[Investigation of genetic polymorphism of the integrase gene in the HIV-1 subtype A populations circulating in the Russian Federation].

The paper presents the data of an investigation of the genetic polymorphism of the pol gene encoding viral integrase (IN) in a HIV subtype A infected population in the Lipetsk Region. The investigators analyzed 32 virus subtype A samples obtained in 2002-2007. Polymorphism at the codons associated with IN resistance to chemicals was observed in 7 virus variants. The found substitutions had a pattern of genetic polymorphism and were unassociated with resistance in 6 patients with the test subtype A population. At the same time, minor RAL resistance mutation was revealed in 1 (3.1%) virus variant while the similar mutations in the subtype G population were about 10%.

[Analysis of HIV-1 integrase gene polymorphism in an HIV-infected population from the nosocomial outbreak of HIV infection in the south of Russia in 1989].

The paper presents the data of an investigation of the polymorphism of the pol gene encoding HIV-1 integrase in a HIV subtype G infected population formed during the 1989 HIV-infection outbreak. The investigators analyzed 41 samples of the viruses obtained in 2005-2007. Polymorphism at codons associated with integrase resistance to chemicals was observed in 11 virus variants. The circulation of mutation viruses that potentially promote the formation of resistance to the integrase inhibitors raltegravir and elvitegravir has been established in untreated patients.

[Genetic characteristics of HIV-1 subtype G reverse transcriptase gene].

The paper presents data of a study of the structure of reverse transcriptase gene in the population infected with HIV subtype G formed during the 1989 HIV infection outbreak in the North-Caucasian region. The authors analyzed 3 samples obtained in 1993-1994 and 17 samples taken in 2000-2001. The phylogenetic analysis indicated that polymerase of the test virus variants belonged to HIV-1 subtype G. The mutations occurring with azidothymidine therapy did not differ from those in subtype B. Analysis of the mutations of resistance to other nucleoside and non-nucleoside reverse transcriptase inhibitors did not show great differences from subtype B either.

[Molecular epidemiologic characteristics of HIV-1 variants isolated in the Lipetsk region].

Molecular epidemiologic study of HIV-1 variants isolated in the Lipetsk region during 1994 - 2006 period was performed. It has been shown that 3 env-subtypes (A, B, and C) and 3 gag-subtypes (A, B, and C) are widespread in the region. The virus was transmitted both sexually and by injecting drug users. Phylogenetic analysis of gag and env genes nucleotide sequences was performed, which revealed that 4 variants of HIV-1 with genotypes gagA/envA, gagB/envB, gagC/envC, and recombinant gagA/envB are circulating in the region.

[Prevalence of mutations of resistance to HIV-I reverse transcriptase inhibitors among HIV-infected patients in the Southern Federal District].

To study the regularities in the spread of drug-resistant HIV-1 strains among HIV-infected patients in the Southern Federal District (SFD), the HIV-1 pol gene site encoded for reverse transcriptase was sequenced in the samples taken from 22 HIV-infected SFD patients who received or did not receive antiretroviral therapy (ARVT). Analysis of the primary sequences of the HIV-1 pol gene in SFD patients untreated with antiviral agents revealed the absence of both primary and secondary mutations of resistance to a nucleoside reverse transcriptase inhibitor (NRTI) and a non-nucleoside reverse transcriptase inhibitor (NNRTI). The group of patients receiving antiviral treatment was found to have different drug resistance mutations in the HIV-1 pol gene: K70R, M184V, K219Q, T215Y/F, L74V, etc. Moreover, the patients on ARVT had higher CD4 T lymphocyte levels and higher immunoregulatory index in the presence of significantly lower HIV replication than the untreated patients. The authors make recommendations how to study HIV resistance in patients who are to be treated and are receiving ARVT and advise to monitor the spread of drug-resistant HIV strains in the SFD.

[Epidemiological characteristics of HIV infection in the Lipetsk Region in 1993-2005].

The spread routes of HIV infection in the Lipetsk Region showing a low incidence of this infection rate are analyzed in detail. The findings indicate that the trend in identification of HIV-infected persons in this region reflects the all-Russian HIV-infection spread situation, but the epidemic process has a number of features.

Molecular epidemiology of HIV-1 in the former Soviet Union: analysis of env V3 sequences and their correlation with epidemiologic data.

OBJECTIVE: To investigate the HIV-1 V3 sequence diversity in the former Soviet Union in 30 subjects infected with HIV-1 via different modes of transmission. PATIENTS: A cohort of children infected after exposure to nonsterile needles during the epidemic in 1988-1989 in southern Russia (Elista, n = 12 and Rostov-on-Don, n = 10), and eight HIV-seropositive subjects from Belarus (Minsk), infected via sexual (n = 7) and parenteral (n = 1) infection. METHODS: The HIV-1 V3 encoding region was amplified by nested polymerase chain reaction on DNA of primary peripheral blood mononuclear cells collected from the study subjects and then cloned and sequenced. RESULTS: The alignment of 127 V3 sequences from 22 patients in the cohort group demonstrated common consensus sequences in both the Elista and Rostov samples. The average means of interperson variation were 5.9 and 6.6% in Elista and Rostov subjects, respectively, and comparable to the mean intraperson variation. The average mean interperson variation between nucleotide sequences of HIV patients infected through sexual transmission was considerably higher (14.9%). CONCLUSION: V3 sequence analysis confirms the epidemiologic data which support the transmission of HIV-1 in children from a single source, and suggests the infection of a mother from her parenterally infected child. Furthermore, the genetic variability of HIV-1 V3 in the noncohort group was particularly divergent indicating the heterogeneity of the virus circulating in the former Soviet Union.

