WHOLE-GENOME SEQUENCING OF SOME UKRAINIAN ISOLATES OF SARS-COV-2 VIRUS AND ANALYSIS OF ITS GENETIC VARIABILITY

pangolin-CoV-2020 was not con ﬁ rmed (Liu P et al, 2020). Seroconversion was observed in racoon dogs, mink and cattle, while for pigs, antibodies were detected only in three out of six references. Transmission of SARS-CoV-2 was observed for mink and rac-coon dogs, but no transmission has been observed for cattle and pigs so far (Dunowska, 2023; Nielsen et al, 2023). Moreover, the possibility of spontaneous infecting of domestic dogs with SARS-CoV-2 coronavirus from humans was demonstrated (Sit et al, 2020) . The genome sequencing showed that the so-called feline SARS-CoV-2 belongs to the phylogenetic cluster A2a along with most recently isolated samples of human to


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a strain of coronavirus that causes COVID-19, a serious and often lethal respiratory disease in man, leading to a pandemic in 2019.This virus is a positive-sense, single-stranded enveloped ribonucleic acid (RNA) virus, related to Sarbecovirus subgenus of Betacoronavirus genus (Rambaut A et al, 2020;Rabi FA et al, 2020, Ahmed R et al, 2021;Ferretti L et al, 2020, Gorbalenya AE et al, 2020).
Epidemics caused by coronaviruses were observed in the past (from early 2000's), among others caused by SARS-CoV and the respiratory syndrome coronavirus (MERS-CoV) after 2012.In the context of zoonotic viral diseases, it is important to mention the dynamics of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which poses a signifi cant public health concern.MERS-CoV is known for its zoonotic transmission, with dromedary camels serving as a crucial reservoir.Understanding the transmission dynamics of MERS-CoV, including its potential to persist in the environment, was essential for devising effective prevention and control strategies for SARS-CoV-2, hich appeared late 2019.Researchers developed experience in the detection and identifi cation of these viruses (Yan Y Chang & Wang L, 2020;Lam T T-Y et al, 2020;Elaswad A et al, 2020;Lu R et al, 2020;Dunowska, 2023).
The pandemic outbreak of coronavirus (named coronavirus 2019 or COVID-19) was caused by a new coronavirus, namely SARS-CoV-2 virus, fi rst diagnosed in humans in December 2019, in China (Wuhan, Hubei province).On January 30, 2020, the WHO announced that the outbreak of the diseases, caused by SARS-CoV-2 coronavirus, was an emergency situation in the fi eld of public health care (Zhang, Y-Z, Holmes EC, 2020).
In Ukraine, COVID-19 was fi rst diagnosed in March 2020 in Chernivtsi, and on March 13, the fi rst lethal case, caused by SARS-CoV-2, was registered (Resolution of Cabinet of Ministry of Ukraine No. 211 dated March 11, 2020).As of early December 2023, according to the offi cial statistics data for Ukraine, there were about 4,380,047 registered and confi rmed cases of Covid-19, including 101,887 lethal ones Ukraine Ministry of Health, 'Update on the spread and prevention of CO-VID-19 coronavirus infection' (Kyiv, Ukraine, 2022).
The pandemic character of coronavirus spreading and the aggressive behavior of its causal agent made the detection of new mutations in its genome a priority (Zi-Wei Ye et al, 2020;Phun T, 2020;MacLean OA et al, 2021, Ridgway et al, 2022).A further fi eld of investigation became the possible recombination of the virus, either of natural or laboratory origin, that can impact pathogenicity and virus contagion (Rabi FA et al, 2020;Zhou P et al, 2020;Lam T T-Y et al, 2020;Liu P et al, 2020;Sia S F et al, 2020;Ridgway et al, 2022).
Phylogenetic analysis demonstrated that the coronavirus of pangolin (pangolin-CoV-2020), along with the group of coronaviruses, circulating in populations of different bat species are genetically related to SARS-CoV-2.For example, the nucleotide sequence identities among the S protein genes between the Bat-CoV-RaTG13 and SARS-CoV-2 were 93.15 %, and between pangolin-CoV-2020 and SARS-CoV-2, 84.52, but the assumption that SARS-CoV-2 originated directly from pangolin-CoV-2020 was not confi rmed (Liu P et al, 2020).Seroconversion was observed in racoon dogs, mink and cattle, while for pigs, antibodies were detected only in three out of six references.Transmission of SARS-CoV-2 was observed for mink and raccoon dogs, but no transmission has been observed for cattle and pigs so far (Dunowska, 2023;Nielsen et al, 2023).Moreover, the possibility of spontaneous infecting of domestic dogs with SARS-CoV-2 coronavirus from humans was demonstrated (Sit et al, 2020).The genome sequencing showed that the so-called feline SARS-CoV-2 belongs to the phylogenetic cluster A2a along with most recently isolated samples of human WHOLE-GENOME SEQUENCING OF SOME UKRAINIAN ISOLATES OF SARS-COV-2 VIRUS SARS-CoV-2 (Michelitsch A et al, 2020;Garigliany M et al, 2020;Ruiz-Arrondo I et al, 2021).
According to Doliff and Martens (2022) cats appear to play only a limited role in the spread of SARS-CoV-2.Although cats are indeed are susceptible to the virus and reverse zoonotic transmission from humans to cats is occurring regularly, there is up till now no evidence that SARS-CoV-2 circulates among cats.
Recently, Lan et al (2020) determined that the structure of spike (S)-protein, which binds the receptor of human angiotensin-converting enzyme 2 (АСЕ2), has 20 key amino acids for the interaction with the receptor-binding site.The studies have already shown that some animals are potentially susceptible to SARS-CoV-2 because they have the same receptor ACE2, which is widespread in different mammal species, such as Syrian hamster (Mesocricetus auratus), ferret (Mustela furo), mink (Neogale vison), cattle (Bos taurus) and raccoon dog (Nyctereutes procyonoides) (Abdel-Moneim and Abdelwhab 2020; Li F, 2013;Nielsen SS, 2023).Mutations in the locus of the receptor binding domain (RBD) impact the effi ciency ACE2, and thus, the susceptibility to SARS-CoV-2 (Jia et al, 2005;Wan Y et al, 2020;Zi-Wei Ye et al, 2020;Fenollar F et al, 2021, Ridgway, 2022;Cherian S et al, 2021;Hoffmann M et al, 2020 ).
The species of animals that have sequences K31, Y41, N90, and K353 are likely to be susceptible to the SARS-CoV-2 infection, while other species that do not have the mentioned fragments will surely be either less susceptible or resistant to the infection (Devaux et al, 2021).
In addition to currently unknown and susceptible animal hosts of SARS-CoV-2, new mutations in SARS-CoV-2 may become fi xed in the virus which may make animal species with low sensitivity to SARS-CoV-2 more susceptible to infection (Gu H et al, 2020;Kumar A et al, 2021 and2023;McAloose, D et al, 2020;Naqvi A et al, 2020;Dunowska, 2023).
A rapid spreading mutation, N501T in the RBD of S-protein was frequently observed in minks (Neogale vison) at mink farms (Elaswad et al, 2020;Domanska-Blicharz K et al, 2021) and in experimentally infected ferrets (Mustela furo) (Richard et al, 2020).This mutation N501T was found in almost all SARS-CoV-2 sequences, obtained from biological material of minks in the USA (99 %).The studies of Cai et al (Cai HY, Cai A, 2021) demonstrated that mutation N501T was fi rst found in humans and only two months later -in minks.These authors assumed mutations and eventually new virus variants may fi rst occur in humans, later penetrating and getting fi xed in the population of susceptible animals.
Progressive virus mutations provoked the rise of new lineages such as Delta, Epsilon and Omicron (Davies NG et al, 2021;Damas J et al, 2020;Kumar et al, 2023;Markov et al, 2023).This requires testing as many samples of SARS-CoV-2 positive samples for timely detection of possible new variants, lines or mutations of the virus.In Ukraine the Ministry of Health has determined that the Delta and omicron variants have largely replaced the others in recent days (Yakovleva et al, 2022).These authors also mapped lineages and mutations occurring in Ukraine using a large database of Ukrainian whole genome data collected over the past years of the pandemic up to 2021.
The aim of the study.The aim was to gain experience of the animal health services to detect and properly identify SARS-CoV-2 virus and its mutations in Ukraine in relation to possible future spread of the virus in animals.

