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Introduction. The emergence of new epidemiologically significant variants of SARS-CoV-2 has shifted emphasis to development of a live vaccine, which would be able to provide protection against a wide range of antigenic variants of the virus. The aim of the study was to obtain SARS-CoV-2 variants attenuated through cold adaptation and to provide their biological characterization.Materials and methods. The Dubrovka laboratory strain of SARS-CoV-2 and its variants were cultured on Vero and Calu-3 cells. The virus quantification was performed by virus titration in Vero cells and by real-time reverse transcription-polymerase chain reaction. SARS-CoV-2 virions were analyzed using transmission electron microscopy. Genome sequences of the virus were identified by nanopore sequencing. The attenuation (att) phenotype of SARS-CoV-2 variants was identified using Syrian hamsters as an animal model for COVID-19. Results. Cold-adapted (ca) SARS-CoV-2 variants – Dubrovka-ca-B4 and Dubrovka-ca-D2 were produced by continued passaging of the Dubrovka strain in the Vero cell culture at the temperature being gradually decreased to 23ºC and by subsequent cloning. Up to 20 nucleotide substitutions and 18 amino acid substitutions were detected in genomes of ca-variants. Ca-variants, as distinct from the parent Dubrovka strain, actively replicated at 23ºC, while the Dubrovka-ca-D2 variant had a temperature-sensitive (ts) phenotype (did not replicate at 39ºC). Ca-variants of the virus replicated poorly at 37ºC in the Calu-3 human lung cell culture, which, along with the ts-phenotype, can be a marker of virus attenuation for humans. In the intranasally infected Syrian hamsters, ca-variants of the virus demonstrated an attenuation phenotype: they did not cause loss of appetite, fatigue, drowsiness, did not slow down weight gain, replicating much more slowly in the lungs and brain compared to the virulent Dubrovka strain. Conclusion. The obtained attenuated SARS-CoV-2 ca-variants, Dubrovka-ca-B4 and Dubrovka-ca-D2, should be studied further as candidate vaccine strains for a live attenuated vaccine against COVID-19.

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.

Introduction. Immunodeficiency underlying the development of severe forms of new coronavirus infection may be the result of mixed infection with SARS-CoV-2 and other pathogens, including Epstein–Barr virus (EBV).The aim is to study the prevalence and epidemiological features of co-infection with SARS-CoV-2 and EBV. Material and methods. A cross-sectional randomized study was conducted in Moscow region from March to May 2020. Two groups were examined for EBV-markers: hospital patients (n = 95) treated for SARS-CoV-2 infection and blood donors (n = 92).Results. With equal EBV prevalence the detection of active infection markers in donors (10.9%) was noticeably lower than in SARS-CoV-2 patients (80%). Significant differences in this indicator were also found when patients from subgroups with interstitial pneumonia with the presence (96.6%) and absence (97.2%) of SARS-CoV-2 in the nasopharyngeal smear were compared with the subgroup of patients with mild COVID-19 (43.3%). The average IgG VCA and IgG EBNA positivity coefficients in donor group were higher than in patient group (p < 0.05). Patients with active EBV infection markers were significantly more likely to have pneumonia, exceeding the reference values of ALT and the relative number of monocytes (odds ratio – 23.6; 3.5; 9.7, respectively).Discussion. The present study examined the incidence and analyzed epidemiological features of active EBV infection in patients with COVID-19.Conclusion. A significantly higher rate of detection of active EBV infection markers in hospital patients indicates a combined participation SARS-CoV-2 and EBV in the development of interstitial pneumonia. Low levels of specific IgG EBV serve as predictors of EBV reactivation. Exceeding the reference values of ALT and the relative number of monocytes in patients should serve as a reason for examination for active EBV infection markers.