Introduction. In response to the COVID-19 pandemic in the Russian Federation, comprehensive response measures were taken. One of these measures was the development of a viral genome aggregation platform (VGARus) to monitor virus variability. The aim of this paper is to describe the role of the VGARus platform in tracking genetic variation in SARS-CoV-2. Materials and methods. VGARus utilizes sequencing data and bioinformatics tools to monitor genetic variations in SARS-CoV-2. The viral genomes were aligned using NextClade, which also translated them into amino acids and identified mutations. The viral variability over time was analyzed by counting the number of amino acid changes compared to the reference sequence. Results. The analysis of data within VGARus enabled the identification of new virus variants, contributing to improved diagnostic tests and vaccine development. The platform allowed for the prediction of epidemiologic trends, facilitating a rapid response to changes in the epidemiologic situation. For example, using VGARus, an increase in COVID-19 incidence was accurately predicted in the summer of 2022 and early 2023, which were associated with the emergence of Omicron subvariants BA.5 and XBB. Data from the platform helps validate the effectiveness of primers and DNA probes to ensure high diagnostic accuracy and reduce the risk of false negatives. Conclusion. VGARus demonstrates the growing role of genomic surveillance in combating COVID-19 and improving preparedness for future infectious disease outbreaks. The platform is a powerful tool for generating evidence-based solutions to combat a pandemic and mitigate its health, economic and societal impacts. It provides the ability to promptly obtain information on the epidemiologic situation in a particular region of the Russian Federation, use genomic data for phylogenetic analysis, compare the mutational spectrum of SARS-CoV-2 sequences with foreign samples. VGARus data allow for both retrospective analysis and predictive hypotheses. For example, we can clearly see the dynamics of the change of different virus variants: sequences belonging to the Alpha, Beta, Delta, Omicron lineages and many less common ones, clearly form the upsurges of morbidity, the interaction of which is reflected in the epidemiological picture. It is also currently being expanded to monitor other pathogens, increasing its public health relevance.

Background The effect of SARS-CoV-2 mutations and viral load on the severity of COVID-19 is not well understood. The possibility of reinfection with SARS-CoV-2 has already been reported, but dual infection with SARS-CoV-2 is poorly described and is currently under discussion. We describe a study of two strains of SARS-CoV-2 detected in the same patient during the same disease presentation. Methods Two nasopharyngeal swabs were obtained eight days apart from the patient in their 90s, diagnosed with lobar pneumonia (J18.1). Both tests were positive for SARS-CoV-2 with high viral load (Ct = 13). We have performed high-throughput sequencing of SARS-CoV-2 genomes from both swabs. Findings Genomic analysis of SARS-CoV-2 revealed the presence of two genetically distant strains in both swabs. Detected strains belong to different phylogenetic clades (GH and GR) and differ in the seven nucleotide positions. The relative abundance of strains was 70% (GH) and 30% (GR) in the first swab, and 3% (GH) and 97% (GR). Interpretation Our findings suggest that the patient was infected by two genetically distinct SARS-CoV-2 strains at the same time. One of the possible explanations is that the second infection occurred in the hospital. Change of the dominant strain ratio during disease manifestation could be explained by the advantage or higher virulence of the strain belonging to the clade GR.

Currently, a lot of attention is given to SARS-CoV-2 subpopulations and their coexistence with different genomic variants within the same patient. In this study, we performed next-generation whole-genome sequencing and assembly of viruses from samples representing swabs or autopsy specimens obtained from patients diagnosed with СOVID-19, which were initially confirmed by the real-time polymerase chain reaction (Ct = 10.4–19.8). Samples were prepared for sequencing by using the SCV-2000bp protocol. The obtained data were checked for presence of more than one SARS-CoV-2 genetic variants in a sample. Variants of nucleotide substitutions, coverage for each variant, and location of the variable position in the reference genome were detected with tools incorporated in the CLC Genomics Workbench program. In our search for variable nucleotide positions, we assumed that the sample had two genetic variants (not more); the threshold value ≥ 90% was set for probability of the identified variant. Variants represented by less than 20% of the reads in the total coverage were not taken into consideration. The obtained results showed that 5 samples had variability, i.e. they had several genetic variants of SARS-CoV-2. In 4 samples, both of the detected genomic variants differed only in one nucleotide position. The fifth sample demonstrated more substantial differences: a total of 3 variable positions and one three-nucleotide deletion. Our study shows that different genetic variants of SARS-CoV-2 can coexist within the same patient.

