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Three recombinant vaccinia viruses containing different fragments of tick-borne encephalitis virus (TBEV) cDNA representing the 5'-noncoding region (5'NCR), all structural and part of the nonstructural regions were constructed. Western blot analysis showed that E and NS1 proteins were expressed and processed correctly in cells infected with recombinant viruses vC-NS1 (coding for C-prM-E-NS1 region) and vC-NS3 (coding for C-prM-E-NS1-NS2A-NS2B-NS3 region). In contrast, in cells infected with recombinant virus v5'C-NS2A (coding for 5'NCR and C-prM-E-NS1-NS2A regions) expression of NS1 protein was greatly reduced and no E protein was detected. Immunization of mice with vC-NS3 induced high levels of TBEV-specific antibodies and protected them against intraperitoneal challenge with 10(7) LD50 of TBEV. The level of protection was very similar to the level of protection achieved by immunization with commercially available inactivated TBEV vaccine. Although the immunization of mice with recombinants vC-NS1 and v5'C-NS2A induced much lower levels of TBEV-specific antibodies, they were still protected against intraperitoneal challenge with 10(4) and 10(3.6) LD50 of TBEV, respectively. The high level of protection against TBEV infection achieved by the immunization of mice with the recombinant vaccinia virus vC-NS3 makes this virus a very attractive candidate for development of a live recombinant vaccine against TBEV.

Comparative analysis of the specific activities of various batches of Hep-A-in-Vac vaccine showed that the immunologic efficacy of the agent directly depended on the content of hepatitis A virus antigen in the vaccine dose. The results of studying the immunogenicity of the agent on volunteers are compatible with the results of experimental studies and indicate that the test of guinea pigs adequately assesses this characteristic. Assessment of the immunogenic properties of Hep-A-in-Vac prepared using improved technology indicates that Russian vaccine injected three times in a dose containing at least 40 U EIA ensures 87.5% seroconversion in adult seronegative volunteers with the mean geometrical titer of antibodies to hepatitis A virus equal to 683 mIU/ml.

Modernization of the protocol and optimization of the technology of preparation of inactivated hepatitis A vaccine helped prepare the experimental lots of inactivated vaccine meeting all the requirements to such preparations and the WHO recommendations.

Sequencing and computer analysis of a variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region has been carried out. Fifty-nine potential open reading frames (ORFs) of over 60 amino acid residues were identified. Structure-function organization of the VAR-IND DNA segment was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple differences between VAR-IND and the strains of VAC but the high identity of VAR-IND with VAR-HAR in the genetic maps are revealed. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

Collase, an enzymatic preparation made from crab hepatopancreas, was used as a dissociative reagent in preparation of primary cell cultures and for detachment of continuous cells from the substrate during reinoculation. Collase was found to increase viable cell harvest by 2 to 2.5 times in comparison with trypsin and had a less detrimental effect on the cells which retained their proliferative activity, morphology, productivity, and sensitivity to viruses.

Computer analysis of a variola major virus (VAR) genomic fragment bounded by the open reading frames (ORFs) D1R and A33L, which is 47,961 bp long, revealed 46 potential ORFs. The VAR proteins were compared to the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA-capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion bodies protein. Possible functions of the analysed viral proteins are discussed.

Sequencing and computer analysis of the nucleotide sequence of the variola virus strain India-1967 (VAR) genome segment (43069 bp) from the region of HindIII C, E, R, Q, K, H DNA fragments has been carried out. Forty-three potential open reading frames (ORFs) have been identified, and the polypeptides encoded by them have been compared with the analogous proteins of vaccinia virus strain Copenhagen (COP). ORF E7R of VAR is much shorter than the COP analog. The other polypeptides coded by the potential ORFs of VAR are highly conserved in comparison with COP. Possible functions of the predicted viral polypeptides are discussed.

Sequencing of variola virus (VAR) genome region of 43069 bp was carried out. This area contains 42 potential genes. Computer analysis of proteins coding for these viral genes was done. We compared VAR proteins with the those of vaccinia virus. The region studied is conservative for orthopoxviruses.

The authors tried to decode the mechanism of influenza viruses species adaptation in the process of host changing. The functionally important replacement in the surface pocket domains were revealed, particularly in the conservative region 221-241, involving fibronectin-like part. Close replacements were revealed in the region 141-161. The method of construction of heteroduplexes between hemagglutinin RNA of duck, pig, and human viruses was used. The method showed that all heteroduplexes formed recombinogene structures. An unexpected effect of directional recombination was elicited for hemagglutinin RNA heteroduplexes in cases of duck-pig and human-pig viruses. During the directional recombination the following processes took place: the receptor-binding site of animal type was transmitted to the duck virus, while the human receptor-binding site was transmitted to the pig virus. According to the experimental data, a new hypothesis is formulated: the cascade mechanism of directional recombination for duck, animal and human viruses makes it possible for the recombinant viruses to overcome interspecies barriers.

