Biomedical subjects
Publications and source records attributed to R F Mar'iankova.
The conditions necessary for fusion from inside (FFWI) of the BHK-21 cell culture affected by the Lassa and Mopeya arenaviruses were studied. The fusion was shown to occur only in the slightly acid medium and at lower pH meanings for the Mopeya virus, than for the Lassa virus.
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Lysosomotropic agents (NH4Cl, amantadine, chloroquine, monensin) which prevent acidification of intracellular vacuoles, when introduced into the culture medium before or during inoculation of cells (BHK-21, Vero) with arenaviruses inhibit reproduction of these viruses completely or significantly. Mozambique virus proved to be 10 times more sensitive to the effect of lysosomotropic agents than Pichinde and Lassa viruses. Thus, arenaviruses have a pH-dependent stage at the beginning of the reproduction cycle which is indirectly indicative of their penetration into cells by receptor endocytosis.
In experimental infection of mice with Lassa virus, the infectious virus could be detected in all the organs and brain tissues tested. Histopathological lesions were demonstrated in cerebral and spinal cord tissues only. Roentgen irradiation in a dose of 500 R and cyclophosphamide protected mice against a lethal Lassa virus dose. Cyclosporin A in various doses exerted no effect on the outcome of the acute infection. The adoptive transfer of splenocytes from mouse donors inoculated intraperitoneally prevented the development of lethal disease symptoms and death of mice-recipients. It is suggested that immunocompetent cells are involved in the development and outcome of experimental infection of mice with Lassa virus.
Ultrafiltration through hollow fibrous filters followed by purification in interrupted and linear urografin gradients yielded a Lassa virus suspension of high concentration. The use of gamma-irradiation for inactivation of the frozen virus suspension (-70 degrees C) caused no apparent structural changes of virions and made it possible to examine Lassa virus in electron microscope by negative staining. The observed virus particles in their morphology and sizes did not differ from previously described particles of other members of the Arenaviridae family. In ultrathin sections of Lassa virus-infected Vero cells, atypical virions were sometimes visible alongside with typical particles. Within one type of such particles no ribosome-like granules could be detected. Such "hollow" particles may possibly be defective virions. Another kind of atypical particles contained homogeneous electron-dense core and resembled mycoplasma. Of greatest interest are the particles with heterogeneous core in which "sandy" granules can be distinguished. The presence of greater amounts of uranophilic material than usually may be explained by getting into the virion in the process of its formation of a greater amount of genetic material than that present in typical virions.
A new modification of the method for cultivation of cells from blood vessel explants has been proposed, and on its basis several cell lines from different vessels of human embryo and adult subjects have been derived. The sensitivity of vessel cells to viruses was studied on a model of aorta cell culture using a wide spectrum of RNA and DNA viruses. All the strains under study with the exception of respiratory-syncytial virus (RSV) and encephalomyocarditis (EMC) virus induced a productive infection in the cells. The infection with RSV and EMC was abortive. The possibility of virus replication in vessel cells was confirmed by isolation of adenovirus from a human renal artery biopsy.
Pathogenicity for randombred and inbred mice of various age groups of the standard Lassa virus and the virus enriched with defective interfering particles (DIP) was studied. The standard Lassa virus inoculated intracerebrally caused 100% death of C3H/Sn mice aged up to 4 weeks and 60%-70% death of randombred white mice aged 3-4 weeks. BALB/C mice were found to be nonsusceptible to the virus, and its lethality for C57BL/6 and AKR mice varied within the range of 30%-60%. Lassa virus enriched with DIP caused no death of the susceptible animals and showed poor protective activity against the standard virus.
The method of Porterfield and Allison was adapted for titration of the infectious activity of Lassa virus by the plaque formation in Vero cells. The virus was cloned, and the effect of the time of adsorption, pH, temperature, as well as polycations (DEAD-dextran, protamine sulphate) dimethylsuphoxide (DMSO), and trypsin added during adsorption or into the agar overlay on the effectiveness of plaque production by Lassa virus (virus titres, plaque size) were studied. The optimal adsorption time was found to be 1 1/2-2 hours, pH 8.0. The number of plaques produced by the virus was approximately similar at 35 degrees C. The substances under study did not enhance the efficacy of plaque formation, on the contrary, DMSO and high concentrations of polycations decreased plaque size.
Production of Lassa and Machupo viruses in Vero and BHK-21 cells was studied in relation to various conditions of the infected cell cultivation and as a function of different multiplicities of infection. The highest titers (expressed in PFU/ml) were obtained when the cells were grown in roller bottles with daily changes of the medium. The maximum titer in Lassa virus-infected cells was over 10(6), in Machupo virus-infected cells over 10(7). The effect of the autointerfering factor on the growth of Machupo virus was demonstrated. An increase in the multiplicity of infection led to a decrease in the yield of Machupo virus. Production of Pichinde and Machupo viruses in a monocyclic growth experiment was studied. The maximum yield of cell-associated and extracellular Pichinde virus was obtained at 24-32 hours postinfection, and that of extracellular Machupo virus at 32-40 hours postinfection.
Acute and persistent infections of Vero cells with a cloned Lassa virus were studied. After acute infection at a multiplicity of 2 PFU/cell the latent period was 12-16 hrs and the maximum yield of virus particles in the growth medium was obtained 28-32 hours postinfection. A persistently infected cell line was established by inoculating Vero cells with the cloned Lassa virus followed by subcultivation of the infected cells. The infected cells underwent 36 passages. Lassa virus production by the infected cells followed a cyclic pattern varying from 10(2) to 10(5) PFU/ml. Neither the plating efficiency of the virus nor its growth curves changes at 35 degrees C and 39 degrees C. The replication of the cloned virus in infected cells (superinfection) was markedly depressed in comparison to that of normal Vero cells infected with Lassa virus. The role of defective interfering particles in the establishment of persistent infection is discussed.
The early stages of infection of Vero-E6 cell culture with Marburg virus, a member of filovirus family, highly pathogenic for man, were studied. Virus multiplication was completely or significantly inhibited by lysosomotropic agents (LTA) of two types: weak base (ammonium chloride) and ionophore monensin. The level of the inhibiting effect was proportional to LTA concentration and was maximal when the drug was introduced into the culture medium before virus inoculation. Complete inhibition of Marburg virus replication in Vero-E6 cells in the presence of 20 (30) mM ammonium chloride ("lysosomotropic blocking") was overcome by a short-time treatment of the cell culture with the virus adsorbed on it using a medium with a weak-acid pH (4.0-5.0). The results are discussed from the point of view of the mode of this virus penetration into eukaryotic cells.