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Introduction. The ability of SARS-CoV-2 antibodies to neutralize the virus is the primary indicator of their specific activity. The test for virus neutralizing antibodies (NAbs) is much needed in different biomedical studies.The aim of the study is to find optimum conditions for microscopic and spectrophotometric detection of SARSCoV-2 NAbs by inhibition of cytopathic effect (CPE) in cell cultures.Materials and methods. Blood sera collected from COVID-19 convalescent patients and healthy individuals (n = 96) were tested using the ELISA method. The SARS-CoV-2 coronavirus, Dubrovka strain (GenBank accession no. MW514307.1) was grown in culture medium of Vero cell line CCL-81 (ATCC). Real-time RT-PCR, ELISA, and Sanger sequencing were used for detection of the virus. The results of the neutralization test (NT) were assessed through the microscopic examination for CPE and by the methyl thiazolyl tetrazolium (MTT) assay.Results. SARS-CoV-2 was isolated from a COVID-19 patient and adapted to grow in cell culture. At a low dose of infection (MOI = 0.00001), the virus caused a pronounced CPE with the cell viability less than 3%, thus making it possible to assess NT results by CPE inhibition. The NT and ELISA-based comparative study of sera showed positive correlation between virus NAb titers and Nab titers to S-protein RBD (Spearman’s r = 0.714; p < 0.001). The results of NAbs microscopic and spectrophotometric detection (the MTT assay) also demonstrated positive correlation (Spearman’s r = 0.963; p < 0.05).Conclusion. The SARS-CoV-2 virus adapted to Vero cell culture served to develop a NAb titer assessment system, which can be used both in microscopic studies and for an MTT assay in spectrophotometric studies. The MTT assay provides automated reading of NT results, optimizes the statistical analysis of the obtained data, and minimizes subjectivity in assessment of results. Being a vital dye, MTT penetrates only viable cells, thus contributing to the reliability of the obtained results compared to other dyes.

The aim of the study was to analyze genetic characteristics of strains belonging to group A rotaviruses (RVA) circulating in Moscow in 2015–2020, including rare strains non-typeable by polymerase chain reaction (PCR).Materials and methods. A total of 289 stool samples were tested; the samples were collected from children aged 1 month to 17 years, hospitalized with acute gastroenteritis. Immunochromatography and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assays were used for detection of rotaviruses in the samples. The rotavirus genome sequencing was performed using the Sanger technique and nanopore sequencing.Results and discussion. RVA RNA was detected in 131 clinical samples, and the G/[P] genotype was identified in 125 samples. The general profile showed prevalence of RVA strains with the G9P[8]I1 genotype (37%) followed by G3P[8]I2, G4P[8]I1, G2P[4]I2, G1P[8]I1, and G3P[8]I1 variants (18, 15, 11, 5, and 2%, respectively). Seven (5%) isolates were identified as GxP[8]. In 2015–2020, the region reported a decline in G4P[8]I1 genotype prevalence (from 39% to 9%) and an increase in the proportion of the G9P[8]I1 genotype (from 6% to 37%) as compared to 2009–2014. In 2018–2020, a large number of cases with the previously unknown DS-1-like reassortant strain with the G3P[8]I2 genotype were reported; the above strain has become widely common worldwide in the recent years. Nanopore sequencing was performed to analyze the genome of the G3P[8]I2 strain and the rare G4P[6]I1 strain. It was found that the G4P[6]I1 strain was phylogenetically related to porcine rotaviruses.Conclusion. In the recent years, the genetic diversity of RVA circulating in the Moscow Region has changed significantly. The obtained results prove the importance of continuous monitoring of rotavirus infection and selective sequencing of RVA genes to fine-tune data of the type-specific real-time RT-PCR. The ever-changing genetic composition of the circulating RVA strains calls for regular optimization of RVA genotyping systems based on real-time RT-PCR.

Introduction. Due to the high clinical significance of herpesvirus diseases, the searching of fast and effective methods for their diagnosis remains relevant.The aim of the study was to evaluate the diagnostic efficiency of the loop-mediated isothermal amplification of DNA with real-time fluorescent detection (RT-LAMP) with SYTO-82 dye on a model of herpes simplex virus (HSV) infection.Materials and methods. A total of 44 urogenital swabs containing type 1 and type 2 HSV DNA and 43 swabs without HSV DNA, including 33 samples containing the DNA of cytomegalovirus, Epstein-Barr virus and herpesvirus type 6, were studied. For RT-LAMP, Bst 2.0 WarmStart DNA polymerase, SYTO-82 dye, LAMP primers were used.Results. The high efficiency of HSV DNA detection in the RT-LAMP reaction with SYTO-82 dye was shown. RT-LAMP in optimal conditions allowed to reduce reaction time for 2-3 times compared to real-time PCR (to 35 minutes). Analytical sensitivity of HSV type 1 and 2 detection in RT-LAMP was 103 copies of DNA/ml. The diagnostic sensitivity and specificity of the RT-LAMP diagnosis of HSV infection were 96% and 100%, respectively.Discussion. RT-LAMP method has a high sensitivity and specificity comparable to RTPCR, while the risk of false positive results obtaining is minimal.Conclusion. Thus, the reaction of RT-LAMP with SYTO-82 dye allows quickly, with high sensitivity and specificity to detect HSV DNA in clinical material and can be considered as a promising point-of-care testing method.

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