Somatic coliphages are conservative indicators of SARS-CoV-2 inactivation during heat and alkaline pH treatments.

High concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described in wastewater and sewage sludge. It raises the question of the security of land sludge disposal practices during a pandemic. This study aimed to compare SARS-CoV-2's resistance to the main inactivating factors in sludge treatments, pH and heat, to that of native wastewater somatic coliphages. The latest can be easily used as an indicator of treatment efficiency in the field. The effects of heat treatment and pH on the survival of SARS-CoV-2 and somatic coliphages were investigated in simple media. The T90 value (time required for a 90% reduction in the virus or a 1 × log10 decline) at 50 °C was about 4 min for infectious SARS-CoV-2, and around 133 min for infectious somatic coliphages, with no decrease in SARS-CoV-2 genome. For infectious SARS-CoV-2, a slight decrease (<1 log10 unit) was observed at pH 9 or 10 for 10 min; the decrease was over 5 log10 units at pH 11. However, both SARS-CoV-2 genome and infectious somatic coliphages decreased by less than 1 log10 unit at pH 12. All thermal or pH-based treatments that can remove or significantly reduce infectious somatic coliphages (>4 log10) can be considered efficient treatments for infectious SARS-CoV-2. We concluded that somatic coliphages can be considered highly conservative and easy to use indicators of the inactivation of SARS-CoV-2 during treatments based on heat and alkaline pH.

Interaction between norovirus and Histo-Blood Group Antigens: A key to understanding virus transmission and inactivation through treatments?

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.

Transmission of hepatitis E virus by water: An issue still pending in industrialized countries.

Hepatitis E virus (HEV) is an enteric virus divided into eight genotypes. Genotype 1 (G1) and G2 are specific to humans; G3, G4 and G7 are zoonotic genotypes infecting humans and animals. Transmission to humans through water has been demonstrated for G1 and G2, mainly in developing countries, but is only suspected for the zoonotic genotypes. Thus, the water-related HEV hazard may be due to human and animal faeces. The high HEV genetic variability allows considering the presence in wastewater of not only different genotypes, but also quasispecies adding even greater diversity. Moreover, recent studies have demonstrated that HEV particles may be either quasi-enveloped or non-enveloped, potentially implying differential viral behaviours in the environment. The presence of HEV has been demonstrated at the different stages of the water cycle all over the world, especially for HEV G3 in Europe and the USA. Concerning HEV survival in water, the virus does not have higher resistance to inactivating factors (heat, UV, chlorine, physical removal), compared to viral indicators (MS2 phage) or other highly resistant enteric viruses (Hepatitis A virus). But the studies did not take into account genetic (genogroups, quasispecies) or structural (quasi- or non-enveloped forms) HEV variability. Viral variability could indeed modify HEV persistence in water by influencing its interaction with the environment, its infectivity and its pathogenicity, and subsequently its transmission by water. The cell culture methods used to study HEV survival still have drawbacks (challenging virus cultivation, time consuming, lack of sensitivity). As explained in the present review, the issue of HEV transmission to humans through water is similar to that of other enteric viruses because of their similar or lower survival. HEV transmission to animals through water and how the virus variability affects its survival and transmission remain to be investigated.

Persistence of Hepatitis A Virus in Fresh Produce and Production Environments, and the Effect of Disinfection Procedures: A Review.

