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.

Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis

Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).

O vírus Rocio (ROCV) foi responsável por uma explosiva epidemia de encefalite que ocorreu nos anos 70 afetando cerca de 1.000 habitantes de 20 municípios litorâneos do Estado de São Paulo, Brasil. ROCV foi isolado em 1975 de cerebelo de caso humano fatal de encefalite. As manifestações clínicas da doença são semelhantes àquelas descritas para encefalite St. Louis. ROCV apresenta intensa reatividade cruzada com os vírus do Complexo da Encefalite Japonesa (JEV), particularmente com o vírus Ilhéus (ILHV) e com os vírus das encefalites St. Louis, Murray Valley e West Nile. Neste estudo, relatamos o desenvolvimento de um teste de RT-PCR específico para diagnóstico de ROCV e a caracterização molecular das cepas SPAn37630 e SPH37623. Foi realizada a análise filogenética das seqüências parciais dos genes NS5 e E, de ambas as cepas. Os resultados indicaram que essas cepas são intimamente relacionadas ao complexo JEV, mas formando um subgrupo com o ILHV, de acordo com os resultados recentemente publicados por MEDEIROS et al. (2007).

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

A partir de outubro de 2001, o Instituto Adolfo Lutz tem recebido amostras de líquido vesicular e crostas de lesões de pele de pacientes das regiões do Vale do Paraíba, Estado de São Paulo e do Vale do São Patricio, Estado de Goiás. Os dados clínicos e epidemiológicos sugeriam que os surtos poderiam ser causados por Cowpox virus ou Vaccinia virus. A maioria dos pacientes era ordenhadores que tinham lesões vesicopustulares nas mãos, braços, antebraços e alguns na face. A análise por microscopia eletrônica direta (MED) detectou partículas com morfologia de vírus do gênero Orthopoxvirus em amostras de 49 (66,21%) pacientes dos 74 analisados. Os vírus foram isolados em membrana corioalantóide (MCA) de ovo embrionado de galinha e em linhagem celular Vero com confirmação por MED e PCR. Das 21 amostras de lesões submetidas ao PCR utilizando iniciadores para o gene da hemaglutinina (HA), 19 foram positivas. A digestão por enzima de restrição TaqI resultou em quatro fragmentos característicos de Vaccinia virus. A análise nucleotídica do seqüenciamento revelou que esses vírus apresentam 99,9% de similaridade com o Cantagalo virus, descrito como uma cepa de Vaccinia virus, havendo apenas alteração de um nucleotídeo na posição 616 com mudança de um aminoácido na proteína na posição 206. A análise filogenética mostrou que os isolados se agruparam junto aos Cantagalo virus, outras cepas de Vaccinia virus e Rabbitpox virus.