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Aim: Study of circulating 02_AG recombinant form HIV-1 isolates that have been rapidly spreading in Novosibirsk region during 3 recent years., Materials and Methods: WHO protocol for primary HIV isolation was used, automatic sequencer was used for genetic characterization of isolates. Virus specific RNA were isolated and env HIV-1 region DNA fragments were processed. Phylogenetic analysis was also performed., Results: CRF_02AG HIV-1 isolated from peripheral blood of HIV-1 positive patients belonged to CCR5 tropic viruses and had various reproduction characteristics. Most of the HIV isolated were rapidly replicating virus variants characterized by an ability to accumulate high levels of virus protein p24 in cultural fluid. Infectivity and reproductive properties of HIV isolates were confirmed in experimental infection by using clarified cultural liquid of mononuclear cells from healthy donors. Phylogenetic analysis of CRF02_AG HIV-1 variants isolated in Novosibirsk region in 2007 - 2010 showed the formation of a separate outbreak in the area caused by emergence of CRF02_AG HIV-1 in human population., Conclusion: A collection of genetically and biologically characterized CRF02_AG HIV-1 isolates that has not been spreading previously in Russia.

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.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

The most potent antigen among HSV-1 proteins are glycoproteins gB(UL27) and gD(US6). Multiple amino acid sequence alignment of these proteins shows that gD protein is the most specific for HSV-1. Analysis of gD protein epitopes detected the main antigenic determinants not cross-reactive with antigens of other viruses. Virus was isolated and genome DNA was prepared from morphological elements of a patient with herpes simplex infection. US6 gene fragment was cloned in pUC19 vector. Cloning in bacterial expression vectors helped obtain beta-galactosidase-fused recombinant HSV-1 gD protein with 6-histidines affine target for high-performance chromatography purification. ELISA with a set of HSV-1-positive and negative donor sera and a commercial panel of HSV-1 sera (Vektor-Best) showed that recombinant gD can be used as an antigen to HSV-1-specific IgG.

To identify the hantaviruses causing hemorrhagic fever with renal syndrome (HFRS) in the Far East of Russia, blood samples collected from HFRS patients in 1994-1998, were examined by reverse transcription-polymerase chain reaction. In addition, 36 sera were tested by an immunofluorescence assay for antibodies against Hantaan, Seoul, Puumala, and Khabarovsk viruses, and 54 samples were tested by plaque reduction neutralization test. With both serological assays, the highest antibody titers were to Hantaan and/or Seoul viruses. Of 110 blood samples 36 were found RT-PCR positive. Phylogenetic analysis the sequences of a 256-nucleotide (nt) fragment of the hantavirus M genome segment revealed at least 3 genetically distinct hantavirus lineages. Nucleotide sequence comparison showed that two of the lineages, designated as FE and Amur (AMR), differed from one another by 15.9-21.2% and from Hantaan virus by 9.8-17.5%. The third lineage, VDV, differed from Seoul virus by 2.6-5.1%. All S segment sequences were from FE lineage, and differed from Hantaan virus by 10.7-12.6%. Thirty of the 36 (83%) analyzed sequences were found to be the FE genotype, which is very similar to that of Hantaan virus, strain 76-118. Of the remaining hantaviruses, 11% were the AMR genotype, and 6% the VDV genotype, which are genetically novel genotypes of Hantaan or Seoul viruses, respectively.

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific., (Copyright 2000 Academic Press.)

Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.

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 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 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.

Full-length DNA copies of M-gene of remantadine-sensitive and remantadine-resistant variants of the influenza virus strain A/FPV/Weybridge (H7N7) have been synthesised and cloned. Complete nucleotide sequences of both cDNAs were determined by the Maxam-Gilbert method. There are three nucleotide substitutions, two of which lead to amino acid changes in M1 and M2 proteins. The existence of M3 protein, a polypeptide 68 amino acids long, encoded by the negative strand of RNA, is suggested. Amino acid changes in M2 and M3 proteins and their relation to the remantadine resistance are discussed.

A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.

The complete primary structure of cDNA for hemagglutinin gene of influenza virus A/FPV/weybridge/27 subtype H7 has been determined. Its comparison with the structures of analogous genes from other strains of the same subtype has shown 75% of base changes resulting in silent mutations. This suggests the weak immunological pressing in course of evolution of this subtype strains. The reason for apathogenicity of this avian strain is supposed to be elimination of a glycosylation site present in the strain A/FPV/Rostock/34. The possibility of using the obtained data for construction of the new generation of vaccines is discussed.