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At present, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is the selective destruction of cancer cells with the minimal destructive effect on the healthy cells of the body. Orthopoxviruses provide a promising basis for the design of oncolytic drugs. They have a number of advantages over other viral vectors, and one of these advantages is the large capacity of their genomes. It allows them to be furnished with genes encoding proteins with antitumor properties. In this study, we compare the replicative properties of ten variants of the vaccinia virus (LIVP strain of VACV) tested on cultures of human glioblastoma cells and simian CV-1 kidney cells. Some of these viruses have additional genes, such as those encoding the granulocyte-macrophage colony-stimulating factor, apoptosis-inducing protein TRAIL, and green fluorescent protein. We have also studied VACV with five virulence genes deleted, namely, the genes encoding hemagglutinin, the γ-interferon-binding protein, thymidine kinase, the complement-binding protein, and the Bcl2-like inhibitor of apoptosis. The reactogenicity and neurovirulence of this disarmed derivative are much less than in the original LIVP strain. It has been found that derivatives of the vaccinia virus with the deleted thymidine kinase gene most intensely replicate in the glioblastoma cell culture.

Gene therapy can offer a new approach to arthritis treatment which acts at an inflammation site. Numerous studies show high efficacy of gene therapy in different models of arthritis in humans. Even a single injection of a recombinant vector results in a stable prolonged expression of a therapeutic gene and a longterm therapeutic effect. In contrast to biologic therapy involving numerous systemic injections of recombinant anti-inflammatory proteins, gene therapy does not produce systemic side effects. Vectors based on retroviruses, adenoviruses, adeno-associated viruses, and recombinant plasmids could provide delivery of target genes. Of significant importance is the development of noninvasive methods of gene therapy: intranasal and peroral. The current state of research in arthritis gene therapy is discussed in this review.

The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections.

Complete nucleotide sequences of the mitochondrial DNA of Wistar rats and premature aging rats OXYS, sensitive to cataractogenic effect of galactose, from the Center for collective usage «Gene pool of laboratory animals» of the Institute of Cytology and Genetics (ICG), Siberian Branch, Russian Academy of Sciences, were investigated (nucleotide sequences were submitted to GenBank). It was shown that two additional unique nucleotide substitutions between rats OXYS (ICG) and Wistar (ICG) are not found in other known nucleotide sequences of mitochondrial DNA of rats and do not result in amino acid substitutions, so that all differences in phenotypic data of these lines are determined only by differences in the genomic DNA. Comparative analysis showed that Wistar (ICG) rats have a significant number of nucleotide substitutions compared to rat strains derived from outbred rat Wistar colony, which contradicts possible relationship of Wistar (ICG) rats to baseline Wistar rat line or its descendants. It was assumed that the Sprague Dawley rat line is the progenitor of Wistar (ICG) rats.

Fundamentals of the development of transplastome plants and structural features of genetic constructs for incorporation into chloroplast genomes are reviewed. Major achievements in the transformation and expression of alien genes in chloroplasts are considered. The potential of this method for significant increase in the production of alien proteins in transplastome plants is substantiated.

In the course of evolution, viruses have mastered various molecular mechanisms to evade defense reactions of the host organism. The understanding of these mechanisms would promote better comprehension of the crucial reactions directed against infectious agents and further insights into their organization and functioning. A considerable contribution to this field of study can be made by investigating orthopoxviruses pathogenic for humans, such as variola, monkeypox, cowpox, and vaccinia viruses. The experimental data reviewed here suggest that variola virus and other orthopoxviruses, in comparison to other virus families, possess an unsurpassed set of genes whose protein products efficiently modulate the diverse defense reactions of the host.

Orthopoxviral genomes bear genes for a series of homologous secreted proteins binding tumor necrosis factor (TNF). Orthopoxvirus species have different sets of these proteins. Variola virus has only one protein of this series, CrmB. Although CrmB protein sequences are similar to each other, their physicochemical and biological properties show certain species-specific features. We constructed 3D models of complexes formed by TNF-binding domains of variola and cowpox viruses with murine and human TNFs. We also constructed corresponding models with a mutant human TNF. In this mutant TNF, the arginine residue at position 31 involved in receptor binding was replaced by glutamine, characteristic of murine TNF. Analysis of the models showed that the least stable complex should be that formed by cowpox virus CrmB with human TNF, and the Arg31/Gln substitution should significantly stabilize the interaction between cowpox CrmB and mutant human TNF. Experimental comparison of the abilities of recombinant variola and cowpox CrmB proteins to inhibit the cytotoxic action of TNFs confirmed the predictions.

Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTalpha, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.

Two segments of the variable terminal regions of the variola virus (VARV) genome were sequenced in 22 strains from the Russian collection, including about 13.5 kb of the left segment and about 10.5 kb of the right segment. The total length of the sequences was over 540 kb. Phylogenetic analysis of the new and published data determined the relationships among 70 VARV strains, the character of their clustering, and the intergroup and intragroup variation of the strain clusters. Loci with the highest polymorphism rate were identified and proved to map to noncoding regions or to regions of damaged open reading frames, characteristic of the ancestral virus. These loci offer attractive possibilities for developing a strategy of VARV strain genotyping. Recombination analysis by different methods did not detect, except for a single case, significant recombination events in the VARV strains examined.

A combinatorial immune library of human single-chain antibodies (scAbs) was constructed using the genes coding for the variable domains of the heavy and light chains of human immunoglobulins. The genes were cloned from lymphocytes of four subjects vaccinated with the vaccinia virus (VACV). The library included 3 · 107 independent clones. After enrichment with clones producing scAbs against a recombinant analog of the variola virus envelope protein prA30L, the library was used to select a panel of scAbs binding both prA30L and VACV. All scAbs selected were tested for virus-neutralizing activity, and two scAbs proved to suppress VACV plaque formation in monolayers of Vero E6 cells. The specificity of antigen binding was verified by ELISA and Western blotting. The amino acid sequences of the virus-neutralizing scAbs were determined by sequencing their genes.