Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, São Paulo / Estudo longitudinal da ocorrência de adenovírus e vírus da hepatite A em esgoto doméstico na cidade de Limeira, SP

The aim of this study was to verify the presence and annual distribution of adenoviruses and hepatitis A virus in domestic sewage in the city of Limeira, São Paulo. Fifty samples with a volume of 8 liters each were collected weekly from December 2004 to December 2005. The viruses were concentrated by filtration through positively charged ZP60S filter membranes, followed by ultracentrifugation. Human adenoviruses (HAdV) were detected by PCR followed by nested-PCR and screening for species F was done by restriction of the PCR product with TaqI endonuclease. Virus infectivity assays were performed by inoculation of concentrates onto HEp-2 cell monolayers. RT-PCR was used for the detection of hepatitis A virus. HAdV were detected in all samples, and 64% of samples were positive for infectious virus. Species F was present in 82% of the samples. Hepatitis A virus was detected in 48% of the samples. These results demonstrate that HAdV and HAV were present in the domestic sewage of Limeira throughout the period of study, demonstrating the importance of an adequate treatment before the disposal in the environment.

O objetivo do estudo foi verificar a ocorrência e a distribuição anual de adenovírus humanos e vírus da Hepatite A (VHA) no efluente doméstico da cidade de Limeira, São Paulo, ao longo do período de Dezembro de 2004 e Dezembro de 2005, com vistas à futura implementação de sistemas de tratmento de água de esgoto. Cinquenta amostras de efluente bruto com volume de 8L cada foram colhidas semanalmente e os vírus concentrados por filtração em membrana eletropositiva ZP60S, seguida de ultracentrifugação. Adenovírus foram detectados por PCR e nested-PCR. Adenovírus da espécie F foram distinguidos das demais por restrição do produto da PCR com endonuclease TaqI. Ensaios de infectividade viral foram realizados em culturas de células HEp-2. A presença do vírus da hepatite A também foi pesquisada nas mesmas amostras, fazendo-se uso de método de RT-PCR. Adenovírus foram detectados em todas as amostras, sendo a espécie F identificada em 82% destas. Sessenta e quatro por cento dos adenovírus detectados ainda estavam infecciosos. O vírus da Hepatite A foi detectado em 48% das amostras examinadas. Estes resultados evidenciam a presença e a circulação de Adenovírus humano e VHA nas águas de esgoto doméstico de Limeira ao longo do período de estudo, demonstrando a importância de um tratamento adequado desse material antes da disposição no meio ambiente.

A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins