Whole Genome Sequencing of SARS-CoV-2 in Cats and Dogs in South Korea in 2021

Simple Summary Forty SARS-CoV-2-positive swab samples from 40 animals owned by confirmed SARS-CoV-2-infected owners in Korea in 2021 were analyzed using next-generation sequencing (NGS) to identify the genomic lineage of the viruses. Among the 40 animal samples, eight Pango lineages (B.1.1.7) (Alpha variant), B.1.429 (Epsilon variant), B.1.470, B.1.497, B.1.619.1, B.1.620, AY.69 (Delta variant), and AY.122.5 (Delta variant) were identified. This study provides the first reported cases of six lineages (B.1.470, B.1.497, B.1.620, B.1.619.1, AY.69 (Delta variant), and AY.122.5 (Delta variant)) in cats and dogs. Our results emphasize the importance of monitoring SARS-CoV-2 in pets because they are dynamic hosts of variant Pango lineages of SARS-CoV-2. Abstract SARS-CoV-2 infections have caused unprecedented damage worldwide by affecting humans and various animals. The first reported animal infection was observed in a pet dog in Hong Kong in March 2020. 36 countries reported 692 SARS-CoV-2 infections in 25 different animal species by 31 August 2022. Most outbreaks were caused by contact with SARS-CoV-2 infected humans. In South Korea, the first SARS-CoV-2 infection in an animal was reported in a cat in February 2021. As of 31 December 2021, 74 dogs and 42 cats have been confirmed to have SARS-CoV-2 in South Korea. Here, we identified various SARS-CoV-2 genomic lineages in SARS-CoV-2 confirmed cats and dogs. Among the 40 animal samples sequenced for lineage identification, a total of eight Pango lineages (B.1.1.7 (Alpha variant), B.1.429 (Epsilon variant), B.1.470, B.1.497, B.1.619.1, B.1.620, AY.69 (Delta variant), and AY.122.5 (Delta variant)) were identified. The dominant lineages were AY.69 (Delta variant; 37.5%), B.1.497 (35.0%), and B.1.619.1 (12.5%). This study provides the first reported cases of six lineages (B.1.470, B.1.497, B.1.620, B.1.619.1, AY.69 (Delta variant)), and AY.122.5 (Delta variant) in cats and dogs. Our results emphasize the importance of monitoring SARS-CoV-2 in pets because they are dynamic hosts of variant Pango lineages of SARS-CoV-2.


Introduction
Coronavirus disease 2019 (COVID-19) has caused unprecedented global damage by affecting not only humans, but also various animals. COVID-19 was first reported in humans in Wuhan, China, in December 2019, and was caused by SARS-CoV-2 [1]. As of October 2022, about 630 million people have been infected with SARS-CoV-2, and about 6.5 million of them have been confirmed dead [2]. SARS-CoV-2 is a positive-stranded RNA virus belonging to the family Coronaviridae of the order Nidovirales [3]. Coronaviruses are known to have a virus mutation rate about 10 times lower than that of influenza viruses because they have an exonuclease enzyme that lowers the replication error rate [4]; however, similar to many other RNA viruses, the SARS-CoV-2 genome has been constantly mutating to adapt to the host immune system since its emergence [5,6]. The evolution of the SARS-CoV-2 gene has resulted in tens of thousands of mutations in the SARS-CoV-2 genome, which has caused continuous public health difficulties such as vaccines, treatments, and application of diagnostic technologies [7]. The World Health Organization (WHO) has classified SARS-CoV-2 with mutations of high impact as variants of concern (VOC), and to date, five major VOC have been detected: the Alpha (B. 1 SARS-CoV-2 spillover from humans to domestic cats, dogs, and other animals has been reported globally [8]. The first reported animal infection was observed in Hong Kong in March 2020 when a pet dog was infected with SARS-CoV-2 [9]. In total, 36 countries had reported 692 SARS-CoV-2 infections in 25 different animal species (cat, dog, mink, otter, pet ferret, lion, tiger, puma, snow leopard, gorilla, white-tailed deer, fishing cat, binturong, South American coati, spotted hyena, Eurasian lynx, Canada lynx, hippopotamus, hamster, mule deer, giant anteater, west Indian manatee, blacktailed marmoset, common squirrel monkey, and mandrill) as of 31 August 2022 [8]. The actual number of animal infections was expected to be higher than 692 as the number of SARS-CoV-2 sequences uploaded in Global Initiative for Sharing All Influenza Data (GISAID) was >2400 as of 8 December 2022 [10]. Most of these infections are believed to be spillovers from humans, except for a few cases such as mink-to-human infections in European countries [11][12][13] and hamster-to-human infections in Hong Kong [14][15][16].
In South Korea, the first case of SARS-CoV-2 infection in animals was reported on 29 January 2021, in a domestic cat in Gyeongnam province, residing in a religious facility, where multiple individuals (120 residents and visitors) were confirmed to have SARS-CoV-2 infection [17]. The virus lineage was B.1.497 (Pango v.2.3.2), and the virus genomes found in the owner and the cat were identical. Based on an epidemiological investigation of the facility, it was believed that the owner transmitted the virus to cat. This event raised public concern, and guidelines for the management of COVID-19 in pet cats and dogs were established by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) in South Korea in February 2021. The guidelines specified that pets owned by COVID-19 confirmed patients should be tested in provincial government veterinary laboratories if the animals had been in contact with the confirmed patients and showed suspected clinical symptoms of COVID-19. Between February and December 2021, 116 animals (42 cats and 74 dogs) were confirmed to be infected with SARS-CoV-2 (MAFRA internal document, 2022). In this study, 40 SARS-CoV-2-positive swab samples from 40 animals were analyzed using next-generation sequencing (NGS) to identify the genomic lineage of the viruses. Among the 40 animal samples, a total of eight Pango lineages (B.

