Journal of microbiology, epidemiology and immunobiology

Relevance. Influenza viruses have a high potential for genetic change. These viruses are monitored annually around the world, including Russia, to determine the dominant genetic groups and select the strains to be included in influenza vaccines.

Objectives of the study include: analysis of influenza virus circulation in Russia in 2019–2023, phylogenetic and molecular analysis of hemagglutinin (HA) sequences of influenza viruses, detection of mutations associated with drug resistance to neuraminidase (NA) inhibitors and M2-protein (M2) ion channel inhibitors.

Materials and methods. Biological samples containing RNA of influenza viruses were studied: 410 A(H1N1)pdm09, 147 A(H3N2) and 167 B(Victoria). Sequencing of the HA, NA, M fragments was performed on the 3500xL Genetic Analyzer (Applied Biosytems). Data processing and analysis were carried out using DNASTAR, Nextclade, FluSurver and BioNumerics v.6.6 software.

Results. Influenza A(H1N1)pdm09, A(H3N2), B(Victoria) viruses circulating in 2019-2023 were investigated. The highest variability of HA was observed in A(H3N2) viruses. All influenza A(H1N1)pdm09 viruses in the 2022–2023 season had a previously unknown mutation E224A in HA, which increases its affinity for α-2,3 sialic acids — receptors localized in the human lungs, to which the virus binds via HA. 2 and 3% of influenza A(H1N1)pdm09 viruses in 2019–2020 and 2022–2023, respectively, had the D222N mutation in the receptor-binding site of HA, which is associated with more severe disease. The oseltamivir and zanamivir resistance mutation H275Y in NA was detected in 2.3% of influenza A(H1N1)pdm09 viruses in 2022–2023. No oseltamivir and zanamivir resistance mutations in NA were detected in all tested influenza A(H3N2) and B viruses. Sequencing data revealed a mutation of adamantane resistance S31N in M2 in all studied influenza viruses A(H1N1)pdm09 and A(H3N2).

Conclusions. The detection of amino acid substitutions in HA antigenic sites and resistance mutations in NA and M2 confirms the evolution of influenza viruses and the necessity for continuous genetic surveillance. The vast majority of currently circulating viruses remain sensitive to NA inhibitors.

Introduction. Since early May 2022, more than 90,000 cases of monkeypox virus infection have been reported in more than 70 countries around the World. This is the largest outbreak of monkeypox ever recorded outside of Africa.

The aim of the study is to confirm the first case of monkeypox in Russia, to isolate and sequence a new strain of monkeypox virus (MPXV), and to assess its sensitivity to the 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo-[3.2.2.0^2,4]non-8-en-6-carboxylic acid (NIOCH-14) antipox drug.

Materials and methods. The biological materials obtained from the affected area of the skin (contents of vesicles), a nasopharyngeal smear, sputum and venous blood from a patient with suspected monkeypox were used. The disease was confirmed by PCR followed by determination of the nucleotide sequence of viral DNA by sequencing. Isolation of the new MPXV strain from clinical samples was carried out in Vero E6 cells. The antiviral effectiveness of NIOCH-14 against the new MPXV strain was assessed using an adapted spectrophotometric method.

Results. A diagnostic study of the biological samples of a patient who returned from a tourist trip to European countries with complaints of skin rashes all over the body revealed MPXV DNA. A new strain of MPXV was isolated from vesicles in Vero E6 cells, and the genomic sequence MPXV-pustule S45 was assembled using high-throughput parallel sequencing (NGS).

Discussion. The effectiveness of the finished dosage form of NIOCH-14 against the new strain of MPXV based on the results of determining the 50% virus inhibitory concentration (IC₅₀) was 0.02 μg/mL, and the selectivity index (SI) was > 15,000.

Conclusion. In this study, the pathogen of monkeypox was detected and identified using real-time PCR, NGS and electron microscopy, and the first imported case of this disease in Russia was confirmed. It has been proven that the drug NIOCH-14 exhibits high antiviral activity in vitro against the new MPXV strain.

Introduction. Many different vaccines for the prevention of COVID-19 have received emergency use approval in the shortest possible time. Due to the high rate of variability of the pathogen, in this study we analyzed the variability of the structural proteins of the SARS-CoV-2 virus and compared their protective potential in protecting animals from COVID-19.

The aim of the study was to compare the protective potential of the SARS-CoV-2 structural proteins in protecting animals from COVID-19.

Materials and methods. The SARS-CoV-2 virus was used in the study. Transgenic mice B6.Cg-Tg(K18-ACE2)2Prlmn/J (F1) were used as model animals. Recombinant adenoviral vectors rAd5-S, rAd5-N, rAd5-M were used for immunization of animals. Various genetic, virological and immunological methods, as well as methods of working with animals, were used in the study.

Results. The largest number of amino acid substitutions in the structural proteins of different SARS-CoV-2 variants was detected in glycoprotein S, the smallest — in nucleoprotein N. In the COVID-19 animal model, it was shown that only the use of glycoprotein S as a vaccine antigen allows to form protective immunity that protects 100% of animals from a lethal infection caused by the SARS-CoV-2 virus, while the use of protein N protects 50% of animals from a lethal infection, and protein M does not have a protective potential.

