[Analysis of resistance-associated substitutions in hepatitis C virus sequences from Kyrgyzstan].

INTRODUCTION: The countries of Central Asia, including Kyrgyzstan, are characterized by high prevalence and morbidity of HCV infection. Identification of HCV genotype and mutations associated with resistance to direct-acting antiviral (DAA) plays an important role either in conducting molecular epidemiological studies or choosing the treatment tactics. The aim of the work was to research of the genotype diversity of HCV variants circulating in Kyrgyzstan and the identification among them the mutations associated with the development of resistance to DAA. MATERIALS AND METHODS: 38 serum samples from HCV-infected residents of Kyrgyzstan were analyzed in this study. The nucleotide sequences of viral gene fragments (NS3, NS5A, NS5B) were determined by Sangers sequencing and deposited in the international GenBank database under the numbers ON841497ON841534 (NS5B), ON841535ON841566 (NS5A), and ON841567ON841584 (NS3). RESULTS: The HCV subtypes 1b (52.6%; 95% CI 37.367.5%), 3a (44.8%; 95% CI 30.260.2%) and 1a (2.6%; 95% CI 0.513.4%) are circulating in Kyrgyzstan. 37% (95% CI 1959%) of subtype 1b isolates had C316N mutation in the NS5A gene; 46% (95% CI 2370%) had F37L mutation in the NS5A gene; 45% (95% CI 2272%) had Y56F mutation in the NS3 gene. Among subtype 3a isolates, resistance-associated mutations in NS5B fragment were not found. 22% (95% CI 945%) of subtype 3a sequences had a Y93H mutation in the NS5A gene. A combination of Y56F + Q168 + I170 mutations was identified among all sequences of NS3 gene. DAA resistance mutations were not found in NS3, NS5A, NS5B genes of subtype 1a sequence. CONCLUSION: A rather high prevalence of mutations associated with resistance or significant decrease in sensitivity to DAA among HCV sequences from Kyrgyzstan was shown. Updating of data on HCV genetic diversity is necessary for timely planning of measures to combat epidemic.

Consensus Integrase of a New HIV-1 Genetic Variant CRF63_02A1.

The high genetic variability of the human immunodeficiency virus (HIV-1) leads to a constant emergence of new genetic variants, including the recombinant virus CRF63_02A1, which is widespread in the Siberian Federal District of Russia. We studied HIV-1 CRF63_02A1 integrase (IN_CRF) catalyzing the incorporation of viral DNA into the genome of an infected cell. The consensus sequence was designed, recombinant integrase was obtained, and its DNA-binding and catalytic activities were characterized. The stability of the IN_CRF complex with the DNA substrate did not differ from the complex stability for subtype A and B integrases; however, the rate of complex formation was significantly higher. The rates and efficiencies of 3'-processing and strand transfer reactions catalyzed by IN_CRF were found to be higher, too. Apparently, all these distinctive features of IN_CRF may result from specific amino acid substitutions in its N-terminal domain, which plays an important role in enzyme multimerization and binding to the DNA substrate. It was also found that the drug resistance mutations Q148K/G140S and G118R/E138K significantly reduce the catalytic activity of IN_CRF and its sensitivity to the strand transfer inhibitor raltegravir. Reduction in sensitivity to raltegravir was found to be much stronger in the case of double-mutation Q148K/G140S.

[A single-tube real-time RT-PCR assay for the RNA detection and quantification of genetically diverse HIV Including rare non-M group of the HIV-1 and HIV-2].

The RT-PCR method with real-time fluorescence detection was used for development of φ prototype of diagnostic kit for reliable diagnosis of genetic variants of RNA of the HIV-1 of groups M, N, O, P and RNA of the HIV-2 in blood plasma and serum. The kit is stable against nucleotide defects, provides broad linear range of concentration of the HIV RNA, 100% analytical specificity and adequate analytical sensitivity: 42 ME/ml (HIV-1 of group M), 45 copies/ml (HIV-2), 92 copies/ml (HIV-1 of group O), 90 copies/ml (HIV-1 of group N). The kit provided successful detection and measurement of HIV RNA concentration in the samples of the international reference panel of the HIV-1 genotype. The results of the test correlate with results of commercial tests.

