Rotavirus vaccine stability in the aquatic environment.

AIMS: To evaluate the thermal and length of stability of the Rotaviruses (RV) vaccine (RotaTeq) in the aquatic environment. METHODS AND RESULTS: Surface freshwater, brackish and drinking water were spiked with RV vaccine strain and stored at 22 and 4°C. The virus infectivity and genome persistence were evaluated by plaque assay and RT-qPCR, respectively, up to 180 days. Infectious RV vaccine particles showed to be less stable in the brackish water matrix than in surface and drinking water either at 22 or 4°C. The estimated T90 values obtained by the linear regression model were 18, 55 and 59 days, respectively for brackish, surface and drinking water stored at 22°C and 68, 154 and 240 days at 4°C. As expected, the genome persistence showed to be less affected by length and temperature of storage in all the matrices evaluated. CONCLUSIONS: The evidence of high stability of the RV vaccine in water matrices reinforces the importance for surveillance of RV vaccines strains in the environment regarding the potential occurrence of unexpected infections and virus genomic reassortments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of reassortants and the shedding of the live attenuated vaccine strains after vaccination can compromise the vaccine safety by introducing new viral variants in the environment.

Waterborne diseases classification and relationship with social-environmental factors in Florianópolis city - Southern Brazil.

This study aimed to investigate and classify the occurrence of waterborne diseases in Florianópolis city, Santa Catarina State, Southern Brazil and to correlate these diseases with the following social-environmental indicators of the local population: type of water supply, adequate collection and sewage treatment, areas of flooding and domestic water tank cleaning. Reports of outpatients were analyzed for surveillance of waterborne diseases during the period of 2002 to 2009. Waterborne diseases were classified into four groups: Group A: diarrheal diseases; Group B: parasitological diseases; Group C: skin diseases and Group D: eye diseases. The diarrheal, parasitological and skin diseases were the most frequently reported. Waterborne diseases belonging to Group A in all sites were correlated with other waterborne diseases groups, which can be an indicator of the circulation of other waterborne diseases. Regarding the social-environmental indicators assessed, the most correlated with waterborne diseases were the origin and quality of the water supply, followed by inadequate collection and treatment of sewage, frequent flooding, and finally the lack of cleanliness of the water reservoir. The results highlight the need for policies aiming for improvement of the sanitation service in the maintenance of human, animal and environmental health.

Enteric viruses in surface water and sediment samples from the catchment area of Peri Lagoon, Santa Catarina State, Brazil.

This paper aims to quantify human adenovirus (HAdV), rotavirus species A (RVA), and hepatitis A virus (HAV) in surface water and sediments and to determine the viability of HAdV in these samples. Water and sediment samples were collected, and HAdV, RVA, and HAV were quantified by real-time polymerase chain reaction (PCR); HAdV was also evaluated for infectivity by a plaque assay (PA). For the water samples, HAdV was detected in 70.8% of the summer collections, with 82.4% containing infectious HAdV; the HAdV incidence in winter was 62.5%. For the sediment samples, the incidence of HAdV was 37.5% in the summer collections, with 66.7% containing infectious HAdV; the HAdV incidence in winter was 37.5%. RVA was detected in 20.8 and 45.8% of surface water samples collected in summer and winter, respectively, and 8.3 and 12.5% of sediment samples collected in summer and winter, respectively. HAV was detected only in surface waters, with 54.8 and 12.5% positivity in summer and winter samples, respectively. This study demonstrated that enteric viruses are present in water and sediments and that the presence of infectious viruses should be investigated whenever possible for quantitative microbial risk assessment studies. Combined analyses of water and sediments are important for reliable public health risk analysis of recreational and lagoon waters.

Stability of human enteric viruses in seawater samples from mollusc depuration tanks coupled with ultraviolet irradiation.

AIMS: To evaluate the stability in seawater of human adenovirus (HAdV2), murine norovirus (MNV-1) and hepatitis A virus (HAV) in a shellfish depuration system with and without ultraviolet (UV) treatment. METHODS AND RESULTS: Seawater was seeded with viruses and disinfected using a 36 W lamp. Samples were collected at 24, 48, 72, 96 and 120 h; viruses were concentrated and the viral decay was evaluated using molecular and cell culture methods. Based on the molecular results, at 120 h of disinfection, there was a reduction of more than 3 log(10) for HAdV2 and HAV; MNV-1, a 4.5 log(10) reduction was observed at 72 h. Infectious MNV-1 was not detected after 72 h of treatment; while HAdV2 remained infectious. Seawater not treated demonstrated a progressive viral reduction for the three viruses tested. CONCLUSIONS: The UV reduced the number of viral particles, and the results indicate there is natural and gradual decrease of viral load and viability in seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: UV irradiation is the method of choice for shellfish depuration in many countries; this work showed useful information about the viral stability in seawater and application of UV to water disinfection to be used in shellfish depuration tanks.

