Christophe Gantzer

Surveillance of adenoviruses and noroviruses in European recreational waters

Abstract Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

ABSTRACT Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Aggregation and surface properties of F-specific RNA phages: Implication for membrane filtration processes

We report an experimental investigation of the electrokinetic properties and size variations of four F-specific bacteriophages of the types MS2, GA, Qbeta and SP (21-30 nm in diameter) over a broad range of pH values (1.5-7.5) and NaNO3 electrolyte concentrations (1-100 mM). The results obtained by dynamic light scattering show that the aggregation of SP and GA particles takes place over the whole range of pH and ionic strength conditions examined. For MS2 phages, the aggregation of MS2 particles is not observed for pH higher than the isoelectric point (pI) and large ionic strengths for which interparticular repulsive electrostatic interactions are however expected to be sufficiently screened. Aggregation of the MS2 phages, dispersed in 1 and 100 mM electrolyte concentration, occurs at pH 4, which basically corresponds to the pI as determined by electrophoresis measurements. The Qbeta particles suspended in solutions of low electrolyte concentrations aggregate at low pH values (pI approximately 3) and, unlike MS2, at large ionic strengths over the whole range of pH conditions considered in this study. These elements allow the determination of the hydrophobic sequence for the four phages SP approximately GA>Qbeta>MS2. Close inspection of the electrokinetic results reveals small to significant variations of the pI values-depending on the phage considered-with respect to the concentration of indifferent NaNO3 electrolyte. This indicates that features other than chemical and electrostatic in nature play a key role in determining the pI and more generally the electrophoretic mobility mu of viral particles. A qualitative interpretation is given and is based on the consideration of inner electro-osmotic flow within the isolated or aggregated particles. The impact of the flow properties within the particles is further in agreement with recent theoretical formalism developed for the electrokinetics of soft multiplayer particles, the phages analyzed here being some illustrative examples. The determination and qualitative interpretation of the surface properties of the viral particles as reported in the current study are commented within the context of water treatment especially concerning viral removal by membrane filtration processes.

Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers

ABSTRACT Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qβ) were exposed to UV radiation from 0 to 150 mJ · cm−2. PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qβ having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ · cm−2. In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ · cm−2 and 60 mJ · cm− 2, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.

Comparison of Coliforms and Coliphages as Tools for Assessment of Viral Contamination in River Water

ABSTRACT The aim of the study was to evaluate the presence of pathogenic viruses in the Moselle River and to compare the usefulness of thermotolerant coliforms and somatic coliphages as tools for river water quality assessment in terms of viral contamination. Thermotolerant coliforms and somatic coliphages were enumerated by standardized methods in 170 samples of river water drawn from five sampling sites along the Moselle River (eastern France). BGM cell culture and integrated cell culture-reverse transcription-PCR DNA enzyme immunoassay were used to determine the presence of pathogenic viral genome (Enterovirus and Norovirus genogroup II [GGII]) and infectious Enterovirus spp. in 90 1-liter samples. No infectious Enterovirus spp. were isolated, but Enterovirus and Norovirus GGII genomes were detected in 38% of the samples. Norovirus GGII genome was mostly detected in winter, whereas Enterovirus genome was mostly detected in summer and fall. Somatic coliphages appeared to be less sensitive to higher river water temperature than thermotolerant coliforms. Furthermore, the number of river water samples positive for pathogenic viral genome increased with increasing concentration of somatic coliphages, whereas coliform concentration was unrelated to viral genome contamination. Consequently somatic coliphages, which are less sensitive to environmental factors than thermotolerant coliforms in river water, would provide a promising tool for assessment of river water quality in terms of fecal and viral pollution.

Impact of Chemical and Structural Anisotropy on the Electrophoretic Mobility of Spherical Soft Multilayer Particles: The Case of Bacteriophage MS2

We report a theoretical investigation of the electrohydrodynamic properties of spherical soft particles composed of permeable concentric layers that differ in thickness, soft material density, chemical composition, and flow penetration degree. Starting from a recent numerical scheme developed for the computation of the direct-current electrophoretic mobility (mu) of diffuse soft bioparticles, the dependence of mu on the electrolyte concentration and solution pH is evaluated taking the known three-layered structure of bacteriophage MS2 as a supporting model system (bulk RNA, RNA-protein bound layer, and coat protein). The electrokinetic results are discussed for various layer thicknesses, hydrodynamic flow penetration degrees, and chemical compositions, and are discussed on the basis of the equilibrium electrostatic potential and hydrodynamic flow field profiles that develop within and around the structured particle. This study allows for identifying the cases where the electrophoretic mobility is a function of the inner structural and chemical specificity of the particle and not only of its outer surface properties. Along these lines, we demonstrate the general inapplicability of the notions of zeta potential (zeta) and surface charge for quantitatively interpreting electrokinetic data collected for such systems. We further shed some light on the physical meaning of the isoelectric point. In particular, numerical and analytical simulations performed on structured soft layers in indifferent electrolytic solution demonstrate that the isoelectric point is a complex ionic strength-dependent signature of the flow permeation properties and of the chemical and structural details of the particle. Finally, the electrophoretic mobilities of the MS2 virus measured at various ionic strength levels and pH values are interpreted on the basis of the theoretical formalism aforementioned. It is shown that the electrokinetic features of MS2 are to a large extent determined not only by the external proteic capsid but also by the chemical composition and hydrodynamic flow permeation of/within the inner RNA-protein bound layer and bulk RNA part of the bacteriophage. The impact of virus aggregation, as revealed by decreasing diffusion coefficients for decreasing pH values, is also discussed.

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).

Presence of Viral Genomes in Mineral Water: a Sufficient Condition To Assume Infectious Risk?

ABSTRACT 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.

Occurrence, Survival, and Persistence of Human Adenoviruses and F-Specific RNA Phages in Raw Groundwater

ABSTRACT Detection of specific genetic markers can rapidly identify the presence of enteric viruses in groundwater. However, comparison of stability characteristics between genetic and infectivity markers is necessary to better interpret molecular data. Human adenovirus serotype 2 (HAdV2), in conjunction with MS2 phages or GA phages, was spiked into raw groundwater microcosms. Viral stability was periodically assessed by both infectivity and real-time PCR methods. The results of this yearlong study suggest that adenoviruses have the most stable persistence profile and an ability to survive for a long time in groundwater. According to a linear regression model, infectivity reductions of HAdV2 ranged from 0.0076 log10/day (4°C) to 0.0279 log10/day (20°C) and were significantly lower than those observed for phages. No adenoviral genome degradation was observed at 4°C, and the reduction was estimated at 0.0036 log10/day at 20°C. Occurrence study showed that DNA of human adenoviruses could be observed in groundwater from a confined aquifer (7 of the 60 samples were positive by real-time PCR), while no fecal indicators were detected. In agreement with the persistence of genetic markers, the presence of adenoviral DNA in groundwater may be misleading in term of health risk, especially in the absence of information on the infective status.

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments

Aims:  To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro‐organisms by primary and secondary wastewater treatment processes.