Resumo Os poliomavírus humanos JC e BK (JCPyV e BKPyV) são virus ubíquos, espécie-específicos, pertencentes à família Polyomaviridae. Estes vírus são conhecidos por serem excretados pela urina humana, sendo considerados potenciais indicadores de contaminação por águas residuais urbanas. Buscando acessar a distribuição de JCPyV e BKPyV em amostras de águas coletadas de uma estação de tratamento de esgoto e de um arroio canalizado de Porto Alegre, Brasil, duas técnicas de nested-PCR foram otimizadas e aplicadas às amostras coletadas. Os amplificados obtidos foram submetidos ao sequenciamento e suas sequências analisadas com base em sequências de poliomavírus humanos previamente depositadas no GenBank. Doze de 30 amostras de água (40%) foram positivas para JCPyV, enquanto 6 amostras (20%) foram positivas para BKPyV. Os resultados do sequenciamento confirmaram a presença dos subtipos 1 e 3 de JCPyV, enquanto apenas os BKPyV Ia e Ib foram encontrados. Este estudo demonstra pela primeira vez a presença de poliomavírus humanos em águas superficiais e em amostras coletadas em uma estação de tratamento de esgoto na região sul do Brasil.

Abstract The human polyomaviruses JC and BK (JCPyV and BKPyV) are ubiquitous, species-specific viruses that belong to the family Polyomaviridae. These viruses are known to be excreted in human urine, and they are potential indicators of human wastewater contamination. In order to assess the distribution of both JCPyV and BKPyV in urban water samples collected from a sewage treatment plant (STP) and from a canalized water stream of Porto Alegre, Brazil, two nested-PCR assays were optimized and applied to the samples collected. The amplicons obtained were submitted to sequencing, and the sequences were analyzed with sequences of human polyomaviruses previously deposited in GenBank. Twelve out of 30 water samples (40%) were JCPyV positive, whereas six samples (20%) were BKPyV positive. The sequencing results confirmed the presence of JCPyV subtypes 1 and 3, whereas only BKPyV Ia and Ib were found. This study shows for the first time the presence of human polyomaviruses in surface water and in samples collected in a sewage treatment plant in southern Brazil.

Resumo O presente estudo analisou a eficiência do processo de fotoeletrooxidação como metodologia para a degradação e inativação de adenovírus em água. A concepção experimental emprega uma solução preparada a partir de água estéril contendo 5,107 cópias genômicas/L (gc/L) de uma amostra padrão de adenovírus humano tipo 5 (HAdV-5), dividida em duas partes iguais, uma para servir como controle e outra tratada por fotoeletrooxidação (PEO) durante 3 horas e com uma corrente de 5A. As amostras recolhidas durante o processo de exposição foram analisadas por PCR quantitativo em tempo real (qPCR) para identificação e quantificação do genoma viral. Antes da extração de ácidos nucleicos, um passo de tratamento com DNAse paralelo foi realizado para avaliar a integridade das partículas virais. Um ensaio de qPCR integrado à cultura de células (ICC-qPCR) permitiu analisar a viabilidade de infecção em uma cultura de células. O processo mostrou-se eficaz testada para a degradação viral, com uma redução de 7 log10 da carga viral após 60 minutos de tratamento. As amostras tratadas com DNAse exibiram redução completa da carga viral após uma exposição de 75 minutos ao processo, e a análise de ICC-qPCR mostrou partículas virais completamente não-viáveis em 30 minutos de tratamento.

The present study analyzed the efficiency of the photo-electro-oxidation process as a method for degradation and inactivation of adenovirus in water. The experimental design employed a solution prepared from sterile water containing 5.107 genomic copies/L (gc/L) of a standard strain of human adenovirus type 5 (HAdV-5) divided into two equal parts, one to serve as control and one treated by photo-electro-oxidation (PEO) for 3 hours and with a 5A current. Samples collected throughout the exposure process were analyzed by real-time polymerase chain reaction (qPCR) for viral genome identification and quantitation. Prior to gene extraction, a parallel DNAse treatment step was carried out to assess the integrity of viral particles. Integrated cell culture (ICC) analyses assessed the viability of infection in a cell culture. The tested process proved effective for viral degradation, with a 7 log10 reduction in viral load after 60 minutes of treatment. The DNAse-treated samples exhibited complete reduction of viral load after a 75 minute exposure to the process, and ICC analyses showed completely non-viable viral particles at 30 minutes of treatment.

The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines.