key: cord-0712464-fxs2eu6t authors: Medkour, Hacène; Catheland, Sébastien; Boucraut‐Baralon, Corine; Laidoudi, Younes; Sereme, Youssouf; Pingret, Jean‐Luc; Million, Matthieu; Houhamdi, Linda; Levasseur, Anthony; Cabassu, Julien; Davoust, Bernard title: First evidence of human‐to‐dog transmission of SARS‐CoV‐2 B.1.160 variant in France date: 2021-11-08 journal: Transbound Emerg Dis DOI: 10.1111/tbed.14359 sha: 539fc976dacad71ade49e2e11891589637da627d doc_id: 712464 cord_uid: fxs2eu6t Since the start of the coronavirus disease of 2019 (COVID‐19) pandemic, several episodes of human‐to‐animal severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) transmission have been described in different countries. The role of pets, especially domestic dogs, in the COVID‐19 epidemiology is highly questionable and needs further investigation. In this study, we report a case of COVID‐19 in a French dog living in close contact with its owners who were COVID‐19 patients. The dog presented rhinitis and was sampled 1 week after its owners (a man and a woman) were tested positive for COVID‐19. The nasal swabs for the dog tested remained positive for SARS‐CoV‐2 by reverse transcription quantitative real‐time PCR (RT‐qPCR) 1 month following the first diagnosis. Specific anti‐SARS‐CoV‐2 antibodies were detectable 12 days after the first diagnosis and persisted for at least 5 months as tested using enzyme‐linked immunoassay (ELISA) and automated western blotting. The whole‐genome sequences from the dog and its owners were 99%–100% identical (with the man and the woman's sequences, respectively) and matched the B.1.160 variant of concern (Marseille‐4 variant), the most widespread in France at the time the dog was infected. This study documents the first detection of B.1.160 in pets (a dog) in France, and the first canine genome recovery of the B.1.160 variant of global concern. Moreover, given the enhanced infectivity and transmissibility of the Marseille‐4 variant for humans, this case also highlights the risk that pets may potentially play a significant role in SARS‐CoV‐2 outbreaks and may transmit the infection to humans. We have evidence of human‐to‐dog transmission of the Marseille‐4 variant since the owners were first to be infected. Finally, owners and veterinarians must be vigilent for canine COVID‐19 when dogs are presented with respiratory clinical signs. Since its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [the aetiological agent of coronavirus disease of 2019 ] has spread across the entire planet and caused a pandemic resulting, at the end of August 2021, in more than 216 million cases and 4.5 million deaths [www.worldometers.info/ coronavirus]. While its origins remain unclear, it is accepted that it emerged from an animal reservoir, possibly bats (Zhou et al., 2020) , and may have involved an intermediate host such as an animal in which the virus accumulated mutations making it more suitable for transmission to humans (T. Zhang et al., 2020) . Coronaviruses are widespread in animals (including birds, pigs, ruminants, dogs and cats) (Alluwaimi et al., 2020) . Experimentally, researchers have shown that the SARS-CoV-2 multiplies easily in ferrets and hamsters, and several animal species may be susceptible to the infection, including Rousettus aegyptiacus (fruit bats), pangolins, felines, mink, dogs and rabbits (do Vale et al., 2021) . Other species susceptible to infection are likely to be discovered. In addition, genetic variants of SARS-CoV-2 have been emerging and circulating around the world throughout the COVID-19 pandemic (Centers for Disease Control & Prevention, 2021) . Some of these variants, however, have caught scientists off guard by suddenly moving in an unexpected direction. Is this due to failures in virus surveillance or does it mean that SARS-CoV-2 is able to circulate unnoticed in an animal reservoir before returning to humans? In any case, the circulation of this pathogen must be carefully examined, and it is important to understand the role that animals play in the epidemiology of the disease . During the recent years, several episodes of human-to-animal transmission have been described in different countries. These episodes were mainly related to cats and also to other species, such as dogs and minks (Decaro, Vaccari, et al., 2021; Oude Munnink et al., 2021) . Moreover, several cases of SARS-CoV-2 infection have also