Vol 15, No 6 (2025)

Respiratory viral infections pose a serious public health issue resulting in high morbidity and mortality, as well as profound socioeconomic losses. Therefore, it accounts for a need to research respiratory viral infections immunopathogenesis, development of effective vaccines and antiviral drugs, as well as measures to monitor viral infections. The aim of the review is to analyze current methods for modeling respiratory viral infections ex vivo. Material and methods. There were analyzed current data regarding development and application of models based on primary epithelial cells (PECs) derived from various anatomical sites of the human respiratory tract (RT) and 3D cell cultures. For this, there were assessed 158 publications retrieved from the main databases (Web of Science, PubMed, Scopus, Elsevier, Google Scholar and RSCI until January 2025) by querying the keywords: respiratory viruses; primary airway epithelium cultures; organoids; immunopathogenesis; tropism; cellular receptors; cytokines. Results and discussion. The analysis showed that models based on PECC are widely used in virological studies of respiratory viral infections, which, however, is coupled to certain disadvantages. More advanced are RT 3D models (organoids or mini-organs, spheroids, “organs on a chip”), which allow not only to reproduce infectious processes, but also to study immunopathogenesis taking into account the immunometabolic and immunoneurological status. Using RT PECs and 3D models, the properties of a number of respiratory viruses actual in the first quarter of the XXI century (influenza and parainfluenza, pneumoviruses, coronaviruses, rhinoviruses, bocaviruses, adenoviruses) such as tissue tropism, receptor interactions and innate immune response were assessed. Moreover, we also present information on promising models for respiratory viral infections that reproduce essential aspects of RT physiology. Conclusion. The primary biotechnological aim for virological studies of respiratory viral infections is to generate a multiparameter, reproducible and cost-effective RT modeling system that imitates its morphological and functional structure.

Introduction. In the structure of the norovirus surface VP1, it consists of the S domain which N-terminal sequence is responsible for capsid formation, and P domain that extends beyond the capsid. Possibility of using S domain for preparing chimeric virus-like particles is shown. The aim of the work is to obtain a recombinant VP1 protein S domain and to characterize its immunogenic and antigenic properties. Materials and methods. Cloning of the sequence encoding VP1 protein S domain, modeling the structure of proteins, microbiological methods for working with E. coli cells producing recombinant proteins, polymerase chain reaction, electrophoresis of nucleic acids in an agarose gel and proteins in a polyacrylamide gel, affinity chromatography, enzyme immunoassay, electron microscopy were applied during the study. Results. A genetic construct encoding the recombinant VP1 norovirus genotype GII.4 protein S domain optimized for highly efficient expression in E. coli is obtained. E. coli Rosetta 2 cells were transformed with the genetic construct. Conditions for protein production and purification were optimized. A soluble recombinant protein reproducing norovirus VP1 protein S domain and forming virus-like particles with a diameter of 30 nm is obtained. Immunization of mice with the obtained protein caused specific antibody production including low-avidity IgM antibodies. When assessing antigenic properties, it was shown that antibodies interacting with the recombinant S domain are found in the blood samples collected from volunteers. Antibody titers, but of lower avidity, specific to the latter corresponded to those against full-length VP1. Likely, it was due to the localization of the S domain inside the viral particle. Conclusion. Antigenic and immunogenic properties of recombinant protein reproducing structure of norovirus VP1 S domain are characterized, and its ability to form virus-like particles is shown.

