[Rapid spread of the HIV-1 circular recombinant CRF02-AG in Russia and neighboring countries].

The spread of the HIV-1circular recombinant CRF02-AG in countries of the former Soviet union (Commonwealth of Independent States, CIS) was studies using partial and full genome sequences. The full-genome sequence of the CRF02-AG recombinant circulating in Russia was obtained for the first time. A Global phylogenetic tree of CRF02-AG full-genome sequences was constructed. Three distinct groups of the sequences were detected as clustered by the geographical location (CIS, South Korea, and France), which is indicative of the single-virus introduction in each of the regions mentioned above. The CIS cluster exhibiting minimum genetic diversity was, therefore, relatively young. The phylogenetic analysis of the env gene sequences within the CIS cluster made it possible to clearly discriminate three branches: two of Russian and one of Uzbek origin. The low genetic diversity within the two Russian subclusters provides evidence of at least two recent independent introductions of the CRF02-AG recombinant from Central Asia into Russia. This work was performed within the framework of the 7th Federal Research Program (FP&), Project EURIPRED (European Research Infrastructures for Poverty Related Diseases), grant agreement No.312661.

In Vitro Antiviral Activity of a New Indol-3-carboxylic Acid Derivative Against SARS-CoV-2.

The coronavirus disease (COVID-19) pandemic has brought into sharp relief the threat posed by coronaviruses and laid the foundation for a fundamental analysis of this viral family, as well as a search for effective anti-COVID drugs. Work is underway to update existent vaccines against COVID-19, and screening for low-molecular-weight anti-COVID drug candidates for outpatient medicine continues. The opportunities and ways to accelerate the development of antiviral drugs against other pathogens are being discussed in the context of preparing for the next pandemic. In 2012-2015, Tsyshkova et al. synthesized a group of water-soluble low-molecular-weight compounds exhibiting an antiviral activity, whose chemical structure was similar to that of arbidol. Among those, there were a number of water-soluble compounds based on 5-methoxyindole-3-carboxylic acid aminoalkyl esters. Only one member of this rather extensive group of compounds, dihydrochloride of 6-bromo-5-methoxy-1-methyl-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy) carbonylindole, exhibited a reliable antiviral effect against SARS-CoV-2 in vitro. At a concentration of 52.0 µM, this compound completely inhibited the replication of the SARS-CoV-2 virus with an infectious activity of 106 TCID50/mL. The concentration curves of the analyzed compound indicate the specificity of its action. Interferon-inducing activity, as well as suppression of syncytium formation induced by the spike protein (S-glycoprotein) of SARS-CoV-2 by 89%, were also revealed. In view of its synthetic accessibility - high activity (IC50 = 1.06 µg/mL) and high selectivity index (SI = 78.6) - this compound appears to meets the requirements for the development of antiviral drugs for COVID-19 prevention and treatment.

Agent-Based Simulation of the COVID-19 Epidemic in Russia.

The COVID-19 pandemic has created a public health emergency in Russia and across the world. The wavelike spread of the new coronavirus infection, caused by newly emerging variants of the coronavirus, has led to a high incidence rate in all subjects of the Russian Federation. It is becoming extremely topical to get the opportunity to manage the development of the epidemic and assess the impact of certain regulatory measures on this process. This will help government agencies make informed decisions to control the burden on healthcare organizations. It is often impossible to obtain such assessments without using modern mathematical models.

[Molecular epidemiology of HIV-1 strains in the Moscow Region].

The Moscow Region is one of the HIV-1-affected subjects of the Russian Federation; there were 34613 HIV-1-infected subjects as of October 31, 2009. To characterize the molecular epidemiology of HIV-1 in the Moscow Region, the investigators obtained and studied HIV-1 variants from 61 infected subjects of the region, who were major risk groups: intravenous drug users (IDUs) and hetero- and homosexually infected persons. Genetic analysis of HIV-1 variants was carried out by sequencing the gag genes (729 nucleotides in length, including full-length protein p17 and partial p24) andlor env (270 nucleotides in length, V3 region) with further phylogenetic analysis. The findings demonstrated that HIV-1 subtype A variants are dominant in the Moscow Region and detectable in 93.5% of IDUs and 100% of heterosexually infected persons. Phylogenetically (and accordingly epidemiologically) unrelated HIV-1 subtype B strains were revealed in 4 patients, including 2 IDUs.

The Differential Anti-HIV Effect of a New Humic Substance-Derived Preparation in Diverse Cells of the Immune System.

The anti-HIV activity of a new humic substance-derived preparation has been studied in individual pools of immune cells (CD4+ T lymphocytes, macrophages, dendritic cells). Near-complete inhibition of the HIV infection (by more than 90%) was achieved by treating each of the abovementioned cell types with non-toxic concentrations of the preparation. The inhibitory effect demonstrates the possibility of preventing the depletion of a significant portion of functionally important immune cells. A comparative study of infection inhibition in individual cell pools has allowed us to reveal the differences in the preparation's effectiveness in each of the cell populations. A R5-tropic HIV-1 infection in macrophages exhibited maximum sensitivity to the preparation: 90% and 50% inhibition of the infection were observed in the presence of concentrations as low as 1.4 and 0.35 µg/ml, respectively. A 15- and 19-fold higher concentration was required to achieve the same extent of inhibition in dendritic cells infected with the same strain. The effectiveness of the drug in CD4 + T lymphocytes is quite comparable to its effectiveness in macrophages. The drug is universally effective for both the T- and M-tropic variants of HIV-1.

[The molecular epidemiology of HIV-1 in Belarus in (1996-2004): a predominance of subtype A variants and circulation of subtype B variants].

To study the molecular epidemiology of HIV-1 in Belarus, the genetic sequences of HIV-1 variants were obtained from 50 infected persons, which represented the main stages, risk groups, and geographic areas of the epidemic. The env and gag sequences were studied for HIV-1 variants from 31 persons, the env sequences were for HIV-1 variants from 18 persons, and the gag sequence was for HIV-1 variant from 1 person. Phylogenetic analysis indicated that the sequences of HIV-1 variants from 46 persons were homogenic and evolutionally closely related to IDU-A strains specific for other epidemics in the former Soviet Union are dominating in the epidemic in Belarus. Circulation of epidemiologically unrelated subtype B viruses was also established.

Analysis of DNA from human adenovirus type 6 with restriction endonucleases HindIII, BglIII and BamHI.

The genome of the type 6 human adenovirus has three restriction sites for R.BamHI, thirteen for R.HindIII and ten for R.BglII. The terminal fragments of DNA cleaved by each of the enzymes have been determined by means of terminal nucleotidyl transferase and by analysis of the DNA-terminal protein complex. The sequence of the cleaved fragments has been determined by partial cleavage of DNA, simultaneous digestion of DNA with various combinations of enzymes and secondary digestion of individual isolated fragments with other enzymes. The following order of the cleaved fragments in the adenovirus type 6 genome has been found (the figures in brackets are the weights in mega-daltons): for R.BamHI-B(7.1)-D(3.0)-C(4.05)-A(8.5); for R.HindIII-F(1.7)-C1(2.14)-A(3.44)-M(0.046)-I(1.24)-J(0.77)-D(2.1)-E(1.96)-B(3.18)-H(1.36)-L(0.18)-C2(2.14)-G(1.44)-K(0.16); for R.BglII-E(2.07)-B(3.58)-A(4.8)-C(3.36)-I(0.78)-D(3.25)-G(1.37)-J(0.21)-F(1.85)-K(0.17)-H(0.94).

Simultaneous introduction of distinct HIV-1 subtypes into different risk groups in Russia, Byelorussia and Lithuania.

