[The molecular epidemiological aspects of hiv-infection spreading in Perm region].

Retrospective analysis of HIV-infection spreading in Perm region in conjunction with the genetic characterization of viral subtypes circulated on this territory from 1988 (when 1st case of infection was detected) until 2005 was performed. Analysis of epidemic process allowed to determine three periods of its development basing on both epidemic intensity and nature of circulating HIV-1 subtypes. During 1988 - 1996 (first period), when viral population was heterogenous (simultaneous circulation of three HIV-1 subtypes) with multiple routes of transmission, the epidemic process was characterized by low intensity. High incidence of HIV-infection among injection drug users and high homogeneity of circulated HIV-1 variants (98% of isolated variants belonged to HIV-1 subtype A with low level of genetic variability) were characteristics of the second period lasted from 1997 to 2001. Decrease in HIV-infection incidence in 2002-2005 was accompanied by the increase of HIV-1 transmission through heterosexual contacts and continuation of subtype A predominance between isolates. However increase in heterogeneity of viral population during this period, which manifested as increase of env and pol genes polymorphism, was detected.

[Molecular and epidemiological characterization of HIV-infection in the Altai Territory].

The genetic analysis of the variants of HIV, type 1, circulating in the Altai Territory was made. The results obtained with the use of the serological analysis and the method of the comparative evaluation of the electrophoretic mobility of heteroduplexes demonstrated that almost all analyzed samples (98.3%) belonged to subtype A. Genetic differences between these viruses did not exceed 9.20%. Moreover, 86.8% of them contained mutation V771 in the protease-coding area. Thus, HIV of subtype A, characteristic of CIS countries and containing mutation V771, may be regarded as the dominating viruses in the Altai Territory and not the viruses of subtypes B, C or A/E, typical of comparatively less remote China.

[Protease and reverse transcriptase genetic polymorphism in HIV type 1 subtype A variants predominating in cis countries].

To define frequencies of drug resistance mutations among HIV-1 variants circulating within the territory of Russia, subtype A HIV-1 nucleotide sequences encoding protease and reverse transcriptase were analyzed. The analysis was carried out in 141 antiretroviral-naive individuals. Low frequency (less than 1%) of primary drug resistance mutations was shown. However, high frequencies of secondary mutations V77I in protease and A62V in RT (67% H 63%, respectively) linked to each other in most cases were observed. The HIV-1 isolates bearing both substitutions (MutV77I/A62V) were also characterized by the presence of several synonymous mutations, suggesting common origin for these viruses. HIV Biochip Hybridization microarray and/or Restriction fragment-length polymorphism analyses were performed to characterize gene pol polymorphism in additional 178 subtype A HIV-1 isolates. Among total 319 samples studied, Mutv77IA62V variant accounted for 56%, and was found to predominate in Russia in terms of both its geographical distribution and number of cases caused. Moreover, these viruses were prevalent in the regions known to have highest incidence of HIV-1 infection (Irkutsk, Samara, and Moscow regions). In addition, three other variants were found: viruses not containing the substitutions V77I or A62V, and variants bearing only one of them. Evolutional relationships between all four HIV-1 variants, as well as potential impact of the gene pol polymorphism on HIV-1 replicative fitness and drug resistance development are discussed.

[Anti-CD4 autoimmunity in HIV-infected persons in Russia].

The frequency of anti-CD4 antibodies was determined in the sera or plasma derived from the patients infected with HIV-1 belonging to different genetic subgroups. The anti-CD4-antibodies in a dilution of > or = 1:1000 were found in 14% of the patients infected with the gagA/envA virus characteristic for injectable drug users in East Europe. The frequency of autoimmune antibodies among the HIV-infected patients with envB virus was substantially less (4.4%). Competitive ELISA using monoclonal antibodies to different CD4 domains demonstrated that irrespective of the viral genotype, the autoimmune epitope is located within the D4 or D3/D4 domains of CD4 receptor.

[Biological properties of HIV-1 variants circulating among drug users in Russia].

Human immunodeficiency virus type 1 variants belonging to subtype A, as well as recombinant gaga/engvB variants, derived from HIV-infected patients living in the Moscow and Perm Regions, were isolated and characterized. Intravenous administration of psychoactive drugs was a major risk factor of the infection for all the patients. All the examined isolates of HIV-1 types A and A/B were shown to be characterized by a low virus-specific activity and to be used as secondary CCR5 and CXCR4 protein receptors. The findings suggest that the domination of subtype A variant in this risk group is unassociated with fundamental differences in biological properties between the isolates of this subtype and recombinant viruses.