Sample Collection and Detection of SARS-CoV-2
In total, 110 SARS-CoV-2 positive domestic animal (41 cats and 69 dogs) sample sets (oropharyngeal and rectal swabs, as well as nasopharyngeal swabs) were collected in seven South Korean municipalities (Seoul, Daegu, Gwangju, Sejong, Gyeonggi-do, Gyeongnamdo, and Jeonbuk-do) between February and December 2021 and sent to the Animal and Plant Quarantine Agency (APQA) for whole genome sequencing (Supplementary Table S1). All owners of the sampled animals were confirmed to be SARS-CoV-2 positive by the Ministry of Health, and the animal samples were collected and tested the day of, or one to two days after confirmation. To identify the presence of the SARS-CoV-2 genome, commercially available real-time RT-PCR assays (Kogene Biotech, Seoul, South Korea) targeting the RNA-dependent RNA polymerase (RDRP) gene and envelope (E) protein gene were performed according to the manufacturer's instructions. Briefly, viral RNA was extracted using the Maxwell ® RSC Total Nucleic Acid kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Real-time RT-PCR was performed using a Bio-Rad thermocycler (Bio-Rad, Hercules, CA, USA). Animal samples with a cycle threshold (Ct) value of 20-30 were then subjected to NGS of the whole genome of the virus.

Next-Generation Sequencing and Assembly
RNA was converted to cDNA using the PrimeScript RT reagent kit (Takara Bio Europe SAS, Saint-Germain-en Laye, France) with a combination of oligo-dT and random hexamer methods, following the manufacturer's protocol. cDNA was used for viral DNA enrichment using the Qiagen SARS-CoV-2 Primer Panel (Qiagen, Hilden, Germany). The amplified PCR products were used for sequencing-ready library preparation using an Illumina DNA LibraryPrep kit (Illumina, San Diego, CA, USA). The prepared libraries were loaded onto the Illumina Mini-Seq platform and a 150 bp paired-end sequencing kit (300 cycles).

Genotyping and Phylogenetic Analysis
Lineage classification was performed using the Pangolin tool (v.4.1.3) based on the Pango Dynamic Nomenclature System [24,25]. Phylogenetic analysis of the whole-genome sequencing data of the samples was performed by multiple sequence alignment of the sequences with the complete genomes available in the GISAID database. A total of 26,373 complete genome sequences of SARS-CoV-2 strains reported in South Korea were retrieved from the GISAID database on 16 October 2022. Among the 26,373 complete genome sequences, 800 were selected by blasting each animal sample sequence and choosing the 20 sequences showing the highest homology with each sample sequence. After removing redundant sequences, 421 were selected (Supplementary Table S3). The 461 (421 and 40) sequences were aligned using MAFFT v7.508 and a phylogenetic tree was constructed using IQ-TREE v.2.2.0 to apply the GTR + F model of nucleotide substitution with default heuristic search options and bootstrapping with 1000 replicates [26,27].

SARS-CoV-2 Whole Genome Sequencing in Cats and Dogs
All samples collected from SARS-CoV-2 confirmed pets of SARS-CoV-2 confirmed owners were tested for SARS-CoV-2 genomic surveillance. Forty samples (25 cats and 15 dogs) showed Ct values of approximately 20 to 30 in real-time RT-PCR, and they were subjected to whole genome sequencing using Illumina. The run produced an average of 3,825,036 reads and the average coverage was 2126.8 with a read depth of 20x covering 97.0% of the full-length genome of the reference genome (Supplementary Table S2). Forty full-genome sequences were generated, with an average genome size of 29,891 bp.