The complete genetic map of the variola major virus strain India-1967 is built basing on the sequence data. The suggested map is compared with the maps of the sequenced genomic regions of Copenhagen and Western Reserve strains of vaccinia virus and Harvey strain of variola major virus. The principle differences revealed in the genomic organization of these viruses are discussed.

Analysis of variola virus nucleotide sequence revealed proteins belonging to several families which provide the virus with the possibility of overcoming the barriers of specific and non-specific host defence against viral infection. The complement-binding proteins, lymphokine-binding proteins, and serine protease inhibitors can be assigned to this type, as can the proteins providing the orthopoxviruses with resistance to interferon. The revealed differences between the genes (proteins) of variola and vaccinia viruses under study are discussed.

Computer analysis of full coding sequences of variola major virus strain India-1967 genome and vaccinia virus strain Copenhagen genome have been carried out. A wide set of proteins containing ankyrin-like repeats have been identified for both viruses. Only three proteins of this family of the studied viruses are highly homologous. The rest of the proteins are different. The possible role of such proteins in determination of virus tissue tropism is discussed.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.

The 10466 nucleotide long sequence of the cDNA copy of the tick-borne encephalitis strain 205 viral genome has been determined. It includes the 5'-nontranslating region, the genes for structural as well as nonstructural proteins and the first 93 nucleotides of 3'-nontranslating region. The difference in amino acid sequences of structural and nonstructural proteins of strains 205. Sofjin and Neudoerfl of the tick-borne encephalitis virus and the nucleotide changes in 5'- and 3'-nontranslating of these strains are discussed.

The preparations of purified Marburg virus were isolated from blood plasma of infected guinea pigs and characterized. Viral RNA was extracted from the virions. The cDNA was synthesized on the isolated RNA matrix by the reverse transcriptase with the use of dissipated priming. The obtained cDNA was inserted into the plasmid pBR322 by the connector technique and the resulting recombinant plasmids were cloned in Escherichia coli cells. The specific clones selected by molecular hybridization method were analyzed by the restriction mapping and cross-hybridization. Four overlapping cDNA clones were found and the virus specific 5012 bp fragment of the viral genome was sequenced. Three open reading frames were found and the preliminary analysis of the coded amino acid sequence and corresponding genes was fulfilled.

A hybrid vaccinia virus expressing a chimeric protein consisting of thymidine kinase and the encephalitogenic determinant, S1, from guinea pig myelin basic protein was constructed. Infection of guinea pigs with the virus resulted in the development of allergic encephalomyelitis.

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.

The nucleotide sequences of the cDNAs of the genes for the structural proteins C, preM, M and E of the tick-borne encephalitis viral strain 205 have been determined. The complete nucleotide sequence of the 5'-end nonstructural region of the viral genome has been studied for the first time. The difference in the amino acids sequences of the structural proteins from different strains (205, Sofiin and Neidorf) of the virus is discussed.

Survival and morphogenesis of the enucleated fragments of Acetabularia has been studied after their reconstruction from the cell membrane and the endoplasm. Membranes and endoplasms of different species as well as of the same species but from plants in different functional states and with different regenerative abilities were combined. It was shown that inability of plants for growth and morphogenesis was due to the membrane state. When reconstructing anuclear fragments from membranes and endoplasms of different species or the same species but from plants in different functional states, a phenomenon of membrane-endoplasm incompatibility was found.

The ribosomal RNA transport from a nucleus to a perinuclear cytoplasm and its following distribution in the cytoplasm of Acetabularia mediterranea cells were studied using transplantation of RNA-labeled rhizoid into unlabeled stalk. In addition rifamycin treatment was used for inhibition of cytoplasmic RNA synthesis. Acetabularia nuclei contain the stable RNA fractions similar to those present in some other eukaryotes. Nuclear 25S and 17S ribosomal RNA rapidly enter the rhizoid cytoplasm whereas the following trasfer of them to other regions of the cell is a very slow process. Within two days only an insignificant part of 25S and 17S ribosomal RNA is transferred from the rhizoid to the stalk and is distributed there over the base-apical gradient. No preferential transfer of the nuclear ribosomal RNA to the apical region was observed.

The biosynthesis of nuclear RNA, its transport from the nucleus to the cytoplasm and distribution in the cytoplasm were studied in Acetabularia mediterranea under different light conditions. It was shown that the nuclear RNA incorportate 3H-uracil more rapidly in the darkness and the transport of labeled RNA from the nucleus slowed down after the transfer of plants in the cold medium in the darkness. To study the distribution of nuclear RNA in the cytoplasm, the 3H-uracil labeled nuclei were transplanted in the rhizoids of unlabeled plants, the dikaryons obtained were kept for different time in the light and in the darkness and the content of 3H-RNA was determined in different stem regions. It was shown that the transport of 3H-RNA in the cytoplasm is slowed down in the darkness and it is distributed by the basal-apical gradient. RNA is rapidly accumulated in the apical stem zone in the light and redistributed afterwards in the basal stem zones. The problem of relationship between the polarity and nuclear RNA distribution in Acetabularia is discussed.