Vol 100, No 2 (2023)

Introduction. The antigen complex of opportunistic pathogenic bacteria (ACOPB) has a protective effect against avian influenza viruses, herpes virus type 2, and other viruses that cause acute respiratory viral infections. In the context of the COVID-19 pandemic, an important task is to find out whether ACOPB has a protective effect against SARS-CoV-2.

The purpose of the study was to evaluate in vitro the ACOPB virus-inhibitory activity against the Dubrovka laboratory strain of SARS-CoV-2.

Materials and methods. The study was performed using Vero cell line CCL-81, human peripheral blood mononuclear cells (PBMCs), mouse monoclonal anti-idiotypic antibodies structurally mimicking biological effects of human interferons (IFNs), the Dubrovka laboratory strain of SARS-CoV-2. The infectivity of the virus was assessed by two methods: by virus titration using cell cultures and the limiting dilution method when the results are assessed by a cytopathic effect; the second method was a plaque assay. The in vitro virus inhibition test was performed using the cell culture susceptible to SARS-CoV-2; the mixture containing a specific dose of the virus and a two-fold dilution of ACOPB was transferred to the cell culture after the ACOPB medication had interacted with the virus at 4ºC for 2 hours. The ACOPB virus-inhibitory activity against SARS-CoV-2 was assessed by the functional activity of α/β and γ IFN receptors (R_IFN) in human PBMCs induced in vitro by ACOPB and the ACOPB mixture with the specific dose of SARS-CoV-2. The R_IFN expression level was measured by the indirect membrane immunofluorescence test.

Results. Hemagglutination assay using chicken, mouse, guinea pig, and human red blood cells was performed for detection of the SARS-CoV-2 inhibitory protein. The lysate of Vero CCL-81 cells infected with SARS-CoV-2 Dubrovka demonstrated the highest hemagglutination activity with guinea pig red blood cells and low titers of hemagglutination in the virus-containing fluid. The virus inhibition test in the Vero CCL-81 cell culture demonstrated that ACOPB inhibited 10 doses of SARS-CoV-2 Dubrovka with the titer 1 : 32, providing 100% protection of the cell culture for 8 days (the monitoring period). ACOPB induced α/β and γ R_IFN expression on membranes of human PBMCs in in vitro cultures and decreased R_IFN α/β and γ expression after its interaction with SARS-CoV-2 Dubrovka.

Conclusion. The experimental studies including the virus inhibition test in the cell culture susceptible to SARS-CoV-2 Dubrovka and the indirect membrane immunofluorescence assay using monoclonal anti-idiotypic antibodies mimicking IFN-like properties demonstrated that ACOPB had both an immunomodulatory and a virus-inhibitory effect.

Introduction. Bacillus anthracis is the causative agent of anthrax, a pathogen characterized by high genetic monomorphism that complicates differentiation of strains. Thus, molecular methods for pathogen typing require the improvement.

The aim of the study. To select marker SNPs for new genetic groups of B. anthracis and to develop a method for their laboratory identification using HRM PCR.

Materials and methods. The core genome of 222 strains of B. anthracis from the GenBank database and 66 strains from the collection of pathogenic microorganisms of the Stavropol Anti-Plague Institute was aligned using the parsnp software. A dendrogram based on 7242 core genome SNPs was built in MEGA X software. The strains for validation of the HRM method included representatives of various genetic groups. The HRM PCR reaction was performed using the "Type-it HRM PCR Kit" and "KAPA HRM FAST qPCR Kit" and a Rotor Gene DNA thermocycler with HRM function. Data analysis and visualization were performed using custom scripts in the Python and R development environments.

Results and discussion. Marker SNPs for 6 genetic groups have been identified, which make it possible to determine whether strains belong to one of 7 new subclusters. Pairs of primers were selected for the loci containing them, HRM PCR parameters were optimized for discrimination of different alleles of SNP loci, and an analysis scheme was developed.

Conclusion. Thus, marker SNPs were selected to determine the genetic subclusters A.Br.CEA, A.Br.STI, A.Br.Tsiankovskii, B.Br.Europe, B.Br.Siberia, B.Br.Asia, B.Br.018, and a new laboratory method was developed for molecular subtyping of B. anthracis using HRM PCR.

Introduction. Antimicrobial resistance is a global public health concern. Salmonella spp., which can be transmitted to humans through contaminated food, are among the most important foodborne pathogens worldwide.

Materials and methods. The antimicrobial resistance of 358 bacterial isolates collected from food and water in the Republic of Belarus (Belarus) in 2018–2021 was studied by analyzing phenotypic and genotypic characteristics of antibiotic bacterial resistance. MALDI-TOF mass spectrometry was used to classify and identify bacteria. Phenotypic antimicrobial susceptibility of bacteria was measured by the minimum inhibitory concentration method using a Sensititre automated bacteriological analyzer and the disk diffusion test for 45 antimicrobial agents. Antimicrobial resistance genes in multidrug-resistant Salmonella isolates were identified by whole-genome sequencing.

Results. The in vitro testing of phenotypic bacterial susceptibility showed high susceptibility to fluoroquinolones (97.2%), third-generation cephalosporins (93.9%), carbapenems (98.0%), ampicillin (81.8%), aminoglycosides (97.5%), tetracyclines (87.5%), chloramphenicol (93.8%), trimethoprim/sulfamethoxazole (co-trimoxazole) (95.3%) and colistin (85.2%). It was found that the antibiotic resistance mechanism in S. enterica was associated with the presence of genes blaTEM-1B (82%), blaTEM-1C (7.7%), blaSHV-12 (2.6%), blaDHA-1 (2.6%), blaCMY-2 (7.7%), qnrB2 (9.1%), qnrB4 (9.1%), qnrB5 (9.1%), qnrB19 (72.7%), aac(6’)-Ib-cr (9.1%), aac(6’)-Iaa (100%), aadA1 (13.2%), aadA2 (8.8%), tetB (74.3%), tetA (25.7%), tetM (2.9%), tetD (28.6%), mcr-9 (1.5%).

