Journal of microbiology, epidemiology and immunobiology

Introduction. As a result of routine testing of HIV-1 drug resistance (DR), a significant amount of viral nucleotide sequences and epidemiological data of HIV-infected individuals have been collected. Combined with the increasing use of bioinformatics methods in practice, it has become possible to study the features of HIV-1 resistant variants spread using molecular clustering analysis.

The aim of the study was to validate the molecular clustering analysis in a pilot region of Russia using a significant number of nucleotide sequences to study the features of the spread of HIV-1 resistant variants.

Materials and methods. HIV-1 nucleotide sequences were obtained from 899 HIV-infected patients who were registered at the Oryol AIDS Center in 2016–2021. HIV-1 genetic variants were determined using the Stanford University database, REGA and HIV BLAST. Resistance mutations and prognostic HIV-1 DR were determined using the Stanford University database. Phylogenetic analysis was carried out using the MEGA program. HIV-1 molecular clusters were identified using Cluster Picker software.

Results. In the pilot region, sub-subtype A6 dominated (85.7%); an increase in the share of CRF63_02A6 was noted. HIV-1 resistance was found in 13.6% of patients without antiretroviral therapy (ART) experience and in 52.0% with ART experience. Molecular clusters were more often formed by HIV-1 nucleotide sequences from ART-naïve patients. HIV-1 DR variants were less likely to fall into molecular clusters. The sources of transmitted mutations were more often patients with ART experience. The most actively and efficiently transmitted mutations were K103N, V179E/T, Y181C and G190S, associated with virus resistance to efavirenz and nevirapine.

Background. Q fever is the one of the best-studied zoonoses, which is widespread throughout almost the entire territory of Africa, excluding the territory of the Sahara. However, the current data on the incidence of coxiellosis and the circulation of Coxiella burnetii on this continent are limited and vary according different sources.

In 1980–1990, the Soviet-Guinean Research Virology and Microbiology Laboratory conducted studies to estimate the distribution of the Q fever pathogen, assess the herd immunity in humans and identify specific antibodies in the sera of livestock. However, in subsequent years, the research was suspended.

The aim of this study is to obtain up-to-date data on the distribution of C. burnetii in all landscape and geographical zones of the Republic of Guinea.

Materials and methods. The study was carried out in the laboratory of the Russian-Guinean Center for Epidemiology and Prevention of Infectious Diseases (Kindia, Republic of Guinea). The study involved 332 sera of febrile patients and 3156 sera from practically healthy volunteers, 1074 blood samples of livestock, 1648 suspensions of ticks, 319 specimens of small mammals and 298 of bats. The study was carried out using ELISA and PCR methods, selected samples were subjected to in-depth genetic analysis.

Results and discussion. The study of the distribution of C. burnetii on the territory of all landscape-geographical zones of the Republic of Guinea was carried out. For the first time, an officially registered case of human Q fever case has been identified. The role of livestock, small mammals and bats in the circulation of the pathogen has been established. It has been shown that the main vectors in Guinea are ixodid ticks of the Amblyomma variegatum, Hyalomma truncatum and Rhipicephalus decoloratus species. Employing molecular methods, C. burnetii strains carrying the QpH1 plasmid, capable of causing diseases in humans and animals were identified. For the first time, the complete nucleotide sequence of 16S rRNA of the Q fever pathogen (OQ152497–OQ152500) identified on the territory of Guinea was determined and registered in the GenBank database.

Conclusion. Taking into account the high epidemiological significance of Q fever, the study of the specifics of C. burnetii circulation in Guinea remains an urgent task. Regular monitoring and assessment of risk factors for diseases caused by coxiella are necessary for the development of an algorithm for laboratory diagnosis and recommendations for clinicians.

Introduction. Obtaining stabilized recombinant HIV-1 Env trimers that have a close to the native conformation is one of the directions in the field of development of vaccines against HIV-1.

The aim of the study was to obtain and characterize the stabilized trimer Env of HIV-1 SOSIP.664 based on the circulating genetic variant of the recombinant form CRF63_02A6.

Materials and methods. Bioinformatics resources were used to design the trimer Env gene based on the HIV-1 recombinant genetic variant CRF63_02A6. The designed gene was synthesized and cloned as part of an integration plasmid vector. A stable producer of trimer Env was obtained by transfection of the CHO-K1 cell line using the developed plasmid vector. Purification of the protein complex was performed using affinity chromatography and gel filtration. Antigenic properties of trimer Env were studied using immunochemical analysis using broadly neutralizing HIV-1 monoclonal antibodies (bnAbs).

Results. A new variant of the stabilized trimer Env of the surface glycoprotein of the recombinant form CRF63_02A6 HIV-1 SOSIP.664 was designed, including additional stabilizing modifications. Based on the CHO-K1 cell line, a stable producer was obtained and a purification protocol for the designed trimer Env was developed. It was found that the trimer Env CRF63_02A6 SOSIP.664 is effectively recognized by bnAbs 2G12, VRC01 and PGT126.

Conclusion. The obtained results indicate the prospects for further study of the structural features of the trimer Env CRF63_02A6 SOSIP.664, as well as its immunogenicity and the possibility of using it as a vaccine antigen.