OBJECTIVE: To investigate genotypes and serotypes of HIV-1 variants in Russia, Byelorussia and Lithuania. PATIENTS AND METHODS: Sera from 20 HIV-1-infected individuals were tested in an enzyme-linked immunosorbent assay (ELISA) with 19 V3 synthetic peptides, and serum HIV-1 V3 RNA was amplified and sequenced. RESULTS: Sequence comparison of the envelope V3 region among specimens tested revealed a 2-29% range nucleotide divergence, with a mean of 19%. Phylogenetic analysis from the homosexual men were shown to belong to subtype B, and all of the heterosexually infected individuals to subtype C. Sequences from the parenterally infected individuals were more heterogeneous. IOn the peptide ELISA three reactivity patterns were found. Serum samples from six out of seven homosexual men showed reactivity to peptides p108 or p110 representing V3 amino-acid sequences found in US/West European HIV-1 isolates. Serum samples from six of seven individuals who had acquired HIV-1 through heterosexual contacts were reactive to peptide p169. Four out of six parenterally infected patients had peak reactivity to p168. CONCLUSION: Distinct HIV-1 variants were found in Russia, Byelorussia and Lithuania, which were introduced simultaneously in the mid-1980s. This diversity was shown to be associated with the route of transmission. Homosexual men appeared to be infected with subtype B and heterosexually infected individuals with subtype C HIV-1 variants. HIV-1 subtypes A, C, D and G were found among parenterally infected individuals.

PIP: HIV-1 variants show a relatively high level of genomic and antigenic diversity. This heterogeneity is particularly high in the V3 domain of envelope glycoprotein gp120, which is implicated in a number of biological properties of HIV-1, including cell tropism, infectivity, and cytopathogenicity; it is also a target for both humoral and cellular immune response. At least nine subtypes of HIV-1 have been identified. Subtypes A-H are phylogenetically equidistant and shown to be geographically associated with different subcontinents. Subtype B is most prevalent in North and South America and western Europe. Subtypes A, C, D, G, and H are found frequently in sub-Saharan countries, while subtype C is also found in India. Subtype E sequences have been found in patients from Thailand and Central Africa, and subtype F has been described in Romania and Brazil. This study reports findings from an investigation of genotypes and serotypes of HIV-1 variants in Russia, Byelorussia, and Lithuania. Sera from 15 HIV-1-infected men and 5 HIV-1-infected females were tested by ELISA with 19 V3 synthetic peptides with amplified and sequenced serum HIV-1 V3 RNA. Sequence comparison of the envelope V3 region among specimens tested revealed a 2-29% range of nucleotide divergence, with a mean of 19%. Distinct variants of HIV-1 were found in Russia, Byelorussia, and Lithuania, which were introduced simultaneously in the mid-1980s. The diversity was shown to be associated with the route of transmission. Homosexual men appeared to be infected with subtype B compared to heterosexually infected individuals with subtype C HIV-1 variants. HIV-1 subtypes A, C, D, and G were found among parenterally-infected individuals. These findings are based upon the three peptide reactivity patterns identified by ELISA. Serum samples from six out of seven homosexual men showed reactivity to peptides p108 or p110 representing V3 amino-acid sequences found in US/West European HIV-1 isolates. Serum samples from six out of seven individuals who had acquired HIV-1 through heterosexual contacts were reactive to peptide p169, and four out of six parenterally infected patients had peak reactivity to p168.

EcoRI activity: enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.

Investigation of human immunodeficiency virus fusion peptides. Analysis of interrelations between their structure and function.

The N-terminal region of the human immunodeficiency virus type 1 (HIV-1) gp41 appears to be involved in virus-cell membrane fusion. To study the influence of fusion domain structure on gp41 interaction with artificial lipid membranes, two families of peptides were synthesized. The peptides of the first family starting from the C-terminal Gly-532 of gp160 (BRU isolate) were assembled in a stepwise manner to N-terminus of gp41(Ala-517). These hydrophobic peptides, containing 10-16 amino acid residues (a.a.), were able to form channel-like current fluctuation through planar lipid membranes, and the longest 15-16 a.a. peptides lysed the liposomes. Peptides of the second family beginning from the C-terminal Arg-538 and continuing to Val-510 contained several hydrophilic amino acid residues. These 15-22 a.a. peptides also increased the conductance of planar lipid bilayers and lysed liposomes. The degree of liposome lysis depended upon peptide length and concentration. The attachment of gp120 C-terminal amino acid or peptides to N-terminus of 517-538 peptide resulted in complete loss of activity. The effects of the second family of peptides on membranes were reduced to a great extent at acidic pH. The conjugation of 22 a.a. Lys peptide with bovine serum albumin decreased its lytic activity. The circular dichroism study of these peptides revealed alpha-helix configuration in hydrophobic and aqueous media only for deca- and longer peptides. The electron microscopy of 22 a.a. peptide performed in the aqueous medium showed large spherical aggregates about 0.5-0.7 micron in diameter consisting of long filaments approximately 5 nm in diameter. Other tested peptides could generate only short strings. Thus, the effects of fusion peptides on lipid membranes depends on their sequence and length, secondary and tertiary structures, and freedom of their N-terminus.