Although information is limited, it is evident that prolonged persistence of infectious Hepatitis A virus (HAV) is a factor in the transmission of the virus via fresh produce. Consequently, data on persistence of the virus on produce, and in environments relevant to production, such as soils, water and surfaces, are required to fully understand the dynamics of transmission of HAV via foods. Furthermore, information on effective disinfection procedures is necessary to implement effective post-harvest control measures. This review summarises current information on HAV persistence in fresh produce and on relevant disinfection procedures. On vegetables, HAV can remain infectious for several days; on frozen berries, it can persist for several months. HAV can remain infectious on surfaces for months, depending on temperature and relative humidity, and can survive desiccation. It can survive for several hours on hands. Washing hands can remove the virus, but further data are required on the appropriate procedure. Chlorination is effective in water, but not when HAV is associated with foodstuffs. Bleach and other sodium hypochlorite disinfectants at high concentrations can reduce HAV on surfaces, but are not suitable for use on fresh produce. There is only limited information on the effects of heating regimes used in the food industry on HAV. HAV is resistant to mild pasteurisation. Some food components, e.g. fats and sugars, can increase the virus' resistance to higher temperatures. HAV is completely eliminated by boiling. Quantitative prevalence data are needed to allow the setting of appropriate disinfection log reduction targets for fresh produce.

Molecular features of Hepatitis E Virus circulation in environmental and human samples.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is emerging but its circulation between humans and the environment remains misunderstood. HEV ORF2 gene encodes the capsid playing a key role in viral interactions with surfaces, ORF3 products are involved in the viral cycle. Our aim was to study the molecular characteristics of ORF2 and ORF3 which could favor HEV fitness in patients and the environment. STUDY DESIGN: Samples from 69 patients with hepatitis (blood/stools), 20 urban wastewaters, 20 effluents of a pig slaughterhouse, 22 farm pigs (stools), 20 wild boars (liver/stools) were collected in North-Eastern France. HEV strains were analyzed by direct sequencing within the ORF2 M region, of ORF2/ORF3, for phylogeny and physicochemical prediction and for ORF2 by ultra-deep sequencing. RESULTS: The results showed frequent HEV-positive samples: 9.1% of the patient bloods, 23.1% of their stools; 25.0% of wastewaters, 75.0% for the slaughterhouse, 10.0% of the boar livers, 5.3% of their stools. The strains were classified as HEV genotype 3. In ORF2, HEV highlighted one homogeneous major viral variant within quasispecies and a decrease in predicted antigenicity for two minor mutations (D442G, V402A). A cysteine signature at position 81 in ORF3 was observed in the boars. CONCLUSIONS: HEV RNA genotype 3 was detected in patients and in animals, in a slaughterhouse effluent and in wastewater. Moreover, the low variability of amino acids in the ORF2 M region and molecular features in ORF2 and ORF3 suggested that HEV strains could be advantageous for key properties.

F-Specific RNA Bacteriophages, Especially Members of Subgroup II, Should Be Reconsidered as Good Indicators of Viral Pollution of Oysters.

Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated.IMPORTANCE This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality.

The effect of chlorination and hydrodynamic shear stress on the persistence of bacteriophages associated with drinking water biofilms.

AIMS: This work aimed to assess at pilot scale the effect of chlorination and water flushing on 2-month-old drinking water biofilms and, above all, on biofilm-associated F-specific RNA bacteriophages MS2, GA and Qß. METHODS AND RESULTS: Chlorination (4 mg l(-1) ) was applied first with a hydrodynamic shear stress of 1 Pa and second with an increase in hydrodynamic shear stress to 10 Pa. Despite a rapid decrease in the number of biofilm bacteria and associated phages, infectious phages were still detected on surfaces after completion of the 150 min cleaning procedure. The resulting sequence of phage removal was: GA > Qߠ≫ MS2. CONCLUSIONS: The effect of chlorine on biofilm bacteria and biofilm-associated phages was limited to the upper layers of the biofilm and was not enhanced by an increase in hydrodynamic shear stress. A smaller decrease was observed for MS2 than for GA or Qß after completion of the cleaning procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences observed between the three phages suggest that the location of the viral particles in the biofilm, which is related to their surface properties, affects the efficiency of chlorine disinfection.

Relevance of F-Specific RNA Bacteriophages in Assessing Human Norovirus Risk in Shellfish and Environmental Waters.