Introduction. The importance of noroviruses in human infectious pathology and the danger of large epidemic outbreaks in organized groups determine the need to develop means of specific prevention of infection.

The aim of the study was to obtain recombinant norovirus VP1 protein and analyze its immunogenic and antigenic properties .

Materials and methods. Computer analysis of nucleotide and amino acid sequences, molecular cloning, polymerase chain reaction, electrophoresis of nucleic acids in agarose gel and proteins in polacrylamide gel, affinity chromatography, enzyme immunoassay.

Results and discussion. A genetic construct encoding recombinant VP1 of the GII genotype norovirus with codons optimized for highly effective expression in Escherichia coli has been created. The strain of E. coli Rosetta 2 (DE3) has been transformed by genetic construct. VP1 expression was carried out in E. coli cells, conditions for its production, purification and renaturation were optimized. A purified soluble recombinant VP1 protein forms virus-like particles with a diameter of 30–50 nm. Immunization of BALB/c mice by protein lead to antibodies production with a titer greater than 1 : 1000. When evaluating antigenic properties, it was shown that human IgG, IgM, and IgA antibodies interact with recombinant VP1. The total antibody detection rate was 47,4%. The results indicate the possibility of using recombinant VP1 for development of domestic vaccine for the prevention of norovirus infection.

Introduction. There is a large variety of licensed influenza vaccines worldwide, but their common limitation is rather narrow specificity and inability to protect against antigenic-drift variants of influenza virus. Therefore, optimization of immunogenic and cross-protective properties of licensed influenza vaccines is an urgent priority of public health agenda. One such approach is to modulate the immunogenic properties of live attenuated influenza vaccine (LAIV) by truncating the open reading frame of influenza virus non-structural protein 1 (NS1). The main objective of this study is to evaluate the immunogenic properties of the H1N1 seasonal LAIV strain by truncation of the NS1 protein to 126 amino acides.

Materials and methods. Using reverse genetics technique, two H1N1 LAIV strains with full-length and truncated NS1 protein with three consecutive stop codons added after the 126^th amino acid residue were obtained.C57BL/6J mice were immunized intranasally with the vaccine candidates, twice at a three-week interval. Seven days after the second immunization, cells were isolated from spleen and lung tissues and stimulated with whole wild-type H1N1 influenza virus. Levels of systemic and tissue-resident cytokine-producing CD4⁺ and CD8⁺ memory T cells were assessed by intracellular cytokine staining assay with flow cytometry. Replication of engineered vaccine strains in in vitro and in vivo systems was also evaluated.

Results. Truncation of NS1 protein of the LAIV strain significantly increased the levels of virus-specific CD4⁺ effector memory T cells in spleens and the levels of CD4⁺ tissue-resident memory T cells in lungs of mice after two-dose immunization, indicating a higher potential for protection against influenza infection of the LAIV NS₁₂₆ vaccine strain compared to the classical variant of LAIV. Importantly, the LAIV NS₁₂₆ strain also had a more pronounced attenuated phenotype in mice than its classical counterpart.

Background. Disinfectants are effective means of non-specific prevention of infections associated with the provision of medical care. Violation of disinfectant use regimes leads to the formation of microorganism resistance to them. To monitor the spread of clinically significant microorganisms resistant to disinfectants, the development of methods for their detection, including molecular genetic methods, remains relevant.

The aim of the study was to develop a multiplex real-time PCR for the identification of qacA/B and smr genes, the determinants of resistance to cationic biocides, in Gram-positive bacteria.

Materials and methods. Conserved regions of qacA, qacB and smr genes were searched, and primers and probes were designed using BLASTN, GeneRunner, and Multiple Primer Analyzer programs. To evaluate the analytical sensitivity of the multiplex PCR, plasmids pTZ57-qacA/B, pTZ57-smr, and pTZ57-16S containing qacA/B, smr and 16S rRNA gene fragments of 197 bp, 127 bp, and 287 bp, respectively, were constructed. The method was tested on clinical isolates of Gram-positive bacteria (n = 30).

Results. A multiplex real-time PCR using TaqMan probes was developed for the detection of qacA/B and smr genes in Gram-positive bacteria. The 16S rRNA gene was used as an internal amplification control. The sensitivity of the multiplex PCR was 10³ copies for all genes. Multiplex PCR validation showed that qacA/B genes were present in 30%, smr genes were present in 10% of the isolates tested. The reproducibility of the results was 100%.

Conclusion. The developed multiplex PCR differs from existing assays by high specificity and short turnaround time, as well as by the presence of an internal amplification control. It can be used for the detection of Gram-positive bacteria potentially resistant to cationic biocides.

The aim of the review is to describe existing laboratory methods for determining the sensitivity of bacteria to a combination of antibiotics and bacteriophages. However, more and more often there are scientific papers in which their combined action is described as synergism. The mechanisms of this phenomenon have not been fully studied, but it has been proven that not only virulent but also moderate phages can enter into synergy with antibiotics, allowing the minimum inhibitory concentration of the antibiotic to be reduced several times. Since synergy cannot yet be empirically predicted, microbiological laboratories use various in vitro methods, most of which are labor-intensive. The development of a new technique that can be introduced into the daily practice of microbiological laboratories is relevant.