PIP: In 1988, an HIV-1 epidemic occurred in Elista, Kalmyk Republic, Russia, among 90 children in two hospitals after exposure to blood contaminated needles from an HIV infected infant. A few months later, a similar HIV-1 outbreak in children occurred in Rostov-on-Don, Russia, probably a result of transporting children from Elista to Rostov-on-Don hospitals. In Rostov-on-Don, it appears that seven HIV infected infants transmitted HIV to their mothers during breast feeding. Health workers collected blood samples from 22 HIV-1 infected subjects in Elista (n = 12) and Rostov-on-Don (n = 10 including 1 mother-child pair) and from 8 control subjects who became infected with HIV-1 via sexual (7) and parenteral (1) transmission from Minsk, Belarus. Researchers wanted to determine the extent of the diversity of proviral DNA encoding the V3 loop from different patients in the children cohort. They used nested polymerase chain reaction on DNA of primary peripheral blood mononuclear cells and then cloned and sequenced them to detail the HIV-1 V3 encoding region. The Elista and Rostov-on-Don samples shared common consensus sequences (127 nucleotide sequences) in the V3 region. The average mean interperson variation between the nucleotide sequences of HIV patients infected through sexual transmission from Minsk was 14.9%, which was much higher than those for Elista and Rostov HIV patients infected through parenteral transmission (5.9% and 6.6%, respectively). The major nucleotide sequence in the mother in the Rostov group, who was presumably infected with HIV by her HIV infected infant during breast feeding, matched that of her daughter. The mother had no history of blood transfusion or any other risk factors except breast feeding. These findings confirm that the Elista and Rostov groups shared a common HIV source. They also suggest that breast feeding was the route of HIV transmission for the mother. The genetic variability of HIV-1 V3 in the control group demonstrated the heterogeneity of HIV-1 in the former USSR.

Identification of an env G subtype and heterogeneity of HIV-1 strains in the Russian Federation and Belarus.

OBJECTIVE: To identify HIV-1 envelope sequence subtypes in infected individuals from the Russian Federation and Belarus. PATIENTS: A cohort of children infected after exposure to non-sterile needles during the 1988-1989 HIV-1 epidemic in southern Russia (n = 20) and HIV-1-seropositive individuals from Russia (n = 1) and Belarus (n = 7) infected via sexual transmission. METHODS: DNA samples derived from peripheral blood mononuclear cells were analysed for their HIV-1 genotypes by the heteroduplex mobility assay (HMA). The 1.3 kilobase-pair env gene fragments encoding a portion of gp120 were amplified by nested polymerase chain reaction, cloned and sequenced. The env sequences derived from these patients were aligned and phylogenetic neighbour-joining and maximum parsimony-derived trees generated. RESULTS: The env sequences derived from eight individuals infected in Russia and Belarus belong to subtype A (one), B (four), C (two), and D (one). Sequences derived from children, infected during parenteral manipulations in southern Russia, and one mother were closely related, but highly divergent, as a group, from all prototypic strains (genetic divergence, 17.2-22.9%). However, they clustered together with env sequences of the V1525 and LBV21-7 isolates from Gabon, recently described to be members of a new HIV-1 env subtype G. CONCLUSION: Extensive heterogeneity of HIV-1 subtypes was evident in the Russian Federation and Belarus. Our data also support the existence of an HIV-1 env genetic subtype G, and such isolates are now apparently present on both the African and European continents. These variants were identified through V3 peptide enzyme-linked immunosorbent assay screening and subsequent HMA analysis. The combination of these techniques represents a model for screening HIV variants within a large population.

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.

Identification of HIV-1 subtype G from Uganda.

PIP: HIV-1 subtype G was identified by the authors in the former Soviet Union, and is related to a sequence VI525 from Gabon. All told, subtype G isolates have been identified in the former Soviet Union, Gabon, Zaire, and Nigeria. A subtype G gag sequence has also been reported in Taiwan. This paper reports the first HIV-1 subtype G to be isolated from Uganda. Uganda has the highest reported incidence of HIV-1 infection in Africa, with HIV-1 subtypes A, B, C, and D having already been isolated in the country. The authors isolated and sequenced HIV-1 genetic subtype G from a Ugandan female patient with CDC stage IV disease and a CD4 count of 20/cu. mm. Although recently immigrated to the UK from Uganda, there is epidemiologic evidence that the patient was infected with HIV-1 in Uganda. The V3 region of the isolate had 35 amino acids, with the common African tetrapeptide GPGQ at the tip of the loop. There were deletions relative to other G subtype sequences in the V4 and V5 env regions. The phylogenetic relationship of UGJW3 with other HIV-1 strains was inferred using nucleotide sequences and the PHYLIP package. The C2-V5 env region of the isolate was found to cluster with the subtype G isolates from the former Soviet Union and the Gabon. These findings confirm the need for further examination of the distribution of HIV-1 genetic subtypes and the exploration of their biologic significance.^ieng

Immunogenic properties of reverse transcriptase of HIV type 1 assessed by DNA and protein immunization of rabbits.