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.

Surveillance of human viral contamination and physicochemical profiles in a surface water lagoon.

The present study evaluated the contamination of a surface water lagoon (Peri Lagoon) in Florianópolis, Santa Catarina, Brazil, by human adenovirus (HAdV), polyomavirus JC (JCPyV), hepatitis A virus (HAV) and rotavirus species A (RVA). Efforts were driven to determine the correlation between viral presence and the physicochemical parameters of the lagoon and measure the distribution of these viruses throughout the year (June 2010 to May 2011). A total of 48 samples were collected, concentrated and analyzed by qPCR (quantitative polymerase chain reaction). Approximately 96% of the samples were positive for HAdV (46/48), 65% were positive for RVA (31/48), 21% were positive for JCPyV (10/48) and 12% were positive for HAV (6/48). The presence of JCPyV was positively correlated with that of NO(2)(-)N, and also there was a positive correlation between the presence of each one of the viruses (HAdV, HAV and RVA) in winter. Samples from water dedicated for human consumption and recreation tested positive for HAdV by qPCR. These samples were also subjected to viral integrity and viability assays: 83% (10/12) contained intact viral particles and 66% (8/12) contained infectious particles. Our results demonstrate the release of human waste into water sources, justifying the urgent need to add viral parameters to water quality surveillance.

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.

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.

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.

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods.

The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20% of the AdV, 90% of the RV and 100% of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8% of the AdV and 90% of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.

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.

Microcosm environment models for studying the stability of adenovirus and murine norovirus in water and sediment.

Microcosms are useful tools for understanding the survival and fate of enteric viruses in aquatic environments. This study set out to determine the stability of infectious enteric viruses in an aquatic environment using a laboratory scale microcosm. Sediment and overlaying water were collected from a lagoon and inoculated with known concentrations of recombinant adenovirus (AdV-GFP) and murine norovirus (MNV-1). Infectious particles of these viruses were measured using fluorescence microscopy (AdV-GFP) or the plaque assay method (MNV-1), over 85 days in two different conditions: under natural sunlight and in fully darkened environments. The time required to reach one log reduction in viral titres (T90) of viable viruses in a natural condition microcosm for AdV-GFP and MNV-1 was shorter than in a dark condition microcosm. There was also a negative correlation between the temperature and infectivity of these viruses in both water and sediment samples. Considering that microcosms aim to mimic natural environment conditions and that AdV-GFP and MNV-1 are excellent surrogates for measuring the infectivity of the respective viruses strains, the results presented here have the potential to be applied in future health hazard studies and also would be useful for future climate scenarios.

Detection of hepatitis A virus (HAV) in oysters (Crassostrea gigas).

Because shellfish (oysters, clams, and mussels) are filter-feeders, pathogens become concentrated within them, and human consumption of raw, or under-cooked shellfish can result in disease outbreaks. Identification of hepatitis A virus (HAV) in shellfish has been difficult for several reasons: the concentration of virions in shellfish tissues are very low, detection methods based on in vitro propagation are unreliable, recovery of virions from shellfish tissues is inefficient, and PCR inhibitors in shellfish tissues limit the success of RT-PCR. These facts underlie difficulties in determining cause and effect relationships between hepatitis A outbreaks and detection of HAV contamination in shellfish samples. We have developed a reliable and highly sensitive method for detection of HAV in oyster tissues at low levels (0.001 FFU/ml-fluorescent focus units per milliliter). Our method combines dissection of the gastrointestinal oyster tract, organic extraction before PEG precipitation, and RNA extraction with Trizol LS, followed by RT-PCR and hybridization using a digoxigenin-labeled HAV cDNA probe. Our results will benefit both public health officials concerned about hepatitis A infections caused by consumption of HAV-contaminated oysters and shellfish producers who require reliable methods for quality control of commercial oyster production.

Spatial distribution of enteric viruses and somatic coliphages in a Lagoon used as drinking water source and recreation in Southern Brazil.