UNLABELLED: Human noroviruses (HuNoVs) are the main cause of shellfish-borne gastroenteritis outbreaks. In the absence of routine technical approaches allowing infectious particles to be detected, this viral pathogen is currently targeted by genome research, leading to difficult interpretations. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as fecal and viral contamination indicators in shellfish and water from a local harvesting area. FRNAPH were also used as microbial source tracking tools. Constraints imposed by detection limits are illustrated here by the detection of infectious FRNAPH in several samples in the absence of FRNAPH genomes. The opposite situation was also observed, likely explained by the persistence of the genomes being greater than infectivity. Similar considerations may be applied to HuNoVs, suggesting that HuNoV genome targeting is of limited relevance in assessing infectious risks. While FRNAPH did not provide any benefits compared to Escherichia coli as fecal pollution indicators in water, novel observations were made in shellfish: contrary to E. coli, a seasonal trend of infectious FRNAPH concentrations was observed. These concentrations were higher than those found in water, confirming bioaccumulation in shellfish. This study also underlines a relationship between the presence of HuNoV genomes and those of human-specific FRNAPH subgroup II (FRNAPH-II) in shellfish collected throughout Europe. Further research should be undertaken to evaluate FRNAPH potential as an indicator of the presence of infectious HuNoVs. To this end, shellfish involved in HuNoV-caused gastroenteritis outbreaks should be analyzed for the presence of infectious FRNAPH-II. IMPORTANCE: This work provides new data about the use of F-specific RNA phages (FRNAPH) as a tool for evaluating fecal or viral contamination, especially in shellfish. In our case study, FRNAPH did not provide any benefits compared to E. coli as fecal pollution indicators in water but were found to be very useful in shellfish. Their concentrations in shellfish were higher than those found in the surrounding water, confirming bioaccumulation. This study also underlines a relationship between the presence of human norovirus genomes (HuNoVs) and those of FRNAPH subgroup II (FRNAPH-II). Considering that the two virus types have similar behaviors and since FRNAPH infectivity can be investigated, the specific detection of infectious FRNAPH-II could be regarded as an indication of the presence of infectious HuNoVs. The contribution of infectious human FRNAPH targeting for assessing the viral risk associated with HuNoVs in shellfish should thus be investigated.

Rapid, simple and efficient method for detection of viral genomes on raspberries.

In recent years, foodborne viruses, especially human noroviruses (NoV) and hepatitis A virus (HAV), have been increasingly reported as the causes of foodborne disease outbreaks. Soft red fruits, especially raspberries, have a high incidence among the types of food concerned. Due to low infectious doses and low concentrations of enteric viruses in food samples, it is necessary to have an efficient and rapid detection method to implement prevention measures. A standard method for virus detection and quantification in food, including raspberries (XP CEN ISO/TS 15216-1 and -2, 2013) is currently available. This method proposes a consensus detection approach by RT-real time PCR (RT-qPCR) but also a virus extraction procedure based on the elution-concentration principle. In this study, an alternative method of extraction in which RNAs are directly extracted from food matrices (based on direct RNA extraction) has been optimized. First, each step was improved to make it a highly rapid, specific and simple method. Second, the standard virus concentration method was compared with the optimized direct RNA extraction one. Human enteric viral surrogates, Murine Norovirus (MNV) and F-specific RNA bacteriophage GA, were selected according to their adhesion properties and resistance to pH close to our main targets (NoV and HAV). Raspberries were artificially contaminated using two different techniques (immersion and spotting) in order to define a recovery rate and the amounts of virus recovered. Results showed that the direct RNA extraction method revealed significantly higher viral extraction efficiency (46.2%) than the elution-concentration method (20.3%), with similar proportions of inhibitors for both. In the same way with inoculation by spotting, the best recovery rate of GA phage (39.7% against 0.7%) and MNV (42.8% against 0.5%) was observed by direct RNA extraction. For the lowest concentrations of phage and virus in the immersion bath, only the direct RNA extraction method allowed their recovery. Direct RNA extraction proved to be more effective (best recovery rate), faster (<8h) and simpler (fewer steps) than the one proposed in the CEN ISO standard method when it came to detecting enteric viruses on raspberries.