Genetic characterization of an isolate of HIV type 1 AG recombinant form circulating in Siberia, Russia.

Before 2008, HIV-1 subtype A was the predominant genetic variant in the Novosibirsk oblast of Russia as well as in most parts of this country. However, a rapid spread of the recombinant HIV-1 02_AG form has been reported in Novosibirsk since 2009. We have analyzed the genome of the 10.RU.6637 isolate, a HIV-1 02_AG recombinant form, which represents a monophyletic cluster of the HIV-1 variants widespread in this region. Phylogenetic analysis has shown that the Siberian 10.RU.6637 isolate displays the highest sequence identity to the HIV-1 subtype AG forms circulating in Uzbekistan. However, recombination analysis of 10.RU.6637 has demonstrated that this isolate is a recombinant form between HIV-1 subtype A and CRF02_AG, differing in its genetic structure from both the CRF02_AG reference sequences and the Central Asian variants of HIV-1 02_AG.

[Study of biological properties of HIV-1 variants resistant to antiretroviral preparations].

AIM: Separation of HIV-1 isolates from HIV infected patients who had received antiretroviral therapy courses. Analysis of genetic and replicative properties of the separated isolates, study of pol gene mutation stability sustentation that is responsible for the emergence of drug resistance. MATERIALS AND METHODS: HIV isolate separation was carried out by co-cultivation ofperipheral blood mononuclears of HIV infected patients with previously stimulated phytohemagglutinin cells of healthy donors. Virus replication was evaluated by the level of p24 virus specific protein accumulation determined in enzyme immunoassay. HIV-1 subtype identification, detection of HIV-1 genome mutations were carried out by pol gene nucleotide sequence determination and subsequent analysis of the data obtained - by using specialized program resources. RESULTS: 14 infectious HIV-1 subtype A, B and CRF02_AG isolates were separated containing various sets of mutations determining resistance to widely used in clinical practice nucleoside and non-nucleoside reversetranscriptase inhibitors. Comparative analysis of mutation specter detected in HIV-1 variant genomes before isolation and after their cultivation showed that during HIV-1 cultivation in mononuclear blood cells without the addition of antiretroviral preparations not only partial loss of mutations is observed but also emergence of new drug resistance mutations; and most of the mutation causing virus resistance to antiretroviral preparations remain. CONCLUSION: High reproductive properties of the HIV-1 isolates separated allow to use them to evaluate effectiveness of the drugs being developed against HIV-1 resistant to antiretroviral preparations.

[Prevalence of mutations responsible for resistance to antiretroviral preparations among HIV-1 variants circulating in Novosibirsk region].

AIM: Analyze the diversity and prevalence of mutations in human immunodeficiency virus type 1 (HIV-1) genome emerging in response to antiretroviral therapy isolated from HIV-infected individuals of Novosibirsk region in 2010, 2011. MATERIALS AND METHODS: Detection of mutations in HIV-1 genome responsible for the resistance to. antiretroviral preparations (ARVP) was carried out by determination of pol gene nucleotide sequence and subsequent analysis ofthe data obtained by program HIVdb: Genotypic Resistance Interpretation Algorithm. RESULTS: HIV-1 resistance mutations to antiretroviral preparations were detected in 23.6% of the total number of the studied samples. The most prevalent mutations are those conditioning resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (M184V, Y181C and K103N). In studies of HIV-1 isolated from 4 patients who had not received antiretroviral therapy (ARVT) transmission of HIV-1 resistant to various groups of preparations was detected. CONCLUSION: The detected facts of ARVP resistant HIV-1 circulation among patients who had not received ARVT and the data obtained on the mutations emerging in response to therapy underline the relevance of administration of HIV-1 resistance profile study during both decrease of ARVT effectiveness and primary administration of therapy to HIV infected patients.

