BRIEF RESEARCH REPORT published: 31 March 2022 doi: 10.3389/fvets.2022.826991 The Finding of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) in a Wild Eurasian River Otter (Lutra lutra) Highlights the Need for Viral Surveillance in Wild Mustelids Miguel Padilla-Blanco 1† , Jordi Aguiló-Gisbert 2† , Vicente Rubio 3 , Víctor Lizana 2,4 , Eva Chillida-Martínez 2 , Jesús Cardells 2,4 , Elisa Maiques 5* and Consuelo Rubio-Guerri 1* Edited by: 1 Department of Pharmacy, Facultad de Ciencias de la Salud, Universidad Cardenal Herrera-CEU, Valencia, Spain, 2 Servicio Levon Abrahamyan, de Análisis, Investigación, Gestión de Animales Silvestres (SAIGAS), Facultad de Veterinaria, Universidad Cardenal Université de Montréal, Canada Herrera-CEU, Valencia, Spain, 3 Instituto de Biomedicina de Valencia del Consejo Superior de Investigaciones Científicas, Reviewed by: Centro de Investigació Biomédica en la Red sobre Enfermedades Raras, Instituto de Salud Carlos III, Valencia, Spain, 4 Martin Beer, Wildlife Ecology & Health Group (WE&H), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 5 Department of Friedrich Loeffler Institute, Germany Biomedical Sciences, Facultad de Ciencias de la Salud, Universidad Cardenal Herrera-CEU, Valencia, Spain Stefania Lauzi, University of Milan, Italy Animals have been involved in the three known outbreaks of severe respiratory *Correspondence: Elisa Maiques syndromes due to coronaviruses (years 2005, 2012, and 2019). The pandemic nature emaiques@uchceu.es of the SARS-CoV-2 outbreak increases the likelihood of infection from humans of Consuelo Rubio-Guerri susceptible animal species that, thus, could become secondary viral hosts and even consuelo.rubio@uchceu.es disease reservoirs. We present evidence of spillover infection of wild mustelids by † These authors have contributed reporting the presence of SARS-CoV-2 in a Eurasian river otter found near a water equally to this work reservoir in the Valencian Community (Spain). We detected the virus using two different Specialty section: commercial RTqPCR assays on RNA extracted from the nasopharynx (swabbing) This article was submitted to and from lung tissue and mediastinal lymph node homogenates. The corresponding Veterinary Infectious Diseases, a section of the journal samples from two additional otters from distant sites tested negative in identical Frontiers in Veterinary Science assays. The diagnosis in the positive otter was confirmed by two-tube RT-PCR assay Received: 01 December 2021 in which RNA was first retrotranscribed, and then specific regions of the spike (S), Accepted: 14 February 2022 nucleocapsid (N), and ORF10 genes were separately amplified from the produced Published: 31 March 2022 cDNA, followed by electrophoretic visualization and Sanger sequencing. The sequences Citation: Padilla-Blanco M, Aguiló-Gisbert J, of the amplified products revealed some non-synonymous changes in the N and Rubio V, Lizana V, Chillida-Martínez E, ORF10 partial sequences, relative to the consensus sequence. These changes, identified Cardells J, Maiques E and Rubio-Guerri C (2022) The Finding of already in human patient samples, point to human origin of the virus, although their the Severe Acute Respiratory specific combination was unique. These findings, together with our previous report Syndrome Coronavirus (SARS-CoV-2) of SARS-CoV-2 infection of feral American mink, highlight the need for SARS-CoV-2 in a Wild Eurasian River Otter (Lutra lutra) Highlights the Need for Viral surveillance of wild or feral mustelids to evaluate the risk that these animals could become Surveillance in Wild Mustelids. SARS-CoV-2 reservoirs. Front. Vet. Sci. 9:826991. doi: 10.3389/fvets.2022.826991 Keywords: SARS-CoV-2, RT-PCR, Lutra lutra, wildlife, Spain Frontiers in Veterinary Science | www.frontiersin.org 1 March 2022 | Volume 9 | Article 826991 Padilla-Blanco et al. SARS-CoV-2 in a Wild Otter INTRODUCTION (Aonyx cinereus) infection by SARS-CoV-2 in a North-American aquarium (Georgia, USA) (24). With the goal of contributing to A zoonotic origin of the severe acute respiratory syndrome the knowledge of the extent of SARS-CoV-2 