MATERIALS AND METHODS
Sixteen SARS-CoV-2 positive samples, not sequenced before, were provided by the Center for Public Health of the Ministry of Health of Ukraine.These samples were obtained from hospitalized 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.All samples were transported in a cryobox with dry ice (-70 ºС), and after the delivery to the laboratory prior to the tests kept at -70 ºС without any protectant added.
Combined DNA/RNA isolation from the samples of biological material was conducted using IndiSpin Pathogen kit (Indical Bioscience) according to the manufacturer's recommendations, using 200 ul of sample.Effi cacy of this kit was verifi ed in our laboratory, using an internal control RNA sample provided by Seegene, South Korea as a part of 2019-nCoV Assay kit (Ref RV10248X, LOT RV9120H23) (synthetic specifi c fragments of viral RNA to be used as positive control for RT-qPCR assay for SARS-CoV-2 detection).
The amount of RNA isolated was determined using Qubit RNA HS assays kits (Life Technologies, Carlsbad, California, USA) on a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, California, USA) according to manufacturer's manual.The presence of SARS-CoV-2 in the purifi ed RNA samples for the downstream steps was estimated by fl uorescence detection of RdRp and N genes using the RT-qPCR kit Allplex SARS-CoV-2 Assay (Seegene, South Korea), Ref RV10248X, LOT RV9120H23 according to manufacturer's protocol on a plate-type amplifi er Bio-Rad tm 1000 with module CFX96 tm using the built-in software to evaluate the result.
The cDNA was obtained using a SuperScript tm VI-LO tm cDNA synthesis kit (Invitrogen, Carlsbad, CA, the USA) according to the manufacturer's protocol and amplicons was produced using the Ion AmpliSeq SARS-CoV-2 panel and a protocol published by Ther-moFisher.The samples selected for sequencing had the cDNA concentration of 12-15ng/μl) measured using a Qubit 3.0 fl uorometer (ThermoFisher Scientifi c, USA) with Qubit™ dsDNA HS Assay Kit (ThermoFisher Scientifi c, USA).
All libraries were prepared using the Ion One Touch tm instrument (ThermoFisher Scientifi c, USA), and subjected to templating runs.The library quality was checked and confi rmed by standard agarose gel electrophoresis.
Sequencing of SARS-CoV-2 virus was conducted by NGS using the Ion GeneStudio™ S5 System and Ion 520™ Chip Kit with 6 barcodes per chip (ThermoFisher Scientifi c, USA).
To conduct the phylogenetic analysis, we used Nextclade 2.3.0 (Aksamentov I et al, 2021) to locate the 6 sequenced samples on the global phylogenetic tree.We also determined phylogenetic relations between our 6 sequences and 495 verified sequences of high quality, reported in Ukraine and deposited in the GISAID EpiCoV™ database, (https://gisaid.org/).

RESULTS
The fi rst stage of the studies involved the confi rmation of the state and concentration of the target virus RNA and QC check and the presence of SARS-CoV-2 virus using the RT-qPCR test kit.The best quality samples had an RNA concentration 12-15 ng/which was used to prepare the libraries for sequencing.
From the 16 samples we obtained, six sequences with suffi cient quality were deposited in the database https:// gisaid.org,inventory number EPI_SET_230516yp (https://doi.org/10.55876/gis8.230516yp).Other samples either had a very low RNA concentration or were not suitable for further sequencing due to considerable degradation.
According to the PANGO nomenclature (Aine O'Toole et al, 2021) 2).According to the data of the global database of the sequenced GISAID isolates, the virus of Delta variant was fi rst detected in India on October 2020.One of our six isolates was related to Omicron variant (line BA.1.18),which was fi rst detected in South Africa and Botswana in November 2021.This variant is characterized by multiple mutations in RGB region, more than observed in the Delta variant.

