product with a mass change of +6 Da. These results shed light on the photochemical reactions that take place in RNA and suggest that the analytical techniques used to detect RNA reactivity could bias the observed reaction kinetics.
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During an outbreak of Ebola virus disease (EVD), hospitals' connections to municipal wastewater systems may provide a path for patient waste bearing infectious viral particles to pass from the hospital into the wastewater treatment system, potentially posing risks to sewer and wastewater workers. To quantify these risks, we developed a Bayesian belief network model incorporating data on virus behavior and survival along with structural characteristics of hospitals and wastewater treatment systems. We applied the model to assess risks under several different scenarios of workers' exposure to wastewater for a wastewater system typical of a mid-sized U.S. city. The model calculates a median daily risk of developing EVD of approximately 6.1×10(-12) (90% confidence interval: 1.0×10(-12) to 5.4×10(-9) ; mean 1.8×10(-6) ) when no prior exposure conditions are specified. Under a worst-case scenario in which a worker stationed in the sewer adjacent to the hospital accidentally ingests several drops (0.35 mL) of wastewater, median risk is 5.8×10(-4) (90% CI: 8.8×10(-7) to 9.5×10(-2) ; mean 3.2×10(-2) ) . Disinfection of patient waste with peracetic acid for 15 minutes prior to flushing decreases the estimated median risk to 3.8×10(-7) (90% CI: 4.1×10(-9) to 8.6×10(-5) ; mean 2.9×10(-5) ). The results suggest that requiring hospitals to disinfect EVD patient waste prior to flushing may be advisable. The modeling framework can provide insight into managing patient waste during future outbreaks of highly virulent infectious pathogens.
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To obtain detailed information on the diversity of infectious norovirus in oysters (Crossostrea gigas), oysters obtained from fish producers at six different sites (sites A, B, C, D, E, and F) in Japan were analyzed once a month during the period spanning October 2015-February 2016. To avoid false-positive polymerase chain reaction (PCR) results derived from noninfectious virus particles, samples were pretreated with RNase before reverse transcription-PCR (RT-PCR). RT-PCR products were subjected to next-generation sequencing to identify norovirus genotypes in oysters. As a result, all GI genotypes were detected in the investigational period. The detection rate and proportion of norovirus GI genotypes differed depending on the sampling site and month. GII.3, GII.4, GII.13, GII.16, and GII.17 were detected in this study. Both the detection rate and proportion of norovirus GII genotypes differed depending on the sampling site and month. In total, the detection rate and proportion of GII.3 were highest from October to December among all detected genotypes. In January, the detection rates of GII.4 and GII.17 reached the same level as that of GII.3. The proportion of GII.17 was relatively lower from October to December, whereas it was the highest in January. To our knowledge, this is the first investigation on noroviruses in oysters in Japan, based on a method that can distinguish their infectivity.
Chapter
Enteric viruses excreted in high concentrations in the feces of infected individuals are the main target of investigations in environmental virology. The discharge of those agents in different environmental matrices represents a risk of infection for the population through several routes of infection, particularly in countries where sanitation is deficient. Studies in environmental virology were initiated in the beginning of the year 1940 and still present many challenges, related not only to methodologies and establishment of parameters but also to the use of such studies as a tool for public policies. This chapter is a short review of the progress made in the area featuring the contributions of work conducted in Latin America to solidify the progress of knowledge in the region.
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Avian influenza remains a threat to human wellbeing. Hypochlorite derivatives are commonly used as disinfectants to prevent the spread of the disease. The World Health Organization has listed chlorine dioxide (ClO2) as an A1-level, safe, and efficient disinfectant. In this study, we tested the efficacy of ClO2, in aqueous solution and gas forms, against avian influenza A (H7N9) virus. The virus suspension was mixed with ClO2 aqueous solutions of various concentrations and for various time intervals. Aliquots of the mixture were then serially diluted, and the 50% tissue culture infective dose (TCID50) was measured with a hemagglutination test on MDCK cells. ClO2 gas produced from generators was introduced in a chamber containing the virus suspension in a Petri dish. The infective activity of the surviving virus was measured by the hemagglutination test. An aqueous solution of ClO2 at 126 µg/mL for 15 seconds was effective given that no surviving virus was detected with the hemagglutination test. ClO2 gas at >5 µL/mL sustained for 1 h inactivated the virus effectively, while at 2.5 µL/mL for 1 h, it only partially inactivated the virus. ClO2 as gas or aqueous solution at a certain concentration is effective in inactivating the H7N9 virus, and can be applied for the decontamination and disinfection of environments.