Clostridioides difficile is a spore-forming enteropathogenic anaerobic bacterium and one of the most common opportunistic human pathogens. Its pathogenicity relies on the production of toxins, sporulation, biofilm formation, and the ability to withstand numerous stresses encountered in the host environment. In addition to these well-known mechanisms, C. difficile possesses a remarkably complex CRISPR-Cas system characterized by two cas operons and multiple CRISPR arrays. While CRISPR-Cas systems are primarily studied as adaptive immune mechanisms against bacteriophages and mobile genetic elements, accumulating evidence suggests they may also be integrated into broader regulatory networks that contribute to bacterial physiology, adaptation, and virulence. However, the regulation and functional dynamics of this system in C. difficile remain largely unexplored. In this study, we investigated the regulation of the C. difficile CRISPR-Cas system under biofilm-inducing factors. Quantitative PCR analysis revealed the induction of several CRISPR arrays and the partial cas operon under high intracellular levels of the secondary messenger cyclic di-guanosine monophosphate, a key regulator of bacterial phenotypic shifts. These results were partially confirmed by interference efficiency assays. A secondary bile salt, sodium deoxycholate, known to trigger biofilm formation, also increased both cas operons and one CRISPR array expression, suggesting its role for CRISPR-Cas system regulation during host-associated stress Moreover we identified glucose as a regulatory factor for C. difficile CRISPR-Cas system. Elevated glucose concentration in the medium induced the expression of the partial cas operon and CRISPR 3–4 arrays. However, at the same time it functionally suppressed the interference efficiency of the system. Together, our findings demonstrate that the C. difficile CRISPR-Cas system is responsive to biofilm-inducing signals and nutrient availability, linking its regulation to key aspects of bacterial physiology and adaptation to the host. This work also highlights the potential for non-canonical regulatory roles of CRISPR-Cas in C. difficile survival and pathogenesis.

Human herpesvirus 6A (HHV6A) and human herpesvirus 6B (HHV6B) are ubiquitous viruses that infect more than 95% of the population. Clinical manifestations of HHV6 infection and associated diseases are diverse, which may depend on virus molecular genetic characteristics (genovariants). Estimating the significance of the molecular genetic diversity is complicated due to the lack of proper classification. The aim of the study was to develop an intraspecies HHV6A and HHV6B classification. Using 50 and 207 HHV6A and HHV6B full-genome sequences retrieved from the NCBI Nucleotide database, various fragments of virus genome were analyzed. Multiple sequence alignment was performed using MAFFT L-INS-i algorithm; F81 nucleotide substitution model and maximum likelihood method were used to construct dendrograms. Nucleotide substitutions were determined relative to reference sequences X83413 (HHV6A) and AF157706 (HHV6B). Genovariants were defined based on the nucleotide substitutions in variable positions of the genomic fragment. The results were confirmed by constructing dendrograms. An opportunity of using fragments of HHV6A and HHV6B genomes to construct an intraspecies classification was assessed. Fragments U90(part206) and U90В(part431) were selected as optimal. Based on the nucleotide sequences of the fragments, the intraspecies classification for HHV6A and HHV6B was constructed, including seven genovariants of each virus. The genovariants were characterized by unique nucleotide composition in the signature positions. A minimum (0.001 or less for both viruses) nucleotide diversity within the isolated genovariants was established. The classification reflects the phylogenetic relationships of circulating and inherited chromosomally integrated forms of HHV6A and HHV6B: divergence of HHV6A genovariants depending on its persistence form and integration site and coevolution of two HHV6B forms within several genovariants. Further studies on virus molecular genetic diversity in different regions of Russia and abroad may supplement the classification. The method of HHV6A and HHV6B classification is characterized by simplicity, technological accessibility and can be implemented in laboratories of different levels of technical equipment. The classification can be used to analyze an effect of virus molecular genetic diversity on the clinical characteristics of associated diseases, optimize the epidemiological surveillance system and develop new approaches for diagnostics, prevention, and treatment of HHV6 infection.

