[The localization and preliminary characteristics of antigenic sites (B epitopes) in the nucleocapsid protein of the Lassa virus]. / Lokalizatsiia i predvaritel'naia kharakteristika antigennykh saitov (B-épitopov) v belke nukleokapsida virusa Lassa.

Nucleocapsid protein of Lassa virus contains 4 epitope sites. Three of them were localized in amino acid position 123-1 27, 337-346, and 518-527. Monoclonal antibody 1108 specific to Lassa virus NP-protein reacted with 123-127 synthetic peptide in solid-phase ELISA and precipitated nucleocapsid proteins of Machupo, Tacaribe, and Mopeia arenaviruses in RIP.

[The immunogenic activity of the Lassa virus structural proteins]. / Immunogennaia aktivnost' strukturnykh belkov virusa Lassa.

The immunogenic properties of Lassa virus GP1, GP2, and NP polypeptides were studied in rabbits. Lassa virus NP polypeptide, in contrast to GP1 and GP2 polypeptides, was shown to induce the highest titres of antibodies determined by IFA and ELISA tests. Moreover, the antibody relative avidity experiment showed that the anti-NP antibodies has ELISA index of 0.63 whereas anti-GP1 and anti-GP2 antisera had those of 0.49 and 0.28, respectively.

[The detection of the Marburg virus antigen by solid-phase immunoenzyme analysis]. / Vyiavlenie antigena virusa Marburg metodom tverdofaznogo immunofermentnogo analiza.

Comparative studies of two variants of the enzyme-linked immunosorbent assay (ELISA) were carried out to determine the sensitivity of the detection of Marburg virus antigens in Vero cells. Both competitive and two-antibody ELISA variants detected as little as 5 ng of Marburg virus antigen. The Vero cell monolayer was found to produce 5-50 ng/0.05 ml of the virus-specific proteins at 6 to 8 days postinfection.

[The biological properties of the HIV isolated from a virus carrier living in the Byelorussian SSR]. / Biologicheskie svoistva VICh, vydelennogo ot virusonositelia, prozhivaiushchego v BSSR.

Biological properties of an AIDS agent first isolated from a native citizen in the USSR are presented. The source of the virus was a young Byelorussian woman who in the near past had had sexual contacts with a citizen from one of the Central Africa countries. The isolate is thought to be of HIV-I type. It replicated perfectly in many continuous lymphocyte lines and had HIV-characteristic morphology. The protein spectrum of the isolate was gp120, gp41, p65/51, p55, p32, p24, p17. Reverse transcriptase activity was detected in the culture fluid of the virus-containing cell cultures. The isolate was designated HIV-IZ.

[The isolation and characteristics of recombinant monoclonal antibodies to the Lassa virus]. / Poluchenie i kharakteristika rekombinantnykh monoklonal'nykh antitel k virusu Lassa.

Recombinant monoclonal antibodies to Lassa virus were produced. The reactivity of the monoclonal antibodies was studied by indirect fluorescence antibody (IFA) technique, enzyme-linked immunosorbent assay (ELISA), and radioimmunoprecipitation method. The observed reactivity did not correlate with IgG isotyping groups.

[Virus-specific proteins and RNA of the virus of hemorrhagic fever with renal syndrome]. / Virusspetsificheskie belki i RNK virusa gemorragicheskoi lokhoradki s pochechnym sindromom.

Virus-specific proteins G1, G2, and N with molecular weights of 70, 55-57, and 50 kilodaltons, respectively, were detected by radioimmunodiffusion tests in VERO E-6 cells infected with strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the European USSR from a patient with HFRS, a fatal human case of HFRS (the strains K-27 and P-360) and from a bank vole (strain CG-1820). The sera from human convalescents after HFRS in the European USSR and rat sera prepared with the CG-1820 strain precipitated proteins possessing similar electrophoretic characteristics from a lysate of cells infected with the CG-1820, K-27 and P-360 strains. The sera from human HFRS convalescents in the Far East did not precipitate protein Gl. The viral RNA derived by immunosorption method from intracellular nucleocapsids of CG-1820 strain and strain 4590 (isolated from Ap. Peninsulae in the Far East) contained 3 classes of molecules: L, M, and S. L- and M-RNA of these strains had the same molecular weight (1.62 and 1.38 megadaltons). The molecular weight of S-RNA of the strain 4590 was 0.76 megadalton and that of the CG-1820 strain 0.83 megadalton. It is assumed that there are species differences among the viruses, causative agents of HFRS, circulating in the European and Far East regions of the USSR.