Occurrence of and Sequence Variation among F-Specific RNA Bacteriophage Subgroups in Feces and Wastewater of Urban and Animal Origins.

F-specific RNA bacteriophages (FRNAPH) have been widely studied as tools for evaluating fecal or viral pollution in water. It has also been proposed that they can be used to differentiate human from animal fecal contamination. While FRNAPH subgroup I (FRNAPH-I) and FRNAPH-IV are often associated with animal pollution, FRNAPH-II and -III prevail in human wastewater. However, this distribution is not absolute, and variable survival rates in these subgroups lead to misinterpretation of the original distribution. In this context, we studied FRNAPH distribution in urban wastewater and animal feces/wastewater. To increase the specificity, we partially sequenced the genomes of phages of urban and animal origins. The persistence of the genomes and infectivity were also studied, over time in wastewater and during treatment, for each subgroup. FRNAPH-I genome sequences did not show any specific urban or animal clusters to allow development of molecular tools for differentiation. They were the most resistant and as such may be used as fecal or viral indicators. FRNAPH-II's low prevalence and low sequence variability in animal stools, combined with specific clusters formed by urban strains, allowed differentiation between urban and animal pollution by using a specific reverse transcription-PCR (RT-PCR) method. The subgroup's resistance over time was comparable to that of FRNAPH-I, but its surface properties allowed higher elimination rates during activated-sludge treatment. FRNAPH-III's low sequence variability in animal wastewater and specific cluster formation by urban strains also allowed differentiation by using a specific RT-PCR method. Nevertheless, its low resistance restricted it to being used only for recent urban pollution detection. FRNAPH-IV was too rare to be used.

Impact of the virus purification protocol on aggregation and electrokinetics of MS2 phages and corresponding virus-like particles.

Previous experimental and theoretical studies have established that electrokinetic and aggregation properties of soft MS2 phages are not only governed by the physico-chemical features of their proteinaceous outer surface but are also significantly impacted by those of their inner RNA component (Dika et al. Appl. Environ. Microbiol., 2011, 14, 4939-4948). These conclusions contradict the recent findings of Nguyen et al. (Soft Matter, 2011, 7, 10449-10456) who reported identical electrokinetic and aggregation characteristics for MS2 and corresponding virus like particles (VLPs) that lack the internal RNA component. We demonstrate here that this contradiction originates from the different purification methods adopted prior to measurements. More generally, we show that stability and electrohydrodynamics of viruses differ according to purification by (i) dialysis, (ii) isopycnic centrifugation in the cesium chloride gradient, and (iii) precipitation using polyethylene glycol (PEG). Methods (i) and (iii) lead to aggregation of MS2 phages at pH ≤ 4 and pH ≤ 6 in 1-100 mM NaNO3 solutions, respectively, while under such conditions aggregation is not observed for MS2 and VLP suspensions prepared according to (ii). In addition, VLPs prepared following methods (i) and (iii) aggregate only at the isoelectric point (pH ~ 3-4) in 1 mM NaNO3 solution. Electrophoretic mobility data of stable MS2 and VLP particles were further examined using a recent formalism for electrokinetics of soft multilayered colloids. The analysis qualitatively shows how the purification protocol may affect either the outer particle surface properties and/or the inner particle content. Finally, the non-DLVO aggregation behavior of MS2 and VLPs purified via the above protocols is discussed in terms of the possible change in corresponding interparticular interactions.

The impact of temperature on the inactivation of enteric viruses in food and water: a review.