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Inactivation of poliovirus type 1 by 1 N HCl, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCl, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 10(2) PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCl, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.
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The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5' nontranslated regions (5'NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5'NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5'NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.
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During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.
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Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log10 within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.
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Appropriate interpretation of a positive reverse transcription-PCR is an important issue for virus-related health hazard assessment because viral genomes and infectious viruses exhibit different behavior patterns in water. In this context, using Poliovirus 1 and Feline calicivirus f9 as examples of enteric viruses, first we demonstrated that the stability of infectious viruses is greatly affected by the temperature of mineral water (10, 20, and 35°C) and that, in contrast, temperature has little effect on the corresponding genomes. Second, we demonstrated that infectious particles are degraded more rapidly than viral genomes at all temperatures studied. At 35°C, Poliovirus 1 infectivity was reduced 4 logs after only 19 days, while an equivalent reduction would have taken 75 years (according to the model applied) for the viral genome. Contradictory conclusions can also be drawn concerning the sensitivity of viral serotypes depending on whether the infectious virus or the viral genome is considered. The Feline calicivirus f9 genome is more resistant than the Poliovirus 1 genome, whereas the opposite is true for the corresponding infectious viruses. Thus, we concluded that a positive test for a viral genome in mineral water must be interpreted with utmost caution because of the lack of a correlation between the presence of viral genomes and viral infectivity. Detection of viral genomes may be necessary to identify infectious risk for the human population, but it cannot be considered sufficient.
Article
Nucleic acid (NA) amplification techniques are useful to detect viruses in water and other environmental samples because they are highly sensitive, specific and can detect fastidious enteric viruses that do not grow well or not at all in cell cultures. However, RT-PCR was found to detect inactivated viruses. In terms of risks to public health this constitutes a false positive result, as inactivated viruses are no longer infectious. When poliovirus type 1 and coliphage MS2 were studied for (a) persistence in water and sewage and (b) inactivation in water by free chlorine, chlorine dioxide and UV radiation, RT-PCR assays underestimated virus inactivation. The use of multiple RT-PCR amplification sites, larger RT-PCR genomic targets and immunocapture RT-PCR sometimes reduced, but did not eliminate, the discrepancy between loss of infectivity and loss of RT-PCR titre. Virus presence based on RT-PCR detection must be interpreted with caution when predicting human health risks.
Article
The reduction of Norwalk virus (NV) by a 2 mg/L dose of pre-formed monochloramine was determined at pH 8 and 5°C in bench-scale, batch disinfection experiments using quantitative RT-PCR for NV assays. Two other enteric viruses, poliovirus 1 (PV1) and coliphage MS2, were included for comparison and assayed by infectivity as well as RT-PCR. After 3h, reductions of PV1 and MS2 by infectivity assays were about 1 log10 but there were no reductions of these viruses by RT-PCR assays. Hence, RT-PCR underestimated virus inactivation by monochloramine. However, NV reduction by monochloramine was about 1 log10 by RT-PCR assay, suggesting that it is more susceptible to monochloramine than the other two viruses tested. Based on RT-PCR titre reduction, the CT99 value for NV was about 775 mg-min/L. If the reduction of NV infectivity by monochloramine is ever greater than the reduction of RT-PCR signals, the CT99 value would be smaller. However, the results of this study indicate that NV and the other enteric viruses tested are not rapidly and extensively reduced by disinfection with pre-formed monochloramine.
The degradation of a mixture of four 5′-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1–0.5 mg/I) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.
Article
The reduction of Norwalk virus (NV) by a 2 mg/L dose of pre-formed monochloramine was determined at pH 8 and 5°C in bench-scale, batch disinfection experiments using quantitative RT-PCR for NV assays. Two other enteric viruses, poliovirus 1 (PV1) and coliphage MS2, were included for comparison and assayed by infectivity as well as RT-PCR. After 3h, reductions of PV1 and MS2 by infectivity assays were about 1 log10 but there were no reductions of these viruses by RT-PCR assays. Hence, RT-PCR underestimated virus inactivation by monochloramine. However, NV reduction by monochloramine was about 1 logl0 by RT-PCR assay, suggesting that it is more susceptible to monochloramine than the other two viruses tested. Based on RT-PCR titre reduction, the CT99 value for NV was about 775 mg-min/L. If the reduction of NV infectivity by monochloramine is ever greater than the reduction of RT-PCR signals, the CT99 value would be smaller. However, the results of this study indicate that NV and the other enteric viruses tested are not rapidly and extensively reduced by disinfection with pre-formed monochloramine.
