Estudos ultra-estruturais e imunocitoquímicos da infecção pelo vírus da hepatite C em macacos rhesus / Ultrastructural and immunocitochemistry studies of the infection for the virus of hepatites C in rhesus monkey

Alterações morfológicas do tecido hepático de macaco rhesus (Macaca mulatta) infectado experimentalmente com o vírus da hepatite C (HCV) foram analisadas utilizando técnicas em microscopia eletrônica. A localização do RNA e de proteínas específicas do HCV nos hepatócitos foi demonstrada por meio das técnicas de hibridização in situ e imuno-microscopia eletrônica. As modificações no tecido hepático mais marcantes foram observadas entre a quinta e sétima semana pós-inoculação. Numerosos hepatócitos apresentaram, progressivamente, citoplasma rarefeito e vacuolizado, culminando com a citólise na sétima semana. Na oitava semana alguns hepatócitos já exibiam sinais regenerativos, indicados pelo aumento de células binucleadas e recuperação da individualização celular. A proliferação reticular de membranas e vésículas derivadas do retículo endoplasmático rugoso (REr) observada no citoplasma dos hepatócitos do macaco rhesus infectado evidenciou semelhança com a do HCV encontrado em chimpanzé, em culturas celulares que abrigam os réplicons do HCV. Para a localização das proteínas virais, cortes de amostras de fígado incluídas em LR-gold, foram incubados com anticorpos monoclonais contra a proteína do nucleocapsídeo e a proteína NS5A e com anticorpo policlonal contra as proteínas E1, E2, NS3 e NS4, seguido de incubação com o conjugado proteína A-ouro coloidal. A proteína do nucleocapsídeo foi localizada sobre membranas do REr que envolvem as mitocôndrias e diretamente associada à membrana externa da mitocôndria. As proteínas não-estruturais foram localizadas em vesículas e cisternas dilatadas do REr. Na localização do RNA viral pela técnica de hibridização in situ foram utilizadas sondas de fita negativa complementar e fita positiva, utilizando para ambas, oligonucleotídeos específicos. As observações ultra-estruturais evidenciaram marcações específicas sobre membranas do REr e sobre vesículas dentro das cisternas do REr que se formam durante a infecção por HCV...

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque.

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report

This preliminary report describes human and cow cases of poxvirus that recently ocurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11). Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures

Para detecçäo de micoplasmas em soros e culturas de células foi aplicada a microscopia eletrônica de transmissäo. Os soros foram centrifugados em alta rotaçäo, enquanto que aos detritos celulares aplicou-se uma centrifugaçäo mais lenta. Os sedimentos ressuspendidos foram preparados por meio de contrastaçäo negativa. Por esta técnica detectou-se contaminaçöes por micoplasmas com maior freqüência do que pela aplicaçäo do corante fluorescente direto (DAPI) em culturas celulares com micoplasmas, servindo de controle. A aparência das bordas celulares dos micoplasmas e de seu conteúdo fornece alguns dados sobre a viabilidade das partículas