[Analysis of the cell tissue culture contamination with the bovine viral diarrhea virus and mycoplasmas].

Different cell tissue cultures and commercial fetal calf sera (FTS) used in biological and virological research were screened for the bovine viral diarrhea virus (BVDV, Pestivirus genus, Flaviviridae family) and mycoplasma contamination. BVDV was detected using RT-PCR and Indirect immunofluorescence (with monoclonal antibodies) methods in 33% cases of the studied cell lines and in > 60% cases of FCS. BVDV was shown to present and reproduce in high spectra of human cell lines, as well as in monkey, pig, rabbit, goat, dog, and cat cells at high levels (up to 100-1000 genome-equivalent copies per cell) and reached up to 10(3)-10(7) genome-equivalent copies per serum ml. The molecular mechanisms of the long virus persistence without definite signs of destruction should be studied.

[Formation of transfecting DNA in acute cellular infection with the tick-borne encephalitis virus mediated by an oncogenic virus]. / Obazovanie transfitsiruiushchei DNK pri ostroi infektsii kletok virusom kleshchevogo éntsefalita, oposredovannoe onkogennym virusom.

To prove the association of the transfecting activity of nucleic acid from cells co-infected with TBE and SV40 viruses with DNA, experiments were carried out using fractionation of nucleic acid preparations in cesium sulphate gradient an on a column with HAP. The experiments led to a conclusion that the infectivity of the preparations used was associated with double-stranded DNA. Investigation of nucleic acid infectivity form cytochalasine-enucleated cells indicated that the function of the nucleus was necessary for formation of transfecting DNA in mixed TBE and SV40 infection of cell. No formation of transfecting DNA was observed in experiments of blocking replication and transcription of cellular genome. The retention of the transfecting activity of DNA during 6 passages of cells at approximately the same level indicated the lack of selective preferences or inhibition of cells containing transfecting DNA.

[Changes in natural killer activity in the influenza virus infection of mice and in treating lymphocytes with the influenza virus and its proteins in vitro]. / Izmenenie aktivnosti estestvennykh killerov pri zarazhenii myshei virusom grippa i pri obrabotke limfotsitov virusom grippa i ego belkami in vitro.

Changes in the activity of natural cytotoxic lymphocytes (natural killers, NK) were studied in the course of infection of CBA mice with a mouse-adapted or unadapted (epidemic) variants of influenza A/USSR/90/77 virus. The activity of NK against K562 target cells in the peripheral blood, lungs, and spleens was found to increase considerably 18 hours after intranasal inoculation with any of the variants and to persist at a high level for up to 72 hours. Virus replication in the lungs and peripheral blood was observed only in mice infected with the adapted virus variant. A conclusion was drawn that the increase in NK activity in the organs of influenza virus-infected mice was nonspecific providing no protection against a fatal outcome after infection with the mouse-adapted (pathogenic) virus. Treatment of lymphocytes recovered from the peripheral blood, lungs, and spleens of infected and non-infected mice in vitro with preparations of intact viruses and isolated viral proteins exerted different effects on the NK activity: enhancement after treatment with intact viruses and inhibition by isolated viral proteins. The effect did not depend on the virulence of the strain from which viral proteins were isolated. The importance of the phenomenon of different modulating effect of influenza virus and its structural proteins on the NK activity is discussed.

[Methods of isolating and selecting recombinant vaccinia viruses expressing heterogenetic viral antigens]. / Metody polucheniia i selektsii rekombinantov virusov ospovaktsiny, ékspressiruiushchikh chuzherodnye virusnye antigeny.

The paper describes a method using plasmid construction pSC11 for generation of recombinant vaccinia viruses supporting coexpression of heterologous genes and beta-galactosidase. The Ca2+-phosphate method of cell transfection by recombinant DNAs generated on the basis of pSC11, and selection of recombinant viruses from blue plaques of virus-infected cells in the presence of X-gala are reported at length.

[Persistence of the Japanese encephalitis virus in L929 cells. Correlation of viral and cellular factors]. / Persistentsiia virusa iaponskogo éntsefalita v kletkakh L929. Korreliatsiia virusnykh i kletochnykh faktorov.

The paper presents the results of the study of the first stage of Japanese encephalitis (JE) virus persistence in cultures of L929 cells. The main parameters of the establishment and development of JE virus persistence in these cells characterizing the system as a chronically infected one. A possible role in the mechanism of persistence of various cellular and viral factors: interferon, ts-mutants, defective particles, was studied. Interferon was shown to be the main factor of virus carrier state perpetuation in the L-JE system. The role of defective particles, ts-mutants, and possible association of JE virus genome with nuclear DNA of L929 cells in the mechanism of persistence is discussed.

