Dengue virus identification by transmission electron microscopy and molecular methods in fatal dengue hemorrhagic fever.

Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.

Characterization of the HCV core virus-like particles produced in the methylotrophic yeast Pichia pastoris.

Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly.

Plasmid DNA-recombinant Opc protein complexes for nasal DNA immunization.

The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMVbeta-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMVbeta-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA-protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against beta-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to beta-galactosidase induced by DNA-protein complexes was also detected.

Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli.

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli.

Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1).

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.

Characterization of a virus isolated from the cerebrospinal fluid of patients with epidemic neuropathy.

A previously unknown disease, termed epidemic neuropathy (EN), occurred in Cuba between 1991 and 1993. When samples of cerebrospinal fluid (CSF) from 45 patients with EN and 11 controls were inoculated into cultures of VERO cells, almost all (93%) of the samples from the cases of EN but only one (9%) of the control samples produced a slowly progressing cytopathological effect (CPE). Although the results of other studies indicated the presence of a picornavirus-like virus in CSF samples from EN cases, the CPE and other physico-chemical characteristics observed were not those expected of picorn-viruses. Several aetiological factors may have contributed to EN but at least one virus could have played a major role.

Effect of policosanol on foam-cell formation in carrageenan-induced granulomas in rats.

Policosanol is a new cholesterol-lowering drug isolated and purified from sugar-cane wax, which prevents the development of lipofundin-induced lesions and foam-cell formation in New Zealand rabbits and Wistar rats. This study was conducted to examine the effects of policosanol on foam-cell formation in carrageenan-induced granulomas in rats. Eighteen Wistar rats were randomly distributed in three experimental groups which received orally for 20 days Tween 20 H2O as vehicle (control group) or policosanol at 2.5 or 25 mg kg-1. At the 11th day, lipofundin was injected intraperitoneally for 8 days to induce formation of foam cells in the granuloma. At day 13, carrageenan was injected subcutaneously for granuloma induction and seven days later animals were killed. A significant reduction of the foam-cell formation in granulomas of policosanol-treated rats was observed. It is concluded that policosanol prevents the development of foam cells in carrageenan-induced granulomas (extravascular medium) in rats.

Effect of policosanol on lipofundin-induced atherosclerotic lesions in rats.

Policosanol is a mixture of higher aliphatic alcohols isolated from sugar cane wax, showing cholesterol-lowering effects and preventing the development of lipofundin-induced lesions in New Zealand rabbits. This study was conducted to determine whether policosanol orally administered to rats also protects against the development of lipofundin-induced atherosclerotic lesions. Fifty four male Wistar rats were randomly distributed amongst a negative control group, a positive control group intravenously injected with lipofundin for eight days, and four experimental groups also injected with lipofundin, but orally receiving policosanol at 0.5, 2.5, 5 and 25 mg kg-1, respectively. Policosanol treatment was orally administered once-a-day for eight days, while control groups similarly received equivalent amounts of vehicle. A significant reduction of the atherosclerotic lesions in the treated animals was observed. It is concluded that policosanol has a protective effect on lipofundin-induced aortic lesions in Wistar rats.