Temperature is considered as the major factor determining virus inactivation in the environment. Food industries, therefore, widely apply temperature as virus inactivating parameter. This review encompasses an overview of viral inactivation and virus genome degradation data from published literature as well as a statistical analysis and the development of empirical formulae to predict virus inactivation. A total of 658 data (time to obtain a first log(10) reduction) were collected from 76 published studies with 563 data on virus infectivity and 95 data on genome degradation. Linear model fitting was applied to analyse the effects of temperature, virus species, detection method (cell culture or molecular methods), matrix (simple or complex) and temperature category (<50 and ≥50°C). As expected, virus inactivation was found to be faster at temperatures ≥50°C than at temperatures <50°C, but there was also a significant temperature-matrix effect. Virus inactivation appeared to occur faster in complex than in simple matrices. In general, bacteriophages PRD1 and PhiX174 appeared to be highly persistent whatever the matrix or the temperature, which makes them useful indicators for virus inactivation studies. The virus genome was shown to be more resistant than infectious virus. Simple empirical formulas were developed that can be used to predict virus inactivation and genome degradation for untested temperatures, time points or even virus strains.

A comparison of different concentration methods for the detection of viruses present in bottled waters and those adsorbed to water bottle surfaces.

This study aimed to provide a tool for selecting the best approach to virological testing of bottled waters. Different methods were investigated. Method A examined the recovery of virus RNA following in situ lysis of virus particles in the aqueous phase and of those adhered to the bottle wall, method B examined the recovery of virus RNA following lysis of virus particles in the aqueous phase, and method C examined the recovery of intact virus particles. Method C generated the lowest genome recovery rate regardless of the water and virus type used, therefore comparison was mainly conducted between methods A and B.The effects of independent variables on the viral RNA recovery rate were determined by full factorial design. These independent variables included three waters (differing in mineral composition), four viruses (poliovirus 1, hepatitis A virus, Norovirus, and the MS2 phage), three incubation times (0, 10, and 20 days), and two methods (A and B). According to the results, each factor influenced the recovery rate of viral RNA with the exception of incubation time. Statistical analysis identified interactions between the factors. The strongest interactions involved the water and virus types, as well as the methods. The results suggested that method A should be used for the concentration and detection of hepatitis A virus, regardless of the divalent cation concentration of the bottled water. Method A was most suitable for water with the highest mineral content (divalent cation concentration of 250 mgL(-1)) and for the analysis of viruses capable of adsorbing onto the bottle walls (Poliovirus 1). Method B could be recommended for the analysis of water whose cation concentration is unknown.

Impact of internal RNA on aggregation and electrokinetics of viruses: comparison between MS2 phage and corresponding virus-like particles.

We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO(3) electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggregation profiles for MS2 and MS2 VLPs. Under the given pH and concentration conditions, MS2 VLPs exhibit electrophoretic mobility larger in magnitude than that of MS2, and both have similar isoelectric point (IEP) values (∼4). The electrokinetic results reflect a greater permeability of MS2 VLPs to electroosmotic flow, developed within/around these soft particles during their migration under the action of the applied electrical field. Results also support the presence of some remaining negatively charged component within the VLPs. In addition, MS2 phage systematically forms aggregates at pH values below the IEP, regardless of the magnitude of the solution ionic strength, whereas MS2 VLPs aggregate under the strict condition where the pH is relatively equal to the IEP at sufficiently low salt concentrations (<10 mM). It is argued that the stability of VLPs against aggregation and the differences between electrokinetics of MS2 and corresponding VLPs conform to recently developed formalisms for the stability and electrohydrodynamics of soft multilayered particles. The differences between the surface properties of these two kinds of particles reported here suggest that VLPs may not be appropriate for predicting the behavior of pathogenic viruses in aqueous media.

Aldicarb and carbofuran transport in a Hapludalf influenced by differential antecedent soil water content and irrigation delay.

