Enteric viruses excreted in feces from infected individuals dispersed in aquatic environments by sewage discharge. Among these viruses, the norovirus (NoV) is actually considered the main cause of gastroenteritis outbreaks worldwide, resulting from the ingestion of contaminated food and water as well as is also associated with hospitalizations. This research aimed to detect and partially characterize the human NoV (GI/GII) in different water matrices and in untreated sewage from Metropolitan Region of Belem. The study involved superficial waters from bay (Ver-o-Peso), river (Acai’s Port), stream (Tucunduba) and two lakes (Bolonha and Agua Preta), as well as treated water (WTP-Bolonha) and untreated sewage (SLP-UNA), monthly collected over two years . The water and sewage (2 liters) were initially concentrated on filtering membranes to obtain a final volume of 2 mL. The nucleic acid was extracted by silica method and submitted to semi nested RT-PCR (reverse transcription Polymerase chain reaction) using NoV GI and GII specific primers. The cDNA obtained after reverse transcription was also used to investigate the GI/GII by TaqMan® real time PCR. The positive samples for both molecular methods were analyzed for 5’end ORF2 by nested (for GI) and semi nested (for GII) in order to obtain amplicon for identification of circulating strains, being further purified using a commercial kit and submitted to molecular characterization in the automated sequencer. The obtained sequences were edited, aligned and compared to others available in gene bank (NCBI) and in the site NoV genotyping tool. In the period of November 2008 to October 2010, 168 water and sewage samples were collected and analyzed for NoV presence, obtaining a positivity of 33.9% (57/168) of which 21.1% (12/57) were positive only by TaqMan® real time PCR, 19.3% (11/57) only by semi nested and 59.6% (34/57) for both. Considering the two methodologies used, in the positive cases GI (82.5% - 47/57) was most frequent than GII (79.0% - 45/57). However, in most samples there was coexistence of the two genogroups (61.4% - 35/57), mainly in the Tucunduba and SLP-UNA samples, considered the most NoV contaminated sites. On the other hand, in WTP-Bolonha this agent was not found. Of 57 positive samples by TaqMan® real time PCR and/or semi nested RT-PCR, 53 were retested for 5’end ORF2, since four samples showed insufficient quantity of material which allowed a new analyze, so, in 47.2% (25/53) the NoV genome was detected, of these 12% (3/25) belonging to GI, 24% (6/25) to GII and 64% (16/25) for both. The most frequent GI and GII genotypes were GI.8 (n=8) and GII.4 (n=12), respectively, but others genotypes were also observed with lower incidence as GII.6 (n=3), GII.9 (n=2), GII.12 (n=1), GII.14 (n=1), GI.1 (n=1) and GI.4 (n=2). Due to low quality of sequences obtained, eight samples could not be genotyped for GI and three for GII. Of 96 samples with concentration of thermotolerant coliforms above the recommended, 34 (35.4%) were also NoV positive. Increase on conductivity and total dissolved solids was observed in materials from Ver-o-Peso and Tucunduba, as well as the turbidity was notably higher in these places and the Acai’s Port. In the less rainy period (July to November) there was a trend in positivity increasing for NoV, and in the highest rainfall (December to June) a decrease in the incidence of this agent was noted. The results obtained in the present study indicate the circulation of NoV GI and GII in aquatic environments in Belem, revealing the degradation that these water bodies have suffered, as a result of poverty or lack of sanitation in our city, allowing the permanence of pathogens in these ecosystems, along with its effluents.
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