Virus reproduction and the time course of changes in liver and kidney functions and in the blood clotting system were studied in the visceral organs of green monkeys and baboons infected with Ebola virus (subtype Zaire). It was shown that monocytes and macrophages were the first cells to be infected with the virus, followed by hepatocytes, adrenocorticocytes, fibroblasts, and endotheliocytes. The early and late pathologic changes in the monkey organs are described. Biochemical data on changes in blood clotting and liver and kidney functions in the course of the infection are presented. The responses of blood clotting and vascular permeability were species specific: Fibrin deposited in blood vessels in green monkeys, while hemorrhages developed in baboons. The results show that species-specific features of monkeys must be taken into account when choosing an experimental model for studying Ebola virus infection. immune complexes, malonic dialdehyde, fibrin, fibrinogen, thrombin, prothrombin, and fibrin degradation products [7][8][9]. Materials and MethodsEBO (subtype Zaire) that was passaged two times in monkeys was used. The virus stock was a 10% suspension of green monkey Results liver, with an activity of 10 5 LD 50 for newborn mice. Suspension was prepared in Eagle MEM with antibiotics. Target cells for EBO in monkeys. Electron microscopicGreen monkeys (Cercopithecus aethiops), baboons (Papio haexamination showed that monocytes and macrophages were the madryas), and outbred newborn mice were used. Twelve green primary targets for EBO in green monkeys (figure 1). Infected macrophages were first detected in liver sinusoids 2 days after infection. On day 3, infected Kupffer's cells were observed in liver sections. EBO virus reproduction in hepatocytes and adre-

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis.

An approach combining virology with light and electron microscopy was used to study the organs of guinea pigs during nine serial passages of Ebola virus, strain Zaire. It was observed that the wild type of Ebola virus causes severe granulomatous inflammation in the liver and reproduces in the cells of the mononuclear phagocyte system (MPS). Based on morphological characterization, two types of virus-cell interactions were demonstrated. The obtained data evidenced for heterogeneity of the population of wild type of Ebola virus. The virus accumulated in the liver of the infected animals, and the lesions became more pronounced with passage. Degenerative changes appeared, and their severity was increased with passage in the other organs as well. The set of target cells diversified and, as a result, not only the MPS cells, but also hepatocytes, spongiocytes, endotheliocytes and fibroblasts became involved in the reproduction of Ebola virus. The possible role of granulomatous inflammation in the development of the adaptive mechanism of Ebola virus to guinea pigs is discussed.

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP)have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.Marburg virus (MBGV) and Ebola virus (EBOV) make up the family of Filoviridae, which belongs to the order Mononegavirales. MBGV causes a severe hemorrhagic disease in humans and nonhuman primates (22). The recent outbreak of MBGV hemorrhagic fever in the Democratic Republic of the Congo underlines the emerging potential of this pathogen (25). MBGV is an enveloped virus with a nonsegmented negativestrand RNA genome 19.1 kb in length, which encodes seven structural proteins (5, 8). The nucleocapsid of the virion is composed of the viral RNA and four proteins, the nucleoprotein (NP), P (formerly called VP35), the viral protein VP30, and the catalytic subunit of the polymerase (L). Two putative matrix proteins, VP24 and VP40, are located between the nucleocapsid and the envelope, which is decorated with the surface protein (GP) (2,3,7,13,15,20).Viral reproduction takes place in the cytoplasm, and the FIG. 1. IEM analysis of ultrathin section of MVA-T7-infected HeLa cells expressing recombinant NP. Immunogold labeling was carried out using a monoclonal antibody directed against MBGV NP (dilution, 1:10) and a goat anti-mouse antibody conjugated with colloidal gold (bead diameter, 5 nm) (dilution, 1:50). NP aggregates appeared as two long sheets of a reticular network of electron-dense material heavily labeled with gold particles. The NP aggregates are in close association with the membrane of the rER. Bar, 150 nm.