ABSTRACT
The application of viral metagenomic techniques and a series of PCRs in a human fecal sample enabled the detection of two novel circular unisense DNA viral genomes with 92% nucleotide similarity. The viruses were tentatively named circo-like virus-Brazil (CLV-BR) strains hs1 and hs2 and have genome lengths of 2526 and 2533 nucleotides, respectively. Four major open reading frames (ORFs) were identified in each of the genomes, and differences between the two genomes were primarily observed in ORF 2. Only ORF 3 showed significant amino acid similarities to a putative rolling circle replication initiator protein (Rep), although with low identity (36%). Our phylogenetic analysis, based on the Rep protein, demonstrated that the CLV-BRs do not cluster with members of the Circoviridae, Nanoviridae or Geminiviridae families and are more closely related to circo-like genomes previously identified in reclaimed water and feces of a wild rodent and of a bat. The CLV-BRs are members of a putative new family of circular Rep-encoding ssDNA viruses. Electron microscopy revealed icosahedral (~23 nm) structures, likely reflecting the novel viruses, and rod-shaped viral particles (~65-460 × 21 × 10 nm in length, diameter, and axial canal, respectively). Circo-like viruses have been detected in stool samples from humans and other mammals (bats, rodents, chimpanzees and bovines), cerebrospinal fluid and sera from humans, as well as samples from many other sources, e.g., insects, meat and the environment. Further studies are needed to classify all novel circular DNA viruses and elucidate their hosts, pathogenicity and evolutionary history.
Subject(s)
Circoviridae/genetics , Circoviridae/ultrastructure , DNA, Viral/chemistry , DNA, Viral/genetics , Genome, Viral , Virion/ultrastructure , Brazil , Circoviridae/classification , Circoviridae/isolation & purification , Cluster Analysis , Feces/virology , Female , Humans , Microscopy, Electron , Middle Aged , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Humans , Animals , Male , Female , Child , Adolescent , Adult , Middle Aged , Chick Embryo , Disease Outbreaks , Vaccinia , Vaccinia virus , Age Distribution , Base Sequence , Brazil , Chlorocebus aethiops , Genes, Viral , Microscopy, Electron , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Vaccinia virus , Vero CellsABSTRACT
Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.
Subject(s)
Disease Outbreaks , Vaccinia virus/isolation & purification , Vaccinia/epidemiology , Adolescent , Adult , Age Distribution , Aged , Animals , Base Sequence , Brazil/epidemiology , Chick Embryo , Child , Chlorocebus aethiops , Chorioallantoic Membrane , Female , Genes, Viral/genetics , Humans , Male , Microscopy, Electron , Middle Aged , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Vaccinia/diagnosis , Vaccinia virus/classification , Vaccinia virus/genetics , Vaccinia virus/ultrastructure , Vero CellsABSTRACT
As linhagens celulares obtidas a partir de larvas do mosquito Aedes albopictus apresentam-se naturalmente infectadas por vírus. O clone C6/36 dessas células, obtido por Igarashi em 1978, mostrou estar livre de vírus e tem sido usado para isolamento do vírus Dengue na maioria dos laboratórios especializados. Em nossos estudos com esse clone, após diferentes períodos de incubaçäo, detectamos a presença de um vírus endogéno. Sobrenadante de cultura de células C6/36, após o 12§ dia de repique, analisado por coloraçäo negativa em microscopia eletrônica, revelou a presença de partículas virais sem envoltório, medindo cerca de 20nm. Esse mesmo teste, quando realizado com células após o 2§, 6§ e 10§ dias de incubaçäo, resultou negativo. As células também foram submetidas à imunofluorescência indireta utilizando anti soros para vários grupos de arbovírus. Os resultados foram negativos, descartando a possibilidade de contaminaçäo laboratorial. A presença de vírus endógeno em células C6/36 pode ocorrer em diferentes laboratórios, sem detectada. Nossos resultados mostram que esse vírus endógeno näo influencia o isolamento do vírus Dengue e a replicaçäo das células. Em razäo da ausência de dados, o emprego dessas células para isolamento de outros vírus deve ser cuidadosamente avaliado