Background. Sepsis is a severe and life-threatening clinical syndrome characterized by a dysregulated host immune response to infection. This maladaptive response promotes widespread endothelial injury and abnormalities in coagulation, often progressing to multi-organ dysfunction and death. Mortality rates remain high, highlighting the urgent need for reliable biomarkers to enable early identification of patients at high risk. Such tools are particularly valuable in low- and middle-income countries (LMICs), where access to advanced diagnostic and therapeutic resources is limited. Monocyte Chemoattractant Protein-1 (MCP-1) is a chemokine that reflects hyperactivation of the innate immune system, while D-dimer indicates activation of coagulation and fibrinolysis. Although both pathways are central to the pathophysiology of sepsis, data evaluating their combined prognostic value in LMIC settings remain scarce. This study aimed to assess the prognostic significance of MCP-1 and D-dimer, both individually and in combination, for predicting 28-day mortality in patients with sepsis. Materials and methods. We conducted a prospective cohort study involving 83 adult patients with newly diagnosed sepsis at Dr. Saiful Anwar General Hospital, Malang, Indonesia. Serum MCP-1 levels were measured using enzyme-linked immunosorbent assay (ELISA), and plasma D-dimer levels were determined by immunoturbidimetry at the time of diagnosis. Patients were followed for 28 days, and survival outcomes were evaluated using Kaplan–Meier survival analysis and Cox proportional hazards regression models. Results. Among the 83 participants, 58 patients (70%) died within 28 days. Non-survivors demonstrated significantly higher MCP-1 and D-dimer levels compared with survivors. An MCP-1 concentration ≥ 123.03 pg/mL was strongly associated with increased mortality (HR 2.664, p = 0.005). Elevated D-dimer (≥ 43.5 mg/L FEU) showed a weaker individual association, but when combined with MCP-1, predictive accuracy for mortality was significantly enhanced (HR 3.986, p = 0.037). Conclusion. The concurrent elevation of MCP-1 and D-dimer identifies patients with sepsis who are at markedly increased risk of death. These findings support the potential utility of integrating inflammatory and coagulation biomarkers for early risk stratification. Moreover, they highlight the central role of the inflammation–coagulation axis in sepsis pathophysiology, with particular relevance for clinical practice in resource-limited settings.

Background. Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by the immune-mediated destruction of pancreatic beta cells, leading to insulin deficiency. Inflammatory cytokines, particularly interleukin-6 (IL-6), play a central role in this pathological process by promoting pro-inflammatory immune responses. Recent evidence highlights the involvement of microRNAs, especially miR-146a and miR-155, in regulating immune cell activation and cytokine signaling pathways. Dysregulation of these microRNAs may disrupt immune homeostasis and contribute to the progression of T1DM. This study aimed to investigate the expression levels of miR-146a and miR-155 in patients with T1DM and to examine their association with serum IL-6 concentrations. Materials and methods. This case-control study included 150 participants, comprising 100 individuals diagnosed with T1DM and 50 healthy controls. Peripheral blood samples were collected to evaluate fasting blood glucose, glycated hemoglobin, and IL-6 levels using an enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction was used to assess the expression of miR-146a and miR-155, normalized to miR-16 as the internal control. Results. The results revealed significantly elevated levels of fasting blood glucose, glycated hemoglobin, and IL-6 in patients compared to controls (p < 0.0001). Additionally, miR-146a expression was increased by a 3.1-fold change, and miR-155 showed a 1.58-fold increase in patients with T1DM compared to healthy individuals. Conclusions. The significant overexpression of microRNAs miR-146a and miR-155, in parallel with elevated serum levels of the pro-inflammatory cytokine IL-6, highlights their crucial role in the immunopathogenesis of T1DM. These findings suggest that miR-146a and miR-155 may act as key regulators in modulating immune responses, contributing to the autoimmune destruction of pancreatic beta cells. Moreover, the combined assessment of these microRNAs and IL-6 may serve as valuable molecular biomarkers for early diagnosis, disease prognosis, and the development of novel immunomodulatory therapeutic strategies in T1DM management.

