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.Although water is recognized as the most precious natural resource on our planet, human activities disregard this fact by continually polluting freshwater bodies. Increasing worldwide awareness of the poor quality of potable water has occurred mainly due to the significant increase in human morbidity and mortality. More than 2.2 million people die every year from diseases associated with poor quality water and sanitary conditions, mostly in developing countries. The presence of pathogenic enteric microorganisms in aquatic environments reveals how human health can be affected by contamination from sewage discharge into surface waters.

Multiply primed rolling-circle amplification is a novel technology that uses bacteriophage phi29 DNA polymerase to amplify circular DNA molecules, without the need for prior knowledge of their sequences. In an attempt to detect Torque teno virus (TTV), rolling-circle amplification was used to amplify DNA extracted from eight human and four pig serum samples. All samples gave high molecular weight (>30 kb) amplification products. By restriction endonuclease digestion, these products generated DNA fragments whose sizes were consistent with those of human TTV (3?8 kb) and swine TTV (Sd-TTV; 2?9 kb) genomes. Two TTV isolates derived from a single AIDS patient, as well as two Sd-TTV isolates derived from a single pig, were characterized by complete nucleotide sequencing. One of the Sd-TTV isolates showed very low (43-45 %) nucleotide sequence similarity to the other Sd-TTV isolate and to the prototype isolate Sd-TTV31, and could be considered the prototype of a novel genogroup.Torque teno virus (TTV) is a non-enveloped, singlestranded, circular DNA virus with a genomic length of 3?4-3?9 kb (Nishizawa et al., 1997;Miyata et al., 1999;Mushahwar et al., 1999) and has been recently classified into a novel, floating genus called Anellovirus (Biagini et al., 2005). TTV is found in the plasma of >80 % of the human population worldwide (Prescott & Simmonds, 1998;Takahashi et al., 1998;Niel et al., 1999). Co-infection of single individuals with multiple TTV isolates is frequent (Takayama et al., 1999;Niel et al., 2000). TTV has a wide genetic diversity and virus isolates have been classified into five main phylogenetic groups (1-5) with low nucleotide sequence similarity between them (Peng et al., 2002). Anelloviruses are not restricted to human hosts and have also been detected in non-human primates, tupaias, cats, dogs and pigs Verschoor et al., 1999;Okamoto et al., 2001Okamoto et al., , 2002. However, few complete nucleotide sequences from animal TTVs have been reported.In their natural replication cycle, some DNA viruses, like circoviruses, employ a rolling-circle mechanism to propagate their circular genomes. Multiply primed rolling-circle amplification is a novel technique able to amplify circular DNA molecules such as plasmids with great efficiency (Dean et al., 2001). The method utilizes bacteriophage phi29 DNA polymerase, a high-fidelity enzyme, with a strong strand-displacing capability, high processivity and proofreading activity (Garmendia et al., 1992;Esteban et al., 1993). Unlike PCR, the primers used in the amplification reaction are random hexamers. Previous knowledge of the nucleotide sequences to be amplified therefore is not necessary. Furthermore, phi29 DNA polymerase is very stable, with linear kinetics at 30 uC for over 12 h, eliminating the need for thermal cycling. The reaction products are high molecular weight, linear, double-stranded, tandem-repeat copies of the input DNA that can subsequently be digested with restriction endonucleases.In this study, multiply primed rolling-circle amplification was ...

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Aims:  A one‐year survey was conducted to examine hepatitis A virus (HAV) prevalence, distribution of genotypes and their relationship to bacterial indicators in raw and treated sewage samples. Methods and Results:  Fifty sewage samples (raw = 25 and treated = 25) were collected twice monthly from one sewage treatment plant in Rio de Janeiro. Virus concentration was performed by adsorption to an electronegative membrane followed by ultrafiltration. Viral RNA was detected by nested reverse transcriptase‐polymerase chain reaction (RT‐PCR) and real‐time PCR and positive products were directly sequenced. Total and faecal coliform concentrations were also determined. By nested RT‐PCR, HAV RNA was detected in 16/50 (32%) and eight (16%) of them were found in treated sewage samples. By real‐time PCR, HAV RNA was detected in 46/50 (92%) samples and 24 were from treated sewage. Phylogenetic analyses classified nine isolates (56%) as subgenotype IA and seven (44%) as IB. Conclusions:  Real‐time PCR was more sensitive than nested RT‐PCR; the presence of subgenotypes IA and IB was described and bacterial indicators cannot be used to predict HAV presence in sewage. Significance and Impact of the Study:  These results demonstrated that HAV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV infection.