Article
The changes in the concentrations of infectious coxsackie B3 virus (detected by cell culture), and the viral genome (detected by RT semi-nested PCR), when the virus is kept at 25°C in sterile phosphate buffered saline (PBS) and in PBS plus clay was monitored. The viral genome survived for twice as long as the infectious virus. The survival of the genome was greatly prolonged by adding 200 mg l−1 Na–montmorillonite to the PBS, while the survival of the infectious virus was only slightly increased. The T90 of the viral genome was at least 3.5 times longer that the T90 of the infectious virus. Thus, the rate of decay of the viral genome is different from that of the infectious virus.
Article
The inactivation mechanism of tobacco mosaic virus (TMV) in a phosphate buffer (pH 6.9) by ozone was studied. We previously reported that the damage of naked TMV-RNA occurred at the guanine moiety of RNA (Shinriki et al., Biochim. biophys. Acta655, 323, 1981). In this paper, we clarified the mode of the inactivation of TMV by using tritium-labeled TMV (TMV∗) prepared by the reconstitution of tritium-labeled TMV-RNA (TMV-RNA∗) and coat protein of TMV. It was found that the amount of extracted TMV-RNA∗ from ozone-treated TMV∗ decreased with the advance of ozonization, and that there was good correlation between the loss of infectivity and the decrease of recovery of TMV-RNA∗. When TMV lost its infectivity due to ozone, tryptophan and tyrosine of the coat protein were also degraded by ozone. Polyacrylamide gel electrophoretic analysis of the substance produced during ozonization showed that the coat protein subunits were aggregated with each other and cross-linked with TMV-RNA∗. From these results, it was concluded that the inability of uncoating is the major cause of the TMV inactivation by ozone.
Article
Studies were conducted to evaluate chlorine dioxide reactivity with proteins and the role of these reactions in the inactivation of the bacterial virus f2 with chlorine dioxide. The effect of chlorine dioxide on the ability of f2 to specifically attach to its host Escherichia coli K13 was compared to the inactivation of virus during the initial seconds of contact time. At pH 7.2 and and 5°C, the virus was rapidly inactivated with 0.6 mg l−1 of chlorine dioxide. Approximately 2 log units of inactivation were observed within 30 s. The loss of protein specific attachment function nearly paralleled intact virus inactivation with 1.2 log units of attachment inhibition occurring within 30 s. The inactivation of virus and the inhibition of specific attachment both increased with increasing pH and increasing disinfectant concentration.Inactivation of f2 was hypothesized to occur as the result of chlorine dioxide reacting with discrete chemical moieties in the viral protein. Cysteine, tyrosine and tryptophan reacted with chlorine dioxide within a time frame that could affect viral inactivation. In denatured f2 capsid protein monomers, these amino acids were almost totally degraded within 2 min by chlorine dioxide. Only tyrosine reacted with chlorine dioxide following treatment of the intact virion with disinfectant. Even though the degradation of tyrosine residues occurred at a much slower rate than the rate of virus inactivation, the protein component of f2 virus appeared to be the site of the lethal lesion produced by chlorine dioxide. These tyrosine reactions with chlorine dioxide appeared to alter the virus such that specific attachment was inhibited.
Article
Nucleic acid (NA) amplification techniques are useful to detect viruses in water and other environmental samples because they are highly sensitive, specific and can detect fastidious enteric viruses that do not grow well or not at all in cell cultures. However, RT-PCR was found to detect inactivated viruses. In terms of risks to public health this constitutes a false positive result, as inactivated viruses are no longer infectious. When poliovirus type 1 and coliphage MS2 were studied for (a) persistence in water and sewage and (b) inactivation in water by free chlorine, chlorine dioxide and UV radiation, RT-PCR assays underestimated virus inactivation. The use of multiple RT-PCR amplification sites, larger RT-PCR genomic targets and immunocapture RT-PCR sometimes reduced, but did not eliminate, the discrepancy between loss of infectivity and loss of RT-PCR titre. Virus presence based on RT-PCR detection must be interpreted with caution when predicting human health risks.
