Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome.

The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk.

Presence of viral genomes in mineral water: a sufficient condition to assume infectious risk?

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 degrees 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.

Recovery of feline calicivirus infectious particles and genome from water: comparison of two concentration techniques.

The aim of this work was to determine the recovery rate of feline calicivirus (FCV-F9) infectious particles and genome from water after a concentration step using either adsorption elution on glass wool or filtration through an electropositive membrane. The results showed that the membrane filtration technique allowed a 75% recovery rate of FCV-F9 infectious particles while the yield was only 5.3% for FCV-F9 genome.

Microbial agents associated with waterborne diseases.

Many classes of pathogens excreted in feces are able to initiate waterborne infections. There are bacterial pathogens, including enteric and aquatic bacteria, enteric viruses, and enteric protozoa, which are strongly resistant in the water environment and to most disinfectants. The infection dose of viral and protozoan agents is lower than bacteria, in the range of one to ten infectious units or oocysts.

Evaluation of bacteriophages during the treatment of sludge.

The aim of this work was to determine the effect of liming and composting on the fate of three bacteriophages (somatic coliphages, F-RNA phages, Bacteroides fragilis phages) considered as potential indicators of viral contamination. It was shown that the three bacteriophages studied exhibited variable densities in sludge. Somatic coliphages were most abundant (10(4) to 10(5) x 10 g(-1) DM) then F-RNA bacteriophages (10(2) to 10(4) x 10 g(-1) DM) and Bacteroides fragilis phages (10(1) to 10(2) x 10 g(-1) DM).

Fates of bacteriophages and bacterial indicators in the Moselle river (France).

It has been suggested that bacteriophages can provide useful information about the pathogenic microorganisms, particularly enteric viruses, present in water. This information is complementary to that obtained from bacterial indicators of faecal contamination, which would be of great value for evaluating the risks associated with the use of certain types of water. Before bacteriophages can be used as indicators of faecal contamination, we need to confirm that bacteriophages give a different response to that given by the well-known bacteria indicators and to determine what happens to bacteriophages in river water.

Bacteroides fragilis and Escherichia coli bacteriophages in human faeces.

Some bacteriophages found in human faeces are being evaluated as possible indicators of viral contamination of water. These bacteriophages include somatic coliphages and Bacteroides fragilis phages. The aims of this study were to determine the occurrence and concentrations of somatic coliphages and Bacteroides fragilis phages in the stools of a human population residing in eastern France (n = 193).

The effect of liming on the microbiological quality of urban sludge.

Treatments applied to sludge in order to stabilise and dehydrate them may give notable inactivation of microorganisms. This is observed when sludge is exposed either to high temperature or drastic pH when residual sludge is limed. The control of virological, parasitological and bacteriological sludge quality by detecting pathogenic microorganisms is slow and too expensive to be commonly practised.

Best viral elution method available for quantification of enteroviruses in sludge by both cell culture and reverse transcription-PCR.

The aim of this study was to select one or several virus extraction techniques that enable simultaneous detection of enterovirus genomes and infectious particles in different types of urban sludge. Eight techniques were compared by using 16 different liquid and solid sludge samples. The numbers of infectious enteroviruses in cell cultures were determined by using the most-probable-number method.

Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.

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.

Enterovirus genome detection in wastewater: multi centric evaluation of a commercial kit.

A multi-centric study was carried out in three laboratories, to evaluate the efficiency of a standardized kit for the detection of enterovirus genome in wastewater. Twenty one samples of 20 liters of wastewater were analyzed before and after concentration through glass wool. Each sample was analyzed with the Amplicor kit as well as with techniques developed independently in each laboratory.

Comparative study of techniques used to recover viruses from residual urban sludge.

Eight virus extraction techniques were compared on three types of residual urban sludge for simultaneous detection of infectious enteroviruses, somatic coliphages, F-specific RNA bacteriophages and Bacteroides fragilis bacteriophages. The highest virus counts were found in extracts obtained using three extraction techniques described by respectively using a 10% beef extract solution at pH 9 and sonication, using a 0.3 M NaCl/7% beef extract solution at pH 7.

Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification.

When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. Seven techniques for extracting hepatitis A virus (HAV) from stool and shellfish samples were compared, in order to identify the protocol most suited to both types of sample and with the best extraction yield. The protocols tested were either techniques for the recovery and purification of total RNA, such as RNAzol, PEG-CETAB, GTC-silica and Chelex, or techniques for isolating specifically HAV using a nucleotide probe or a monoclonal antibody.

Detection of infectious enteroviruses, enterovirus genomes, somatic coliphages, and Bacteroides fragilis phages in treated wastewater.

In this study, three types of treated wastewater were tested for infectious enteroviruses, the enterovirus genome, somatic coliphages, and Bacteroides fragilis phages. The aim of this work was to determine whether the presence of the two types of bacteriophages or of the enterovirus genome was a good indicator of infectious enterovirus contamination. The enterovirus genome was detected by reverse transcription-polymerase chain reaction.

