Development and application of an RT‒PCR assay for the identification of the delta and omicron variants of SARS-COV-2.

The emergence of mutations in the coronavirus genome provides opportunities for occurrence new strains with higher transmissibility, severity and duration of the disease poses. In 2020, a new variant of the coronavirus SARS-COV-2 - Delta was identified in India. This genetic variant has spread rapidly and became dominant in many countries, including Russia. In November 2021, a new outbreak of COVID-19 occurred in Africa driven by a variant SARS-COV-2 named later Omicron. Both variants had increased transmissibility compared to previously encountered variants and quickly, replacing its around the world. To promptly monitor the epidemiological situation in the country, to assess the spread of dominant genetic variants of the virus and to take appropriate measures, we have developed an RT‒PCR reagent kit for the identification of Delta and Omicron by detecting a corresponding combination of major mutations. The minimum set of mutations was chosen which allows to differentiate Delta and Omicron variants, in order to increase the analysis productivity and reduce costs. Primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Delta and Omicron. Similar approach can be implemented for the rapid development of assays for differentiating important SARS-COV-2 variants or for other viruses genotyping for epidemiological surveillance or for diagnostic use in order to assist in making clinical decisions. It was demonstrated that the results of VOC Delta and Omicron detection and their typical mutations were concordant with genotyping based on WGS results for all 847 samples of SARS-CoV-2 RNA. The kit has high analytical sensitivity (1х103 copies/mL of SARS-CoV-2 RNA) for each of the detected genetic variants and possesses 100% analytic specificity for microorganism panel testing. The diagnostic sensitivity (95% confidence interval) obtained during pivotal trials was 91.1-100% for Omicron and 91.3-100% for Delta, while the diagnostic specificity with a 95% confidence interval was 92.2-100%. The use of a set of reagents in combination with sequencing of SARS-CoV-2 RNA as part of epidemiological monitoring made it possible to quickly track the dynamics of changes in Delta and Omicron prevalence in the Moscow region in the period from December 2021 to July 2022.

Probability of Infectious Disease in Humans during Epidemic

Popular SIR models and their modifications used to generate predictions about epidemics and, specifically, the COVID-19 pandemic, are inadequate. The aim of this study was to find the laws describing the probability of infection in a biological object. Using theoretical methods of research based on the probability theory, we constructed the laws describing the probability of infection in a human depending on the infective dose and considering the temporal characteristics of a given infection. The so-called generalized time-factor law, which factors in the time of onset and the duration of an infectious disease, was found to be the most general. Among its special cases are the law describing the probability of infection developing by some point in time t, depending on the infective dose, and the law that does not factor in the time of onset. The study produced a full list of quantitative characteristics of pathogen virulence. The laws described in the study help to solve practical tasks and should lie at the core of mathematical epidemiological modeling.Copyright © 2022 Obstetrics, Gynecology and Reproduction. All rights reserved.

N PROTEIN BASED VACCINE AGAINST SARS-COV-2 PRODUCES A STRONG T CELL IMMUNE RESPONSE TO N PROTEIN OF NOVEL STRAINS

The second generation COVID-19 vaccines should produce the long-term protective immune response to the existing and novel strains of SARS-CoV-2. The Convacell® vaccine was designed to produce such immune response by using N protein as an antigen. N-protein is not susceptible to fast accumulation of mutations and is highly homologous to nucleocapsid proteins of other β-coronaviruses. The study was aimed to perform in vitro assessment of the Convacell® vaccine ability to produce immune response to the Wuhan, Delta, and Omicron strains. Mononuclear cells of vaccinated volunteers and survivors were subjected to N protein stimulation. After that specific activation of the cells was assessed by flow cytometry. The results showed that a sibstantial percentage of CD4 and CD8 cells produced IFNγ and IL2 in response to stimulation. No significant reduction of the response to strains Delta and Omicron compared to the Wuhan strain was revealed. The findings support the direction of the N protein based vaccine design towards creation of the universal vaccine. © 2022 Federal Medical Biological Agency Publishing Group. All Rights Reserved.

DEVELOPMENT OF PCR TEST FOR DETECTION OF THE SARS-COV-2 GENETIC VARIANTS ALPHA, BETA, GAMMA, DELTA

The emergence of novel SARS-CoV-2 genetic variants with increased transmissivity and reduced antibody neutralization efficiency is a threat to global public health. Reverse transcription polymerase chain reaction (RT-PCR) with the use of fluorescent probes, which make it possible to detect the single nucleotide substitutions, is a technique suitable for screening the SARS-CoV-2 RNA-containing samples for the already known functionally significant mutations in the S-gene, identification of which allows to define and differentiate the most epidemiologically significant genetic variants. The study was aimed to develop an assay for the large-scale monitoring of the spread of the SARS-CoV-2 top-priority variants. Based on the whole-genome alignment of the SARS-CoV-2 sequences, deposited in the GISAID database, primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Alpha, Beta/Gamma and Delta variants of concern (VOC). The developed reagent kit for detection of the key mutations in the SARS-CoV-2 S gene by the real time RT-PCR has good analytical and diagnostic characteristics and was authorized as a medical device (reagent) for in vitro use. The results of detecting the VOC and the key mutations with the use of the developed reagent kit were consistent with the data of the whole genome sequencing of 1,500 SARS-CoV-2 RNA samples. The developed reagent kit and the subsequent SARS-CoV-2 RNA sequencing assay used to perform the epidemiological monitoring of SARS-CoV-2 variants made it possible to promptly report the emergence of the Delta genetic variant in Russia, and to trace the dynamic changes in the prevalence of Delta in Moscow Region in April-September 2021. © 2022 Obstetrics, Gynecology and Reproduction. All rights reserved.