Human adenovirus in tissues of freshwater snails living in contaminated waters.

Human adenovirus (HAdV) is resistant to environment and can be used as a marker to detect fecal contamination. Considering the importance of freshwater snails in the aquatic environment, their use as concentrators for HAdV is a complementary tool for viral analysis of water. The goal of the study was to detect HAdV in snails and surface water collected from wetlands of the Sinos River (Rio Grande do Sul, Brazil) basin and to compare rates and viral loads found in both samples. HAdV was detected through real-time PCR. Total and fecal coliforms were detected by Colilert® kit, and viral infectivity of positive samples of the DNA genome was performed in A549 human cell line. All wetlands presented bacterial and viral contamination, but no viral particle was considered viable. The wetland that showed lower fecal coliform mean was Campo Bom, and São Leopoldo (both cities in Rio Grande do Sul) was representative of the highest mean. HAdV was detected in water samples (53%), gastropods' hemolymph (31%) and tissues (16%). Wetlands proved to be environments already altered by human action. Water samples exhibited a higher frequency of HAdV detection; however, in some instances, the target viral genomes were only found in gastropod biological samples. This was a pioneer study in the use of freshwater snails for human enteric viral assessment thus demonstrating that the human organism can retain fecal contamination, complementing and assisting in microbiological water analyzes.

Adenovirus, enterovirus and thermotolerant coliforms in recreational waters from Lake Guaíba beaches, Porto Alegre, Brazil.

In the present study, molecular detection of human adenoviruses (HAdV) and enteroviruses (EV) was performed in surface water samples collected from beaches Ipanema and Lami, located on the shores of Lake Guaíba, city of Porto Alegre, RS, southern Brazil. Furthermore, water safety was evaluated by counting thermotolerant coliforms (TC), following local government regulations. A total of 36 samples were collected monthly from six different sites along the beaches. Viral genomes were found in 30 (83.3%) samples. The higher detection rate was observed for HAdV (77.8%), followed by EV (22.2%). Although low concentrations of TC have been found, the occurrence of viral genomes in water samples was frequent and may pose a potential risk of infection for people bathing in these beaches.

Human adenovirus (HAdV), human enterovirus (hEV), and genogroup A rotavirus (GARV) in tap water in southern Brazil.

The effects of viral gastroenteritis are more devastating in children than in any other age category. Thus, children exposed to the consumption of low quality water are at an increased risk of infection, especially in regions where sanitation is inadequate. The present study aimed to provide a survey of the occurrence of representative enteric viruses: human adenovirus (HAdV), human enteroviruses (hEV), and genogroup A rotavirus (GARV) in tap water samples collected in public schools located at six municipalities of Rio Grande do Sul, southern Brazil. Seventy-three schools were included in the study and tap water samples were analyzed by conventional PCR for the presence of HAdV, hEV, and GARV genomes. hEV showed the highest detection rate (27.4%), followed by HAdV (23.3%), and GARV (16.4%). New approaches to water monitoring should be considered to promote a better water quality and reduce the risk of waterborne diseases, especially considering drinking water to be served to vulnerable individuals.

Surveillance of human and swine adenovirus, human norovirus and swine circovirus in water samples in Santa Catarina, Brazil.

Animal and human wastewater can potentially contaminate water sources and the treatment of drinking water may not effectively remove all contaminants, especially viruses. The purpose of the present study was to evaluate the viral contamination of water used for human and animal consumption in the city of Concórdia, located in southern Brazil. Porcine circovirus type 2 (PCV2), porcine adenovirus (PAdV), human adenovirus (HAdV) and human norovirus (NoV) were searched for using quantitative polymerase chain reaction (qPCR). HAdV-positive samples were tested for viral infectivity by plaque assay. The qPCR results showed that PAdV, PCV2 and HAdV genetic material were present in all sampling sites. NoV was absent in all samples. The presence of genetic material from PAdV and PCV2 was detected in 30% and 45% of the 36 analyzed samples, respectively, with an average of 10(2) gc mL(-1) for PAdV and 10(4) gc mL(-1) for PCV2. HAdV was present in 100% of the samples, with an average of 10(4) gc mL(-1). However, in plaque assay, only 36% of the samples were positive. As viable particles of HAdV were found in drinking water, these results confirm that swine manure and human sewage impact surface water and groundwater, endangering water quality and indicating a potential risk to public health.

Survival of adenovirus types 2 and 41 in surface and ground waters measured by a plaque assay.

To manage artificial recharge systems, it is necessary to understand the inactivation process of microorganisms within aquifers so that requirements regarding storage times and treatment strategies for ground and surface waters can be developed and modeled to improve water management practices. This study was designed to investigate the survival of representative adenoviruses in surface- and groundwaters using a cell culture plaque assay with human lung carcinoma cells (A549) to enumerate surviving viruses. Adenovirus types 2 (Ad2) and 41 (Ad41) were seeded into 50 mL of three sterilized surface waters and groundwaters, and incubated at 10 and 19 °C for up to 301 days. Concentrations of Ad2 and Ad41 were relatively stable in all waters at 10 °C for at least 160 days and in some instances up to 301 days. At 19 °C, virus concentrations were reduced by 99.99% (4 log) after 301 days in surface water. There was approximately 90% (1 log) reduction of both viruses at 19 °C after 160 days of incubation in groundwater samples. There was no overall difference in survival kinetics in surface waters compared to groundwaters. The relatively high stability and long-term survival of adenoviruses in environmental waters at elevated temperatures should be considered in risk assessment models and drinking water management strategies.

