An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21–36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.

No abstract

No abstract

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.