In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

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We report the prevalence of Siberian and Far Eastern subtypes of tick-borne encephalitis virus (TBEV) in Ixodes persulcatus and Ix. pavlovskyi ticks collected in Tomsk and its suburbs during 2006-2008. The TBEV was detected in 5.7% ticks collected in the city, where Ix. pavlovskyi ticks were dominated and 7.5% ticks from suburban foci with prevalence Ix. persulcatus ticks. Genotyping of the virus showed that Siberian subtype (89.5%) is predominant in individual ticks of Tomsk suburbs; however, the proportion of Far Eastern subtype in two urban sites reached 47%. Phylogenetic analysis demonstrated that Siberian subtype variants from individual ticks were quite divergent and original. Only one subclade was found to be similar to Zausaev strain of TBEV, which is the etiological agent of lethal chronic form of tick-borne encephalitis infection. The average level of homology of 5' noncoding region (5'-NCR) of TBEV in the individual ticks was 95% for Far Eastern subtype and 89% for Siberian subtype of TBEV. Multiple substitutions in 5'-NCR were found in viral RNA derived from individual ticks. The A2 and C1 elements of Y-shaped structure and putative site for viral RNA polymerase were most variable regions for TBEV 5'-NCR. The B1 and B2 elements and the start codon were practically conserved. The viral RNA from three TBEV-infected pig kidney embryo cells after three passages (out of 21 polymerase chain reaction-positive ticks) were found to multiple substitutions in 5'-NCR in comparison with viral RNA from individual parent tick. However, these three variants did not replicate efficiently in pig kidney embryo cells that may be connected with a considerable modification of Y-shaped structure of 5'-NCR. The efficiently replicating isolate Kolarovo had only seven substitutions in the 5'-NCR and typical Y-shaped structure for Siberian subtype of TBEV. Our data support the idea that hypervariability of the 5'-NCR reflects viral strategy to select the fittest RNA molecule for productive viral infection in mammalian and tick cells.

Objective: isolation of coronavirus SARS-CoV-2 from clinical sample of patient with COVID-19 in Novosibirsk; obtaining a purified and inactivated viral antigen and study of its antigenic properties. Materials and methods: virus isolation was carried out in Vero cell culture from nasopharyngeal swab positive on SARS-CoV-2 RNA. The efficiency of SARSCoV-2 replication in cell culture was assessed on the appearance of cytopathic effect (CPE) and the presence of viral RNA in cultural medium with reverse transcription – polymerase chain reaction (RT-PCR). Purification, concentration and inactivation of the viral preparation were carried out according to standard methods. The purity of the purified preparation and the profile of viral proteins were determined by electrophoresis in 10% polyacrylamide gel (PAG) with the addition of sodium dodecyl sulfate (SDS). The presence and specificity of viral proteins were detected using COVID-19 convalescent’s sera with enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results: SARS-CoV-2/human/ RUS/Nsk-FRCFTM-1/2020 isolate was obtained after passage on Vero cells from a virus-containing clinical sample. A purified, concentrated, inactivated, whole-virion antigen was obtained. It contains three structural proteins: glycoprotein S (approximately 200 kDa), nucleoprotein N (48 kDa), and matrix protein M (20-25 kDa). All viral proteins were detected with serum antibodies of COVID-19 convalescents. Conclusion: SARS-CoV-2 coronavirus can be isolated in Vero cell culture. The antigenic specificity of the three structural viral proteins (S, N, and M) is preserved in the purified inactivated viral preparation. The inactivated whole-virion antigen of SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 isolate can be used to study the antigenic immunomodulating properties of viral proteins, to obtain immune sera of laboratory animals, and also as a component of test systems for the detection of specific antibodies with ELISA and immunoblotting.