Near‐complete genome of SARS‐CoV‐2 Delta variant of concern identified in a symptomatic dog (Canis lupus familiaris) in Botswana

Abstract We sought to investigate whether SARS‐CoV‐2 was present, and to perform full‐length genomic sequencing, in a 5‐year‐old male crossbreed dog from Gaborone, Botswana that presented overt clinical signs (flu‐like symptoms, dry hacking cough and mild dyspnoea). It was only sampled a posteriori, because three adult owners were diagnosed with SARS‐CoV‐2 infection. Next‐generation sequencing based on Oxford Nanopore Technology (ONT) was performed on amplicons that were generated using a reverse transcriptase real‐time polymerase chain reaction (RT‐qPCR) of confirmed positive SARS‐CoV‐2 nasopharyngeal and buccal swabs, as well as a bronchoalveolar lavage with mean real cycle threshold (qCt) value of 36 based on the Nucleocapsid (N) gene. Descriptive comparisons to known sequences in Botswana and internationally were made using mutation profiling analysis and phylogenetic inferences. Human samples were not available. A near‐full length SARS‐CoV‐2 genome (∼90% coverage) was successfully genotyped and classified under clade 20 O and Pango‐Lineage AY.43 (Pango v.4.0.6 PLEARN‐v1.3; 2022‐04‐21), which is a sublineage of the Delta variant of concern (VOC) (formerly called B.1.617.2, first detected in India). We did not identify novel mutations that may be used to distinguish SARS‐CoV‐2 isolates from the dog and humans. In addition to Spike (S) region mutation profiling, we performed phylogenetic analysis including 30 Delta sequences publicly available reference also isolated from dogs. In addition, we performed another exploratory analysis to investigate the phylogenetic relatedness of sequence isolated from dog with those from humans in Botswana (n = 1303) as of 31 March 2022 and of same sublineage. Expectedly, the sequence formed a cluster with Delta sublineages – AY.43, AY.116 and B.1.617.2 – circulating in same time frame. This is the first documented report of human‐associated SARS‐CoV‐2 infection in a dog in Botswana. Although the direction of transmission remains unknown, this study further affirms the need for monitoring pets during different COVID‐19 waves for possible clinically relevant SARS‐CoV‐2 transmissions between species.

Next-generation sequencing based on Oxford Nanopore Technology (ONT) was performed on amplicons that were generated using a reverse transcriptase real-time polymerase chain reaction (RT-qPCR) of confirmed positive SARS-CoV-2 nasopharyngeal and buccal swabs, as well as a bronchoalveolar lavage with mean real cycle threshold (qCt) value of 36 based on the Nucleocapsid (N) gene. Descriptive comparisons to known sequences in Botswana and internationally were made using mutation profiling analysis and phylogenetic inferences. Human samples were not available.
A near-full length SARS-CoV-2 genome (∼90% coverage) was successfully genotyped and classified under clade 20 O and Pango-Lineage AY.43 (Pango v.4.0.6 PLEARN-v1.3;2022-04-21), which is a sublineage of the Delta variant of concern (VOC) (formerly called B.1.617.2, first detected in India). We did not identify novel mutations that may be used to distinguish SARS-CoV-2 isolates from the dog and humans. In addition to Spike (S) region mutation profiling, we performed phylogenetic analysis including 30 Delta sequences publicly available reference also isolated from dogs. In addition, we performed another exploratory analysis to investigate the phylogenetic relatedness of sequence isolated from dog with those from humans in Botswana (n = 1303) as of 31 March 2022 and of same sublineage. Expectedly, the sequence formed a cluster with Delta sublineages -AY.43, AY.116 and B.1.617.2 -circulating in same time frame. This is the first documented report of human-associated SARS-CoV-2 infection in a dog in Botswana. Although the direction of transmission remains unknown, this study further affirms the need for monitoring pets during different COVID-19 waves for possible clinically relevant SARS-CoV-2 transmissions between species.

INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic virus under genus betacoronavirus (β-CoV) and the major aetiology of a pathological respiratory infection common in humans called coronavirus disease 2019 (COVID-19) (Li et al., 2020). SARS-CoV-2 is postulated to have originated from either horseshoe bats (Rhinolophus affinis) or Malayan pangolins (Manis javanica), in China in 2019 (Andersen et al., 2020;Barua et al., 2021;Seyran et al., 2021;Sreenivasan et al., 2021). Strong support for this zoonotic origin is the fact that bat betacoronavirus was linked to the 2002−2003 SARS epidemic (SARS-CoV), which was thought to be transmitted to humans either directly or through an intermediate host that is yet to be identified (Zhou et al., 2020;Zhou et al., 2020). Prior to the discovery of SARS-CoV-2, pets such as dogs could be infected with an Alphacoronavirus (α-CoV), canine enteric coronavirus (CCoV), known to have mild symptoms and are not life-threatening field (Decaro et al., 2021).