The site-specific deletion in plasmid pBR322.

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII. The endpoints of the deletion in one of the pBR322 delta 1 plasmids occurred at positions 375 and 16666 bp from the EcoRI site, as determined by sequence analysis. Formation of pBR322 delta 1 is most probably due to site-specific recombination between the sequence in the 1666-1670 bp region and the BamHI end of the linear pBR322 molecule. THe deletion was not controlled by the recA system of the host bacteria.

Silent mutation in the V3 region characteristic of HIV type 1 env subtype B strains from injecting drug users in the former Soviet Union.

New independent states of the former Soviet Union are facing a rapidly growing epidemic of HIV-1 among injecting drug users (IDUs). This epidemic is caused by three HIV-1 populations, one belonging to HIV-1 subtype A (IDU-A), another to subtype B (IDU-B), and the third being a recombinant of the IDU-A and IDU-B viruses (IDU-A/B, gagA/envB). Each of these populations is characterized by a high level of genetic homogeneity. We identified a unique synonymous nucleotide substitution in the first isoleucine codon at the IHIGPGR motif (ATT), which was observed in the env subtype B V3 sequences derived from IDUs in Russia and the Ukraine. This substitution was observed in none of 179 sequences obtained from IDUs in western Europe, northern America, and Asia. Molecular epidemiological analysis of HIV-1 strains based on this sequence pattern could be useful for tracing the origin and spread of the IDU-B viruses to other countries and risk groups.

Serological approaches to subtyping of HIV-1 in injecting drug users in Russia: evidence of subtype homogeneity at the main sites of the epidemic.

The aim of this study was to develop and evaluate a simple V3 peptide-based enzyme immunoassay (PEIA) for large-scale serotyping of HIV-1 specimens derived from injecting drug users (IDUs) in the Russian Federation. Two synthetic peptides were evaluated, named P1 (RKSIHIGPGRAFYATGD) and P2 (RTSVRIGPGQVFYKTGD), in an PEIA on 63 HIV-1 IDUs sera for which genotypes had been determined by heteroduplex mobility assay (HMA) and sequencing. The sensitivities of P1 (subtype B) and P2 (subtype A) were 87% and 75% respectively. Specificity of the assay was 100% for both peptides, with 100% predictive values of a monoreactive positive test for both peptides. Using the PEIA with peptides P1 and P2, we have serotyped 375 of 477 serum samples derived from IDUs in 4 main sites of the HIV-1 epidemic in Russia. The results demonstrated a high level of subtype homogeneity in all regions studied. In 3 of 4 territories, Tver' (n=345), and Rostov-on-Don (n=61) regions, and Krasnodar Kray (n=27), 100% of typable sera were found to belong to env subtype A. On the other hand, all specimens serotyped in the Kaliningrad region (n=38) belonged to env subtype B, and there is strong evidence that the recombinant gagAenvB virus which has caused the largest outbreak of HIV-1 in Russia is located in this region. At the present time another parental strain with gagBenvB genotype is of minor importance in the IDUs HIV-1 epidemic in Russia.

[Comparative inhibitory analysis of DNA biosynthesis catalyzed by retrovirus reverse transcriptase]. / Sravnitel'nyi ingibitornyi analiz biosinteza DNK, kataliziruemogo obratnymi transkriptazami retrovirusov.

Comparative study of DNA biosynthesis inhibition, catalyzed by avian myeloblastose virus (AMV) reverse transcriptase (RT), human immunodeficiency virus (HIV) recombinant and native RT, has been performed. 3'-Azido-2',3'-dideoxythymidine 5'-triphosphate (AzTTP); 3'-azido-2',3'-dideoxythymidine 5'-methylenephosphonate-diphosphate: 3'-azido-2',3'-dideoxythymidine 5'-phosphate-phosponoacetate; 3'-azido-2',3'-dideoxythymidine 5'-phosphate-dibromomethylenephosphonate; 2',3'-O-isopropylidenecytidine 5'-methylenephosphonate-diphosphate (rC-IP-MPDP) were used as inhibitors. AzTTP proved to by the most active inhibitor (its activity against HIV RT is higher than against AMV RT), although not selective as the phosphonates; only rC-iP-MPDP has low selectivity.

[Expression of the gene for the gp46 surface glycoprotein from the human T-cell leukemia virus type I (HTLV-I) in bacteria]. / Ekspressiia gena poverkhnostnogo glikoproeida gp46 virusa T-kletochnogo leikoza cheloveka tipa I (HTLV-I) v bakteriiakh.