Conclusion. The data obtained, as well as the analysis of the epidemiological efficacy of various mRNA and vector vaccines, demonstrate that the use of the SARS-CoV-2 glycoprotein S as an antigen allows to form the highest level of protection. Due to the constant change in circulating variants of the SARS-CoV-2 virus, the decrease in the effectiveness of the vaccines with the original antigen composition against new variants of the virus and the continuing high incidence of COVID-19, it is necessary to continuously monitor the effectiveness of vaccines against new variants of the virus and promptly update the antigen composition of vaccines when a decrease in effectiveness is detected.

Relevance. Studies on model systems have confirmed the effectiveness of antisense oligonucleotides, including those that contain photoactive groups, for the modification of nucleic acids. However, this strategy has not yet found wide application due to the lack of successful methods for the intracellular delivery. The development of effective preparations capable of acting on target nucleic acids in cells is an urgent task.

The aim of the study is to create nanocomplexes consisting of aminopropylsilanol nanoparticles and short interfering RNA (siRNA) to study their effect on target nucleic acids by the example of inhibition of influenza A virus replication in vitro.

Materials and methods. MDCK cells, influenza virus A/chicken/Kurgan/05/2005 (A/H5N1), aminopropylsilanol nanoparticles, and native and modified siRNA molecules.

Results and discussion. We have prepared unique Si~NH₂/siRNA nanocomplexes, which consist of aminopropylsilanol nanoparticles and siRNA molecules, which enable cell penetration and selective interaction with target nucleic acids, respectively. The antiviral activity of the proposed nanocomplexes has been studied on MDCK cells infected with the influenza A/H5N1 virus. It has been shown that the double-stranded siRNA molecules in the nanocomplexes, which act by the RNA interference mechanism, are more efficient in inhibiting the replication of the influenza virus than the corresponding single-stranded RNA fragments. The most effective nanocomplex that contained siRNA targeted at the chosen region of mRNA segment 5 of the viral genome reduced virus replication in the culture by a factor of 630. We have shown that non-agglomerated and water-soluble aminopropylsilanol nanoparticles are low-toxic, capable of delivering siRNA into cells and protecting siRNA in the Si~NH₂/siRNA nanocomplexes from hydrolysis by cellular nucleases.

Conclusion. The biological activity of the created nanocomplexes has been demonstrated by the example of highly effective selective suppression of influenza A/chicken/Kurgan/05/2005 virus replication in the cellular system.

Introduction. Streptococcus agalactiae are gram-positive, non-motile and encapsulated cocci. On blood agar, they produce an narrow zone of beta-haemolysis. This pathogen causes invasive bacterial diseases in newborns, including sepsis, meningitis, septicaemia, and pneumonia, when transmitted from infected mothers. Since S. agalactiae is a pathogen of primary concern for public health, this research has been conducted on it.

The objective of the study is the isolation and molecular detection of virulence gene of S. agalactiae group B (GBS), and evaluation of the percentage of mother-to-child transmission of the pathogen.

Materials and methods. A prospective cohort study was designed that included 300 pregnant women who were at more than 35 weeks of pregnancy. The gynaecologist collected 300 vaginal swabs from all participants in this study and followed up on all GBS-positive pregnant women after delivery to take swabs from their neonates. Traditional microbiological and molecular approaches were used to study isolated bacteria.

Result. Sixty (20%) of three hundred pregnant women and 16 (26.6%) of their newborns were enrolled in this study. GBS was detected via culture methods and was confirmed by PCR with primers employed for the detection of atr gene (housekeeping gene). Positive isolates were 100% susceptible to antibiotics such as ceftriaxone, penicillin, and vancomycin, 93% were sensitive to chloramphenicol, 83% to erythromycin, and only 13% to tetracycline.

Conclusion. Our data showed a high frequency of GBS infection in pregnant women and their newborns. A mandatory screening test and preventative medicine should be adopted to minimize the potentially fatal repercussions of this sickness.

Introduction. One of the reasons for spreading antibiotic-resistant microorganisms is the uncontrolled use and inadequate empirical prescription of antibiotics which is not based on the results of the pathogen sensitivity testing. The broth dilution method and one of its implementation options, the reference microdilution method, in contrast to the disk diffusion method, allows testing virtually all pathogen-antibiotic combinations. To realize the method, a production technology of Russian Mueller–Hinton broth (MHB-Obolensk) has been developed under the import substitution program.

The aim. To evaluate the quality of the developed domestic Mueller–Hinton broth in comparative tests with its imported analog BD BBL (MHB-BD) in testing clinical strains of microorganisms, including microorganism–antibiotic combinations pairs which cannot be reliably investigated by the disc diffusion method.

Materials and methods. The study investigated the sensitivity of 47 clinical strains of Gram-positive and Gram-negative bacteria to antibiotics of various functional groups using the broth microdilution method with MHB-Obolensk and MHB-BD.

Results. The MICs values of antibiotics for clinical strains obtained with the developed and control media did not practically differ from each other or differed by +/– one dilution. The difference by two two-fold dilutions was noted when testing Enterococcus faecium–ampicillin, Klebsiella pneumoniae–meropenem, Pseudomonas aeruginosa–levofloxacin and Staphylococcus aureus–ciprofloxacin combinations. For the first two combinations, the MIC values were lower in MHB-Obolensk, and for the last two, they were higher than in MHB-BD. The differences obtained did not affect the clinical categories of sensitivity.

Conclusion. The antibiograms of clinical strains in developed Russian Mueller–Hinton broth was obtained, which did not differ from those for the comparison medium. MHB-Obolensk complies with the requirements of national and international standards and can be used to reliably test, among other things, current combinations of microorganism–antibiotic pairs that cannot be studied using the disk diffusion method.