[Molecular-genetic characteristic of HIV-1 A and B subtypes variants isolated in Novosibirsk region].

AIM: Study phylogenetic interconnections of HIV-1 subtype A and B variants circulating in Novosibirsk region (NSR). MATERIALS AND METHODS: 268 HIV-1 variants isolated in 2007 - 2010 from blood samples of HIV infected patients in NSR, Samara, Congo and Moscow. HIV-1 variant genotyping was performed by analysis of 1.3 kb long pol gene nucleotide sequences. Phylogenetic analysis of nucleotide sequences was carried out by program Mega version 4.1 by constructing phylogenetic trees by nearest neighbor method. Nucleotide distances were calculated by Kimura method. RESULTS: The studied HIV-1 subtype B variants form separate phylogenetic groups with a low HIV-1 nucleotide sequence homology level combined based on territorial principle and/or time of HIV infection in a territory but not possessing interconnection with a specific population risk group. Subtype A HIV-1 is a fairly homogenous monophyletic group. Phylogenetic differences during studies of HIV-1 isolated from risk group patients - injection drugs users and individuals infected through sexual contacts were not detected. HIV variants isolated from patients infected in Moscow and Samara generally grouped with HIV variants circulating in the European part of Russia. CONCLUSION: An independent circulation of genetically separate HIV-1 subtype B groups is observed on the territory of siberian region which is a result of multiple independent introductions of distant variants of the virus. The confirmed limited spread of this HIV-1 genetic variant with a subsequent territorial separateness creates a possibility of formation of genetically different virus populations. The studies of subtype A viruses performed confirm the high level of homogeneity detected earlier in other Russia territories of HIV-1 belonging to this genetic variant. Monophyly of subtype A HIV variants is explained by imposition of 2 factors - territorial mobility of the population inside the country and lack of specific transmission routes for HIV-1 subtype A.

[Properties of CRF02_AG HIV-1 isolates circulating in Novosibirsk region].

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.

[New recombinant variant of human immunodeficiency virus of type 1, subtype envB/envA, isolated in Novosibirsk]. / Novyi rekombinantnyi variant subtipa envB/envA VICH-1, vydelennyi v Novosibirske.

The nucleotide sequence of the variant of human immunodeficiency virus of type 1 (HIV-1), mostly widespread on the territory of the Novosibirsk region, was determined. The analysis of the nucleotide sequence confirmed that this variant belonged to HIV-1 of subtype A. The HIV-1 recombinant variant of subtype envB/envA with the recombination area within the second conservative region C2 of gene env, so far unknown, was detected and characterized. In HIV-1 the area at the beginning of gene env (5'-env) was found to belong to subtype B and the sequence at the end of gene env (3'-env), to subtype A. The analysis of the amino acid sequence of the third variable region of gene env demonstrated that the viruses under study belonged to macrophagotropic "slow/low" variants, characterized by low replication speed. The analysis of nucleotide sequences of the isolated variants of HIV-1 revealed their close genetic relationship with HIV-1 isolates circulating on the territory of Ukraine.

[Molecular epidemiological analysis of the spread of human immunodeficiency virus (HIV) infection in the Novosibirsk region]. / Molekuliarno-biologicheskii analiz rasprostraneniia VICH-infektsii v Novosibirskoi oblasti.