[Incorporation of a DNA replica of the genome of the tick-borne encephalitis virus into cellular DNA]. / Vstraivanie DNK-kopii genoma virusa kleshchevogo éntsefalita v kletochnuiu DNK.

DNA from mouse cells chronically infected with tick-borne encephalitis (TBE) virus was treated with restrictases, the resulting fragments were fractionated by size by gel electrophoresis, denaturated, and transferred from gel on nitro-cellulose filters. The fragments containing virus-specific sequences were detected by hybridization with 32P-DNA replicas of TBE genome RNA synthesized using reverse transcriptase. The presence of virus-specific sequences in DNA fragments from chronically infected cells proves the possibility of integration of DNA-replicas of TBE virus genome and genome of chronically infected cells.

[The isolation and identification of the Sindbis virus from migratory birds in Estonia]. / Vydelenie i identifikatsiia virusa Sindbis ot pereletnykh ptits v Estonii.

The data on isolation from birds and identification of two strains of alphaviruses in Estonia in the territory of Vilsandy natural reserve are presented. Electron microscopy of purified virions allowed the isolates to be classified into the family of togaviruses, and serological identification (neutralization test, CFT) using polyvalent sera and monoclonal antibody showed them to belong to Sindbis virus.

[Sindbis viruses of various geographic origin and differentiation of them from Western equine encephalomyelitis viruses using the polymerase chain reaction]. / Virusy sindbis raznogo geograficheskogo proiskhozhdeniia i differentsirovanie ikh ot virusov zapadnogo éntsefalomielita loshadei metodom polimeraznoi tsepnoi reaktsii.

Comparison of Sindbis virus strains isolated in different regions of the world (in Africa, Australia, and Europe, including Russia and its nearest neighbors) in the polymerase chain reaction (PCR) by the primary gene structure of proteins NSP1 and E1 and in the neutralization test showed the greatest similarity between geographically close strains isolated in Northern Europe (KFL, Karelia, 1381 and 1388, Estonia). Sindbis strains AR339 and Babanki isolated in Africa were similar to each other and to strains from Northern Europe by the examined gene sites but different from the Northern variants in the neutralization test. Geographically remote strains F-720 (Armenia and Southern Europe) and Whataroa (New Zealand) were close to Sindbis virus from Africa and Northern Europe by only one of the genes examined (F-720 by NSP1 and Whataroa by E1). PCR was carried out using oligonucleotide primers containing nucleotide sequences identical to genes NSP1 and E1 sites of Sindbis strains HRSP, Okelbo, and KFL, but different from gene sites of other known representatives of alphaviruses by at least 5 positions. PCR analysis showed that the appurtenance of the geographic variants to Sindbis group can be ascertained only after investigating the homology of at least two genes coding for the replicative and structural proteins. Such a procedure of PCR permits the detection of Sindbis viruses of different geographic origin with changes in their primary structure and allows the differentiation between Sindbis viruses and Western equine encephalomyelitis viruses within the serological complex.

[Similarities and differences between western equine encephalomyelitis viruses with respect to genes for nonstructural protein NSP2 and structural proteins C and E2]. / Skhodstvo i razlichie virusov zapadnogo éntsefalomielita loshadei po genam nestrukturnogo belka NSP2 i strukturnykh belkov C i E2.

Genetic relationships of geographical isolates of the members of WEE virus serocomplex (McMillan, Fort Morgan, Highlands J, and Y62-33) were assessed by the polymerase chain reaction (PCR) and restriction analysis of the PCR products. Oligonucleotide primers (21 nucleotides in length) were chosen for NSP2, nucleocapsid C, and E2-E1 protein genes based on the known primary structure of the McMillan 16310-5614 genome (L. Uryvayev et al., 1994, 1995). These primers were shown to differentiate well the WEE and SV-like strains of the serocomplex. Y62-33 virus (Udmurtia, Russia) was identical to McMillan strain in three studied regions of NSP2, C, and E2-E1 genes. NSP2 gene could be detected in all the studied geographical isolates and was characterized by the same restriction patterns as endonucleases; it appeared to be the most conservative. The structural genes were less conservative. Fort Morgan virus (Colorado, USA) genome reliably differed from McMillan virus (California, USA) and was negative in PCR with primers to C and E2 gene regions. Highlands J genome (Florida, USA) was positive in PCR with the primers to E2-E1 gene regions but differed from McMillan strain by the nucleocapsid gene. An additional comparative PCR analysis of the C-E2 region in the McMillan and Highlands J genomes showed some, but not complete identity. The origin of these two viruses might be due to the selection of different forms of recombinant viruses. A good correlation of structural genes in PCR and the infectivity neutralization test was noted with the primers and polyclonal antibodies to the closely related strains. High specificity of PCR permits a more accurate detection of the virus origin and relationships.