This study aimed to evaluate the contamination level of the Peri Lagoon, the main freshwater reservoir of Santa Catarina Island, Southern Brazil, for human adenovirus (HAdV), hepatitis A virus (HAV), rotavirus species A (RVA), and somatic coliphages (SOMCPH). Viruses were also investigated in sediments and their sensitivity against natural sunlight was analysed by studying their spatial distribution in different depths of the water column. A total of 84 water samples and 48 sediment samples were examined by qPCR or RT-qPCR. Infectivity of HAdV and SOMCPH was determined and quantified by plaque assay method. A sum of 64% and 48% of water and sediment samples were positive for HAdV, respectively. RVA was present in 33% and 18% of water and sediment samples, and 25% of water samples were positive for HAV. HAdV were infectious in 76% of water and 83% of sediment samples that were positive by qPCR. SOMCPH could be detected in 42% and 18% of water and sediment samples, respectively. The data pointed a variation of viruses' prevalence according to the different water column depths. These results demonstrated that water sources and sediments contaminated by human wastes could play an important role in the recontamination of water columns harvested for further treatment or used for recreational purposes. These data can be of great value for future risk assessment analysis.

Settling and survival profile of enteric pathogens in the swine effluent for water reuse purpose.

The present study evaluated the pathogens persistence and settling profile in swine effluent. We determined the enteric pathogens settling characteristics, their survival and inactivation profile in swine effluent (for water reuse purpose) and in sludge (generated after aerobic treatment - during secondary settling process). The study was performed in laboratorial-scale and in full-scale (manure treatment plant). Enteric viruses and enteric bacteria were used as biomarkers. Results showed that these enteric pathogens were significantly reduced from swine effluent during secondary settling process, and enteric viruses removal was correlated with the suspended solids decantation. The design of secondary settlers can be adapted to improve pathogens removal, by diminishing the solids loading rate per area and time, ending in higher hydraulic retention times.

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.

Antiviral evaluation of plants from Brazilian Atlantic Tropical Forest.

The antiviral activity of six medicinal plants from Brazilian Atlantic Tropical Forest was investigated against two viruses: herpes simplex virus type 1 (HSV-1) and poliovirus type 2 (PV-2). Cuphea carthagenensis and Tillandsia usneoides extracts showed the best antiherpes activity. T. usneoides dichloromethane, ethyl acetate and n-butanol extracts, and Lippia alba n-butanol extract showed inhibition of HSV-1, strain 29R/acyclovir resistant. In addition, only L. alba ethyl acetate extract showed antipoliovirus activity. These results corroborate that medicinal plants can be a rich source of potential antiviral compounds.

Human and animal enteric virus in groundwater from deep wells, and recreational and network water.

This study was designed to assess the presence of human adenovirus (HAdV), rotavirus-A (RVA), hepatitis A virus (HAV), and porcine circovirus-2 (PCV2) in groundwater from deep wells, and recreational and network waters. The water samples were collected and concentrated and the virus genomes were assessed and quantified by quantitative PCR (qPCR). Infectious HAdV was evaluated in groundwater and network water samples by integrated cell culture using transcribed messenger RNA (mRNA) (ICC-RT-qPCR). In recreational water samples, HAdV was detected in 100 % (6/6), HAV in 66.6 % (4/6), and RVA in 66.6 % (4/6). In network water, HAdV was detected in 100 % (6/6) of the samples (these 83 % contained infectious HAdV), although HAV and RVA were not detected and PCV2 was not evaluated. In groundwater from deep wells, during rainy period, HAdV and RVA were detected in 80 % (4/5) of the samples, and HAV and PCV2 were not detected; however, during dry period, HAdV and RVA were detected in 60 % (3/5), HAV in only one sample, and PCV2 in 60 % (4/5). In groundwater, all samples contained infectious HAdV. PCV2 presence in groundwater is indicative of contamination caused by swine manure in Concórdia, Santa Catarina, Brazil. The disinfection of human and animal wastes is urgent, since they can contaminate surface and groundwater, being a potential threat for public and animal health.

Hepatitis A virus detection in oysters (Crassostrea gigas) in Santa Catarina State, Brazil, by reverse transcription-polymerase chain reaction.

Shellfish are readily contaminated with viruses present in water containing sewage because of the concentration effect of filter feeding. Hepatitis A virus (HAV) is the main cause of acute hepatitis worldwide and may lead to severe illness or even death. It is transmitted through fecal and oral routes and causes widespread endemic and asymptomatic infections in young children. Here we describe a method for the detection of HAV RNA in shellfish involving the extraction of total RNA from oyster meat followed by reverse transcription-polymerase chain reaction (RT-PCR). Virus recovery from oyster extracts artificially seeded with HAV strain HM 175 was examined by RT-PCR. The minimum detection limit was 3.3 focus-forming units of HAV, and the recovery rate was 75.7%. This method was used to assess the viral contamination of four shellfish beds in Santa Catarina State, Brazil, over a 1-year period. Six (22%) of 27 samples collected in autumn and winter from one shellfish bed tested positive for HAV.