been reported worldwide in domestic pets (especially cats and dogs), and it has been suggested that these animals became infected by their owners or handlers. Infections of domestic pets mostly result in no to mild digestive and respiratory symptoms (Klaus et al, 2021; Sit et al., 2020 Laidoudi et al., 2021; Temmam et al., 2020) , which reported prevalence from 0% to 15.4%, in line with the incidence of SARS-CoV-2 in humans. The possibility of SARS-CoV-2 transmission between humans and animals, especially pets, remains unknown for some variants. In this study, we report a case of SARS-CoV-2 infection in a dog in France after being infected by their owners, with evidence of human-to-dog transmission. We also conducted whole-genome characterization of viruses from the dog and its owners. Million et al., 2020) , and they were administered these treatments in association with zinc for the man and sodic enoxaparin (due to the concentration of D-dimers < 0.27 µg/ml) for the woman. The family owns a dog. The dog is a West Highland White Terrier female of 13-year-old, which suffered from a respiratory distress. On 13 November 2020, the dog was taken to a veterinary clinic by his owner. A nasal swab and blood samples were performed and sent to Scanelis, a veterinary test laboratory (Colomiers, France). First, nucleic acids were extracted and purified from nasal swabs collected at different times by a silica-based method (Nucleospin RNA Virus, Macherey Nagel). The final elution volume was 100 µl and 2.5 µl aliquots were used for the RT-PCR assays. Duplicates of nucleic acid samples were analyzed in 384 wells plates (10 µl final PCR volume). The efficiency of the purification step and the quality of the nucleic acids were controlled, with two exogenous external controls (synthetic RNA and DNA spiked in the lysis buffer) systematically added to the specimen and co-purified with it. These controls were detected by RT-qPCR or PCR. A control sample (nuclease free water) was systematically included in each purification series (23 specimens maximum) and was used as a process negative control. Several positive standards were included in order to check the limit of detection in each series and to calculate the loads. Analyses were performed on the dog samples for canine pathogens implicated in canine cough or rhinitis (Bordetella bronchiseptica, canine distemper virus, respiratory canine adenovirus type 2, canine herpes virus and canine parainfluenza virus) using Scanelis qPCR or PCR assays (http://www.scanelis.com) and, finally, SARS-CoV-2 was tested. The SARS-CoV-2 test was performed using the SARS-CoV-2 Scanelis test adapted from RT-qPCR designed by the Centers for Disease Control and Prevention, USA to obtain a performant multiplex RT-qPCR assay. The primers and probes were as follows: 2019-nCoV_N1- in duplicate. The mean of the duplicate results was calculated for quantitative results. The detection limit (95%) of the assay was determined in probit analysis as seven copies equivalent viral genome per reaction and the limit of quantification as 10 copies equivalent genome per analysis in duplicate. The linearity range of the multiplex assay is 10-10 7 copies equivalent genome per analysis. The dog was followed up, examined and sampled, by nasal swabs and/or blood, 12, 19 and 28 days later. The virus concentrations were measured at these stages using the RT-qPCR test cited above. All the RNA extracts (days 0, +12, +19 and +28) were sent to the IHU laboratory for further analysis. For further analysis, dog sera were collected at days +12 and +19 and +146 after the first consultation. The sera were tested using enzyme-linked immunoassay (ELISA) and automated western blotting (WB) assays as recently described (Edouard, Jaafar, et al., 2021; . For ELISA, we used ID Screen SARS-CoV-2 Double Antigen Multi-species (Innovative Diagnostics, Grabels, France) following the manufacturer's instructions. The test targets multispecies (i.e. minks, ferrets, cats, dogs, cat-tle, sheep, goats, horses and all other receptive species) antibodies directed against the major nucleocapsid protein of SARS-CoV-2. Plates were sensitized with a purified recombinant N antigen. Optical density (OD) was measured at 450 nm using Multiskan GO software (Thermo Scientific, Waltham, MA, USA). The test was validated when the optical density of positive control (OD PC ) was ≥0.35 and a mean ratio of positive (OD PC ) and negative (OD NC ) control was higher than three. The optical density of each sample (OD N ) was used to calculate the sample to positive (S/P) ratio (expressed as a %) where S/P = 100 × (OD N − OD NC )/ (OD PC − OD NC ). When the S/P score was lower than 50% by ELISA, samples were considered negative. They were considered as positive when it was higher than 60% and doubtful when 50 < P/S score <60%. For WB, the strain SARS-CoV-2 IHUMI2 (lineage 20a) was used to produce SARS-CoV-2 antigens, as previously described (Edouard, Jaafar, et al., 2021) . The Jess Simple Western automated nano- The genomic consensus sequences were generated through mapping on the SARS-CoV-2 genome using the sequence from the Wuhan-Hu-1 reference strain (GenBank accession no: NC_045512.2) with Minimap2 software (Li, 2018) . Then, Samtools software was used to allow both soft-clipping PCR primers and removal PCR duplicates, and Freebayes software was used to detect the variant mutants with a minimum mapping quality of 20. The genomic study was performed for all obtained sequences and hallmarks of mutations were retrieved to identify the variant. Phylogenetic reconstruction was performed using the IQ-TREE software with the GTR Model and 1000 ultrafast bootstrap repetitions after alignment of genomes using MAFFT v.7 . The tree was visualized with the iTOL (Interactive Tree Of Life) software as previously described (Katoh & Standley, 2013 ). The main clinical sign observed on the dog was rhinitis (a severe bout of acute rhinitis). Except for SARS-CoV-2, all the other tests for canine F I G U R E 1 Results of the automated western blotting assay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the dog from France at 12, 19 and 146 days following the presentation of the clinical sign (rhinitis) pathogens implicated in canine cough or rhinitis were negative. The dog was positive for SARS-CoV-2 with a viral load of 16,000 SARS-CoV-2 copy genome equivalent per 1 µl extracted RNA (3.19E+06 copies of viral RNA per swab) using RT-qPCR (Table 1) . This viral load is equivalent to viral loads usually detected in humans during the symptomatic phase of the disease . The virus concentrations detected at 12, 19 and 28 days later to the first test were 95, 119 and <4 SARS-CoV-2 copy genome equivalent per 1 µl of extracted RNA, respectively, using the same RT-qPCR. No clinical signs were observed during these consultations (Table 1) . For ELISA, when the S/P score was lower than 50%, samples were considered negative. They were considered positive when it was higher than 60% and doubtful when it ranged from 50% to 60%. All dog sera were tested positive (Table 1) . Simultaneously, the entire dog's sera were tested positive by WB (Table 1) (Figure 1) . Phylogeny tree of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. This tree includes one representative of each variant, eight samples of Marseille-4 and all the other samples of this study. All sequences are available through GISAID Virus isolation was only positive from the man owner sample, and it was confirmed by both specific RT-qPCR and whole genome sequencing. Although 100% of whole genome was covered from both nasopharyngeal swab and culture isolate of the man owner, only 83.4% and 51.93% were covered from the first collected dog sample (corresponds to the clinical phase) and woman owner sample, respectively. Regarding the genomic study, all samples were identified as vari- Within the Marseille-4 clades, all samples (woman, man and dog) were closely related and belonged to the same cluster. Both results (genotyping and phylogeny) suggested a human-to-dog transmission since the owner (the woman) was the first to be infected. More than a year after the COVID-19 pandemic began, it is accepted today that pets are not involved in the transmission of SARS-CoV-2 to humans (Decaro, Balboni, et al., 2021) . Cats are more susceptible than dogs and cat-to-cat SARS-CoV-2 transmission has been demonstrated (Halfmann et al., 2020; Hosie et al., 2021) . In France, in April 2020, a cat, whose owner had been infected with the virus 17 days prior, presented clinical signs with anorexia, vomiting and coughing (Sailleau et al., 2020) . Eight days after the onset of symptoms, molecular analysis (SARS-CoV-2 qPCR) showed that the nasopharyngeal swab was negative, but the rectal swab was positive (Ct: 29). PCRs