[Apoptin enhances the oncolytic activity of vaccinia virus].

Chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain L-IVP) genome. The insertion has replaced major part of the viral C11R gene encoding viral growth factor (VGF), which is important for the virulence. The recombinant virus VVdGF-ApoS24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. The expression apoptin gene from a synthetic early-late promoter of vaccinia virus effectively provides accumulation of the protein in the cells infected with the VVdGF-ApoS24/2 virus. Despite the presence of virus growth factor signal peptide at apoptin N-terminal secretion of the recombinant protein into culture medium did not occur. The recombinant virus VVdGF-ApoS24/2 was found to have a significantly greater selective lyticactivity on human cancer cell lines (A549, A431, U87MG, RD and MCF7) as compared with the parent strain L-IVP and its variant VVdGF2/6 with the deletion of the C11R gene. The results suggest that the use of apoptin represents a promising approach for improving the natural anticancer activities of vaccinia virus.

[Study of coding sequences of variable regions in smallpox virus genome].

Potential ORFs were sought in the extended segments of terminal variable regions in the variola virus genome. These ORFs underwent a detailed structural and functional analysis and were compared both between themselves and with homologous ORFs of various orthopoxviruses. The most conservative and heterogeneous ORFs of 70 VARV strains were detected. The unique for VARV ORF (111 a.a) was revealed for Helder and Mary strains of the Russian collection. In addition, only in the Helder strain, ORF D14L was disintegrated into two separate ORFs. A number of ambiguities were found in the current databases for VARV ORF. The dominating type of evolution was ascertained to be stabilizing selection for analyzed ORF. It has been established that VARV ORF C3L, unlike other poxviral orthologs, undergoes an adaptive selection. These findings suggest that this gene plays an important role in human VARV adaptation.

[Differentiation of geographic biovariants of smallpox virus by PCR].

Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

[Comparative analysis of variable regions in the genomes of variola virus].

Nucleotide sequences of two extended segments of the terminal variable regions in variola virus genome were determined. The size of the left segment was 13.5 kbp and of the right, 10.5 kbp. Totally, over 540 kbp were sequenced for 22 variola virus strains. The conducted phylogenetic analysis and the data published earlier allowed us to find the interrelations between 70 variola virus isolates, the character of their clustering, and the degree of intergroup and intragroup variations of the clusters of variola virus strains. The most polymorphic loci of the genome segments studied were determined. It was demonstrated that that these loci are localized to either noncoding genome regions or to the regions of destroyed open reading frames, characteristic of the ancestor virus. These loci are promising for development of the strategy for genotyping variola virus strains. Analysis of recombination using various methods demonstrated that, with the only exception, no statistically significant recombinational events in the genomes of variola virus strains studied were detectable.

[Immunogenic properties of recombinant vaccinia virus containing the human IL-2 gene].

A genetic construct of the human interleukin-2 (IL-2) gene within vaccinia virus (L-IVP strain) has been designed. The authors show the capacity of CV-1 cells infected with the recombinant vaccinia virus VV-SIL2 to secrete human IL-2 into the culture medium. Human IL-2 has been detected by immunoblotting. The sera from the animals immunized with the recombinant virus VV-SIL2 exhibited both human IL-2 and its antibodies throughout the observation period. This recombinant virus immunization induced both humoral and cell-mediated immune responses to human IL-2; the observed changes in the concentrations of cytokines are likely to suggest that the response predominantly followed a Th1 pathway. The study construct was nontoxic at the used concentrations and administration routes. The findings point that it is promising to investigate the adjuvant properties of the recombinant VV-SIL2 vaccine-based preparation for immunization in combination with various vaccines and to study this construct in therapy for cancer diseases.

[Construction of DNA-fragments' libraries with complete genomes of different variola strains].

Libraries of hybrid plasmids carrying DNA fragments of complete genomes of 8 variola virus strain from the Russian Collection belonging to 2 epidemical types and isolated in various geographic regions of the world were obtained. Genomic sequences of variola virus can be thus preserved for a long time in a biologically safe form and provide the research work on studying the genetic organization of this unique virus and on developing modern methods for rapid detection of variola virus and other orthopoxviruses.

[Comparative restriction enzyme analysis of the genome in variola virus strains from the Russian collection]. / Sravnitel'nyi restriktsionnyi analiz genomov shtammov virusa natural'noi ospy iz Rossiiskoi kollektsii.