Article
Nucleic acid hybridization has been used to detect viral nucleic acid in water. This type of assay, in contrast with tissue culture assays, may not distinguish between viable and non-viable viruses. We evaluated, by comparison with tissue culture infectivity assay (plaque forming method), the ability of the gene probe assay to detect viable poliovirus 1 (LSc) in well water, autoclaved well water, filter-sterilized well water and autoclaved phosphate buffered saline kept at 37 and 15°C for 75 days, and in dechlorinated tap water held at room temperature. A gradual decline in numbers of poliovirus was observed in all of the samples by cell culture assay. With the exception of autoclaved well water and phosphate buffer, a parallel decline in virus detectable by gene probe occurred in all other water samples. These results suggest that detection of poliovirus 1, by gene probe, is influenced by the presence of microorganisms or their products and to a lesser extent by temperature. This suggests that in some natural waters, the detection of poliovirus 1, by gene probe, may be expected to correlate to detection by tissue culture.
Article
Studies were conducted with the bacterial virus f2 to determine the reactivity of chlorine dioxide with viral nucleic acid, and to evaluate the role of these reactions in the inactivation of virus with chloride dioxide. The effect of chlorine dioxide on naked infectious RNA was compared to the inactivation of intact virus and to infectious f2 RNA extracted from virus which was treated with chlorine dioxide. At pH 7.2 and 5°C, greater than 4 log units of virus inactivation were observed within 2 min of contact time. Almost no inactivation of infectious RNA extracted from chlorine dioxide treated virus was observed. Treatment of naked infectious RNA with chlorine dioxide yielded less than 1 log unit of inactivation after 5 min of contact time. The rate of inactivation both f2 virus and infectious RNA by chlorine dioxide increased with increasing pH.Inactivation of f2 infectious RNA was attributed to chlorine dioxide reactions with nucleotides. The reaction of chlorine dioxide within yeast RNA was uniquely associated with guanosine monophosphate (GMP). The reaction between chlorine dioxide and GMP may account for inactivation of naked f2 RNA. However, this reaction does not explain the inactivation of intact f2 virus, as the RNA within the treated virion remains infectious despite several log units of virus inactivation.
Article
Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.
Article
MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.
Article
The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.
Article
The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.
Article
The rate of inactivation of poliovirus in water by chlorine is strongly influenced by the pH, which in turn influences the relative amounts of HOCl and OCl- that are present and acting on the virus in the region of pH 6 to 10. The distribution of HOCl and OCl- is influenced to a lesser extent by the addition of NaCl. The major part of the sharp increase in disinfection rate seen with this salt is thought to be due to its effect on the virus itself resulting in an increased chlorine sensitivity, especially at high pH.
Article
The kinetics of inactivation of six enteric viruses plus simian virus 40 and Kilham rat virus by free available chlorine was studied under carefully controlled laboratory conditions. It was found that the different virus types demonstrated a wide range of susceptibility to chlorine disinfection. The rate of inactivation was greater at pH 6 than at pH 10; however, the relative susceptibilities of the different viruses were affected differently by a change in pH, suggesting that the pH influenced both the species of chlorine present and the susceptibility of the different viruses to chlorine. The presence of potassium chloride also affected the susceptibility of viruses to chlorine.
Article
The mechanism of enteroviral inactivation by ozone was investigated with poliovirus 1 (Mahoney) as the model virus. Ozone was observed to alter two of the four polypeptide chains present in the viral protein coat of poliovirus 1. However, the alteration of the protein coat did not significantly impair virus adsorption or alter the integrity of the virus particle. Damage to the viral RNA after exposure to ozone was demonstrated by velocity sedimentation analysis. It was concluded that the damage to the viral nucleic acid is the major cause of poliovirus 1 inactivation by ozone.
Article
Chlorine concentrations below 0.8 mg/liter inactivated poliovirus without causing separation of the viral components. These results indicate that the release of RNA from the capsids is the result, not the cause, of virus inactivation by chlorine.
Article
Chlorine dioxide and iodine inactivated poliovirus more efficiently at pH 10.0 than at pH 6.0. Sedimentation analyses of viruses inactivated by chlorine dioxide and iodine at pH 10.9 showed that viral RNA separated from the capsids, resulting in the conversion of virions from 156S structures to 80S particles. The RNAs release from both chlorine dioxide- and iodine-inactivated viruses cosedimented with intact 35S viral RNA. Both chlorine dioxide and iodine reacted with the capsid proteins of poliovirus and changed the pI from pH 7.0 to pH 5.8. However, the mechanisms of inactivation of poliovirus by chlorine dioxide and iodine were found to differ. Iodine inactivated viruses by impairing their ability to adsorb to HeLa cells, whereas chlorine dioxide-inactivated viruses showed a reduced incorporation of [14C]uridine into new viral RNA. We concluded, then, that chlorine dioxide inactivated poliovirus by reacting with the viral RNA and impairing the ability of the viral genome to act as a template for RNA synthesis.
