Detection of SARS-CoV-2 in a dog with hemorrhagic diarrhea

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA.

Case presentation: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog's acute hemorrhagic diarrhea.

Conclusion: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog's pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.

Keywords: B.1.177; Dog COVID-19; Ile402Val S protein substitution; One Health; SARS-CoV-2; Zoonosis.

Fig. 1

Alignment of the partial S gene sequences. The sequences shown are, from top to bottom, the following ones: consensus early Wuhan (GenBank ID: NC_045512.2); present dog fecal sequence (in bold; GenBank and GISAID IDs: MW872017 and EPI_ISL_1490671, respectively); the positive control used (GISAID ID: EPI_ISL_433320); the variants indicated with their trivial and canonic designations (Alpha, GISAID ID: EPI_ISL_581117; Beta, GISAID ID: EPI_ISL_660605; Gamma, GISAID ID: EPI_ISL_792680). Bases exhibiting complete conservation are marked at the bottom by asterisks. The base deviations from the Wuhan sequence are green-shadowed and squared. The alignment was performed using Bio-Edit [23]

Fig. 2

SARS-CoV-2 genomic coverage obtained by Next-generation sequencing (NGS). The horizontal axis corresponds to the position of the SARS-CoV-2 consensus genome and the vertical one to the depth of the sequence (DS) at a given position. On the top, aligned over the nucleotide positions, the bar shows a linear diagram of the entire genome, showing the span of the ORF1ab, spike (S) and nucleocapsid (N) genes as well as the region amplified of the S gene that was subjected to Sanger sequencing (in black, labeled “partial S sequence”)

Fig. 3

Phylogenetic analysis. Maximum Likelihood Tree based on the complete SARS-CoV-2 genome. The analysis was carried out as reported [7] and is detailed in Supplementary Methods. The evolutionary history was inferred by the Neighbor-Joining method. The optimal tree is shown. The percentage of trees in which the associated taxa clustered together is shown next to branches. The tree is drawn to scale, with branch lengths proportional to the number of substitutions per site. This analysis involved 34 nucleotide sequences. There were a total of 32,122 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [26]. The name of each sequence is composed of four elements separated by vertical lines: the host where the virus was isolated; the virus strain name (for the SARS-CoV-2 sequences isolated from animals other than humans, countries where they were found are shown in brackets); the specific SARS-CoV-2 variant that they belonged to; and the GenBank or GISAID Accession ID. Bold names indicate sequences belonging to the B.1.177 variant. The black dot (●) marks the present dog SARS-CoV-2 sequence

Fig. 4

Comparison of fecal abundance of main bacterial phyla in the present dog with the abundances reported [22] in dogs with acute diarrhea and inflammatory bowel disease. For details on microbiome monitoring, see the text and the Supplementary Methods. Data from our dog stool are shown in red. All other results are from [22]. NHD, acute non-hemorrhagic diarrhea (n = 12); AHD, acute hemorrhagic diarrhea (n = 13); IBD, inflammatory bowel disease (active disease, n = 9; therapeutically controlled and thus clinically insignificant, n = 10). The symbols give the median for the percent abundance in each dog, and the whiskers give the range