Identification of circo-like virus-Brazil genomic sequences in raw sewage from the metropolitan area of São Paulo: evidence of circulation two and three years after the first detection

BACKGROUND Two novel viruses named circo-like virus-Brazil (CLV-BR) hs1 and hs2 were previously discovered in a Brazilian human fecal sample through metagenomics. CLV-BR hs1 and hs2 possess a small circular DNA genome encoding a replication initiator protein (Rep), and the two genomes exhibit 92% nucleotide identity with each other. Phylogenetic analysis based on the Rep protein showed that CLV-BRs do not cluster with circoviruses, nanoviruses, geminiviruses or cycloviruses. OBJECTIVE The aim of this study was to search for CLV-BR genomes in sewage and reclaimed water samples from the metropolitan area of São Paulo, Brazil, to verify whether the first detection of these viruses was an isolated finding. METHODS Sewage and reclaimed water samples collected concomitantly during the years 2005-2006 were purified and concentrated using methodologies designed for the study of viruses. A total of 177 treated reclaimed water samples were grouped into five pools, as were 177 treated raw sewage samples. Nucleic acid extraction, polymerase chain reaction (PCR) amplification and Sanger sequencing were then performed.e FINDINGS CLV-BR genomes were detected in two pools of sewage samples, p6 and p9. Approximately 28% and 51% of the CLV-BR genome was amplified from p6 and p9, respectively, including 76% of the Rep gene. The detected genomes are most likely related to CLV-BR hs1. Comparative analysis showed several synonymous substitutions within Rep-encoding sequences, suggesting purifying selection for this gene, as has been observed for other eukaryotic circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. MAIN CONCLUSION The results therefore indicated that CLV-BR has continued to circulate in Brazil two and three years after first being detected.

Identification of circo-like virus-Brazil genomic sequences in raw sewage from the metropolitan area of São Paulo: evidence of circulation two and three years after the first detection.

BACKGROUND: Two novel viruses named circo-like virus-Brazil (CLV-BR) hs1 and hs2 were previously discovered in a Brazilian human fecal sample through metagenomics. CLV-BR hs1 and hs2 possess a small circular DNA genome encoding a replication initiator protein (Rep), and the two genomes exhibit 92% nucleotide identity with each other. Phylogenetic analysis based on the Rep protein showed that CLV-BRs do not cluster with circoviruses, nanoviruses, geminiviruses or cycloviruses. OBJECTIVE: The aim of this study was to search for CLV-BR genomes in sewage and reclaimed water samples from the metropolitan area of São Paulo, Brazil, to verify whether the first detection of these viruses was an isolated finding. METHODS: Sewage and reclaimed water samples collected concomitantly during the years 2005-2006 were purified and concentrated using methodologies designed for the study of viruses. A total of 177 treated reclaimed water samples were grouped into five pools, as were 177 treated raw sewage samples. Nucleic acid extraction, polymerase chain reaction (PCR) amplification and Sanger sequencing were then performed.e. FINDINGS CLV-BR: genomes were detected in two pools of sewage samples, p6 and p9. Approximately 28% and 51% of the CLV-BR genome was amplified from p6 and p9, respectively, including 76% of the Rep gene. The detected genomes are most likely related to CLV-BR hs1. Comparative analysis showed several synonymous substitutions within Rep-encoding sequences, suggesting purifying selection for this gene, as has been observed for other eukaryotic circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. MAIN CONCLUSION: The results therefore indicated that CLV-BR has continued to circulate in Brazil two and three years after first being detected.

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells.

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

The purpose of this work was to acquire an overview of the infectious cycle of HAdV-41 in permissive HEK 293 cells and compare it to that observed with the prototype of the genus, Human adenovirus C HAdV-2. HEK 293 cells were infected with each virus separately and were harvested every 12 h for seven days. Infection kinetics were analysed using confocal and electronic microscopy. The results show that, when properly cultivated, HAdV-41 was not fastidious. It had a longer multiplication cycle, which resulted in the release of complete viral particles and viral stocks reached high titres. After 60 h of infection, the export of viral proteins from the infected cell to the extracellular milieu was observed, with a pattern similar to that previously described for HAdV-2 penton-base trafficking after 30 h of infection. HAdV-41 had a non-lytic cycle and the infection spread from the first infected cell to its neighbours. The release process of the viral particles is unknown. The results observed for HAdV-41 infection in HEK 293 cells show how different this virus is from the prototype HAdV-2 and provides information for the development of this vector for use in gene therapy.

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.

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira, SÃo Paulo.

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.