Preparation and biochemical characterization of Lassa virus subvirion fractions.

14C-labelled Lassa virus was purified by isopycnic centrifugation and split to subvirion fractions. The purified virus was treated with nonionic detergents Nonidet P-40 (NP-40) and with octylglycoside. After ultracentrifugation in urographin density gradient, two subvirion fractions with buoyant density of 1.24-1.26 and 1.08-1.10 g/cm3 were obtained. The first fraction corresponded to the nucleocapsid of Lassa virus: it contained a protein with molecular mass of 60 kDa, the L and S segments of the genomic RNA. The second one contained a protein with molecular mass of 48 kDa and represented, apparently, envelope fraction of virus particles.

Virus-specific proteins and RNAs from cells infected with hemorrhagic fever with renal syndrome (HFRS) viruses derived from endemic areas of the U.S.S.R.

Cell-associated proteins of rodent (CG-1820 strain) and human (K-27 and 360 strains) hantaviruses isolated in the European endemic areas of the U.S.S.R. are antigenically similar as revealed by immunoprecipitation and immunoblotting assays. Nucleocapsid-associated RNAs of representative hantaviruses of four antigenic groups [Sugiyama et al. (1987) J Gen Virol 68: 979-987] have unique PAGE patterns. "Electropherotyping" of the RNAs isolated from infected cells might be used for identifying and distinguishing hantaviruses.

[Electron microscopic study of Lassa virus]. / Elektronno-mikroskopicheskoe izuchenie virusa Lassa.

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.

[Pathogenic Machupo and Lassa arenaviruses: the biochemical properties of virion RNA and proteins]. / Patogennye arenavirusy Machupo i Lassa: nekotorye biokhimicheskie kharakteristiki virionnykh RNK i belkov.

Lassa virus purified in the isodensity sucrose concentration gradient had the following buoyant densities: 1.17 g/cm3 (sucrose), 1.19 g/cm3 (cesium chloride), 1.16 g/cm3 (urografin). Similar parameters were obtained for Machupo virus. Virion RNAs of these viruses contained 5 sedimentation classes of molecules: 30-31S, 28S, 22-24S, 18S, and 4-6S. Experiments on hybridization of individual sedimentation classes of RNA with an excess of poly(A)-containing RNA from the infected cells as well as inhibition of synthesis of 28S and 18S virion RNAs with low concentrations (0.005-0.5 micrograms/ml) of actinomycin D showed the genetic information for virus proteins to be coded for in two segments: 30-31S and 22-24S. The method of self-annealing demonstrated molecules with complementary sequence ("plus" and "minus" strands) in genome RNAs. In addition to previously described major proteins (78K, 64K, 37K), high performance liquid gel-penetrating chromatography of Machupo virus structural proteins revealed a minor protein with molecular weight of 50 kilodaltons. Pulse-chase experiments demonstrated in the infected cells a precursor-product metabolic bond between glycosylated proteins 78K and 37K. Lassa virus contained 3 structural major proteins with molecular weights 60, 48, and 34 kilodaltons (K). The 60K protein was detected in the nucleocapsid fraction, and 48 K protein in the soluble subvirion fraction. Proteins 60K and 34K were immunoprecipitated in greatest amounts in the infected cells.

[Characteristics of monoclonal antibodies against Lassa virus]. / Kharakteristika monoklonal'nykh antitel k nukleoproteidu virusa Lassa.

Some properties of monoclonal antibodies to the Lassa virus have been characterized. The competitive immunoenzyme analysis has revealed the presence of at least three antigens in the Lassa virus nucleoprotein.

[Characteristics of the subvirion components formed after the treatment of Machupo virus with a nonionic detergent]. / Kharakteristika subvirionnykh komponentov, obrazuiushchikhsia posle obrabotki virusa Machupo neionnym detergentom.

Machupo virus labeled with radioactive precursors of RNA or protein synthesis was purified by isodensity ultracentrifugation in sucrose concentration gradient. The purified virus was disrupted with NP-40 nonionic detergent in the presence of 1 M KCl, and subvirion fractions were separated by ultracentrifugation in urografin density gradient. This treatment resulted in formation of two subvirion components with a buoyant density 1.25-1.26 and 1.10-1.12 g/cm3 in urografin concentration gradient. The subvirion fraction with the density of 1.25-1.26 g/cm3 contained high-molecular virion RNAs, a major protein of molecular weight of 64 kD, and seemed to be the virion nucleocapsid. The solubilised fraction (1.10-1.12 g/cm3) contained glycosylated protein 37 kD which seems to be a surface glycoprotein.