Conclusion. All the bacterial isolates were phenotypically susceptible to first-line antibiotics used in treatment of salmonellosis: fluoroquinolones and third-generation cephalosporins. The whole-genome sequencing of multidrug-resistant Salmonella isolates (19.0%) detected resistance genes for 9 groups of antibiotics: aminoglycosides (100%), beta-lactams (57.4%), fluoroquinolones (16.2%), tetracyclines (51.5%), macrolides (1.5%), phenicols (30.4%), trimethoprim (13.0%), sulfonamides (47.8%) and colistin (1.4%). Thus, epidemiological surveillance of the Salmonella spread through the food chain is of critical importance for the monitoring of antimicrobial resistance among foodborne Salmonella.

Introduction. The main biological raw materials for the production of immunobiological preparations for identification of Bacillus anthracis are its specific antigens, the protective antigen and the EA1 protein.

Purpose. To determine the antigenic identity of immunodominant proteins of different genovariants of B. anthracis isolated by gel chromatography and electrophoresis.

Materials and methods. Culture filtrates of isogenic variants of B. anthracis strain 575/122 (pXO1⁺, pXO2⁺): R01 (pXO1⁺, pXO2^–); R00 (pXO1^–, pXO2^–) were used in the study. Gel chromatographic fractionation and electrophoretic separation were carried out according to standard methods. The antigenic properties of proteins isolated by gel chromatography and electrophoresis were studied by immunodiffusion with polyclonal monospecific sera against the protective antigen and the EA1 protein of the S-layer.

Results. Gel chromatographic separation of B. anthracis 575/122 culture filtrates R01 (pXO1⁺, pXO2^–) and R00 (pXO1^–, pXO2^–)yielded fractions 1 and 5. Sera against EA1 protein and antigens of fraction 1 of strains B. anthracis 575/122 R00 and B. anthracis 575/122 R01 culture filtrates identified the identical antigens. Serum against antigens of fraction 5 of B. anthracis 575/122 R01 contained antibodies to numerous proteins, including the protective antigen isolated by electrophoresis.

Discussion. The antigenic identity of immunodominant proteins isolated by gel chromatography and electrophoresis was identified.

Conclusion. EA1 and PA proteins isolated by electrophoresis and gel chromatography can be used for production of monoclonal and polyclonal monospecific antibodies suitable for the design of diagnostic preparations.

The development of sequencing technologies and bioinformatic analysis made it possible to conduct molecular and epidemiological studies, in which nucleotide sequences of the human immunodeficiency virus (HIV) are used as information added to the patient profile. From a practical perspective, studies of prevalence of HIV drug resistance (HIVDR) are of the highest significance. To promote such studies, different countries use databases that serve as repositories of genetic and epidemiological information. The Russian HIVDR database (https://hivresist.ru/) was created in 2009. Nevertheless, it was characterized by limited applicability for a long time. Since 2021, after the regulatory documents had been revised and updated, the entry of HIVDR research results into the Russian HIVDR database has been mandatory. Therefore, the priority attention has been given to upgrading the database and improving its functional capabilities. Different methods have been developed to enter clinical, epidemiological and genetic data. At the time of this study, the Russian database HIVDR contained 10,626 unique records about patients and 13,126 nucleotide sequences deposited by 10 institutions. The following functions have been provided for data analysis: quality control of the epidemiological and clinical information about a patient, quality control of nucleotide sequences, contamination check, subtyping, detection of DR mutations, identification of viral tropism and generation of standardized reports. The efforts toward further development of the Russian HIVDR database will be focused on designing tools for detection and analysis of molecular clusters, adaptation to routine application for epidemiological surveillance of HIV infection.

Analysis of literature data on the structure of the Lassa virus genome and its strain diversity has shown that the molecular heterogeneity of strains is essential in the design of vaccines and evaluation of their efficacy, which is determined by the relevant WHO recommendations. During virus reproduction, Lassa counteracts cellular immunogenesis by suppressing the expression of the suppressor of signaling proteins, cytokines and the RLR receptor that recognizes viral two-segmented RNA.

The GP protein, which determines the pathogen's infectivity and tropism, should be the main target for vaccines being developed. Other targets are the processes of viral RNA synthesis that determine the features of immunogenesis. A study of the immunogenesis of Lassa fever shows that the preferred candidate would be a replicating apathogenic vaccine capable of inducing an optimal combination of cellular and humoral responses, that is, causing high activity of T-lymphocytes and the production of viral neutralizing antibodies. One of the most important characteristics of a live candidate vaccine should be genetic stability to exclude reversion to a more pathogenic genotype.

Of the more than 130 candidate vaccines against Lassa fever designed, only two being the most promising were tested for immunogenicity and safety in humans (a recombinant on the measles virus platform and a DNA vaccine). A recombinant of Lassa virus with vesicular stomatitis virus and reassortants of Mopeya and Lassa viruses (MOPV/LASV- ML29) and (r3ML29) are promising for further development. A candidate vaccine rVSVΔG/LVGPC, similar in design to the rVSVΔG-ZEBOV-GP vaccine against Ebola, is promising.