Persistence of infectious hepatitis A virus and its genome in artificial seawater.

The stability of the hepatitis A virus (HAV) genome detectable by RT-PCR in artificial sterile seawater seeded with HAV has been compared to that of HAV detectable in cell culture. The HAV genome was detectable by RT-PCR for 232 days while virus particles were detectable in cell culture for only 35 days. This difference in stability indicates that detection of the HAV genome by RT-PCR is not a reliable indicator of the survival of HAV detectable in cell cultures.

Reverse transcriptase PCR detection of astrovirus, hepatitis A virus, and poliovirus in experimentally contaminated mussels: comparison of several extraction and concentration methods.

Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (RT-PCR). Shellfish were experimentally contaminated by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues were processed by using borate buffer, glycine solution, saline beef, and saline beef-Freon extraction methods.

Potentiation of the virucidal activity of sterilized natural waters.

After heat, conductivity has been described as the second most inactivating factor of viruses in surface waters. Using poliovirus-1, we showed that a highly significant linear relationship between virus inactivation rate and water conductivity can be obtained for most of the tested sterilized water samples. Using sterile saline solutions, we demonstrated that this apparent relationship is false, i.

Use of Doehlert matrices for study of poliovirus-1 adsorption.

Experiments designed according to Doehlert matrices were carried out to study poliovirus-1 adsorption to Na-montmorillonite in a complex aqueous environment. Salt concentration and valence, virus load, clay concentration, and organic matter concentration were included in the design as selected parameters for possible or known involvement in viral adsorption in environmental waters. Use of this experimental design not only allowed to detect and quantify direct influence of the tested parameters upon the viral response, but also to reveal the influence of interactions between these tested factors.

Enterovirus genomes in wastewater: concentration on glass wool and glass powder and detection by RT-PCR.

Standard methods for detecting enteroviruses in environmental samples require cell culture, which is time consuming and expensive. The reverse transcription-polymerase chain reaction (RT-PCR) is a rapid, sensitive method for detecting enteroviruses in water. However, environmental samples often contain substances that inhibit PCR amplification of target RNA.

Poliovirus-1 inactivation and interaction with biofilm: a pilot-scale study.

A pilot-scale study was initiated to examine the behavior of viruses pulse injected into a distribution system. The influence of a free-chlorine residual and that of virus preadsorption to clay particles was evaluated by tracing the viruses both in the water flow and after elution from the biofilm. These experiments demonstrated, first, that virus preadsorption on 40 mg of Na-montmorillonite per liter increased the residence time of the viruses within the pilot plant by roughly three times and, second, that preadsorption to clay did not prevent viruses from being inactivated by chlorine.

Effect of heat on the survival of infectious coxsackievirus B3 and its genome in water.

The aim of this study was to estimate the resistance of the viral genome of coxsackie B3 to heat (from 25 degrees C to 95 degrees C) with respect to the infectious virus. The results show that viral genomes were much more resistant to heat than infectious virus. The infectious coxsackie B3 was undetectable following incubation at 55 degrees C for 15 min.

Comparison study on three protocols used to concentrate poliovirus type 1 from drinking water.

The efficiency of three techniques used to concentrate enteric viruses from water media and based on adsorption-elution on glass are tested. The techniques are adsorption on glass wool (GW) at the natural pH of the water and adsorption on glass powder using acidified water (pH 3.5).

Direct sequencing of hepatitis A virus strains isolated during an epidemic in France.

Direct sequencing of PCR products was used to study the VP1 region of the hepatitis A virus (HAV) genome (position 2199 to 2356) of nine strains isolated from human stools collected during a hepatitis A epidemic (western France, 1992), three strains from environmental samples (1990, 1991, and 1992), and two HAV cell culture isolates (the French strain CF53/Lyon and strain CLF). These viruses differed from CF53/Lyon (genotype I) by between 1 and 10.3%, and results indicated the existence of two groups of strains belonging to two different subgenotypes (IA and IB).

Rotavirus quantification: a comparison between the enumerative and the MPN techniques.

A commonly used technique to isolate rotaviruses from a water environment involves the inoculation on cell culture associated with the revelation of the viral multiplication by indirect immunofluorescence. The results of the experiment using this methodology may be expressed either with an enumerative technique or with a MPN technique. We have found that the enumerative method provided the highest yield as opposed to the MPN method by a ratio close to 3.

Virucidal activity of an activated sludge supernatant.

The virucidal activity of the activated sludge aqueous phase was studied from the time of initial inoculation with a poliovirus type 1 suspension and for durations of three and nine days. The mixtures were incubated in presence of a nutritive medium at 26 degrees C and samples were drawn at regular intervals of time for viral titration. The activated sludge supernatant (ASS) caused an important decrease of the titer of the poliovirus type 1 suspension especially after nine days of incubation.