A set of recombinant plasmids containing different fragments of HTLV-I env gene has been constructed on the basis of pUR290-pUR292 vectors. The hybrid proteins containing different fragments of ENV predecessor in the C-terminal of beta-galactosidase differed in stability in Escherichia coli cells. The presence of N-terminal of ENV predecessor in recombinant proteins considerably decreases their resistance to proteases of the bacterial cell. Elimination of this fragment led to obtaining of the recombinant plasmid pESG coding for the high level of synthesis of the env-specific hybrid polypeptide (up to 30% of the total cellular protein). This 134 Kda protein is able to interact efficiently with the HTLV-I positive sera and may be used in the diagnostic test-systems for identification of the HTLV-I infected patients.

[Expression of the gag-precursor of the human T-cell leukemia virus type I in Escherichia coli: study of the stability and antigenic properties of virus-specific hybrid proteins]. / Ekspressiia gag-predshestvennika virusa T-kletochnogo leikoza cheloveka tipa I v kletkakh Escherichia coli: izuchenie stabil'nosti i antigennykh svoistv virusspetsificheskikh gibridnykh belkov.

A set of recombinant plasmids containing sequences of HTLV-I viral gag-gene has been constructed on the basis of pUR290-pUR292 vector plasmids. The resulting hybrid proteins containing different fragments of GAG-precursor in the C-end of beta-galactosidase differed to a large extent in stability in Escherichia coli cells. The presence of an N-end fragment of GAG-precursor in the recombinants decreases drastically their resistance to bacterial proteases. Elimination of the fragment resulted in obtaining the recombinant plasmid pGdN coding for high rate synthesis (up to 30% of total cellular protein) of gag-specific hybrid polypeptide in Escherichia coli HB101 cells. This 145 kDa protein efficiently interacts with HTLV-I positive sera. It can be used in diagnostic test-systems for indicating HTLV-I infected persons.

[Expression of a fragment of the env gene, coding for the GP46 surface glycoprotein of the human T-cell leukemia virus, in Escherichia coli cells]. / Ekspressiia fragmenta gena env, kodiruiushchego poverkhnostnyi glikoprotein GP46 virusa T-kletochnogo leikoza cheloveka tipa II, v kletkakh bakterii Escherichia coli.

Designing of recombinant plasmids pSB2 and pSB3 with the 932 bp HTLV-II env gene inserts encoding the full-length surface membrane glycoprotein gp46 is described. Vectors pGOmpF and pET32a expressing genes cloned under control of the late bacteriophage T7 promoter were used. Western blot analysis of cellular proteins derived from E. coli B834/pSB2 and E. coli B834/pSB3 revealed that 34 kD and 31 kD polypeptides corresponding to the full-length gp46 and its processed form without signal peptide were synthesized under control of these recombinant plasmids. Cytotoxic activity of the recombinant proteins towards bacterial cells was demonstrated. Both polypeptides specifically reacted to sera from humans infected with HTLV-II. High antigenic specificity of P34-HTLV-II proteins makes a promising candidate for diagnostic confirmation tests.

[Frequencies of the CCR2-64I and SDF1-3'A alleles associated with progression of the HIV-1 disease in healthy individuals from Moscow]. / Chastota vstrechaemosti allelei CCR2-64I i SDF1-3'A, vliiaiushchikh na razvitie simptomov pri VICH-infektsii sredi zhitelei g. Moskvy.

The frequencies of two mutations associated with the development of clinical symptoms upon infection with human immunodeficiency virus type 1 (HIV-1) were determined in a cohort of individuals from Moscow. Allelic frequency of the first mutation, CCR2-64I, causing the substitution of valine with isoleucine in the CCR2 chemokine receptor, was 0.1106 (95% confidence interval, 0.0714-0.1498). The frequency of the second mutation the G to A substitution in the 3'-untranslated region of the stromal-derived factor 1 encoding gene, SDF1-3'A, was 0.2125 (95% confidential interval, 0.1608-0.2642). Both values were slightly higher than those obtained earlier for Western European countries. This result can be explained by higher proportion of Asian immigrants, characterized by higher frequencies of these mutations, in the population of Moscow.

[Cloning and expression of the gene for diphtheria toxin and its subunits in Escherichia coli]. / Klonirovanie i ékspressiia gena difteriinogo toksina i ego otdel'nykh sub''edinits v bakteriiakh Escherichia coli.