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes.

Background: The diagnosis of infectious diseases currently relies predominantly on polymerase chain reaction (PCR) assays. However, the range of pathogens that can be detected simultaneously using these methods is limited, and their effective use depends on an a priori etiological hypothesis. Multiplex PCR-based targeted next-generation sequencing (mp-tNGS) is considered a promising complementary approach for expanded viral testing. This study aimed to develop an in-house mp-tNGS method, perform an initial evaluation of its analytical sensitivity, and explore its performance using clinical respiratory specimens. Methods: The method comprised a panel targeting 28 viruses and bioinformatics pipelines for mp-tNGS data processing. Analytical sensitivity was evaluated using positive control samples (PCSs) containing encapsulated RNA or DNA. Performance with clinical specimens was explored using 910 nasopharyngeal and oropharyngeal swab specimens, which were divided into three groups based on the results of prior PCR testing. Results: Of the 28 viral targets, 23 underwent experimental analytical evaluation, whereas five were assessed in silico only. For 19 of the 23 PCSs tested, the estimated limit of detection (LoD) ranged from 6.3 × 10² to 9.4 × 10³ copies/mL. In the group of 216 PCR-positive specimens, PCR and mp-tNGS results were fully concordant in 139 cases; in 28 specimens, mp-tNGS additionally detected other viruses. Among 394 PCR-negative specimens, mp-tNGS detected viruses in 81. In the group of 300 hospital-derived specimens, mp-tNGS detected at least one viral target in 131 specimens, including additional viral detections and coinfections not identified during the initial testing. A substantial proportion of the additional findings involved Epstein–Barr virus (EBV) and cytomegalovirus (CMV), warranting cautious clinical interpretation. Conclusions: The developed mp-tNGS approach demonstrated the potential to simultaneously detect a broad range of known viruses, provide viral typing, and identify coinfections. The method may be used as an adjunctive tool to broaden the laboratory diagnosis of respiratory infections; however, further optimization, validation, and standardization of criteria for result interpretation are required.

Цель исследования – изучить взаимосвязь между состоянием тучных клеток (мастоцитов, или лаброцитов) семенников и сперматогенезом в норме и при различных типах повреждения тестикул. Материалы и методы. Исследования проводились на 35 самцах крыс линии Вистар. Использовали две ýкспериментальные модели повреждения семенников: прокол и компрессию. Для изучения взаимосвязи сперматогенеза и состояния тучных клеток (ТК) использовали морфологические и морфометрические методы исследования. Для оценки функционального состояния тестикул хемилюминесцентным методом проводили исследование уровня общего тестостерона в крови. Результаты. При различных повреждениях тестикул в органе развиваются схожие деструктивные процессы, характеризующиеся наличием некротизированных канальцев, семенных шаров, снижением количества клеток сперматогенного ýпителия, увеличением числа нефункционирующих канальцев, а также изменением ряда морфометрических показателей. Реакция ТК на различные виды повреждения проявляется в усилении их функциональной активности. Так, после прокола на фоне уменьшения количества ТК происходит активация их синтетической функции, в то время как при сдавлении ТК отвечают не только повышением функциональной активности, но и увеличением их числа в органе. Заключение. Нарушение сперматогенеза при различных травмах семенника сопровождается усилением функциональной активности ТК независимо от характера повреждения. Однако увеличение количества ТК в органе происходит только при сохранении гематотестикулярного барьера. Поскольку нормальный сперматогенез осуществляется на фоне достаточно высокой синтетической активности ТК, то ýту реакцию повышенного синтеза и дегрануляции можно рассматривать как компенсаторную.