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This study aimed to assess anthropogenic impact of surrounding population in the Private Reserve of Natural Heritage at Pantanal, the world's largest freshwater wetland ecosystem located in the centre of South America. Viral aetiological agents of acute gastroenteritis as rotavirus A (RVA), noroviruses, human adenoviruses, klassevirus and of hepatitis, as hepatitis A virus, were investigated in different aquatic matrices. Annual collection campaigns were carried out from 2009 to 2012, alternating dry and rainy seasons. Viral particles present in the samples were concentrated by the adsorption-elution method, with negatively charged membranes, and detected by qualitative and quantitative PCR. From a total of 43 samples at least one virus was detected in 65% (28) of them. Viruses were detected in all matrices with concentrations ranging from 2 × 10This study is part of a collaborative project designed to investigate the environmental and health conditions of the Private Reserve of Natural Heritage at Pantanal, the largest seasonally flooded wetland in the world. The project aimed to promote health and quality of human and wildlife extending technical-scientific knowledge about pathogens present in the region. By assessing the occurrence of human enteric viruses in different water matrices we demonstrated the anthropogenic impact of surrounding population and pointed out the potential risk of infection by exposure of susceptible individuals.

The preservation of water resources is one of the goals of the designation of parks that act as natural reservoirs. In order to assess the impact of the presence of humans in an environmental preservation area bordering urban areas, the presence of four pathogenic enteric viruses [group A rotavirus (RV-A), norovirus (NoV), human adenoviruses (HAdV), and hepatitis A virus (HAV)], as well as the physico-chemical parameters, and Escherichia coli levels were assessed in riverine water samples. From June 2008 to May 2009, monthly monitoring was performed along the Engenho Novo River. RV-A, NoV, and HAdV were observed in 29% (31/108) of the water samples, with concentrations of up to 10(3) genome copies/liter. The natural occurrence of infectious HAdV was demonstrated by Integrated Cell Culture-PCR (ICC-PCR). This study confirms the suitability of using the detection of fecal-oral transmitted viruses as a marker of human fecal contamination in water matrices and indicates the spread of pathogenic viruses occurring in an alleged area of environmental protection.

The aim of this study was to determine the occurrences of the group A rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV) in the surface waters of an urban lagoon (Rodrigo de Freitas Lagoon) in the city of Rio de Janeiro, Brazil. During one year of surveillance, water samples were obtained from the lagoon and other interconnected ecosystems (river and beach). The samples were concentrated using an adsorption-elution method with a negatively charged membrane and tested by qualitative and quantitative polymerase chain reaction assays. RVA was the most prevalent virus detected (24.3%) with a viral load ranging from 3.0 x 10¹-5.6 x 10(4) genome copies/L, followed by NoV (18.8%) and HAdV (16.7%). Considering water samples suitable for bathing, according to Escherichia coli criterion (< 2,000 most probable number/100 mL), viruses were detected in 50% (57/114) of them. Physicochemical parameters were also measured and showed possible correlations between turbidity and RVA presence and between pH and NoV presence. These data demonstrate the importance of considering viral parameters to ensure water quality and the utilisation of these parameters as additional tools for the characterisation of environmental contamination.

Polyomavirus JC (JCPyV) is largely excreted by the human population through the urinary route and has been recognized as a potential viral marker for human waste contamination. This study aims to investigate the dissemination of JCPyV in waste water from a sewage treatment plant (STP) located in Rio de Janeiro, Brazil, and to describe the prevalence of JCPyV subtypes currently present in this population. Raw and treated sewage samples were collected bimonthly during one year, and examined for the presence of JCPyV using nested polymerase chain reaction (nPCR) and quantitative real time PCR (qPCR). JCPyV was detected by nPCR in 96% and 43% of raw and treated sewage samples, respectively. The concentration of JCPyV present in the samples ranged from 1.2 × 103 to 3.2 × 105 and 2.6 × 102 to 6.2 × 103 genome copies per 2 ml of concentrated raw and treated sewage sample, respectively. The strains were characterized and the obtained nucleotide sequences indicated that the detected JCPyV strains clustered with subtypes of East African, West African and European origin. To our knowledge, this is the first study describing the incidence and diversity of JCPyV strains in raw and treated sewage in Brazil.