Background. Methamphetamine use compromises both innate and adaptive immune responses, substantially reducing the body’s ability to combat pathogens and preserve mucosal integrity. This immunosuppression heightens susceptibility to opportunistic infections such as herpes simplex virus type 1 (HSV-1), promotes viral reactivation, disrupts the balance and diversity of the oral microbiota, and accelerates inflammatory processes within oral tissues. As a result, it exacerbates a wide range of oral health problems, including gingivitis, periodontal disease, dental caries, oral mucosal lesions, and delayed wound healing within the oral cavity. The aim of the study was to investigate the levels of interferon-alpha (IFNα), interferon-gamma (IFNγ), and interleukin-29 (IL-29) in methamphetamine users with or without HSV-1 infection, and to examine their association with the development of gingivitis. Materials and methods. This case-control study included 88 male participants aged 20–35 years, categorized into four groups: healthy controls, methamphetamine users, methamphetamine users with HSV-1 infection, and patients with gingivitis. Gingival crevicular fluid (GCF) samples were collected from all participants, and the concentrations of IFNα, IFNγ, and IL-29 were measured using enzyme-linked immunosorbent assay (ELISA). Results. IFNα and IFNγ levels were highest in the healthy control group and were significantly reduced in the methamphetamine, methamphetamine with HSV-1, and gingivitis groups (p < 0.05). In contrast, IL-29 levels were significantly elevated in gingivitis patients compared to all other groups. Furthermore, methamphetamine users with HSV-1 infection exhibited markedly higher IL-29 concentrations than methamphetamine users without HSV-1 infection. Conclusion. Methamphetamine use and HSV-1 infection significantly impair immune cytokine responses, reflected by marked reductions in IFNα and IFNγ levels, alongside elevated IL-29. The pronounced increase in IL-29 was most evident in gingivitis patients, suggesting a potential role for this cytokine in amplifying local inflammatory processes and contributing to periodontal tissue breakdown.

Introduction. Specific prevention of vaccine-preventable infections (VPIs) has been the main prerequisite to radically lower their incidence in the Russian Federation. Since the Kherson Region joined the Russian Federation in 2024–2025, no VPI cases have been recorded, likely due to the level of herd immunity. The actual number of individuals immune to VPIs can only be estimated through serologically monitored herd immunity. Study objective was to assess herd immunity to measles, mumps, rubella, and diphtheria in specific Kherson Region districts. Materials and methods. The study involved 2.807 volunteers in seven municipal districts, aged 1 to 70+ years. A volunteer cohort, stratified by age (1–5, 6–11, 12–17, 18–29, 30–39, 40–49, 50–59, 60–69, ≥ 70), was randomized using the Web application. During the study, participants completed a questionnaire, IgG antibodies to specific agents (measles, mumps, rubella, diphtheria toxin) were measured in venous blood samples using ELISA test kits (Russia). Results. The average cohort seroprevalence for measles, rubella, mumps, and diphtheria among volunteers in the region were 83.4%, 95.1%, 65.7%, and 83.4%, respectively. Adults aged 18–29 years were the least protected against measles, rubella, and mumps, with the seropositivity: < 70% for measles; 84.5% for rubella; and 44.6% for mumps. The most protected were people aged ≥ 50 years: 91.5–99.4% for measles; > 98% for rubella; and 74.5–87.1% for mumps. People over 60 were the least protected against diphtheria (63.8–76.5%). In children’s groups, maximum seroprevalence was noted in the group aged 6–11 years: 90.8% for measles; > 96.9% for rubella; 64.6% for mumps; and 90.4% for diphtheria. Most volunteers had low to moderate anti-measles and anti-diphtheria toxin IgG levels; and high anti-rubella IgG levels. The seropositivity of “naive” volunteers (those denying infection or vaccination) indicates insufficient laboratory VPI diagnostics in the Kherson Region. Conclusion. The level of herd immunity in the surveyed areas (Kherson Region) met the criterion for epidemiological well-being only with regard to rubella. Specifically, the percentage of seronegative individuals was below 10%. Insufficient seroprevalence for the measles and mumps viruses, as well as the C. diphtheriae, reflects conditions conducive for the spread of these infections among the population.