Genetic immunization may be one way to prime individuals for a subsequent broad anti-HIV-1 immune response. Reverse transcriptase of HIV-1 (RT) presents a selective target for attempts to arrest replication of HIV-1. Rabbits immunized with a plasmid carrying the gene for reverse transcriptase HIV-1 (RT DNA) developed potent antibody and cellular responses to the gene product. The immunogenic properties of RT DNA and recombinant reverse transcriptase were compared in rabbits. The specific immune responses were similar to those reported previously for HIV-1 infected humans. The array of B and T cell epitopes recognized in RT DNA-immunized rabbits was broader than in rabbits immunized with the recombinant RT. We localized seven novel B and T cell epitopes and concordance between B cell and helper T cell epitopes was observed. B cell epitopes of RT induced proliferation of peripheral blood mononuclear cells and were active as helper T cell epitopes. T cell-proliferative responses to the epitopes of RT preceded or paralleled the production of antibodies of the same specificity. Subdomains of reverse transcriptase involved in the enzymatic activity of RT were highly immunogenic. Anti-RT IgG partially inhibited reverse transcription in vitro.

[Formation of virus-like particles by HIV-1 Gag proteins, expressed by a recombinant vaccinia virus]. / Obrazovanie virusopodobnykh chastits belkov Gag VICh-1, ékspressiruemym rekombinantnym virusom ospovaktsiny.

Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed.

[Synthesis and comparative analysis of antigenic activity of peptides from hypervariable region V3 of human immunodeficiency virus I gp120 surface glycoprotein]. / Sintez i sravnitel'nyi analiz antigennoi aktivnosti peptidov gipervariable'noi oblasti V3 poverkhnostnogo glikoproteida gp120 virusa immunodefitsita cheloveka tipa I.

A set of peptides (amino acid positions 10-23) corresponding to seven most widely spread variants of the gp120 V3 domain in the HIV-infected population of South Russia were prepared by the solid-phase synthesis. A laboratory variant of the indirect enzyme-linked immunosorbent assay (ELISA) was developed for the determination of V3 specific antibodies with use of the peptides synthesized. The analysis of the V3-specific antibodies in HIV-infected using the elaborated test-system revealed a correlation between the V3 variants distribution and the occurrence of antibodies against these variants.

[A new oxa-analog of myristic acid, suppressing replication of the human immunodeficiency virus]. / Novyi oksaanalog miristinovoi kisloty, podavliaiushchii replikatsiiu virusa immunodefitsita cheloveka.

A series of oxaanalogs of myristic acid were synthesized and tested for antiviral activity in MT4 cells infected with human immunodeficiency virus 1 (HIV-1). The synthesized acids have no toxic effect on uninfected MT4 cells at a concentration of 100 microM. 14,14,14-Trifluoro-12-oxatetradecanoic acid substantially (by 75%) inhibits the reproduction of HIV-1. Other compounds synthesized, (7Z)-13-, (9Z)-13-, and (7Z)-11-oxatetradecenoic acids, exhibit no antiviral effect.

[New phospholipids--inhibitors of human immunodeficiency virus reproduction. Synthesis and antiviral activity]. / Novye fosfolipidy--ingibitory reproduktsii virusa immunodefitsita cheloveka. Sintez i antivirusnaia aktivnost'.

Several novel phosphatidic acid derivatives of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine were synthesized, which contained dialkylphosphatidyl, dialkylthiophosphatidyl moieties, as well as diacylphosphatidyl moiety with either 14,14,14-trifluoro-12-oxatetradecanoyl or natural acyl residues inherent in egg yolk phosphatidylcholine. Diacylphosphatidyl derivatives of glycyrrhetinic acid were also prepared. All the synthesized compounds exhibited significant anti-HIV activity. The glycyrrhetinic acid derivatives are of special interest because of their low toxicity and a supposedly different mechanism of action.

[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.

[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.

[The detection of viral antigens and ribosomal RNA in cells by using low-temperature media]. / Vyiavlenie virusnykh antigenov i ribosomal'noi RNK v kletkakh s ispol'zovaniem nizkotemperaturnykh sred.

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl-embedded HIV-infected cells. A genomic probe containing ribosomal sequences and labeled with biotin was used to hybridize rRNA molecules in sections of animal cells embedded in Lowicryl. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression and viral proteins at the ultrastructural level.