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

Abstract

Animals have been involved in the three known outbreaks of severe respiratory syndromes due to coronaviruses (years 2005, 2012, and 2019). The pandemic nature of the SARS-CoV-2 outbreak increases the likelihood of infection from humans of susceptible animal species that, thus, could become secondary viral hosts and even disease reservoirs. We present evidence of spillover infection of wild mustelids by reporting the presence of SARS-CoV-2 in a Eurasian river otter found near a water reservoir in the Valencian Community (Spain). We detected the virus using two different commercial RTqPCR assays on RNA extracted from the nasopharynx (swabbing) and from lung tissue and mediastinal lymph node homogenates. The corresponding samples from two additional otters from distant sites tested negative in identical assays. The diagnosis in the positive otter was confirmed by two-tube RT-PCR assay in which RNA was first retrotranscribed, and then specific regions of the spike (S), nucleocapsid (N), and ORF10 genes were separately amplified from the produced cDNA, followed by electrophoretic visualization and Sanger sequencing. The sequences of the amplified products revealed some non-synonymous changes in the N and ORF10 partial sequences, relative to the consensus sequence. These changes, identified already in human patient samples, point to human origin of the virus, although their specific combination was unique. These findings, together with our previous report of SARS-CoV-2 infection of feral American mink, highlight the need for SARS-CoV-2 surveillance of wild or feral mustelids to evaluate the risk that these animals could become SARS-CoV-2 reservoirs.

Keywords: Lutra lutra; RT-PCR; SARS-CoV-2; Spain; wildlife.

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.

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.