DISCUSSION
The results, obtained in the study, are in agreement with the published data, according to which the fi ve Delta isolates and the one Omicron variant we tested, were characterized by multiple mutations in S gene, which is accompanied by faster spreading and a considerable decrease in virus pathogenicity (Davies NG et al, 2021;Chatterjee S et al, 2023;Jacobs et al, 2023;Liang, 2023;Jung et al, 2022).
The method and equipment we used enables determination of mutations in the SARS-CoV-2 genome, including unknown ones (Elaswad A et al, 2020;Yuan S et al, 2020;Phun T, 2020;Kumar A et al, 2021;Fischhoff IR et al, 2021;MacLean OA et al, 2021).
The volume of sequence reading is crucial because it directly impacts coverage, accuracy, sensitivity, and reliability of sequencing data.Higher volume ensures better coverage, accuracy in variant detection, sensitivity to detect rare variants, and statistical confi dence in results; it should be at least one million readings for SARS-CoV-2 genome, which is minimally suffi cient to obtain a whole sequence of SARS-CoV-2 virus genome.We obtained more than one million readings per sample (Aine O'Toole et al., 2021).The Spike protein mutation H69/V70 which we detected in our Omicron isolate belonging to lineage BA.1.18was reported before in lineage BA.1 by Zaman et al (2022) and Kandeel et al (2023).
The combination of high transmissibility and the presence of a considerable number of people who may be hosts of SARS-CoV-2 virus creates a threat of virus transmission into the populations of animals.Seroconversion was observed in racoon dogs, mink and cattle, while for pigs, antibodies were detected only in three out of six references.Transmission of SARS-CoV-2 was observed for mink and raccoon dogs, and no transmission was observed for cattle and pigs (Dunowska, 2023;EFSA, 2023).In Ukraine according to offi cial statistics the six mink farms united by the Ukrainian Stock Breeders Association raise 1.1 million minks, which is about 95 % of the Ukrainian market.All products are exported.Since government agencies do not test mink farms for the presence of SARS-CoV-2, it is possible that coronavirus outbreaks can occur at any time.These assumptions are in agreement with previous publications (Devaux et al, 2021;EFSA, 2023).
According to Doliff and Martens (2022) cats appear to play only a limited role in the spread of SARS-CoV-2.Although cats are indeed are susceptible to the virus and reverse zoonotic transmission from humans to cats is occurring regularly, there is up till now no evidence that SARS-CoV-2 circulates among cats.Some published data have proposed that the Omicron variant may have arisen from cross-species transmission, given its specifi c combination of mutations, particularly in the spike protein responsible for host cell entry (Sun et al, 2022;Burki, 2022;Kupferschmidt, 2021;Doliff & Martens, 2022;Carabelli et al, 2023).Therefore, it seems unlikely that this hypothesis will be conclusively confi rmed in the future.Nevertheless, these concerns underscore the critical importance of conducting extensive surveillance of potentially vulnerable animal populations.
At present the scientifi c community uses several classifi cation systems of SARS-CoV-2 variants: GISAID, Nextstrain and PANGO.PANGO differs from other systems as it uses dynamic nomenclature, focused on actively circulating lines of the virus and those, spreading to new areas (Rambaut et al, 2020).At the time of the study, the Omicron variant was just spreading, but at the time of publication of this article, the lines that were common in 2020-2022 (Alpha, Beta, Delta) have completely disappeared and been replaced by newer variants (currently, according to https://covid.cdc.gov/covid-data-tracker/, the Omicron JN.1 variant dominates).This process is likely to continue, so the relevance of whole-genome studies aimed at identifying new mutations and new variants will remain (Rambaut et al, 2021;Aine O'Toole, 2021).To facilitate communication beyond the scientifi c audience, the World Health Organization developed a system of marking the virus variants using the letters of the Greek alphabet.At present, this system is for the following virus variants B.1.1.7 (Alpha),B.1.351 (Beta) To refi ne the future goal, the researchers at the veterinary medicine institutions of National Academy of Agrarian Sciences (NAAS) of Ukraine, aim to create and implement a comprehensive survey and monitoring program for SARS-CoV-2 in animal populations.This initiative involves collaborating with relevant veterinary institutions to sample and monitor specifi c animal species susceptible to the virus, mainly minks NYCHYK et al. and cats.The goal is to conduct regular surveillance to detect any potential transmission or mutation events, thereby contributing to the early detection and control of the virus in animal populations.
Adherence to ethical principles.Only viral RNA was used in the study without any connection to any personal data; the methodological approaches complied with the requirements of IEC/ISO 17025 standard.Confl ict of interests.The authors declare the absence of any confl icts of interests.Financing.The study was conducted with the fi nancial support of the State Fund for Research of Ukraine, grant No. 2020.01/0121.

Table 1 .
Characteristics of the sequenced SARS-CoV-2 isolates 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, BSL-2+ biosafety level, ISO/IEC 17025: 2019 accredited by National Accreditation Agency of Ukraine.

Table 2 .
The mutations found in the genome of the investigated SARS-CoV-2 isolates