Pesticide use in agroecosystems can adversely impact groundwater quality via chemical leaching through soils. Few studies have investigated the effects of antecedent soil water content (SWC) and timing of initial irrigation (TII) after chemical application on pesticide transport and degradation. The objectives of this study were to investigate the effects of antecedent soil water content (wet vs dry) and timing of initial irrigation (0h Delay vs 24h Delay) on aldicarb [(EZ)-2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime] and carbofuran [2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate] transport and degradation parameters at a field site with Menfro silt loam (fine-silty, mixed, superactive, mesic Typic Hapludalf) soils. Aldicarb and carbofuran were applied to plots near field capacity (wet) or near permanent wilting point (dry). Half of the dry and wet plots received irrigation water immediately after chemical application and the remaining plots were irrigated after a 24h Delay. The transport and degradation parameters were estimated using the method of moments. Statistical significance determined for SWC included averages across TII levels, and significance determined for TII included averages across SWC levels. For the dry treatment, aldicarb was detected 0.10 m deeper (P<0.01) on two of the four sampling dates and carbofuran was detected at least 0.10 m deeper (P<0.05) on all of the sampling dates compared to the wet treatment. Pore water velocity was found to be higher (P<0.10) in the dry vs wet treatments on three of four dates for aldicarb and two of four dates for carbofuran. Retardation coefficients for both pesticides showed similar evidence of reduced values for the dry vs wet treatments. These results indicate deeper pesticide movement in the initially dry treatment. For aldicarb and carbofuran, estimated values of the degradation rate were approximately 40-49% lower in the initially dry plots compared to the initially wet plots, respectively. When the initial irrigation was delayed for 24h, irrespective of antecedent moisture conditions, a 30% reduction in aldicarb degradation occurred. This study illustrates the deeper transport of pesticides and their increased persistence when applied to initially dry soils.

Atrazine and alachlor transport in claypan soils as influenced by differential antecedent soil water content.

Increased attention to ground water contamination has encouraged an interest in mechanisms of solute transport through soils. Few studies have investigated the effect of the initial soil water content on the transport and degradation of herbicides for claypan soils. We investigated the effect of claypan soils at initial field capacity vs. permanent wilting level on atrazine and alachlor transport. The soil studied was Mexico silt loam (fine, smectitic, mesic Aeric Vertic Epiaqualf) with a subsoil clay content, primarily montmorillonite, of >40%. Strontium bromide, atrazine, and alachlor were applied to plots; half were at field capacity (Wet treatment), and half were near the permanent wilting point (Dry treatment). Soil cores were removed at selected depths and times, and cores were analyzed for bromide and herbicide concentrations. Bromide, atrazine, and alachlor were detected at the 0.90-m depth in dry plots within 15 d after experiment initiation. Bromide was detected 0.15 m deeper (P < 0.05) in the Dry compared with the Wet treatment at 1, 7, and 60 d after application and >0.30 m deeper (P < 0.01) in the Dry treatment at 15 and 30 d after application; similar treatment results were found for atrazine and alachlor, although on fewer dates with significant differences. The mobility order of the applied chemicals was bromide > atrazine > alachlor. The atrazine apparent half-life was significantly longer in the Dry plots compared with the Wet plots. The retardation factor determined from the relative velocity of each herbicide to that of bromide was higher for alachlor than for atrazine. This study identifies the impact that shrinkage cracks have for different moisture conditions on preferential transport of herbicides in claypan soils.

Effects of pH on plaque forming unit counts and aggregation of MS2 bacteriophage.

AIM: The aim of this study was to determine whether aggregation processes in aqueous phase may explain the decrease in plaque forming unit (PFU) counts for pH close to the isoelectric point (pI) of viral particles (MS2 phages). METHODS AND RESULTS: Loss in PFU was observed for pH < or = pI (pI(MS2) = 3.9): for example, at pH 2.5, loss was approx. 3 log(10) PFU. Particle size analysis combining results of dynamic light scattering and flow particle image analysis was then applied to determine the aggregate state of viral suspensions by recording size distributions. The size of major population significantly changed to 30 nm at neutral pH to more than several micrometres when passing below the isoelectric point. CONCLUSIONS: Our study shows that MS2 phages exhibit significant aggregation processes for pH < or = pI leading to aggregate with sizes of few micrometres. This aggregation process can largely explain the decline in PFU counts. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clear that viral aggregation can be a source of significant bias for PFU assays because in the presence of an aggregate the PFU count can be less than the sum of its constituent particles. Therefore, cautions should be taken in terms of conditions of storage (pH far from pI) to avoid such aggregation artefact.