were negative 28 days after the appearance of the clinical signs. Sequencing was performed and showed that SARS-CoV-2 from the cat was comparable to that circulating in humans at the same time. The genome shows the D614G amino acid mutation in the spike glycoprotein, specific to the A2 clade. This confirmed feline case is the first reported in France. Cases of SARS-CoV-2 infection in dogs are considered very rare (86 described worldwide compared to more than 216 million cases reported in human). To date, dog-to-dog SARS-CoV-2 transmission (e.g. in a family home or kennel) and dog-to-human transmission have not been demonstrated . Dogs are therefore assumed to be infected by their owners. This has been well described, particularly, in Italy and Brazil in large population surveys of canines living in households and in contact with COVID-19 infected or uninfected people (Calvet et al., 2021; Patterson et al., 2020) . The SARS-CoV-2 infected dogs have very few clinical signs, most of which are respiratory (Michael et al., 2021) . In Texas, as part of a longitudinal household transmission study of pets living with persons with COVID-19, two pets were confirmed to be infected with the SARS-CoV-2 B.1.1.7 variant of concern (VOC). The pets were a dog and a cat from the same household, sampled two days after their owner was tested positive for COVID-19. The oral, nasal and fur swabs for both pets were tested positive for SARS-CoV-2 by qRT-PCR, and consensus wholegenome sequences from the dog and cat were 100% identical and matched the B.1.1.7 VOC. Sneezing by both pets was noted by the owner in the weeks between initial and follow-up testing (Hamer et al., 2021) . Serological investigations show that dogs in contact with the virus produce antibodies without having any symptoms . In the laboratory, of five dogs inoculated experimentally by the intranasal route, only one had a positive PCR (rectal swab) 6 days post infection . In another study, virus shedding was not demonstrated after the experimental infection of three dogs (Bosco-Lauth et al., 2020) . In the canine case we studied, the first viral load was high when the dog was symptomatic and the viral RNA-carriage period was longer than in the French cat case because the dog was still positive (by qPCR) 28 days after the first positive diagnosis. However, no virus culture was performed on the dog samples, so we cannot confirm that the dog was infectious or even contagious for 1 month. It is impor- epidemiology. In fact, the virus replicates poorly in dogs, particularly due to the fact that they have few ACE2-carrying cells in the respiratory tracts Zhai et al., 2020) . A new 20A variant emerged in June 2020 in agricultural workers in northeast Spain and France in July 2020 (Hodcroft et al., 2020) . In Mar- . This variant could have originated from SARS-CoV-2 passing through an animal, possibly a mink Fenollar et al., 2021) . Based on previous serological studies from France, it was also concluded that transmission from humans to pets (including dogs) was most likely Laidoudi et al., 2021) , but these studies could not confirm on the transmission between humans and animals. In this study, genotyping and phylogeny results both highlight virus transmission between humans and dogs, with evidence of human-to-dog transmission since the woman owner was the first to be infected. We describe here the first confirmed case of COVID-19 in a dog in France. Transmission of the virus from human to dog is therefore possible (Leroy et al., 2020 The authors declare no conflict of interest. All the human data have been generated as part of the routine work at Assistance Publique-Hôpitaux de Marseille (Marseille university hospitals), and this study results from routine standard clinical management. This study has been approved by the ethics committee of our institution (No. 2020-029). Access to the patients' biological and registry data issued from the hospital information system was approved by the data protection committee of Assistance Publique-Hôpitaux de Marseille (APHM) and was recorded in the European General Data Protection Regulation registry under number RGPD/APHM 2019-73. All applicable international and national guidelines for the care of dogs were followed. The owners of the dog gave their consent for the samples to be taken by a veterinarian. 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