Norovirus (NoV) is one of the most important aetiological agents of acute gastroenteritis both in developed and developing countries. NoV is shed in high concentrations by infected persons and contaminates recreational and drinking water through sewage discharge into the environment. The aim of this study was to determine the prevalence, genotypes and removal ratio of NoV by PCR, seminested-PCR and quantitative PCR (qPCR) assays in a sewage treatment plant in Rio de Janeiro city, Brazil, during one year of surveillance. NoV was detected in 7 (15%), 14 (29%) and 28 (58%) samples using PCR, seminested-PCR and qPCR, respectively. The mean removal ratio for the activated sludge process was 0.6 log10 for NoV genogroup I (GI) and 0.32 log10 for NoV genogroup II (GII). The peak NoV concentration was detected in the coldest months, with 53,300 genomic copies/litre. Nucleotide sequencing and phylogenetic analysis revealed that five strains clustered with GI strains and six with GII strains. This study demonstrates that NoV spreads into the environment despite the sewage treatment process and remains a source of waterborne outbreaks of acute gastroenteritis.

Rotaviruses A (RV-A) infection is the most common cause of acute diarrheal diseases in infants and the dissemination of these viruses in the environment represents a public health hazard. The present study aims to evaluate reverse transcription-polymerase chain reaction (RT-PCR) based protocols for the detection of RV-A genes in different types of environmental samples. RV-A were concentrated by the adsorption-elution method using negatively charged membranes associated with a Centriprep Concentrator 50. The RV-A VP4, VP7 and VP6 genes were detected using RT-PCR in river water from the Amazon Hydrographic basin (Northern region) and from wastewater in a sewage treatment plant in Rio de Janeiro (Southeast region), Brazil. RV-A were successfully detected in water environmental samples by the methods used. The detection of the VP6 gene by RT-PCR was the most sensitive for detecting RV-A in environmental samples (44.0%), when compared to the detection of the VP4 (33.3%) and VP7 (25.3%) genes. Based on nucleotide sequence and phylogenetic analysis of the partial VP6 gene, 22 environmental samples were determined to be subgroup II (Wa-like). These results indicate that analysis of environmental samples could possibly make a valuable contribution to studies on the epidemiology of RV-A.

Human astroviruses (HAstV) and noroviruses (NoV) are shed frequently at high concentrations and persist for long periods in the environment, raising a significant health risk of water-related gastroenteritis. The aim of this study was to evaluate an adsorption-elution method with an HA (mixed cellulose esters) negatively charged membrane to determine the best recovery of HAstV and NoV from different environmental waters. As the presence of MgCl(2) affects viral adsorption onto the membrane, three different MgCl(2) concentrations were evaluated. The best recovery of both NoV and HAstV from mineral and river water samples was between 18% and 64%, while recovery from tap water and sea water samples was between 3% and 14%. These results suggest that detection and recovery of each enteric virus with this adsorption-elution method requires a specific MgCl(2) concentration and depends on the source of environmental water tested. The combination of the HA negatively charged membrane to concentrate viruses with quantitative PCR detection allows for the identification of gastroenteritis viruses implicated in acute outbreaks of gastroenteritis.

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

An explosive epidemic of DENV-3 in 2002 was the most severe dengue epidemic reported in Brazil since dengue viruses were introduced., During the summer of 2002, Rio de Janeiro had a large epidemic of dengue fever; 288,245 cases were reported. A subset of 1,831 dengue hemorrhagic fever cases occurred. In this study, performed in the first half of 2002, samples from 1,559 patients with suspected cases of dengue infection were analyzed. From this total, 1,497 were obtained from patients with nonfatal cases, and 62 were obtained from patients with fatal cases. By the use of different methods, 831 (53.3%) cases, including 40 fatal cases, were confirmed as dengue infection. When virus identification was successful, dengue virus type 3 (DENV-3) was obtained in 99% of cases. Neurologic involvement was shown in 1 patient with encephalitis, confirmed by the detection of DENV-3 RNA in the cerebrospinal fluid. This explosive epidemic of DENV-3 was the most severe dengue epidemic reported in Brazil since dengue viruses were introduced in 1986.

This study presents the results obtained in the monitoring of dengue virus (DENV) transmission in the Greater Metropolitan Region of the State of Rio de Janeiro, in the period 2000-2001. A total of 5324 serum samples from suspected cases of dengue were analysed in order to confirm dengue infection. The introduction of DENV-3 to the region in December 2000 resulted in the co-circulation of three serotypes: DENV-1, DENV-2 and DENV-3. In this study, virus isolation and/or reverse transcriptase PCR (RT-PCR) confirmed 52.3% (42/79) of DENV-3 cases, showing the importance of acute serum samples in the virological surveillance of the disease. Despite the introduction of a new serotype, an outbreak due to DENV-1 was observed in the municipality of Niteroi. The restriction site-specific PCR (RSS-PCR) patterns obtained for DENV-1 and DENV-2 isolated in that period showed that those strains belonged to the subtypes previously circulating in the state. DENV-3 RSS-PCR patterns confirmed that these viruses belonged to subtype C (Sri Lanka/India strains), represented by the strain circulating on the American continent. These data showed the importance of an active surveillance programme in countries where dengue is endemic.

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7% of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2% and 4.3% was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.