Whole-genome sequencing of some Ukrainian isolates of SARS-COV-2 virus and analysis of its genetic variability

Aim. The aim was to gain experience of the animal health services to detect and properly identify SARS-CoV-2 virus with whole-genome sequencing method and its genetic variability in Ukraine in relation to possible future spread of the virus in animals. Methods. Sixteen SARS-CoV-2 positive samples, not sequenced before, were pro- vided by the Center for Public Health of the Ministry of Health of Ukraine. These samples were obtained from hos- pitalized patients from early October to mid-November of 2021. The viral RNA was isolated from nasopharyngeal swab samples of SARS-CoV-2 qPCR positive (Ct 21-28) patients (males and females) with moderate and severe symptoms who were being hospitalized. The samples were fully anonymized. The Ion Torrent S5 instrument (Ox- ford Nanopore, the USA) was used to sequence the mentioned SARS-CoV-2 isolates, originating from Ukraine. TorrentSuite 5.16.1 was used for data processing and analysis. Nextclade 2.3.0 was used for phylogenetic analysis to locate the 6 sequenced samples on the global phylogenetic tree. It was determined phylogenetic relations be- tween tested 6 sequences and 495 verified sequences of high quality, reported in Ukraine and deposited in the GI- SAID EpiCoVTM database, (https://gisaid.org/) for the period of January 2020 – December 2022. In the compari- son of sequences obtained, the sequence of SARS-CoV-2 virus isolate Wuhan-Hu-1 (GenBank NC_045512.2) was used as a reference sequence, according to which the sequences were aligned. All studies were carried out in the laboratory of the Research Training Center for Animal Disease Diagnostics at the Institute of Veterinary Medicine of the National Academy of Sciences of Ukraine. Results. Among the 16 isolates tested, all were confirmed to con- tain SARS-CoV-2 RNA, of which only six isolates were sequenced with sufficient quality and could be classified, five of them as Delta variants (two belong to lineage AY.126 (B.1.617.2.33), two to AY.122 (B.1.617.2.122), and one to AY.4.2.3 (B.1.617.2.4.2)), and one isolate as an Omicron variant (BA.1.18). Important mutations detected in our isolates were a S:N501Y substitution and S:H69 deletion in the gene of the virus envelope spike protein. Among the examined isolates, the Omicron variant (BA.1.18) was found to exhibit greater genetic variability, with over 60 mutations compared to previous variants. In our investigation, we identified mutations in the sequenced Delta variants too, ranging from 35 mutations in AY.122 (B.1.617.2.122) to 41 mutations in AY.126 (B.1.617.2.33) in the genome compared to the reference Wuhan-Hu-1 (MN908947) variant. Important mutations found regarding infectivity were 1) for the Delta variants: T478K, L452R mutations in the RBD region, and 2) for the Omicron variant: S371L, G339D, S375F, S373P, K417N, N440K, S477N, G446S, E484A, T478K, Q493R, Q498R, G496S, N501Y, and Y505H mutations in the RBD region. Conclusions. The whole-genome sequencing of 6 isolates of SARS-CoV-2 virus was performed, and three sublines of the Delta variant were found: AY.126 (B.1.617.2.33), AY.122 (B.1.617.2.122), AY.4.2.3 (B.1.617.2.4.2) and one subline for the Omicron variant (BA.1.18), all of which were deposited in the international database GISAID as EPI_SET_230516yp. The data obtained in this study add to the existing ones delivered by the Ministry of Health in Ukraine and can be used in laboratories, (including veterinary ones), detecting the SARS-CoV-2 virus in risk animal populations, in order to prevent the spread of the disease to humans and animals, as well as to detect possible mutational changes in the pathogen genome that may affect infectivity and pathogenicity.