Occurrence and persistence of bacterial and viral faecal indicators in wastewater biofilms.

Biofilms within wastewater treatment plants can capture enteric microorganisms initially present in the water phase immobilising them either definitively or temporarily. Consequently, fates of microorganisms may totally change depending on whether they interact or not with biofilms. In this study, we assessed the stability of wastewater biofilms comparing the evolution of the concentrations of bacteria (heterotrophic plate count [HPC], thermotolerant coliforms [TC]) and viral (somatic coliphages [SC] and F-specific phages [F +]) indicators in the biofilms and in the corresponding wastewaters at 4 and 20 dgrees C. Additionally, we assessed the monthly occurrence of these bacterial and viral indicators as well as of pathogenic protozoa (Cryptosporidium oocysts and Giardia cysts) in three native wastewater biofilms for four months. Our results show that viral indicators (SC and F + ) persist longer in biofilms than in the corresponding wastewaters at 4 degrees C as well as at 20 degrees C. In contrast, persistence of bacterial indicators (TC and HPC) depends on both the temperature and the matrix. Differences between viral and bacterial persistence are discussed. Monthly analysis of native wastewater biofilms shows that bacterial and viral indicators, as well as Cryptosporidium oocysts and Giardia cysts, attach to wastewater biofilms to a concentration that remains stable in time, probably as a result of a dynamic equilibrium between attachment and detachment processes.

Development of a viral concentration method for bottled water stored in hydrophobic support.

Several protocols have been described for the detection of genomes of enteric viruses from water using two-step procedures: membrane filtration and RT-PCR detection. However, these methods, when applied to bottled water, generally consider only the aqueous phase. Such procedures do not take into account the adhesion of viruses onto the hydrophobic container. Potential adhesion results in loss of viral concentration in the aqueous phase and consequently viral pollution is underestimated in such a system. A procedure based on the addition of surfactant to elute viruses followed by membrane concentration was developed to avoid this underestimation. Firstly, using poliovirus 1 as a model, this study demonstrated that the best solution to recover virus and/or viral genome is a mix of sodium dodecyl sulphate, a nonionic detergent and guanidine thiocyanate. Furthermore, temperature has a significant but low effect on elution. A positively charged 0.2 microm inorganic membrane composed of Alumina (Anodisc, Whatman) is also the best membrane to concentrate viral material before the detection by RT-PCR. Finally, the developed protocol gives significantly higher poliovirus 1 recovery rate than a reference protocol previously described (aqueous phase concentration on Zetapore). The difference can be explained by the recovery of the viruses adsorbed onto the water container.

Occurrence of bacterial indicators and bacteriophages infecting enteric bacteria in groundwater in different geographical areas.

AIMS: The aim of this research was to determine the suitability of coliphages (bacteriophages) for assessing the microbial quality of groundwater. METHODS AND RESULTS: The number of several bacterial indicators (faecal coliforms, Escherichia coli, enterococci and spores of sulfite-reducing clostridia) and bacteriophages (somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis) were determined in groundwater of aquifers in various geographical areas. Results show that the relative abundance, determined as percentages of positive detections, of the bacterial indicators and bacteriophages varies depending on the aquifer. CONCLUSIONS: A single bacterial indicator may not be enough to assess microbiological quality in certain aquifers. One bacterial indicator and a bacteriophage parameter provide more information than two bacterial indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Coliphages (CPH) provide different information from that provided by bacterial indicators on the microbial quality of groundwater in different geographical areas. Easy, fast and inexpensive methods for the detection of CPH are feasible in both industrialized and developing countries.