Variability analysis of HIV-1 gp120 V3 region: I. Point estimators for the amino acid distribution characteristics.

Enumerating procedure for symbol sequences is proposed. Relationship between Hamming distance for symbol sequences and Euclidean distance for corresponding enumerations is established, and more universal Hamming-transformed Euclidean measure is constructed. A distribution function of amino acid substitutions and some of its point estimators (consensus, subconsensus, sample mean, sample central moments and asymmetry coefficient) are introduced. Hamming-transformed Euclidean measures between consensus, subconsensus and sample means for ten HIV-1 taxons of gp120 V3 regions are calculated. It is demonstrated that these taxons have a complicated pattern which is significant for their classification.

Dependencies of substitution steps number on Hamming distance are identical for one-parameter discrete models of both direct and parallel genetic diversity.

With the help of previously introduced enumeration procedure (M.Yu. Shchelkanov, A.N. Yudin, A.V Antonov, N.S. Starikov, A.A. Vedenov, E.V. Karamov, J. Biomol. Struct. Dyn. 15, 217-229 (1997)) and probability distribution function for the enumeration after some substitution steps (M.Yu. Shchelkanov, L.A. Soinov, V.V. Zalunin, D.A. Gumennyi, A.N. Yudin, A.A. Natan, V.B. Kireev, E.V. Karamov, J. Biomol. Struct. Dyn. 15, N 4, (1998)) we have demonstrated that dependencies of replication acts number on Hamming distance are identical for one-parameter discrete models of both direct and parallel genetic diversity.

Variability analysis of HIV-1 gp120 V3 region: III. Distinctions between various sets of peptide fragments derived from the sequences belonging to different HIV-1 taxons.

Distinction criterion for various sets of fixed length peptide fragments and integral distinction measure for various sets of peptide fragments with different length and start position ranges have been introduced on the base of an enumeration procedure and a point estimators for the amino acid distribution characteristics introduced previously (M. Yu. Shchelkanov, A. N. Yudin, A. V. Antonov, N. S. Starikov, A. A. Vedenov, E. V. Karamov, J. Biomol. Struct. Dyn. 15, 231-241 (1997)). Differences between 6-10-mer peptides derived from the majority of HIV-1 taxon pairs are demonstrated to be located generally in the vicinity of the V3-loop top. This validates the suitability of V3 top mimicking synthetic peptides for HIV-1 serotyping. A significant difference between E subtype V3 C-terminus peptides and the corresponding peptides derived from the other subtypes has been demonstrated. Taking into account the Langerhans' cells tropism of E subtype virus variants we have hypothesized the influence of mutations in the V3 C-terminus on HIV-1 cell tropism.

Variability analysis of HIV-1 gp120 V3 region: IV. Distribution functions for intra- and inter-subtype amino acid hamming distances.

Distribution functions for intra- and inter- HIV-1 V3-loop subtypes amino acid Hamming distances were calculated (850 V3-loop sequences from the Los Alamos HIV-1 Database (1996) were used). These functions have pronounced bell-like shape. Such shapes of the histograms for HIV-1 V3 intra- and inter-subtype distriutions are discussed to confirm the applicability of different hierarchical cluster procedures for HIV-1 V3 classification. Two-mode distribution for the subtype E could sertificate that this subtype includes two thinner taxons.

One-parameter discrete model of the genetic diversity.