[A comparative analysis of the polypeptides of the the Sindbis and Karelian fever viruses]. / Sravnitel'nyi analiz polipeptidov virusov Sindbis i karel'skoi likhoradki.

In pulse-chase experiments with Karelian fever virus-infected cells, proteins were found with molecular weights of 130, 98, 78, and 62 kD of which the first, second and fourth were classified as polypeptide precursors of the structural proteins of virion. The molecular weights of proteins E1, E2 and C of 52, 47 and 34 kD, respectively, as well as isoelectric points of isolated glycoproteins (pI E1 = 6.3, pI E2 = 8.4) were similar in KFV (strain Leiv-9298) and Sindbis virus (strain AR339). The antigenic similarity of the strains under study in neutralization test with hyperimmune sera, the identity of physicochemical characteristics of the structural proteins of KFV and prototype Sindbis virus strain suggest a close relationship of the Leiv-9298 strain to the Afro-European variants of Sindbis virus.

[Analysis of the primary structure of segments of the Karelian fever virus genome]. / Analiz pervichnoi struktury uchastkov genoma virusa karel'skoi likhoradki.

Primary structure of two parts of Karelian fever virus (KFV) genome (29-57 nt and 10507-11591 nt) cloned in recombinant plasmids has been studied and compared with that of Sindbis (HRSP strain) and Ockelbo viruses. Fifty-four nucleotide substitutes were revealed in the sequenced parts of KFV genome including partially 5' and 3'-nontranslated sites ( a total of 1613 nucleotides, or approximately 13.8% of the genome length), in comparison with the Sindbis virus prototype HRSP strain, this being in good correlation with strain variability. Eighteen nucleotide substitutes (96.4% homology) were detected in the NSP1 gene site (60-557 nt) of KFV in comparison with Sindbis virus and only 5 substitutes (98.8% homology) vs. Ockelbo virus. These data on primary structure of KFV genome reliably and unambiguously indicate the appurtenance of this virus to Sindbis-like viruses.

[Monoclonal antibodies to the Karelian fever virus. The characteristics of their biological properties]. / Monoklonal'nye antitela k virusu karel'skoi likhoradki. Kharakteristika biologicheskikh svoistv.

Biological properties of 6 variants of monoclonal antibodies (MAb) to Karelian fever virus, a member of the alpha-virus serocomplex Sindbis-WEE, produced by the available hybridomas. The productivity of hybridomas of the "Karel" series in tissue culture and in cultivation as ascitic fluid was evaluated. Among the antibodies analysed, all were specific to envelope proteins, of them 2 were against protein E2 and four against protein E1. Comparison of MCA biologic activity (neutralizing, antihemagglutinating activities, participation in immunofluorescence, EIA, and immune blotting) allows one to distinguish four different hybridomas among them producing specific antibodies differing in their properties.

[Demonstration of the influenza virus A RNA by nucleic acid molecular hybridization using biotin-treated probes]. / Vyiavlenie RNK virusa grippa A metodom molekuliarnoi gibridizatsii nukleinovykh kislot s ispol'zovaniem biotinirovannykh zondov.

A probe containing full-size DNA copy of influenza A/USSR/90/70 virus protein gene M labeled with biotin on 32P was used for influenza A virus RNA detection by dot hybridization method. For labeling with biotin, a new method of its administration by chemical modification of nucleic acid was employed. In homologous DNA:DNA hybridization the sensitivity of determinations was less than 1 pg in the biotin-treatment of the probe and 1.25 pg in its radioactive labeling. Hybridization of DNA probe with cytoplasmic RNA isolated from influenza A virus-infected (strains A/USSR/90/77 and A/Texas/77) MDCK cells revealed RNA in the dot corresponding to 4.5-5.5 1g ID50 of virus present in 2 x 10(4) cells. The probe did not bind with negative controls in any dot in all the tests. The results of the study indicate that DNA probes labeled with biotin and 32P and used in dot hybridization for influenza A virus RNA detection in infected cells show the similar sensitivity and specificity.