Assessment of adenovirus, hepatitis A virus and rotavirus presence in environmental samples in Florianopolis, South Brazil.

AIMS: To assess the presence of human adenovirus (HAdV), hepatitis A (HAV) virus and rotavirus A (RV-A) in environmental samples from the Southern region of Brazil and to provide viral contamination data for further epidemiological studies and governmental actions. METHODS AND RESULTS: Water samples from various sources (seawater, lagoon brackish water, urban wastewater, drinking water sources-with and without chlorination and water derived from a polluted creek) and oysters of two growing areas were analysed by enzymatic amplification (nested PCR and RT-PCR), quantification of HAdV genome (qPCR) and viral viability assay by integrated cell culture-PCR (ICC-PCR). From June 2007 to May 2008 in a total of 84 water samples, 54 (64·2%) were positive for HAdV, 16 (19%) for RV-A and 7 (8·3%) for HAV. Viability assays showed nonpositive samples for HAV; though, infectious viruses were confirmed for RV-A (12·5%) and HAdV (88·8%). Oyster samples by PCR were positive for HAdV (87·5%) and RV-A (8·3%), but none for HAV. Quantitative PCR in oysters showed means loads in genomic copies (gc) of 9·1 × 10(4) gc g(-1) (oyster farm south) and 1·5 × 10(5) gc g(-1) (oyster farm north) and in waters ranging from 2·16 × 10(6) (lagoon water) to 1·33 × 10(7) gc l(-1) (untreated drinking water). CONCLUSIONS: This study has shown a widespread distribution of the analysed viruses in this particular region with high loads of HAdV in the environment which suggests the relevance of evaluating these viruses as positive indicators of viral contamination of water. SIGNIFICANCE AND IMPACT OF THE STUDY: The environmental approach in this study provides data concerning the prevalence, viability and quantification of enteric viruses in environmental waters and oysters in the South region of Brazil and has indicated that their presence might pose a risk to population in contact with the environmental samples searched.

Detection of enteric viruses in sewage sludge and treated wastewater effluent.

Sewage sludge and treated wastewater when contaminated with enteric virus and discharged into the environment, could pose a human health risk. The aim of study was to verify the presence and viability of enteric viruses in sewage sludge and treated wastewater at a local sewage plant in Florianopolis city, Brazil. Sewage sludge was concentrated by organic flocculation and polyethylene glycol precipitation and wastewater by electronegative membrane filtration and ultrafiltration by Centriprep Concentrator. Adenovirus (AdV), hepatitis A virus (HAV), and Rotavirus (RV) were examined for all samples for 12 months and Poliovirus (PV) was also tested for in sewage sludge samples. AdV was the most prevalent in both kind of samples, followed by RV, PV (in sludge) and HAV. Viral viability by cell culture (ICC-PCR) was: AdV: 100%, HAV: 16.7%, PV: 91.7%, RV: 25% in sludge and AdV: 66.6%, HAV: 66.6% and RV: 0% in wastewater. IFA for AdV in sludge ranged from 70 to 300 FFU/ml. QPCR for AdV ranged from 4.6 x 10(4) to 1.2 x 10(6) and from 50 to 1.3 x 10(4) gc/ml in sludge and wastewater, respectively. HAV quantification in sludge ranged from 3.1 x 10(2) to 5.4 x 10(2) gc/ml. In conclusion, it was possible to correlate presence and viability of enteric viruses in the environmental samples analyzed.

Assessment of norovirus contamination in environmental samples from Florianópolis City, Southern Brazil.

AIMS: To assess norovirus (NoV) contamination in aquatic ecosystems in the city of Florianópolis, in Southern Brazil, to provide epidemiological data that can support actions for environmental contamination control. METHODS AND RESULTS: An adsorption-elution method, followed by ultrafiltration, was performed to concentrate the viruses. NoV were detected using semi-nested PCR and quantified by real-time PCR. From June 2007 to May 2008, NoV were detected in 23% (22/94) of the samples analysed, including seawater, drinking water, superficial water (creek and brackish lagoon) and treated sewage. The mean viral loads for genogroups (G)I and GII in treated sewage samples were 297 and 440 genomic copies (gc) l(-1) , respectively, whereas creek water samples contained 2603 and 1361 gc l(-1) , respectively. Six samples were sequenced: two samples were GII.4, two were GII.2 and two were GI.3. CONCLUSIONS: NoV were detected in all water types analysed, demonstrating the widespread contamination of this geographical area with several cocirculating strains belonging to GI and GII. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the environmental spread of NoV in environmental waters and highlights the potential hazard for human health following the consumption of or contact with these waters, which could result in waterborne or foodborne acute gastroenteritis.

Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices.

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100%) for both viruses and in recreational lagoon water for HAV (100%). The efficiency recovery was 10% for HAdV and HAV in seawater and 10% for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.

Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100 percent) for both viruses and in recreational lagoon water for HAV (100 percent). The efficiency recovery was 10 percent for HAdV and HAV in seawater and 10 percent for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.