Comparative RFLP analysis was for the first time performed for 21 variola virus (VARV) strains of the Russian collection with 20 amplicons covering the total VARV genome. The amplicons were synthesized in the long polymerase chain reaction. A database useful as a reference for identifying VARV strains was generated. VARV strains isolated in different geographical regions were compared and proved to vary mostly in variable genome regions. Each of the dendrograms constructed included three clusters of African, Asian, and VARV-alastrim isolates. The VARV-alastrim isolates differed to the greatest extent from the other strains. VARV strains isolated during an ecdemic variola burst in Moscow (1960) grouped with Asian isolates. Polymorphism of VARV strains was for the first time observed for a single variola burst with a few affected patients.

[The study of orthopoxvirus genes for Kelch-like proteins. II. Construction of cowpox virus variants with targeted gene deletions]. / Izuchenie ortopoksvirusnykh genov, kodiruiushchikh Kelch-podobnye belki. II. Sozdanie variantov virusa ospy korov s napravlennymi deletsiiami genov.

Integrative plasmids p delta C, p delta D, and p delta G were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chicken embryos.

[Expression of synthetic gene for human angiogenin in E. coli cells]. / Ekspressiia sinteticheskogo gena angiogenina cheloveka v kletkakh E. coli.

A synthetic gene for human angiogenin was cloned into pRTang under control of the Proteus mirabilis recA promoter. After induction with mitomycin C, Escherichia coli cells transformed with pRTang produced an additional 14.2-kDa protein. According to electrophoretic and immunochemical analyses, this protein was identical to human angiogenin.

Comparative studies of gamma-interferon receptor-like proteins of variola major and variola minor viruses.

To study specific properties of the human gamma-interferon (gamma-IFN) receptor-like proteins of the highly virulent and low virulent strains of variola (smallpox) virus (VAR) recombinant plasmids determining synthesis of these proteins in E. coli cells have been constructed. The recombinant viral gamma-IFN receptor-like proteins have been found to have high interferon-neutralising activity with regard to human gamma-IFN but not murine gamma-IFN and human alpha-IFN. The variola major and variola minor proteins under study do not differ in the efficiency of human gamma-IFN antiviral activity inhibition.

[Chemico-enzymatic synthesis, cloning and expression of a gene for an analog of human anaphylatoxin C5a]. / Khimiko-fermentativnyi sintez, klonirovanie i ékspressiia gena analoga chelovecheskogo anafilatoksina C5a.

Chemical-enzymatic synthesis and cloning of a gene for a human anaphylatoxin C5a analog were carried out. Recombinant plasmid pRC5a providing the expression of the synthetic gene in the Escherichia coli cells was obtained. The biological activity of the expression product was demonstrated by the chemotaxis activity test and by the release of myeloperoxidases from rat peritoneal cells that was induced by bacterial cell lysates containing the recombinant protein.

[Effective synthesis and cloning of the human interleukin-2 gene and its analog: expression of the interleukin-2 gene in E. coli cells]. / Effektivnyi sintez i klonirovanie gena interleikina-2 cheloveka i egoanaloga: ékspressiia v kletkakh E. coli gena interleikina-2.

Artificial DNA fragments encoding human interleukin-2 (133 a.a.) and its analogue (deletion of 14 C-terminal a.a.) were prepared by means of the DNA polymerase I mediated extension of synthetic polynucleotides having short overlapping sequences at their 3'-ends. The fragments were cloned in specially designed pFH-type plasmids and then excised by the FokI and other restriction endonucleases to yield the subfragments with the structurally predetermined 5'-unique cohesive ends. The complete synthetic gene was constructed by one or two-step ligation. The expressed IL-2 was tested by analysing the T-cell proliferation activity of E.coli crude lisates containing the pEXIL2 expression plasmid.

[Modeling the process of excitatory transmission from the mechanical portion of the auditory system to the nerve endings of the auditory nerve]. / O modelirovanii protsessa peredachi vozbuzhdeniia ot mekhanicheskoi chasti slukhovoi sistemy k neironnym okonchaniiam slukhovogo nerva.

Sharpening of frequency selectivity of the mechanical part of the hearing system due to the transformation of the signals in the receptor system is investigated. It is proposed that excitation of neuron endings is proportional to the amplitude of the current which was produced by the difference of microphonic potentials in the neighbouring hair cells. It is shown that possible degree of the selectivity sharpening of the cochlea which can be explained by this model is in a good agreement with the known experimental results.