[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.

Contamination of cell cultures with bovine viral diarrhea virus (BVDV).

The incidence of contamination of cell strains used in biological and virological studies and of fetal calf sera (FCS) manufactured by Russian and foreign companies used for cell culturing with noncytocidal bovine viral diarrhea virus (BVDV; Pestivirus, Flaviviridae) was analyzed. The virus was detected by reverse transcription PCR and indirect immunofluorescence with monoclonal antibodies to BVDV virion envelope glycoprotein in 25% of 117 cell strains and 45% of 35 tested FCS lots. The virus multiplied and persisted in a wide spectrum of human cell strains and in monkey, swine, sheep, rabbit, dog, cat, and other animal cells. The levels of BVDV genome RNA in contaminated cell cultures reached 10(2)-10(3) g-eq/cell and in serum samples 10(3)-10(7) g-eq/ml. These facts necessitate testing of cells and FCS for BVDV reproduced in cells without signs of infection detectable by light microscopy. The molecular mechanisms of long-term virus persistence in cells without manifestation of cell destruction are unknown.

[Recombination of alpha-viruses during mixed infection in cultured cells. Formation of hybrid forms of RNA of Venezuelan equine encephalomyelitis virus and Karelian fever virus in the region of genes for nucleocapsid and envelope proteins]. / Rekombinatsiia al'favirusov pri smeshannoi infektsii v kul'turakh kletok. Obrazovanie gibridnykh form RNK virusov venesuél'skogo éntsefalomielita loshadei i karel'skoi likhoradki v oblasti genov belkov nukleokapsida i obolochki.

A recombination of the genomes of VEE and KF viruses, two alphaviruses of different serotypes, takes place in mixed infection of chick embryo fibroblasts and Vero cells with vaccine strain VEE-230 and KF-9298 strain. This recombination resulted in the production of recombinant virions with altered antigenic properties. Polymerase chain reaction (PCR) rapidly and effectively detected the hybrid sites of the genome in recombinant viruses. Short nucleotide sequences (approximately 20 n. p.) of a known primary structure in the RNA genomes, specifically reacting with the complementary PCR primers, were chosen as genetic markers. These data are the first experimental validation of the probable recombinant formation of alphaviruses in nature. They are in good agreement with findings of the recombinant structure of Western equine encephalomyelitis virus genome.

[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.

[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.

[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.

[Use of an immunoperoxidase method in situ for detecting recombinant clones of vaccinia virus containing an expressed gene of influenza viral hemagglutinins]. / Ispol'zovanie immunoperoksidaznogo metoda in situ dlia vyiavleniia rekombinantnykh klonov virusa ospovaktsiny, soderzhashchikh ékspressiruemyi gen gemaggliutinina virusa grippa.

The vaccine L-IVP strain of vaccinia virus (VV) was used to construct the recombinant viral clones containing the influenza A hemagglutinin gene. The recombinant T plasmid was obtained with HA gene inserted in the vector pGS-20 (B. Moss) under the 7.5 K promoter of VV. A homologous recombination technique was used to insert the gene with the flanking TK sequences into vaccinia virus genome. The recombinant clones were selected by dot-hybridization with [32P]-labeled HA-probe. These recombinants were analysed for HA gene expression by the indirect immunoperoxidase method in situ using the peroxidase conjugate of the staphylococcal A-protein. This technique allows to obtain stable stained preparation and analyse the protein behavior at the ultrastructural level.