Michele Muscillo

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.

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 log10 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.

Quantitative real-time PCR of enteric viruses in influent and effluent samples from wastewater treatment plants in Italy

The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment.

Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends

Noroviruses (NoVs) give rise to clinically relevant gastroenteritis in all age groups and are widely distributed in both clinical and environmental settings. NoVs are classified into five genogroups (GI to GV), of which GI, GII and GIV infect humans. While data on the epidemiology of human NoVs GI and GII have been steadily increasing, very little information has been published on the spread of GIV in either the health care system or the environment, resulting in a lack of information about its clinical significance and pathogenesis. In order to investigate the distribution of GIV strains in the environment, we analyzed sewage samples collected from five treatment plants, by using newly designed nested RT-PCR assays. A collection of clinical stool samples, originating from pediatric patients with symptoms of acute gastroenteritis, previously analyzed in our laboratory for the presence of NoV GI or GII, was also analyzed for the presence of GIV norovirus. Results of this work attest to the presence of GIV in both clinical and environmental contexts and underline the importance of routinely screening for this genogroup, along with GI and GII, in order to better understand its distribution, prevalence and role during epidemics, which is probably underestimated.

Molecular identification and genetic analysis of Norovirus genogroups I and II in water environments: comparative analysis of different reverse transcription-PCR assays.

ABSTRACT Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.

Molecular Detection of Hepatitis E Virus in Sewage Samples

ABSTRACT Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. In Italy, the true burden of HEV infection is unknown. Molecular HEV screening of raw sewage samples from 11 wastewater treatment plants yielded 19 positives (16%; 18 genotype I, 1 genotype III) evenly distributed throughout Italy. Evidence that HEV could be establishing itself in our region is accumulating and may justify more active surveillance to monitor its spread.

Hepatitis E virus in Italy: molecular analysis of travel-related and autochthonous cases

Human hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The aim of the present study was to contribute to the body of knowledge available on the molecular epidemiology of acute hepatitis E in Italy. Three sets of HEV-specific primers targeting the ORF1 and ORF2 were used to examine serum samples collected from acute hepatitis patients positive for anti-HEV IgG and/or IgM, between 2007 and 2010. Seventeen patients (39.5%) tested HEV RNA-positive: 12 infections, due to genotype 1, were associated with travel to endemic areas (Bangladesh, India and Pakistan), while five infections, due to genotype 3, were presumably autochthonous. Risk factors identified in this group included exposure to raw seafood, pork liver sausages and wild boar. Results from the present study confirm that human HEV infection in Italy is caused by different genotypes, depending on whether the infection is travel-related or autochthonous.

Detection and molecular characterization of noroviruses from five sewage treatment plants in central Italy.

Noroviruses (NoVs) are the most frequent etiological agents of non-bacterial gastroenteritis. These viruses are transmitted through the fecal-oral route, leading to high viral loads in sewages. The objective of this paper was to study the environmental occurrence of the most prevalent NoV strains in different wastewater treatment plants. In addition, molecular characterization of the isolated strains was performed. Two different PCR-based methods were carried out and a novel strategy was used to verify the level of RT-PCR inhibition. From May to September 2007, a total of 97 inflow and outflow samples were collected from five wastewater treatment plants in central Italy. We detected NoV by nested PCR in 96.9% of influent samples: 89.1% contained both genogroups; 4.7% contained only GI and 3.1% only GII. In effluents, we detected NoV in 78.8% of samples: 30.3% contained both genogroups, and 48.5% contained only GI. The major genotypes detected by sequencing analyses were GI/2, GI/5, GII/b, GII/4 and GII/6. This work confirms the wide circulation of NoVs in Italy with a predominance of GI strains, and the widespread distribution of NoV variants in both raw and treated wastewater.

Enteric virus detection in Adriatic seawater by cell culture, polymerase chain reaction and polyacrylamide gel electrophoresis

Abstract Forty samples of sea and estuary water were collected from a 40 km strip along the Adriatic coast of Italy between June 1994 and September 1995. Each sample consisted of 10 l of water. Routine bacteriological analyses were carried out and viral particles concentrated on cross-flow membranes; the concentrated water samples, equally divided into two parts, were used to infect both BGM and Hep-2 cells. Lysates from all cell cultures were further tested for the presence of enteroviruses by reverse-transcribed polymerase chain reaction (RT-PCR) and reoviruses by polyacrylamide gel electrophoresis (PAGE). The results showed widespread viral contamination of the waters tested, particularly in late summer. Under our experimental conditions, BGM cells were more efficient than Hep-2 in recovering viruses. In fact, enteroviruses were detected in up to 33% and reoviruses in 80% of BGM infected with seawater, compared to 8% and 53%, respectively, for the Hep-2 cells. In estuarine samples, enteroviruses were detected in 30% and reovirus in 54% of BGM, compared to 23% and 30% of Hep-2. Twenty nine out of 40 samples showed the presence of infectious particles on the basis of the CPE appearance; after identification of the isolated viruses, only 13 turned out to be specifically contaminated by enteroviruses. Of the latter, five were below the bacteriological standards set by the Italian legislation in line with the EEC Directive 76/160 IEEC for bathing waters.

Molecular detection and genetic diversity of norovirus genogroup IV: a yearlong monitoring of sewage throughout Italy

Noroviruses cause acute viral gastroenteritis worldwide. They are classified in five genogroups, of which GI, GII, and GIV infect humans. Little information is available on the prevalence and clinical effects of GIV noroviruses. We conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 11 wastewater treatment plants throughout Italy, with the aim of studying the circulation of GIV NoV, as well as its genetic diversity. Eight percent of samples tested positive, and sequence analysis showed a considerable degree of genetic variability. These results imply the need for further studies to elucidate the role of this virus as a gastroenteritis-causing pathogen.