The results of cloning Corynebacterium diphtheriae phi 984 tox gene and its A and B subunits in Escherichia coli are presented. Regulatory sequences of tox gene are capable to promote effective expression in E. coli cells. A set of recombinant plasmids has been obtained which can determine the synthesis of A and B individual subunits and are suitable for constructing immunotoxins by gene engineering. The diphtheria toxin of 62 kDa synthesized in E. coli has enzymatic activity and reacts with antitoxin sera. Some sites for E. coli proteases are present in tox-specific polypeptides.

[Molecular genetic characteristics of HIV-1 in Russia]. / Molekuliarno-geneticheskaia kharakteristika VICh-1 na territorii Rossii.

The paper presents data on the variants of human immunodefficiency virus type 1 (HIV-1) currently circulating in Russia. The subtype A HIV-1 variant dominating is shown to be most widespread among drug-injected users in the most regions under study. By using the results of an analysis of 1,464 blood samples taken in the past 4 years in 69 subjects of the Russian Federation, the authors have estimated that this HIV-1 variant is responsible for 93% of all HIV-infection cases in the country. The greatest regional genetic diversity was observed in Moscow and its mean (2.35(1.59) was found to be comparable to that (2.41(1.85) in the whole country. Penetration of the subtype A IV-1 variant early detectable among drug-users into other risk groups was noted.

[The use of the vif gene expression product of HIV-1 in bacteria for detecting specific antibodies in the sera of infected persons]. / Ispol'zovanie produkta ékspressii gena vif VICh-1 v bakteriiakh dlia obnaruzeniia spetsificheskikh antitel v syvorotkakh infitsirovannykh.

A fragment of the genome of human immunodeficiency virus type 1 (HIV-1) coding for p23 protein, the product of vif gene, was cloned in a plasmid vector pUR291. The resulting recombinant plasmid pLacVif1 was conducive in E. coli cells to the synthesis of a hybrid polypeptide with molecular weight of 136 kDa containing antigenic determinants of p23 protein of HIV-1. The employment of this polypeptide for analysis of HIV-1-positive sera by indirect enzyme immunoassay showed that vif-specific antibodies were found in 53% of the cases and their appearance was not related to the stage of the disease.

[Restriction analysis of the DNA of the toxigenic corynephages BF, phi 984, phi 9 and C]. / Restriktsionnyi analiz DNK toksigennykh korinefagov BF, fi 984, fi 9 i C.

The results of restriction analysis of toxigenic corynephages BF, phi 984, phi 9, and C are presented. Phage BF was shown to be a deletion derivative of phage beta vir, phage phi 984 to be omega-like. Toxigenic corynephages phi 9 and C belong to a new group of corynephages designated phi. DNA of phages phi 9 and C as well as chromosomal DNA of the indicator strains was not hydrolysed by specific Hind III endonuclease, that is likely to be associated with the presence of a modification system in host strains.

[The expression of a fragment of the HIV-1 Nef gene in Escherichia coli bacteria]. / Ekspressiia fragmenta gena Nef VICh-1 v bakteriiakh Escherichia coli.

The presence of antibodies to p27, the product of gene Nef, may be an important diagnostic sign since some sera from subjects of the risk groups negative to HIV-1 structural proteins may contain antibody to p27. The study resulted in construction of a hybrid plasmid determining in E coli bacteria the synthesis of a hybrid protein the N-terminus part of which is represented by full-size beta-galactosidase and the C-terminus by a part of protein p27 with the main immunoreactive epitopes. The resulting polypeptide specifically interacts with sera of the infected subjects and may be used for detection of antibodies to the protein Nef in the blood of virus-carriers.

[Biological and physicochemical properties of Corynebacterium diphtheriae B (Freeman) phages phi 984 and phi 9]. / Biologicheskie i nekotorye fiziko-khimicheskie svoistva fagov Corynebacterium diphtheriae B (Freeman), fi 984 i fi 9.

A scheme for preparative isolation of corynephages and their DNA is described. Study of host specificity, toxigenicity, and of cytotoxic effect induced by the phages BF, phi 9, and phi 984 has shown that phage BF has tox- phenotype, and phages phi 9 and phi 984, tox+ phenotype. These phages differ in host specificity and plaque morphology. Electron-microscopic examination of virions showed similarity of phages BF and phi 984 structures, whereas phage phi 9 was markedly different in the size of its head and tail.

[The isolation of the herpes virus from the white crane and a study of its properties]. / Vydelenie virusa gerpesa ot belogo zhuravlia i izuchenie nekotorykh ego svoistv.

Isolation of a virus agent from sick white cranes in National Oka Preserve is described. The results of virological and serological studies on specimens from the sick birds permitted a conclusion that the infection in the cranes could be induced by avian herpes virus.