Article
An investigation was undertaken to determine the effect of chlorine on a small DNA-containing enteric virus. Parvovirus H-1 was exposed to sodium hypochlorite in a phosphate-buffered saline solution at pH 7. Then, the whole virion, the protein capsid, or the nucleic acid was subjected to analysis. The sedimentation rate of the chlorine-treated whole virus decreased from 110S to 43S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virus demonstrated the formation of higher-molecular-weight aggregates resulting from covalent cross-linking of the capsid proteins. Electron microscopic examination revealed that the DNA was extruded as a taillike structure which remained attached to the virus particle. Furthermore, the DNA was intact and still capable of in vitro replication. The adsorption of the chlorine-treated virions to host cells was inhibited, presumably due to the effect of chlorine on the particular spatial arrangement of the capsid proteins required for adsorption. Specific sites on these proteins had become highly reactive, indicating that the initial action of chlorine on parvovirus H-1 was on the viral capsid.
Article
Ozone-treatment of tRNA resulted in the degradation of guanine residues located on the loop regions, such as the anticodon and D-loop region. In addition, it became evident that the guanine residues in the consecutive sequences, such as G-G-G-G and G-G-m1G in tRNAPro, were the most susceptive to the ozone-treatment. The internucleotidic linkage of the treated tRNA was not cleaved but several fragments were obtained by a gel electrophoretic separation after heating at 60% in 1M aniline-acetate buffer (pH 4.5). Major fragments derived from tRNAPro were the 3'-half and the 5'-half molecules produced by the cleavage at the anticodon region.
Article
The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.
Article
We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.
Article
Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.
Article
Several groups of viruses may infect persons after ingestion and then are shed via stool. Of these, the norovirus (NoV) and hepatitis A virus (HAV) are currently recognised as the most important human foodborne pathogens with regard to the number of outbreaks and people affected in the Western world. NoV and HAV are highly infectious and may lead to widespread outbreaks. The clinical manifestation of NoV infection, however, is relatively mild. Asymptomatic infections are common and may contribute to the spread of the infection. Introduction of NoV in a community or population (a seeding event) may be followed by additional spread because of the highly infectious nature of NoV, resulting in a great number of secondary infections (50% of contacts). Hepatitis A is an increasing problem because of the decrease in immunity of populations in countries with high standards of hygiene. Molecular-based methods can detect viruses in shellfish but are not yet available for other foods. The applicability of the methods currently available for monitoring foods for viral contamination is unknown. No consistent correlation has been found between the presence of indicator microorganisms (i.e. bacteriophages, E. coli) and viruses. NoV and HAV are highly infectious and exhibit variable levels of resistance to heat and disinfection agents. However, they are both inactivated at 100 degrees C. No validated model virus or model system is available for studies of inactivation of NoV, although investigations could make use of structurally similar viruses (i.e. canine and feline caliciviruses). In the absence of a model virus or model system, food safety guidelines need to be based on studies that have been performed with the most resistant enteric RNA viruses (i.e. HAV, for which a model system does exist) and also with bacteriophages (for water). Most documented foodborne viral outbreaks can be traced to food that has been manually handled by an infected foodhandler, rather than to industrially processed foods. The viral contamination of food can occur anywhere in the process from farm to fork, but most foodborne viral infections can be traced back to infected persons who handle food that is not heated or otherwise treated afterwards. Therefore, emphasis should be on stringent personal hygiene during preparation. If viruses are present in food preprocessing, residual viral infectivity may be present after some industrial processes. Therefore, it is key that sufficient attention be given to good agriculture practice (GAP) and good manufacturing practice (GMP) to avoid introduction of viruses onto the raw material and into the food-manufacturing environment, and to HACCP to assure adequate management of (control over) viruses present during the manufacturing process. If viruses are present in foods after processing, they remain infectious in most circumstances and in most foods for several days or weeks, especially if kept cooled (at 4 degrees C). Therefore, emphasis should be on stringent personal hygiene during preparation. For the control of foodborne viral infections, it is necessary to: Heighten awareness about the presence and spread of these viruses by foodhandlers; Optimise and standardise methods for the detection of foodborne viruses; Develop laboratory-based surveillance to detect large, common-source outbreaks at an early stage; and Emphasise consideration of viruses in setting up food safety quality control and management systems (GHP, GMP, HACCP).