The dynamics of the spread of individual subtypes of type 1 HIV (HIV-1), circulating in the Novosibirsk region during the epidemic rise of HIV infection was under study. The epidemic of HIV-1 in Novosibirsk has a pattern similar to that in Russia as a whole. At the initial stage of epidemics multiple sources of virus determine the heterogeneity of the isolated subtypes of HIV-1. Then the parenteral route of HIV transmission, connected with the intravenous use of narcotic drugs, becomes dominant. Recently the spread of HIV-1 from the group of intravenous drug users to other groups of the population has been observed. In the circulation of HIV-1 among drug users the leading role was shown to belong to subtype A, which ensures its rapid spread and dominating role in the epidemic process. Further spread of the HIV-1 epidemic is expected to proceed in parallel to the spread of viral hepatitis, sexually transmitted diseases and drug addiction. Thus, HIV-1, subtype A, may be assumed to be dominant in the Novosibirsk region in the nearest future.

Characterization of recombinant integrase of human immunodeficiency virus type 1 (isolate Bru).

Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virus-encoded integrase protein. The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene. Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli). The main kinetic parameters of HIV-1 integrase (K(m) = (3.7 +/- 0.2).10(-10) M, k(cat) = (1.2 +/- 0.3).10(-7 )sec(-1)) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate. Inhibition of integrase by aurintricarbonic acid ([I](50) = 6.3 +/- 0.4 microM) and dependence of integrase activity on Mg2+ and Mn2+ concentration were studied.

[A new approach to detect a particular DNA sequence by UV-immobilization of its hybridization complex with a highly specific probe resulting from ligation of a tandem of short oligonucleotides in solution]. / Novyi podkhod k vyiavleniiu analiziruemoi posledovatel'nosti DNK putem UF-immobilizatsii ee gibridizatsionnogo kompleksa s vysokospetsifichnym zondom, obrazuiushchimsia pri ligirovanii tandema korotkikh oligonukleotidov v rastvore.

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN8 + pN4 + pN8' Bio) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin-alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was completely complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.

[Chronically HIV-1 infected human monocyte culture U937 as a model for assessing the efficacy of anti-HIV agents]. / Khronicheski infitsirovannaia VICH-1 kul'tura monotsitov cheloveka U937 kak model' dlia otsenki éffektivnosti anti-VICh-preparatov.

Cell culture U937 chronically infected with HIV-1 is suggested as a model for adequate evaluation of antiviral activity of HIV inhibitors. Azidothimidine (AZT) notable decreased HIV-1 reproduction in chronically infected U937 cells to passages 15-18. Glycirrhizic acid (GA) effectively inhibited the virus production during the first four passages, while in subsequent passages (up to passage 20) decreased the virus production by only 60% in comparison with the control. If a combination of AZT and GA (1:1000) was used, p24 was not detected in the culture fluid by passage 20. Culturing of U937 cells with AZT led to a 10-fold decrease in the amount of DNA 2-LTR in comparison with the total content of proviral DNA, the content of HIV-1 DNA 1-LTR remaining virtually unchanged. Culturing of U937 with a combination of AZT and GA resulted in a notable decrease in the content of proviral DNA 2-LTR after passage 3, while after passage 9 this form of HIV-1 DNA was not detected at all.

[Detection of unintegrated forms of proviral DNA in HIV infections]. / Opredelenie kol'tsevykh form provirusnoi DNK pri VICh-infektsii.

The content of 1-LTR and 2-LTR circular forms of HIV-1 proviral DNA was determined by nested PCR. Outer and inner primers for the first and second stages of PCR were selected in the env and gag regions that allowed the authors to simultaneously test 1- and 2 LTR of DNA HIV-1 forms in the analyzed samples. The accumulation of circular species of HIV-1 DNA was shown to occur during HIV infection as well for acutely infected cell lines as chronically infected mononuclear cells. The cell cultures providing productive infection was characterized by higher levels of 2-LTR circular HIV-1 DNA. Analysing the clinical samples demonstrated greater differences in the accumulation of the circular forms of proviral DNA. It was detected in 16 tested clinical samples both of 1-LTR and 2-LTR circular DNA in 6 samples and 1-LTR in 11 samples of 2-LTR of HIV-1 DNA is a minor fraction in most clinical samples, but in some samples the relative content of 1-LTR/2-LTR DNA was shown to be 1:1.