infection in wild or coronavirus 2 (SARS-CoV-2) (1) was suspected from the initial feral mustelids, we now report the presence of SARS CoV-2 in human cases (2), linked to the Huanan seafood market of Wuhan a wild Eurasian otter (Lutra lutra) in the Valencia province of (Hubai province, China), where many types of animals were Spain. We document the presence of SARS-CoV-2 in the airway, sold (1). SARS-CoV-2 was initially proposed (2, 3) to have the lungs, and a mediastinal lymph node of this animal. Our evolved from RaTG13, a coronavirus sampled in 2013 in a bat findings extend to a second otter species the susceptibility to (Rhinolophus affinis) from China with which the Wuhan strain infection by SARS-CoV-2, and they support the need for viral of SARS-CoV-2 shared ∼96.1% genome sequence identity (3). monitoring in feral and wild mustelids including otters. However, as the receptor-binding domain (RBD) of the spike protein (S) is crucial for infection [due to its interaction with the angiotensin-converting enzyme 2, ACE2, that is, the cellular MATERIALS AND METHODS receptor for SARS-CoV-2 (4)], the limited (85%) sequence Sample Collection identity (5) of the RBDs of SARS-CoV-2 and RaTG13 led to The positive animal (from now on index case) was an adult suspect the existence of an intermediate host. Previously, the male Eurasian river otter (Lutra lutra). The animal was found severe acute respiratory syndrome coronavirus (SARS-CoV) and on August 25, 2021, freshly road killed (but not smashed) the Middle East respiratory syndrome coronavirus (MERS-CoV) in a road near the Bellús reservoir of the Albaida river, in had been shown to have emerged in bats but to have evolved in the part belonging to the village of Beniganim (population, civets and camels, respectively, before causing human outbreaks 5,899 inhabitants; GPS coordinates, 30S 718216.80 4313010.69) in 2002 and 2013 (6, 7). For SARS-CoV-2, an initial proposal [Figure 1; (25)]. Since 2015, otters have recolonized this reservoir of the Malayan pangolin (Manis javanica) as an intermediate (26), found in a relatively heavily populated territory (110 host (based on the ∼97% sequence identity of the RBDs of the inhabitants per km2 ) in inland Valencia (a Mediterranean pangolin coronavirus with the Wuhan SARS-CoV-2) (8) lost Spanish province) (25). The dead animal was preserved frozen ground because of low (91%) whole-genome sequence identity (−20◦ C) until necropsy, performed 2 weeks later by a SARS- of these coronaviruses (5). A closer relative to the initial Wuhan CoV-2-negative (RTqPCR of nasopharyngeal samples) staff strain of SARS-CoV-2 may be BANAL-52, a coronavirus recently professional from a wildlife surveillance center. The otter found in cave bats from North Laos (near China), which exhibits presented occipital fracture, splenic laceration with abdominal a whole-genome sequence identity with Wuhan SARS-CoV-2 hemorrhage, and heterogeneous lung discoloration assumed to similar to that of RaTG13 and a much higher identity (almost be also due to trauma. There were no noticeable macroscopic complete identity) in RBD sequence (9). alterations in the respiratory organs. A nasopharyngeal swab, Whichever the animal origin of SARS-CoV-2 is, animals are lung tissue, and a mediastinal lymph node were taken and also important in the context of the SARS-CoV-2 pandemic placed aseptically in plastic tubes containing Sample Preservation because some species could become secondary hosts of the virus Solution (reference number P042T0020100) from JiangSu following transmission from humans. This human-to-animal Mole Bioscience (Taizhou, China; sold in Spain by Palex transmission has been recorded for pets and captive or farmed Medical, Madrid, Spain). This proprietary commercial solution animals such as dogs (Canis lupus familiaris) (10), felines, such contains trisodium citrate dyhydrate, guanidine thiocyanate, as cats (Felis silvestris catus) (11), lions (Panthera leo), and tigers acetic acid, sodium acetate trihydrate and water buffer, and is (Panthera tigris) from zoos (12), and several species of mustelids used to inactivate the virus while preserving the viral RNA. (13–18). In experimental infections (19), pigs and chickens The hermetically sealed tubes, placed at −80◦ C <2 h after appeared non-susceptible, fruit bats became transiently infected, procurement, remained at this temperature until their use in and ferrets presented subclinical infection. The infection of SARS-CoV-2 analyses. wild animals is of concern, as it may escape monitoring, with Two additional river otters found at other locations of the the possibility of viral evolution and re-entry into the human Valencian Community were also tested. They were juvenile population from wild animal reservoirs. This is illustrated in females. Their dates and sites of collections were July 7 at the evolution of SARS-CoV-2 during the pandemic toward mice ′ ′′ ′ ′′ El Hondo, Elx (38◦ 10 55 N 0◦ 45 09 W) and August 2 at the infectiousness, leading to the hypothesis of a mice origin of the ′ ′′ ′ ′′ omicron variant (20). Thus, the One Health concept should also Mijares River, Almassora (39◦ 57 11.77 N, 0◦ 4 16.18 W). Their consider SARS-CoV-2 surveillance in wild animals. The present collection sides were >100 km apart one from the other and article supports the need for this surveillance. This need was from the site of collection of the positive otter (near Beniganim, ′ ′′ ′ ′′ already highlighted by our prior report (21) of SARS-CoV-2 38◦ 56 38 N, 0◦ 26 38 W) in independent watercourses. They infection in feral American mink (Neovison vison) and has been were kept frozen until nasopharyngeal, lung, and mediastinal stressed by the recent report of SARS CoV-2 among free-ranging samples were collected for the positive otter. white-tailed deer near Denver (22). Mustelids are believed to be highly susceptible to SARS-CoV-2 infection (14–19, 23). Thus, RNA Extraction surveillance on wild otter also appeared pertinent, particularly Approximately 100 mg of lung tissue and the entire mediastinal since there is a report of captive Asian small-clawed otter lymph node were defrosted, and immediately homogenized Frontiers in Veterinary Science | www.frontiersin.org 2 March 2022 | Volume 9 | Article 826991 Padilla-Blanco et al. SARS-CoV-2 in a Wild Otter FIGURE 1 | Otter carcass retrieval site and external aspect of the animal. (A) Europe (inset) and (main figure) Iberian Peninsula (Spain and Portugal) with the Valencian region in black, enclosing in a rectangle the area that is enlarged in (B), where the large arrowhead points to the Bellús reservoir, (C) zooming on it and its surrounding area, marking the site where the animal was found. (D,E) Shows, respectively, views of the head and the remainder of the body of the animal. in 0.2 ml of Sample Preservation Solution (see above), using in 50 µl of RNase-free water using the NZY Total RNA Isolation hypochlorite-treated and autoclaved (121◦ C, 15 min) small-sized kit (NZYtech, Lisboa, Portugal) and stored at −80◦ C. We glass Dounce-type homogenizers. RNA in each of these entire repeated RNA extraction on a different day with the swab fluid homogenates as well as in 0.2 ml of Sample Preservation Solution, and the lung tissue, to use the new lots of RNA in replicate in which the nasopharyngeal swab had been placed, was isolated analyses. The same procedures and instrumentation (including Frontiers in Veterinary Science | www.frontiersin.org 3 March 2022 | Volume 9 | Article 826991 Padilla-Blanco et al. SARS-CoV-2 in a Wild Otter TABLE 1 | Fragments of viral genes that were PCR-amplified and sequenced here. Gene Primers used for PCR amplificationa Amplified regionb Start position End position Amplicon size (bp) ′ ′ S Forward: 5 -GGACCTTGAAGGAAAACAGG-3 22,160 22,239 80 ′ ′ Reverse: 5 -GAACCATTGGTAGATTTGCCA-3 ′ ′ N Forward: 5 -GCAGTCAAGCCTCTTCTCGT-3 28,871 28,964 94 ′ ′ Reverse: 5 -TTGAACCAGCTTGAGAGCAA-3 ′ ′ ORF10 Forward: 5 -ATTGCAACAATCCATGAGCA-3 29,556 29,704 149 ′ ′ Reverse: 5 -TAGGGAGGACTTGAAAGAGCC-3 a Primers were designed with Primer3 (https://primer3.ut.ee/). b Start and end positions are consensus genome coordinates for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [GenBank (NC_045512.2)]. the use of the same