Article
The inactivation kinetics of bacteriophage f2 were studied by using ozone under controlled laboratory conditions. The phage were rapidly inactivated during the first 5 s of the reaction by 5 and 7 logs at ozone concentrations of 0.09 and 0.8 mg/liter, respectively. During the next 10 min, the phage were further inactivated at a slower rate in both treatments. The [H]uridine-labeled f2 phage and its ribonucleic acid (RNA) were examined to elucidate the mechanism of ozone inactivation, utilizing adsorption to host bacteria, sucrose density gradient analysis, and electron microscopy. The specific adsorption of the phage was reduced by ozonation in the same pattern as plaque-forming unit reduction. RNA was released from the phage particles during ozonation, although it had reduced infectivity for spheroplasts. Electron microscopic examination showed that the phage coat was broken by ozonation into many protein subunit pieces and that the specific adsorption of the phage to host pili was inversely related to the extent of phage breakage. The RNA enclosed in the phage coat was inactivated less by ozonation than were whole phage, but inactivated more than naked RNA. These findings suggest that ozone breaks the protein capsid into subunits, liberating RNA and disrupting adsorption to the host pili, and that the RNA may be secondarily sheared by a reduction with and/or without the coat protein molecules, which have been modified by ozonation.
Comparative inactivation of Norwalk virus, Poliovirus 1, and coliphages MS2 in water by low pressure U.V. radiation Chlorine dioxide reactivity with proteins
  • S Newland
  • G.-A Shin
  • M D S Sobsey
  • V P Olivieri
Newland, S., Shin, G.-A. and Sobsey, M.D. (2001) Comparative inactivation of Norwalk virus, Poliovirus 1, and coliphages MS2 in water by low pressure U.V. radiation. 1st Interna-tional Congress on Ultraviolet Technology, Washington. Noss, C.I., Hauchman, F.S. and Olivieri, V.P. (1986) Chlorine dioxide reactivity with proteins. Water Res 20, 351–356.
Waterborne pathogens kill 10M–20M people/ year
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Anon (1996) Waterborne pathogens kill 10M–20M people/ year. World Water Environ Eng 6.
Degradation of the Poliovirus 1 genome by
  • Ann Arbor
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Ann Arbor, MI: Ann Arbor Science Publisher, Inc., 1980. Degradation of the Poliovirus 1 genome by ClO 2 J. Simonet and C. Gantzer
The mechanism of Poliovirus inactivation by hypochlorous acid
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  • P C Loh
Tenno, K.M., Fujioka, R.S. and Loh, P.C. (1979) The mechanism of Poliovirus inactivation by hypochlorous acid. In Water Chlorination: Environmental Impact and Health Effects, Vol. 3 ed. Jolley, R.L., Brungs, W.A. Cummings, R.B. pp. 665-675. Ann Arbor, MI: Ann Arbor Science Publisher, Inc., 1980.
Mechanism of chloramine inactivation of Poliovirus: a concern for regula-tor
  • R S Fujioka
  • K M Tenno
  • P C Loh
Fujioka, R.S., Tenno, K.M. and Loh, P.C. (1981) Mechanism of chloramine inactivation of Poliovirus: a concern for regula-tor. In Water Chlorination: Environmental Impact and Health Effects, Vol. 4 ed. Jolley, R.L., Brungs, W.A., Cotruvo, J.A., Cummings, R.B., Mattice, J.S. and Jacobs, V.A. pp. 1067–1076.
Comparative inactivation of Norwalk virus, Poliovirus 1, and coliphages MS2 in water by low pressure U.V. radiation. 1st International Congress on Ultraviolet Technology
  • S Newland
  • G.-A Shin
  • M D Sobsey
Newland, S., Shin, G.-A. and Sobsey, M.D. (2001) Comparative inactivation of Norwalk virus, Poliovirus 1, and coliphages MS2 in water by low pressure U.V. radiation. 1st International Congress on Ultraviolet Technology, Washington.
Waterborne pathogens kill 10M-20M people/ year. World Water Environ Eng 6
  • Anon
Anon (1996) Waterborne pathogens kill 10M-20M people/ year. World Water Environ Eng 6.
Effect of particulates on disinfection of enteroviruses by chlorine dioxide
  • P V Scarpino
  • F A O Brigano
  • S Cronier
  • M L Zink
Scarpino, P.V., Brigano, F.A.O., Cronier, S. and Zink, M.L. (1979) Effect of particulates on disinfection of enteroviruses by chlorine dioxide. EPA-600/2-79-054. Washington, DC. US-EPA, 56 pp.