Granuloma formation represents one of the key stages in tuberculosis pathogenesis, characterized by the development of specialized cellular structures surrounding Mycobacterium tuberculosis within foci. These structures are presented by complex, multicomponent assemblies that both detain the pathogen and simultaneously serve as a niche for its persistence. It has been firmly established that the mycobacterial cord factor is an important driver of granuloma induction, being involved in intercellular interactions and the organization of granuloma three-dimensional scaffold. However, the influence of the resident microbiota on this process, as well as its contribution to the formation of mycobacterial biofilms, remains understudied. This work investigates an effect of Corynebacterium kefirresidentii and Staphylococcus epidermidis lysates (isolated from tuberculoma-related caseous necrosis) on in vitro granuloma formation using C57BL/6 mouse peritoneal macrophages and splenocytes. Additionally, an influence of C. kefirresidentii on vaccine strain M. bovis BCG growth and biofilm development was examined. The study demonstrates that C. kefirresidentii induces the formation of a rapidly growing M. bovis BCG biofilm with characteristic cord-like architecture and a prominent extracellular matrix. This effect was accompanied by a statistically significantly expanded area under the growth curve compared to the control (p < 0.0001). Co-cultivation experiments with the clinical strain M. tuberculosis Beijing B0/W148 and bacterial lysates revealed limited antimycobacterial activity but demonstrated a pronounced stimulatory effect on the formation of granuloma-like structures in the M. tuberculosis-induced in vitro granulemogenesis model. C. kefirresidentii and S. epidermidis lysates significantly propagated granuloma formation as evidenced by markedly increased both cellular aggregate number and size (p < 0.01), including the formation of large clusters containing more than 50 cells. Taken together, these findings suggest that satellite pathobiota derived from caseous necrosis of tuberculous foci might be involved in modulating key mechanisms of infection pathogenesis and point at its potential contribution to unfavorable outcomes in pulmonary tuberculosis.

COVID-19 weakens the body’s immune system and predisposes to bacterial infections. It has been increasingly evident about changes in the etiological pattern of pneumonia pathogens due to altered lung microbiota after viral pneumonia, the effect of SARS-CoV-2 on the immune system, and antibiotics taking as part of preventing secondary bacterial infection. Our study was aimed to analyze the dynamic change in the pattern of the respiratory tract microbiota in patients with severe pneumonia in the years from 2019 to 2023. There were enrolled 304 patients with pneumonia diagnosed after X-ray examination assessed from January 2019 to December 2023 inclusive. During the pandemic, all the investigated patients had a positive SARS-CoV-2 PCR result, and in the period after the pandemic all the examined patients were negative. Sputum samples delivered to the laboratory, where we performed microbiological culture with identification by MALDI-ToF mass spectrometry. In the pre-COVID-19 pandemic, 62 sputum samples were analyzed, among which Klebsiella pneumoniae and Stenotrophomonas maltophilia were the most common found in 21% and 17.7%, respectively. Both Acinetobacter baumanii and Staphylococcus aureus were isolated in 14.5% cases. Streptococcus pneumoniae was found in 8.1% cases. During the COVID-19 pandemic, 122 samples were evaluated allowing to observe that K. pneumoniae accounted for half of all isolated microorganisms. The second most common was A. baumannii (23.8%). In the post-pandemic period, 120 samples were analyzed, from which K. pneumoniae was mainly identified (31.7%). The next most frequent among pathogens were S. aureus and A. baumannii — 23.3% and 18.3%, respectively. In this way, there was a statistically significant change in the frequency of detection of Gram-positive and Gram-negative microorganisms in ICU patients during the study periods. Before the COVID-19 pandemic, the proportion of Gram-negative microorganisms in the pattern of pathogens was 67.7%, during the pandemic — 91.0%, in the post-pandemic period — approached the values of 2019–2020 and amounted to 70.0% (p < 0.001). K. pneumoniae and A. baumannii were the most frequently found, however, the statistical significance of the changes was observed only for K. pneumoniae (p < 0.005). An insignificant decline in the detection rate of pneumococcus was established as well. The frequency of staphylococcal discharge after coronavirus infection exceeded the pre-pandemic magnitude (p < 0.001).