Recent evidence shows that anthroponotic infections of SARS-
CoV-2 between animals and humans exists (Sit et al., 2020). The infection in dogs and cats is biologically feasible since they share the same cellular receptor, the angiotensin-converting enzyme type 2 (ACE2) (Decaro & Lorusso, 2020;Decaro et al., 2021). Even though, dogs have little susceptibility to SARS-CoV-2, and the virus replicates poorly whereas cats and ferrets are more permissive to infection . Often, dogs that get infected with SARS-CoV-2 become asymptomatic with low viral ribonucleic acid (RNA) levels unless the RNA is isolated from nasal, oropharyngeal swabs and rectal swabs within 6 to 9 days post-infection field (Barua et al., 2021;Shi et al., 2020;Sit et al., 2020). SARS-CoV-2 infection has also been reported in wild animals including minks, tigers, lions, and ferrets (Dalton et al., 2022;Decaro et al., 2021;Delahay et al., 2021;Mishra et al., 2021;Sharun et al., 2021). Reservoirs for SARS-CoV-2 including immunocompromised humans and animals are postulated to play a role in spurring the mutations of the virus that potentially leads to new variants of concern in the evolving COVID-19 pandemic; and different disease attributes (Otto et al., 2021;Shi et al., 2020). SARS-CoV-2 has been demonstrated to infect pets and most studies are not from African continent (Molini et al., 2022).
Although COVID-19 has been declared a notifiable disease by the World Animal Health Organisation (OIE), it has not been stated as a notifiable disease in Botswana for animals. Due to this fact, there are no clear guidelines to assess nor manage animals infected with SARS-CoV-2 including pets, and on the management of those in close contact with infected humans. However, there has been lobbying by the Department of Veterinary Services (DVS) to other key stakeholders for the adoption of a One-health approach to assessing human cases of COVID-19, especially especially when wild or domestic animal-human close contact happens (Gibbs, 2014;WHO, O. 2017). Here we report the first case of SARS-CoV-2 in a dog from owners previously confirmed of being infected by SARS-CoV-2. The dog tested positive for SARS-CoV-2 reverse transcriptase real-time polymerase chain reaction (RT-qPCR) in nasopharyngeal swabs. The SARS-CoV-2 genome has been almost completely sequenced and clustered with the sequences that have been circulating among humans, especially from the dates of the dog case.

Sample collection
This

DNA extraction and RT-PCR
Nucleic acid extraction was carried out using a Nucleic Acid Extrac-

Tiling PCR, next-generation sequencing and sequence analysis
Residual RNA from RT-qPCR testing were retrieved for tiling PCR using Superscript IV (Invitrogen, Marseille, France) and the hex-

Sequence sorting and phylogenetic analysis
The downloaded FASTA files were analysed using NextClade to assess the coverage, lineage and quality. For reference sequences, we included all the near-full length Delta VOC sequences from GISAD (n = 31 including from Botswana) at the time of the manuscript. Since there were few sequences, no further sampling strategies. The sequences were aligned using NextAlign, and the ensuing multiple sequence alignment (MSA) was used to construct phylogenetic tree. NC_044512 was used as a reference sequence. The tree topology was inferred by performing maximum likelihood (ML) analyses, and the bootstrap values were set at 1000, and the ModelTest v.3.7 (Posada, 2006) was used to select the simplest evolutionary model that adequately fit the sequence data. ML tree were implemented using FastTree (Price et al., 2009). Seaview tool was used to assess the architectures of the produced trees. The tree was visualised in FigTree v1.4.3 and posterior probabilities above 0.80 were noted as statistically significant.

F I G U R E 1
Full genome representation of a Delta SARS-CoV-2 sequence isolated from a dog, defining mutations with those in the Spike (green). Figure generated by covdb.stanford.edu.

RESULTS
In this report, we give details of the first detection, isolation and

DISCUSSIONS
To our knowledge, no previous SARS-CoV-2 RNA detection from a dog has been reported in Botswana or Southern Africa, and this is the first near-full length nonhuman sequence to be generated from Africa. By the time of the manuscript, there were additional partial SARS-CoV-2 sequences from dogs from Egypt that were deposited in GISAID. This presence of SARS-CoV-2 RNA in dogs of this study has been described in dogs elsewhere (Sit et al., 2020) and the occurrence of SARS-CoV-2 has been linked to a number of factors, including the owner's contact (Jairak et al., 2022). The dog's owners sought veterinary care after noticing overt clinical indicators (flu-like symptoms and weight loss), and the animal's COVID-19 status was confirmed.
Despite being unable to extract a human sequence, the discovered strain -AY.94, a sublineage SARS-CoV-2 Delta VOC (B.1.617.2) -was the most common, adding to the growing body of evidence for human to pet transmission. This is not the first study to report Delta vari- given evidence of relevant pet-to-human or pet-to-pet transmission were SARS-CoV-2 transmission dynamics in animal have been extensively investigate (Cui et al., 2022). Although we confirmed a positive SARS-CoV-2 infection in dog, signs were milder compared to what we have observed in symptomatic humans during the same epidemic wave.
This corroborates previous studies showing pet infections as mostly asymptomatic or self-resolving (Hamer et al., 2020).
We profiled the mutations in the Spike and all mutations agreed with those of Delta variant Delta variant of concern (B. for Innovation in Diagnostics, Grant/Award Number: CV21-0110; International Atomic Energy Agency, Grant/Award Number: INT0098

DATA AVAILABILITY STATEMENT
No.

ETHICS STATEMENT
Verbal consent was obtained from all dog owners after explaining the objectives and benefits of detecting and sequencing the viruses from the pet. Sequencing of SARS-CoV-2 is approved by the Health Research and Development Committee (HRDC).