Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a zoonotic virus believed to have originated from bats or pangolins. While primarily affecting humans, SARS-CoV-2 can infect other animals, including dogs and cats, who share the ACE2 receptor. Previous studies have shown that canine infection is often asymptomatic or mild, with low viral RNA levels unless samples are taken early post-infection. This is biologically feasible since dogs share the same cellular receptor, the angiotensin-converting enzyme type 2 (ACE2), as humans, but they exhibit comparatively little susceptibility to infection and poor viral replication. Infection has been documented in various other animals, including minks, tigers, lions, and ferrets, highlighting its zoonotic potential. Although COVID-19 is a notifiable disease for humans, it lacks this status for animals in Botswana. This lack of guidelines prompted the study of this case, examining the first reported infection of a dog with SARS-CoV-2 in Botswana.

The literature extensively documents SARS-CoV-2 infection in various animal species, primarily focusing on its zoonotic potential and the possibility of spillover events from humans to animals. Studies have highlighted the role of ACE2 receptors in facilitating infection, with varying susceptibility among different species. Dogs have shown a generally low susceptibility, often exhibiting asymptomatic or mild infections. The Delta variant, initially identified in India, has spread globally, raising concerns about its potential for zoonotic transmission. Several studies have reported SARS-CoV-2 infections in dogs worldwide, demonstrating the virus's capability to cross species barriers. The lack of similar reports from the African continent emphasized the need for this study and investigation. The review of existing literature also highlights the need for One Health approaches to manage such instances.

A 5-year-old male crossbreed dog exhibiting clinical signs consistent with COVID-19 (flu-like symptoms, dry cough, dyspnea) and living with three SARS-CoV-2-infected human owners was sampled. Nasopharyngeal and buccal swabs, along with bronchoalveolar lavage, were collected and tested for SARS-CoV-2 using RT-qPCR. The samples had a mean qCt value of 36 based on the Nucleocapsid (N) gene. Positive samples underwent next-generation sequencing using Oxford Nanopore Technology (ONT) on amplicons generated through tiling PCR. Sequence analysis was performed using Genome detective, NextClade, and NextAlign. Phylogenetic analysis involved maximum likelihood (ML) analysis using FastTree and ModelTest v.3.7, comparing the dog's sequence to publicly available Delta variant sequences from GISAID, including those from Botswana and other isolates from dogs internationally (n=30). Additionally, an exploratory analysis compared the dog's sequence to 1303 human sequences from Botswana of the same sublineage to determine the phylogenetic relatedness. The resulting phylogenetic tree was visualized using FigTree, with statistically significant posterior probabilities above 0.80 noted.

A near-complete SARS-CoV-2 genome (∼90% coverage) was successfully sequenced from the symptomatic dog. The genome was classified under clade 20O and Pango-lineage AY.43 (a sublineage of the Delta variant). No novel mutations distinguishing the dog's isolate from human isolates were identified. Mutation profiling revealed several mutations consistent with the Delta variant across Spike, Membrane (N), Nucleocapsid (N), and other regions. Phylogenetic analysis showed that the dog's SARS-CoV-2 sequence clustered with Delta sublineages (AY.43, AY.116, and B.1.617.2) circulating during the same timeframe. The analysis comparing the dog's sequence to human sequences from Botswana confirmed its phylogenetic relationship with human Delta sublineages. This is the first reported case of human-associated SARS-CoV-2 infection in a dog in Botswana and one of the first near-full-length nonhuman sequences generated from Africa.

This study provides the first near-complete genome sequencing of SARS-CoV-2 from a dog in Botswana, confirming Delta variant infection. Although the exact transmission route remains unclear (human-to-dog or environmental), the close phylogenetic relationship to human Delta sequences suggests zoonotic transmission. While the dog exhibited milder symptoms than observed in humans with the same variant, the findings highlight the potential for SARS-CoV-2 transmission between humans and pets. The study’s limitations include the unavailability of human sequences from the owners (diagnosed using rapid antigen tests), precluding direct comparison. The dog's symptoms and the temporal relationship with the owners’ infections strongly suggest a zoonotic event. Future research should focus on detailed transmission studies and expand surveillance programs to include companion animals during COVID-19 outbreaks.

This research presents the first reported near-complete genome sequence of SARS-CoV-2 from a dog in Botswana and Africa. It confirms the presence of the Delta variant in a symptomatic canine, highlighting the zoonotic potential of this virus. The study underscores the need for increased monitoring of companion animals during COVID-19 waves, adopting a One Health approach. Further research is required to understand transmission dynamics and the potential role of animals in SARS-CoV-2 evolution and spread.

The main limitation of this study was the unavailability of human SARS-CoV-2 sequences from the dog's owners due to their initial diagnosis with rapid antigen tests. This prevented a direct comparison of viral sequences to confirm the direction of transmission. The absence of additional symptomatic dogs in the vicinity limited the ability to assess the extent of potential transmission within the canine population. The study was based on a single case, which limits the generalizability of the findings. Further research with larger sample sizes and more complete data would strengthen conclusions.