Discussion
To the best of our knowledge, our study is the first report of SARS-CoV-2 whole genome sequencing of cats and dogs in South Korea. A total of 391 pet samples were tested for SARS-CoV-2, 116 of which were positive (29.7 %), and 110 positive samples were finally submitted to APQA for genome sequencing.
In this study, 40 animal samples from 110 SARS-CoV-2 positive pet cats and dogs of by SARS-CoV-2 confirmed owners were sequenced using Illumina NGS. Most animal samples (102, 92.7 %) were from Seoul, where 18.3% of the total population of South Korea reside and the second biggest pets population (18.4%) out of 2,782,811 pet cats and dogs is registered [28].
In total, eight Pango lineages (B. Since the first human SARS-CoV-2 case in January 2020, 630,783 confirmed human cases, with 5563 deaths, have been reported in South Korea, as of December 2021 [29]. All eight SARS-CoV-2 lineages detected in cats and dogs in this study were also found in humans in South Korea. The Pango lineage B.1.1.7 (Alpha variant) was first detected in the obtained human samples in December 2020, and was last detected in September 2021 in South Korea [10]. The total number of human B.1.1.7 sequences uploaded in GISAID is 862. This lineage has been detected in at least 41 countries, most commonly in the United Kingdom (24.0%), the USA (20.0%), Germany (9%), Sweden (6.0%), and Denmark (6.0%) [30]. B.1.1.7 (Alpha variant) was reported in a cat and dog in the USA in February 2021 [31]. It has also been detected in cats and dogs in Argentina, Bosnia and Herzegovina, France, Italy, Latvia, and Thailand in 2021 [10]. In this study, B.  [32]. This lineage was dynamically associated with visitors to clubs and bars in Itaewon, Seoul, from April 29 to 6 May 2020, and caused one of the major local and long-term epidemics in South Korea [33]. A total of 3180 sequences in Korea have been uploaded in GISAID. The B.1.497 lineage has been detected in at least 11 countries, most commonly in South Korea (98.6%), the USA (0.65%), and Japan (0.06%) [30]. There have been no official reports of confirmed cases of this lineage in cats and dogs. In this study, B.1.497 was detected in a cat in Seoul in February 2021, approximately seven months after the first human B.1.497 case. In total, this study detected this lineage in 14 animals (10 cats and four dogs).
The  [10], and became the dominant lineage (56.7%) together with AY.122.5 (19.7%) from April 2020 to January 2022 [34]. A total of 11,348 AY.69 sequences have been uploaded in GISAD: AY.69 was last detected in January 2022 [10]. This lineage has been detected in at least 16 countries, most commonly in South Korea (100.0%) [30]. There have been no official reports of confirmed cases of this lineage in cats and dogs. In this study, AY.69 (Delta variant) was detected in a cat in Seoul in July 2021, two months after the first human AY.69 (Delta variant) case. Since then, it has been detected in eight cats and six dogs.
The AY.122.5 lineage (Delta variant, alias of B.1.617.2.122.5) was first detected in South Korea in August 2021 [10]. It became dominant among Delta variants in South Korea in December 2021 and was the second most detected lineage (19.5%) in January 2022, while Omicron BA.1.1. was the most detected (53.2%) [34]. AY.122.5 was last detected in February 2022, and a total of 4141 sequences from Korea have been uploaded [10]. This lineage has been detected in at least eight countries, most commonly in South Korea (99.0%) and the USA (15.0%) [30]. There have been no official reports of confirmed cases of this lineage in cats and dogs. In this study, AY.122.5 (Delta variant) was detected in a cat in Seoul in October and in a dog in Gyeongnam-do in December 2021, two to four months after the first human AY.122.5 (Delta variant) case.
Korea Disease Control and Prevention Agency (KDCA) performed genomic surveillance on 17.6% human samples submitted for SARS-CoV-2 diagnosis from April 2021 to January 2022, which is a similar samples collection period to that of the current study [34]. The eight Pango lineages identified in cats and dogs were all grouped into the same South Korean human SARS-CoV-2 lineage branches. The delays between the first human and animal cases were between 2 and 9 months, and all animal cases were confirmed after the same Pango lineages were identified in humans. Therefore, the SARS-CoV-2 viruses that circulated in South Korea are believed to have shared hosts, and the results provide additional evidence for the theory that most animal infections of SARS-CoV-2, especially in cats and dogs, are the spillover from humans. We have confirmed that most of the defining mutations of each lineage exist in each genome. Further study is needed to identify any specific mutations that show species-specific characteristics.
Among the eight lineages detected in cats or dogs in this study, four lineages (B.

Conclusions
We identified various SARS-CoV-2 genomic lineages in cats and dogs. Among  Table S1: SARS-CoV-2 positive animals which samples were provided to APQA from Provincial/City government veterinary authorities for variants monitoring; Table S2: Results of Illumina sequencing of SARS-CoV-2 in dogs and cats; Table S3: 420 Korean human SARS-CoV-2 sequences used in the phylogenetic analysis.