homogenizers, duly cleaned, hypochlorite In all the RTqPCR assays (commercial or two tube), we treated, and autoclaved after each utilization) were employed for carried out in parallel cross-contamination controls and positive processing tissue samples from the positive otter and from the controls (B.1.177 variant of SARS-CoV-2), yielding in all cases two negative ones. respective negative and positive results. We used for all PCR procedures an Aria Mx Real-Time PCR (qPCR) instrument (Agilent Technologies, Santa Clara, CA, USA). The researchers Viral Detection and cDNA Preparation performing all the steps from extraction to detection were SARS- We used for viral detection two one-tube RTqPCR commercial CoV-2 negative. assays intended for human nasopharyngeal samples. One of them (Viasure, from CerTest Biotec, Zaragoza, Spain; sold by Sequencing and Data Analysis Palex Medical) (27) uses 5 µl of the isolated RNAs, amplifying Automated Sanger sequencing was done by a dedicated regions of the ORF1ab and nucleocapsid (N) viral genes, as well service (Genomics Department, Príncipe Felipe Research Centre, as the host RNaseP gene. This last gene, used as an internal Valencia, Spain), using an ABI Prism 3730 sequencer (Applied operational control gene, proved positive in otter as it previously Biosystems, Foster City, CA, USA), utilizing as sequencing was in American mink (21). Diagnosis was confirmed using the primers the oligonucleotides used to amplify gene fragments. commercial NZYtech SARS-CoV-2 One-Step RT-PCR kit, RdRp S, N, and ORF10 gene sequences were deposited in and N genes, CE-IVD, utilizing 8 µl of the RNA preparations. GenBank/EMBL/DDBJ (respective accession numbers This test uses the same fluorophore for measuring the combined OK235485, OK235486, and OK235487) and used for signal from the RdRp and N viral genes, with control RNaseP identification of related SARS-CoV-2 sequences in GenBank and detection with a second fluorophore. GISAID databanks by searching with BLASTN (http://blast.ncbi. For sequence information gathering, we amplified specific nlm.nih.gov). The BioEdit ver. 7.2.5 software (28) was used for regions (Table 1) from the S, N, and ORF10 viral genes, using nucleotide and corresponding amino acid sequence alignment appropriate primers in a two-tube RT-PCR procedure (21) and identity estimation, always including in the alignments the applied to 8 µl of RNA isolated from the mediastinal lymph consensus early Wuhan sequence (GenBank NC_045512.2) as node (chosen because its viral level, assessed in conventional one- reference for identification of non-synonymous substitutions. tube assays, was the highest; see Results Section). cDNA was first For phylogenetic analyses, distance matrices were calculated, and generated with NZY First-Strand cDNA Synthesis kit (NZYtech). tree topology was inferred by the maximum likelihood method Then we amplified separately the three selected gene regions in based on p-distances (bootstrap on 2,000 replicates, generated a mixture containing 10 µl of NZYSpeedy qPCR Green Master with a random seed) using the MEGA X software (29). Mix (2×) (NZYtech), 400 nmol of each primer (Table 1), and 2 µl of the cDNA solution (final volume, 20 µl). The temperature RESULTS protocol was (i) 2 min at 95◦ C; (ii) 40 cycles of a sequence of 5 s at 95◦ C and 30 s at 60◦ C; (iii) 30 s at 95◦ C; (iv) 30 s at We first tested RNA from the nasopharynx (swab) of the index 65◦ C; (v) 30 s at 95◦ C. Generation of the expected amplification case with the Viasure assay and consistently and repeatedly product (Table 1), monitored by SYBR green fluorescence, was obtained positive results. The tests were positive on two proven by electrophoretic visualization of DNA in agarose gels. independent RNA extractions from the swab fluid. We then also We routinely use these primers (Table 1) in the same two-tube used lung tissue with positive results with the same test, repeating RT-PCR procedure applied to human viral samples, consistently the lung tissue RNA extraction and again getting positive results. yielding positive amplifications for 103 viral genome copies/µl The mediastinal lymph node was the less abundant material, (inferred from Ct values in the Viasure test). They are highly allowing a single RNA extraction (see the Materials and Methods specific, giving negative results with otter and human samples Section), and was tested last, yielding, as the other samples, that tested negative in the Viasure assay. positive results in two rounds of Viasure testing, actually at an Frontiers in Veterinary Science | www.frontiersin.org 4 March 2022 | Volume 9 | Article 826991 Padilla-Blanco et al. SARS-CoV-2 in a Wild Otter TABLE 2 | Differences in the partial gene sequences found here with the SARS-CoV-2 reference sequence. Viral gene S N ORF10 Fragment (nucleotides)a 22,160–22,239 28,871–28,964 29,956–29,704 GenBank identifier OK235485 OK235486 OK235487 Nucleotide changesa None 28,877–28,878 28,881–28,883 28,899 29,645 AG>TC GGG>AAC G>T G>T Amino acid changesb None Arg203Lys Gly204Arg Arg209Ile Val30Leu a All the nucleotide positions refer to the whole-genome reference sequence NC_045512.2. b Amino acid positions are those for the encoded protein. earlier cycle in the real-time PCR assay than with the swab and obtained from this positive otter, indicating viral evolution not lung samples. All negative controls carried out simultaneously identified earlier. yielded negative results. Similar samples from two additional The three partial S, N, and ORF10 gene sequences clustered otters from other locations of the Valencian Community (see with SARS-CoV-2 sequences (p-distances of around 0) in Sample Collection in the Materials and Methods Section) gave phylogenetic trees built as indicated in the Materials and Methods negative results, while samples from our positive animal run in Section (bootstrap on 2,000 replicates from a random seed) parallel tested positive. (Figure 2). Other coronaviruses, such as SARS-CoV and MERS- Results were also positive for all the RNA samples from the CoV, formed different clusters. Although the small size of index case when we used another commercial RTqPCR test (from the sequenced regions limits the value of detailed quantitative NZYtech) that does not completely overlap with the Viasure test comparisons based on p-distance values, p-distances of the in the genes detected (see the Materials and Methods Section), partial gene sequences, relative to those of SARS-CoV and MERS- while the results were negative with the RNAs from the two CoV, were much larger for S (13.1 and 18.2, respectively) than for additional otters tested in the same way. N (0.2 and 0.45) and ORF10 (0.07 and 0.67). In summary, in the positive animal, the RNAs extracted from the three samples were positive for the ORF1ab and N viral genes and the internal control (RNaseP) monitored DISCUSSION in the Viasure test, with mean Ct values for ORF10/N viral genes of 36.0/31.7, 34.0/30.9, and 31.3/29.6 for lung tissue, To date, there is only one report of SARS-CoV-2 infection of nasopharyngeal swab, and lymph node RNA, respectively. The otter, affecting captive Asian small-clawed otters (Aonyx cinereus) results of the NZYtech assay confirmed the same scale of relative at an aquarium in Georgia (USA). We now report the finding abundance of SARS-CoV-2 RNA, with Ct values of 33.1, 30.9, of SARS-CoV-2 in a wild otter belonging to a different genus and 29.3 for, respectively, lung tissue, nasopharyngeal swab, and and species, the Eurasian river otter (Lutra lutra), suggesting lymph node RNAs. In the case of the other two otters, there was that all otter species may be susceptible to SARS-CoV-2. Our no amplification in these tests for as long as 40 cycles. positive otter did not present obvious signs of disease, in line We obtained some sequence information on three regions with our finding of relatively low viral loads in the samples of the S, N, and ORF10 genes of the virus, utilizing the from the nasopharynx, lungs, and mediastinal lymph node. RNA extracted from the lymph node of the positive animal The susceptibility to infection of otter by SARS-CoV-2 is not in a two-tube RT-PCR procedure [(21); see the Materials and surprising, as otters belong to the mustelid family, and other Methods Section]. Gel electrophoresis revealed DNA products mustelids, particularly minks and ferrets, have been proven of the expected sizes, while Sanger sequencing confirmed to be quite susceptible to SARS-CoV-2 infection (14–19, 21), that the amplified DNAs corresponded to the intended gene having forced entire mass culling of whole mink farms that were regions. Relative to the consensus Wuhan sequence (GenBank suffering from the infection. Although in most cases infected NC_045512.2), there were none, and six and one nucleotide human caretakers caused the initial animal contagion, these changes in the partial S, N, and ORF10 gene sequences, animals effectively transmit the infection among themselves and respectively. These substitutions (Table 2) caused four non- even back to humans (15–17). All these characteristics make synonymous codon changes, three in N and one in ORF10. of mustelids potential secondary hosts of SARS-CoV-2, with Of the viral genome sequences deposited in GISAID (https:// the possibility of back passage of the virus between species www.gisaid.org/, accessed on October 18, 2021), 795 (195 and humans. of them from Europe) combined the partial S and N Until now, worries about these animals in the context of sequences found in the otter virus. The partial ORF10 sequence the SARS-CoV-2 pandemic have centered on pet and farmed found in the otter is found in thousands of deposited viral mustelids, such as ferrets and mink (13–18). However, we already genomes (most of them related to the B.1.177 variant that found two SARS-CoV-2-infected feral mink that were living in was widely distributed in Europe). However, no GISAID- the wild (21). We now extend our observation to a Eurasian river deposited viral genome sequence that combined the three partial otter living far away from the locations where these minks were sequences of S, N, and ORF10 was found in the viral RNA found, suggesting multiple infecting events most likely derived Frontiers in Veterinary Science | www.frontiersin.org 5 March 2022 | Volume 9 | Article 826991 Padilla-Blanco et al. SARS-CoV-2 in a Wild Otter FIGURE 2 | Molecular phylogenetic analyses based on partial gene sequences. These analyses used our sequences of 80-, 94-, and 149-nucleotide fragments of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S gene (A), N gene (B), and ORF10 gene (C) (respective consensus genome coordinates; GenBank acc number NC_045512.2, nt 22,160–22,239, 28,871–28,964, and 29,556–29,704). The trees include the corresponding sequences in SARS-CoV (SARS), Middle East respiratory syndrome coronavirus (MERS-CoV) (MERS), some bat coronaviral sequences, and SARS-CoV-2 sequences reported in animals or in the original Wuhan isolate and a few subsequent variants. The evolutionary history was inferred using the maximum likelihood method based on the Tamura–Nei model. In each case, the tree with the highest log likelihood (−722, −377.01, and −446.87, for trees of S, N, and ORF10 genes, respectively) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying the neighbor-join and BioNJ algorithms to a matrix of pairwise distance estimated using the maximum composite likelihood (MCL) approach and selecting the topology with superior log likelihood value. The trees are drawn to scale, with branch lengths according to the number of substitutions per site (see scale bar at the bottom). The sequences obtained from the Eurasian otter reported here is highlighted in bold-type. GenBank accession numbers for the different sequences are given at the end of each branch name. GenBank accession numbers for our otter viral sequences are OK235485, OK235486, and OK235487 for the S, N, and ORF10 genes, respectively. from the universal expansion of the disease among humans. The of water transmission. This possibility could fit otters, which fact that the time of the finding of the present infected otter also are aquatic animals, particularly since the Bellús reservoir took place when human 14-day notification rate of COVID-19 is of low microbiological quality (30), with recorded episodes of per 100,000 population in the region was above 1,000 cases (the urban wastewater discharge into it via the Albaida river (31) that fifth wave of COVID-19 in Spain)1 makes particularly plausible have been reprimanded by the European Commission (32). In that the present positive otter could have been infected from any case, the finding in our positive animal of viral RNA not humans. We previously reached the same conclusion in our only in the respiratory tract but also in the lung tissue, and in report of infected feral mink, which occurred at an earlier peak the highly secluded internally, mesenteric lymph node, excludes of infections in the local human population (21). We ignore mere contact with external contaminated water that could carry the transmission path from infected humans to these animals. viral remains as a reason for the positivity of this animal. In the case of the feral mink, we speculated on the possibility The finding that the exact combination of viral sequences identified here appears non-reported in any complete viral 1 https://experience.arcgis.com/experience/42474fe756ee4714b7d8fbb730134b92 genome deposited in the GISAID database suggests viral Frontiers in Veterinary Science | www.frontiersin.org 6 March 2022 | Volume 9 | Article 826991 Padilla-Blanco et al. SARS-CoV-2 in a Wild Otter evolution in infected individuals, either humans or animal analyses. CR-G, EM, and MP-B analyzed the results. EM, hosts, that had escaped detection. Unnoticed evolution in VR, and CR-G were responsible for writing the article, humans is possible, since Spain does not stand out because although all authors contributed to this task, making substantial of its frequency of whole SARS-CoV-2 genome sequencing, intellectual contributions, having read, corrected, and approved particularly during large disease peaks (33). Our findings call for the manuscript. continued surveillance of wild or feral susceptible animals, in particular, of mustelids. Our observations of two minks infected FUNDING among a sample of 13 feral minks (21), and now of one infected individual among a sample of three wild otters warrant further This research received external funding to CR-G and EM study among the feral and wild populations of these animals. from Agencia Valenciana de Innovación: COVID-19. Ayudas de concesión directa a soluciones científico-innovadoras DATA AVAILABILITY STATEMENT directamente relacionadas con la lucha contra la COVID-19, (Ref COVID-19-203) and from the Conselleria de Innovación The datasets presented in this study can be found in online Universidades, Ciencia y Sociedad Digital: Subvenciones a repositories. The names of the repository/repositories grupos de investigación emergentes (Ref, GV/2021/163), and accession number(s) can be found in the and from a grant to VR (PID2020-120322RB-C21) from article/supplementary material. the Spanish Agencia Estatal de Investigación (Plan Estatal de I+D+i). ETHICS STATEMENT ACKNOWLEDGMENTS The animal study was reviewed and approved by Animal Ethics Committees of UCH-CEU University (research permit no. 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Available online at: http://www.argos.gva.es/bdmun/pls/argos_mun/ DMEDB_COMADATOSINDICADORES.DibujaPagina?aNComaId=24& Conflict of Interest: The authors declare that the research was conducted in the aNIndicador=2&aVLengua=c (accesed November 3, 2021). absence of any commercial or financial relationships that could be construed as a 26. La Recuperación de la Nutria (Lutra lutra) en la Comunitat Valenciana. potential conflict of interest. INFORME TÉCNICO 07/2016. Servici de Vida Silvestre, Direcció General de Medi Natural i d’Avaluació Ambiental (2016). Available online at: https:// Publisher’s Note: All claims expressed in this article are solely those of the authors agroambient.gva.es/documents/91061501/162536532/IT07_2016$+$La$+ and do not necessarily represent those of their affiliated organizations, or those of $recuperaci%C3%B3n$+$de$+$la$+$nutria$+$en$+$la$+$CV/c7fb7d5e- d28b-4ae3-872d-7ce438a6e47d (accesed November 3, 2021). the publisher, the editors and the reviewers. 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