Machupo virus polypeptides: identification by immunoprecipitation.

The most abundant protein in purified Machupo virions (Corvallo strain) labelled with 14C-Protein hydrolysate is a 64 K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37 K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78 K polypeptide and a minor 50 K polypeptide have been detected. In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64 K, NP), and the others were glycosylated polypeptides (78 K and 37 K). The synthesis of NP and 78 K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78 K is a precursor for structural glycoproteins of Machupo virions.

[Effect of actinomycin D on the reproduction of the Machupo virus]. / Vliianie aktinomitsina D na reproduktsiiu virusa Machupo.

The effect of inhibitors of mRNA synthesis (actinomycin D and alpha-amanitin) and DNA replication (mitomycin C and ethidium bromide) on Machupo virus reproduction was studied. Actinomycin D (1.0-4.0 micrograms/ml) and alpha-amanitin (10.0 micrograms/ml) added immediately after adsorption reduced the infectious virus titre by 2 lg or more. Actinomycin D inhibited virus reproduction not only at the early but also at the later stages of infection. Mitomycin C and ethidium bromide did not inhibit Machupo virus reproduction. The cells treated with actinomycin D at early and late stages of infection were found to contain immunoprecipitable virus-specific proteins 78, 64, and 37 KD described previously. The total amount of virus-specific proteins in the inhibitor-treated cells was reduced 3.7 and 2,6-fold after addition of actinomycin at 0 and 12 h postinfection, respectively, as compared with untreated cells. The mechanism of action of actinomycin D on the Machupo virus reproduction is discussed.

Ribonucleic acids of Machupo and Lassa viruses.

Sucrose gradient velocity centrifugation, polyacrylamide gel electrophoresis and RNA-RNA hybridization were used to characterize Lassa and Machupo virion RNAs as well as virus-specific RNAs from cells infected with Pichinde and Machupo viruses. Five RNA species: 30-31S, 28S, 22-24S, 18S and 4-6S have been detected in Lassa, Machupo, and Pichinde virion RNAs. Among them 28S, 18S and 4-6S RNAs cosediment and comigrate with respectively cell RNAs. RNase resistance analyses suggest the presence of extensive secondary structures and complementary RNAs in Lassa, Machupo, and Pichinde virion RNAs. Annealing with poly(A)-containing RNA from infected cells has revealed that the bulk of "minus" strands of Machupo virion RNA is located in 22-24S and 28-31S fractions of sucrose gradient. Thus Machupo and Lassa viruses as well as Pichinde virus contain two genomic RNA fragments: "large" (molecular weight of about 2.2 X 10(6] and "small" (molecular weight of about 1.3 X 10(6]. In the cells infected with Pichinde virus and treated with actinomycin D (1.0 microgram/ml) synthesis of 18S, 22-24S and 30-31S RNAs has been registered. At least 22-24S and 30-31S classes comprise "plus" and "minus" strands. In cells infected with Machupo virus in the presence of actinomycin D the synthesis of similar sedimentation classes of RNAs and certain amounts of 28S RNA have been detected.

[Sedimentation analysis of the virion RNA of Machupo virus]. / Sedimentatsionnyi analiz virionnoi RNK virusa Machupo.

Pichinde and Machupo viruses labeled with 3H-uridine were concentrated and purified by different methods. The buoyant density of the viruses was for Pichinde 1.14-1.15 g/cm3 (urographimeter), 1.19 g/cm3 (sucrose), 1.25 g/cm3 (cesium chloride), for Machupo virus 1.25 g/cm3 (cesium chloride). Virion RNAs of Pichinde and Machupo viruses were divided in sucrose concentration gradient into 4 sedimentation classes: 7-10 S, 18 S, 20-22 S, and 28-30 S. Virion 28 S and 18 S RNAs cosedimented with ribosomal cellular RNAs. The RNAs isolated at late stages of infection from the infected cells labeled with 3H-uridine in the presence of actinomycin D (1 microgram/ml) contained two major sedimentation classes of virus-specific molecules: 20-22 S and 30-31 S. Like Pichinde virus, Machupo virus genome is supposed to contain two RNA fragments (30-31 S and 20-22 S) as well as admixtures of ribosomal RNAs.

[Accumulation of certain arenaviruses in transplantable VERO and BHK-21 cells]. / Nakoplenie nekotorykh arenavirusov v perevivaemykh kletkakh VERO i BHK-21.

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.