Lineage variability in surface components expression within Porphyromonas gingivalis.

The periodontopathogen Porphyromonas gingivalis is represented by a spectrum of phenotypes ranging from commensals to pathogenic lineages. Capsule and fimbriae are considered key virulence factors in this specie, involved in colonization and host defenses evasion. Since these virulence traits may not be expressed by certain strains, we aimed to test the hypothesis that certain clusters or genotypes of P. gingivalis correlate with the production of capsule and fimbriae. Sixteen P. gingivalis isolates were evaluated. Capsule (K) was detected by optical microscopy of negatively stained cells. The presence of fimbriae (F) was determined by TEM. Genotypes were determined by NotI macrorestriction fragments analysis through Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST) based on seven house-keeping genes. The phenotypes included F(+)K(+) (n = 4), F(-)K(+) (n = 5), F(+)K(-) (n = 5) and F(-)K(-) (n = 2). The analysis of whole genome macrorestriction fragments revealed 14 different clusters. MLST data also revealed extensive genetic diversity; however, PFGE and MLST profiles showed evident differences. There was no association between P. gingivalis clusters and encapsulated and/or fimbriated phenotypes. Genotyping methods were not able to discriminate isolates according to the production of virulence factors such as capsule and major fimbriae, indicating that recombination played a key role in the expression of capsule and fimbriae in P. gingivalis.

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

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.

Two novel circo-like viruses detected in human feces: complete genome sequencing and electron microscopy analysis.

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.

Rotavirus diarrhea in children and adults in a southern city of Brazil in 2003: distribution of G/P types and finding of a rare G12 strain.

Between May and August in 2003, a total of 251 fecal samples were collected from children and adults with diarrhea (5 inpatients and 246 outpatients) at a private hospital in the city of Ponta Grossa, the state of Paraná, Brazil. Group A rotavirus was detected in 71 of 251 (28.3%) specimens: 55 (77.5%) from children under 5 years of age and 16 (22.5%) from individuals aged 6-72 years. All 71 strains exhibited a "long" RNA pattern when analyzed by PAGE. Sixty-one positive samples that yielded enough RNA were submitted to PCR genotyping. The most frequent G/P genotype combination detected was G1P[8] (86.9%; 53/61) followed by G9P[8] (3.3%; 2/61) and G12P[9] (1.6%; 1/61). Rotaviruses with G2, G3, G4, P[4], or P[6] specificity were not detected. For three strains (4.9%) bearing G1 genotype, the VP4 specificity could no be determined, and two specimens (3.3%) remained G/P non-typeable. One rotavirus strain (HC91) bearing G12P[9] genotype with a "long" electropherotype was isolated from an 11-month-old boy with diarrhea for the first time in Brazil. The cell-culture grown HC91 strain was shown to belong to serotype G12 by neutralization.

Severe acute respiratory syndrome-a global concern-influenza virus isolated from suspected cases in Brazil from April to June 2003.

Severe acute respiratory syndrome (SARS) is a condition associated with substantial morbidity and mortality. Coronavirus has been associated with this severe emerging disease, with a pattern suggesting droplet or contact transmission. From April to June 2003, Institute Adolfo Lutz received 16 respiratory secretions from hospitalized patients with recent history of travel to an area with local transmission of SARS. Rapid antigen detection for influenza A and B, parainfluenza types 1, 2 and 3, respiratory syncytial virus and adenovirus; electron microscopy, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR) and serologic assay were performed. Virus isolation attempts were performed in Hep-2, Vero, MDCK, NCI-H292, MRC-5, LLC-MK2 and FRhK-4. Influenza virus of type A/Panama/2007/99 (H3N2) and A/New Caledonia/20/99 (H1N2) were identified.

Rotavírus, adenovírus, astrovírus, calicivírus e "small round virus particles" em fezes de crianças, com e sem diarréia aguda, no período de 1987 a 1988, na grande São Paulo / Rotavirus, adenovirus, astrovirus, calicivirus and small round virus particles in feces of children with and without acute diarrhea, from 1987 to 1988, in the greater São Paulo

Between 1987 and 1988, 193 faecal specimens from children, with or without diarrhea, were submitted to enzyme immunoassay, polyacrylamide-gel electrophoresis and electronmicroscopy tests for virus detection. The positivity for Rotavirus, Adenovirus, Astrovirus, Calicivirus and Small Round Virus Particles (SRVP) was 11.3, 3.1, 2.1, 1.0 and 4.1, respectively, for the 97 children with acute diarrhea. Of the 96 children without diarrhea, 4.2 were positive for Rotavirus, 1.0 for Calicivirus and 7.3 for SRVP. Of 15 positive specimens for Rotavirus, 14 showed electrophoretic patterns proper to group A and 1 specimen of group C Rotavirus. The analysis of electrophorotypes demonstrated great heterogeneity of electrophoretic patterns and predominance of subgroup 2, "long". The association of virus, bacteria and parasites was present both in children with or without acute diarrhea.

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.

Diagnóstico rápido de vírus do grupo herpes por microscopia eletrônica: - observaçöes atípicas nas amostras de pacientes aidéticos / Rapid diagnostic of herpesvirus group by electron microscopy: atypical observations in the sample of patients with AIDS

A Seçäo de Microscopia Eletrônica do Instituto Adolfo Lutz recebeu no período de 1986-90, 129 amostras de materiais biológicos (líquido vesicular, raspado de lesäo genital, raspado de lesäo anal, saliva e urina) de pacientes aidéticos, com suspeita de infecçäo herpética, para diagnóstico rápido de vírus do grupo Herpes, por microscopia eletrônica. Dessas amostras, 25%(32/129) foram positivas para vírus Herpes, com algumas apresentando aspectos atípicos, tais como: a) grande quantidade de partículas virais; b) detecçäo de mais de um nucleocapsídeo num mesmo envoltório; e c) detecçäo de grandes agregados de nucleocapsídeos sem envoltórios. Estas observaçöes poderiam ser atribuídas às características de imunodepressäo desses pacientes e a sua divulgaçäo foi por nós considerada importante, pois seriam dados adicionais para o estudo de infecçöes virais, nesses pacientes imunodeprimidos

Diagnóstico rápido de vírus por microscopia eletrônica / Rapid virus diagnosis by electron microscopy

A Seçäo de Microscopia Eletrônica(ME) do Instituto Adolfo Lutz(IAL) realiza diagnósticos rápidos de: Herpesvírus, Poxvírus, Papilomavírus, Rotavírus, Adenovírus, Calcivírus, "Small Round Vírus Particles-SRVP", agente de Norwalk, Parvovírus e bacteriófagos em diversas amostras biológicas. Contribui também no controle de qualidade de antígenos virais como Sarampo, Influenza, Rocio, Dengue, Hepatite A e B, HIV e outros. A coloraçäo negativa é uma das técnicas utilizadas no diagnóstico rápido de vários vírus associados às doenças humanas. A morfologia, a subestrutura e o diâmetro säo características importantes para diferenciar esses vírus. Apesar da impossibilidade de se realizar exames em larga escala, do alto custo e da necessidade de técnicos especializados, é uma técnica de fácil execuçäo, com obtençäo rápida do resultado. É útil para detectar vírus de difícil cultivo e é o único método que permite observar mais de um agente numa mesma amostra. Embora existam vários laboratórios de ME em Universidades e em Instituiçöes de Pesquisa, cada laboratório está empenhado em sua linha de pesquisa e säo poucos os que se dedicam à área de diagnóstico rápido de vírus