One-parameter discrete model estimating genetic distance between precursor and descendant nucleotide sequences after several steps of substitution acts is developed. This model based on the previously introduced symbol sequences enumeration procedure (M.Y. Shchelkanov, A.N. Yudin, A.V. Antonov, N.S. Starikov, A.A. Vedenov, E.V. Karamov, J. Biomol. Struct. Dyn. 15, 231-241 (1997)) differs from Jukes-Cantor and Kimura models by the absence of the assumptions usual for continuous Markov's processes. Formula obtained with the help of our model are more preferable since they take into account multiple repetition substitution ability and they are correct in the entire admissible range of parameters.

Variability analysis of HIV-1 gp120 V3 region: II. Hierarchy of taxons.

In the previous work (M. Yu. Shchelkanov, A. N. Yudin, A. V. Antonov, N. S. Starikov, A. A. Vedenov, E. V. Karamov, J. Biomol. Struct. Dyn. 15, 217-229 (1997)) we have introduced the amino acid distribution function within HIV-1 taxons and Hamming-transformed Euclidean measures between their characteristics: consensus, subconsensus and sample mean. In this work the referred characteristics are used for hierarchical classification of amino acid sequences of gp120 V3 region belonging to different HIV-1 taxons. A comparative analysis of the results produced by various classification methods is carried out. Multidimensional scaling of distance matrix for the specified characteristics is used to visualize the pattern of HIV-1 variability.

Detection by immunogold techniques of HIV antigens in Lowicryl ultrathin sections of infected cells.

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl K4M-embedded HIV-infected cells. With serum from an AIDS patient, specific immunogold labelling was obtained exclusively on mature viral extracellular structures. The more intense reactivity was obtained with core antigens. The present immunoelectron microscopy method provides several advantages - high sensitivity of immunodetection, good preservation of cellular morphology, easy preparation procedure - which could lead to the use of this method for HIV-infected human tissues.

[Suppression of the human immunodeficiency virus in cultured cells by 5'-phosphites of 3'-azido-2',3'-dideoxynucleosides]. / Podavlenie virusa immunodefitsita cheloveka v kul'ture kletok 5'-fosfitami 3'-azido-2',3'-didezoksinukleozidov.

5'-Phosphites (5'-hydrogenphosphonates) of 3'-azido-2'-, 3'-dideoxynucleosides are shown to be effective inhibitors of the human immunodeficiency virus (HIV-1) in MT4 cell culture. 5'-Phosphite of 3'-azido-2', 3'-dideoxythymidine was the most active among these compounds and even a little more active as compared to the well-known anti-AIDS drug 3'-azido-2',3'-dideoxythymidine; at the same time 5'-phosphites of 3'-azido-2',3' -dideoxynucleosides with adenine, guanine and cytosine bases were more active than the corresponding nucleosides. The toxicity of all four phosphites was comparatively low and the equimolar mixture of all four phosphites was 2-3 fold less toxic than each of them separately. Data on the decreased toxicity of the phosphite mixture are explained from the viewpoint of a decreased pool disbalance of natural 2'-deoxynucleoside 5'-triphosphates in cells; a significant pool disbalance is developed in the case of 3'-azido-2',3'-dideoxythymidine action.

[Inhibition of reproduction of the human immunodeficiency virus in cell culture by 2',3'-dideoxynucleoside-5'-phosphites]. / Ingibirovanie reproduktsii virusa immunodefitsita cheloveka v kul'ture kletok 5'-fosfitami 2'-3'-didezoksinukleosidov]

5'-Phosphites (5'-hydrogenphosphonates) of 2',3'-dideoxynucleosides (T, A, G, C) were synthesized and studied as inhibitors of human immunodeficiency virus type 1 (HIV-1) in MT4 and CEM13 cell cultures. It was shown that all 5'-phosphites effectively inhibit the production of viral antigens and protect cells from the cytotoxic effect of HIV infection. 5'-Phosphites were more active antiviral compounds than the corresponding nucleosides.

[Identification of immunoreactive epitopes in proteins coded by gag, env, and pol genes of the type I human T-lymphotropic virus (HTLV-I) using synthetic peptides]. / Identifikatsiia immunoreaktivnykh épitopov v belkakah, kodiruemykh genami gag, env i pol T-limfotropnogo virusa cheloveka tipa I (HTLV-I) s ispol'zovaniem sinteticheskikh peptidov.

Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the gag p19, env gp46, and pol proteins of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins.