Highly conserved binding region of ACE2 as a receptor for SARS-CoV-2 between humans and mammals.

Several cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection transmitted from human owners to their dogs have recently been reported. The first ever case of SARS-CoV-2 transmission from a human owner to a domestic cat was confirmed on March 27, 2020. A tiger from a zoo in New York, USA, was also reportedly infected with SARS-CoV-2. It is believed that SARS-CoV-2 was transmitted to tigers from their caretakers, who were previously infected with this virus. On May 25, 2020, the Dutch Minister of Agriculture, Nature and Food Quality reported that two employees were infected with SARS-CoV-2 transmitted from minks. These reports have influenced us to perform a comparative analysis among angiotensin-converting enzyme 2 (ACE2) homologous proteins for verifying the conservation of specific protein regions. One of the most conserved peptides is represented by the peptide "353-KGDFR-357 (H. sapiens ACE2 residue numbering), which is located on the surface of the ACE2 molecule and participates in the binding of SARS-CoV-2 spike receptor binding domain (RBD). Multiple sequence alignments of the ACE2 proteins by ClustalW, whereas the three-dimensional structure of its binding region for the spike glycoprotein of SARS-CoV-2 was assessed by means of Spanner, a structural homology modeling pipeline method. In addition, evolutionary phylogenetic tree analysis by ETE3 was used. ACE2 works as a receptor for the SARS-CoV-2 spike glycoprotein between humans, dogs, cats, tigers, minks, and other animals, except for snakes. The three-dimensional structure of the KGDFR hosting protein region involved in direct interactions with SARS-CoV-2 spike RBD of the mink ACE2 appears to form a loop structurally related to the human ACE2 corresponding protein loop, despite of the reduced available protein length (401 residues of the mink ACE2 available sequence vs 805 residues of the human ACE2). The multiple sequence alignments of the ACE2 proteins shows high homology and complete conservation of the five amino acid residues: 353-KGDFR-357 with humans, dogs, cats, tigers, minks, and other animals, except for snakes. Where the information revealed from our examinations can support precision vaccine design and the discovery of antiviral therapeutics, which will accelerate the development of medical countermeasures, the World Health Organization recently reported on the possible risks of reciprocal infections regarding SARS-CoV-2 transmission from animals to humans.

Cryptic SARS-CoV-2 lineage identified on two mink farms as a possible result of long-term undetected circulation in an unknown animal reservoir, Poland, November 2022 to January 2023.

In late 2022 and early 2023, SARS-CoV-2 infections were detected on three mink farms in Poland situated within a few km from each other. Whole-genome sequencing of the viruses on two of the farms showed that they were related to a virus identified in humans in the same region 2 years before (B.1.1.307 lineage). Many mutations were found, including in the S protein typical of adaptations to the mink host. The origin of the virus remains to be determined.

Zooanthroponotic transmission of SARS-CoV-2 and host-specific viral mutations revealed by genome-wide phylogenetic analysis.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including the selection of adaptive mutations and the emergence of new variant lineages. Here, we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate the transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, ~⅔ were plausibly introduced into these animal species from local human populations, while the remaining ~⅓ were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health.

Pluripotent Stem Cells of Order Carnivora: Technical Perspective.

Human and mouse induced pluripotent stem cells (PSCs) are widely used for studying early embryonic development and for modeling of human diseases. Derivation and studying of PSCs from model organisms beyond commonly used mice and rats may provide new insights into the modeling and treating human diseases. The order Carnivora representatives possess unique features and are already used for modeling human-related traits. This review focuses on the technical aspects of derivation of the Carnivora species PSCs as well as their characterization. Current data on dog, feline, ferret, and American mink PSCs are summarized.

Bird flu spread between mink is a 'warning bell'.

Big outbreak at a Spanish farm reignites fears of an H5N1 influenza pandemic.

One Health Investigation of SARS-CoV-2 in People and Animals on Multiple Mink Farms in Utah.

From July−November 2020, mink (Neogale vison) on 12 Utah farms experienced an increase in mortality rates due to confirmed SARS-CoV-2 infection. We conducted epidemiologic investigations on six farms to identify the source of virus introduction, track cross-species transmission, and assess viral evolution. Interviews were conducted and specimens were collected from persons living or working on participating farms and from multiple animal species. Swabs and sera were tested by SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) and serological assays, respectively. Whole genome sequencing was attempted for specimens with cycle threshold values <30. Evidence of SARS-CoV-2 infection was detected by rRT-PCR or serology in ≥1 person, farmed mink, dog, and/or feral cat on each farm. Sequence analysis showed high similarity between mink and human sequences on corresponding farms. On farms sampled at multiple time points, mink tested rRT-PCR positive up to 16 weeks post-onset of increased mortality. Workers likely introduced SARS-CoV-2 to mink, and mink transmitted SARS-CoV-2 to other animal species; mink-to-human transmission was not identified. Our findings provide critical evidence to support interventions to prevent and manage SARS-CoV-2 in people and animals on mink farms and emphasizes the importance of a One Health approach to address emerging zoonoses.

Hands off the Mink! Using Environmental Sampling for SARS-CoV-2 Surveillance in American Mink.

Throughout the COVID-19 pandemic, numerous non-human species were shown to be susceptible to natural infection by SARS-CoV-2, including farmed American mink. Once infected, American mink can transfer the virus from mink to human and mink to mink, resulting in a high rate of viral mutation. Therefore, outbreak surveillance on American mink farms is imperative for both mink and human health. Historically, disease surveillance on mink farms has consisted of a combination of mortality and live animal sampling; however, these methodologies have significant limitations. This study compared PCR testing of both deceased and live animal samples to environmental samples on an active outbreak premise, to determine the utility of environmental sampling. Environmental sampling mirrored trends in both deceased and live animal sampling in terms of percent positivity and appeared more sensitive in some low-prevalence instances. PCR CT values of environmental samples were significantly different from live animal samples' CT values and were consistently high (mean CT = 36.2), likely indicating a low amount of viral RNA in the samples. There is compelling evidence in favour of environmental sampling for the purpose of disease surveillance, specifically as an early warning tool for SARS-CoV-2; however, further work is needed to ultimately determine whether environmental samples are viable sources for molecular epidemiology investigations.

Severe acute respiratory disease in American mink experimentally infected with SARS-CoV-2.

An animal model that fully recapitulates severe COVID-19 presentation in humans has been a top priority since the discovery of SARS-CoV-2 in 2019. Although multiple animal models are available for mild to moderate clinical disease, models that develop severe disease are still needed. Mink experimentally infected with SARS-CoV-2 developed severe acute respiratory disease, as evident by clinical respiratory disease, radiological, and histological changes. Virus was detected in nasal, oral, rectal, and fur swabs. Deep sequencing of SARS-CoV-2 from oral swabs and lung tissue samples showed repeated enrichment for a mutation in the gene encoding nonstructural protein 6 in open reading frame 1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID-19 and, as such, are uniquely suited to test viral countermeasures.

GPS Tracking of Free-Roaming Cats (Felis catus) on SARS-CoV-2-Infected Mink Farms in Utah.

Zoonotic transmission of SARS-CoV-2 from infected humans to other animals has been documented around the world, most notably in mink farming operations in Europe and the United States. Outbreaks of SARS-CoV-2 on Utah mink farms began in late July 2020 and resulted in high mink mortality. An investigation of these outbreaks revealed active and past SARS-CoV-2 infections in free-roaming and in feral cats living on or near several mink farms. Cats were captured using live traps, were sampled, fitted with GPS collars, and released on the farms. GPS tracking of these cats show they made frequent visits to mink sheds, moved freely around the affected farms, and visited surrounding residential properties and neighborhoods on multiple occasions, making them potential low risk vectors of additional SARS-CoV-2 spread in local communities.

Cryobanking European Mink (Mustela lutreola) Mesenchymal Stem Cells and Oocytes.

The European mink (Mustela lutreola) is one of Europe's most endangered species, and it is on the brink of extinction in the Iberian Peninsula. The species' precarious situation requires the application of new ex situ conservation methodologies that complement the existing ex situ and in situ conservation measures. Here, we report for the first time the establishment of a biobank for European mink mesenchymal stem cells (emMSC) and oocytes from specimens found dead in the Iberian Peninsula, either free or in captivity. New emMSC lines were isolated from different tissues: bone marrow (emBM-MSC), oral mucosa (emOM-MSc), dermal skin (emDS-MSC), oviduct (emO-MSc), endometrium (emE-MSC), testicular (emT-MSC), and adipose tissue from two different adipose depots: subcutaneous (emSCA-MSC) and ovarian (emOA-MSC). All eight emMSC lines showed plastic adhesion, a detectable expression of characteristic markers of MSCs, and, when cultured under osteogenic and adipogenic conditions, differentiation capacity to these lineages. Additionally, we were able to keep 227 Cumulus-oocyte complexes (COCs) in the biobank, 97 of which are grade I or II. The European mink MSC and oocyte biobank will allow for the conservation of the species' genetic variability, the application of assisted reproduction techniques, and the development of in vitro models for studying the molecular mechanisms of infectious diseases that threaten the species' precarious situation.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Manifestation of SARS-CoV-2 Infections in Mink Related to Host-, Virus- and Farm-Associated Factors, The Netherlands 2020.

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

SARS-CoV-2 in a Mink Farm in Italy: Case Description, Molecular and Serological Diagnosis by Comparing Different Tests.

This study described a SARS-CoV-2 infection in minks on an Italian farm. Surveillance was performed based on clinical examination and a collection of 1879 swabs and 74 sera from dead and live animals. The farm was placed under surveillance for 4.5 months, from the end of July 2020, when a man working on the farm tested positive by RT-PCR, till mid-December 2020 when all the animals were sacrificed. Clinical examination revealed no clinical signs or increased mortality rates attributable to SARS-CoV-2, while diagnostic tests detected only four weak PCR-positive samples, but 100% of sera were positive for SARS-CoV-2 anti-S antibodies. The phylogenetic analysis of two SARS-CoV-2 sequences from two minks and the sequence of the worker showed that they belonged to different clades. It could be therefore assumed that two distinct introductions of the virus occurred on the farm, and that the first introduction probably occurred before the start of the surveillance period. From the data collected, and especially from the detection of specific antibodies through the combination of different tests, it can be postulated that syndromic surveillance combined with genome detection by PCR may not be sufficient to achieve a diagnosis in asymptomatic animals. In particular, the serological approach, especially when using tests directed towards the S protein, may be useful for improving the traceability of virus circulation in similar environments.

Experimental Infection of Mink with SARS-COV-2 Omicron Variant and Subsequent Clinical Disease.

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that mink remain positive for viral RNA for days, experience clinical signs and histopathologic changes, and transmit the virus to uninfected recipients. Preparedness is crucial to avoid spread among mink and spillover to human populations.

The basis of mink susceptibility to SARS-CoV-2 infection.

Of all known airborne diseases in the twenty-first century, coronavirus disease 19 (COVID-19) has the highest infection and death rate. Over the past few decades, animal origin viral diseases, notably those of bats-linked, have increased many folds in humans with cross-species transmissions noted and the ongoing COVID-19 pandemic has emphasized the importance of understanding the evolution of natural hosts in response to viral pathogens. Cross-species transmissions are possible due to the possession of the angiotensin-converting enzyme 2 (ACE2) receptor in animals. ACE2 recognition by SARS-CoV-2 is a critical determinant of the host range, interspecies transmission, and viral pathogenesis. Thus, the phenomenon of breaking the cross-species barrier is mainly associated with mutations in the receptor-binding domain (RBD) of the spike (S) protein that interacts with ACE2. In this review, we raise the issue of cross-species transmission based on sequence alignment of S protein. Based on previous reports and our observations, we can conclude that the occurrence of one of two mutations D614G or Y453F is sufficient for infection of minks by SARS-CoV-2 from humans. Unfortunately, D614G is observed in the world's most common line of virus B.1.1.7 and the latest SARS-CoV-2 variants B.1.617.1, B.1.617.2, and B.1.617.3 too.

Histopathology and localization of SARS-CoV-2 and its host cell entry receptor ACE2 in tissues from naturally infected US-farmed mink (Neovison vison).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease in mink similar to human COVID-19. We characterized the pathological findings in 72 mink from US farms with SARS-CoV-2 outbreaks, localized SARS-CoV-2 and its host cellular receptor angiotensin-converting enzyme 2 (ACE2) in mink respiratory tissues, and evaluated the utility of various test methods and specimens for SARS-CoV-2 detection in necropsy tissues. Of SARS-CoV-2-positive animals found dead, 74% had bronchiolitis and diffuse alveolar damage (DAD). Of euthanized SARS-CoV-2-positive animals, 72% had only mild interstitial pneumonia or minimal nonspecific lung changes (congestion, edema, macrophages); similar findings were seen in SARS-CoV-2-negative animals. Suppurative rhinitis, lymphocytic perivascular inflammation in the lungs, and lymphocytic infiltrates in other tissues were common in both SARS-CoV-2-positive and SARS-CoV-2-negative animals. In formalin-fixed paraffin-embedded (FFPE) upper respiratory tract (URT) specimens, conventional reverse transcription-polymerase chain reaction (cRT-PCR) was more sensitive than in situ hybridization (ISH) or immunohistochemistry (IHC) for detection of SARS-CoV-2. FFPE lung specimens yielded less detection of virus than FFPE URT specimens by all test methods. By IHC and ISH, virus localized extensively to epithelial cells in the nasal turbinates, and prominently within intact epithelium; olfactory mucosa was mostly spared. The SARS-CoV-2 receptor ACE2 was extensively detected by IHC within turbinate epithelium, with decreased detection in lower respiratory tract epithelium and alveolar macrophages. This study expands on the knowledge of the pathology and pathogenesis of natural SARS-CoV-2 infection in mink and supports their further investigation as a potential animal model of SARS-CoV-2 infection in humans.

Integrated histopathological, lipidomic, and metabolomic profiles reveal mink is a useful animal model to mimic the pathogenicity of severe COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted on mink farms between minks and humans in many countries. However, the systemic pathological features of SARS-CoV-2-infected minks are mostly unknown. Here, we demonstrated that minks were largely permissive to SARS-CoV-2, characterized by severe and diffuse alveolar damage, and lasted at least 14 days post inoculation (dpi). We first reported that infected minks displayed multiple organ-system lesions accompanied by an increased inflammatory response and widespread viral distribution in the cardiovascular, hepatobiliary, urinary, endocrine, digestive, and immune systems. The viral protein partially co-localized with activated Mac-2+ macrophages throughout the body. Moreover, we first found that the alterations in lipids and metabolites were correlated with the histological lesions in infected minks, especially at 6 dpi, and were similar to that of patients with severe and fatal COVID-19. Particularly, altered metabolic pathways, abnormal digestion, and absorption of vitamins, lipids, cholesterol, steroids, amino acids, and proteins, consistent with hepatic dysfunction, highlight metabolic and immune dysregulation. Enriched kynurenine in infected minks contributed to significant activation of the kynurenine pathway and was related to macrophage activation. Melatonin, which has significant anti-inflammatory and immunomodulating effects, was significantly downregulated at 6 dpi and displayed potential as a targeted medicine. Our data first illustrate systematic analyses of infected minks to recapitulate those observations in severe and fetal COVID-19 patients, delineating a useful animal model to mimic SARS-CoV-2-induced systematic and severe pathophysiological features and provide a reliable tool for the development of effective and targeted treatment strategies, vaccine research, and potential biomarkers.

Mutations that adapt SARS-CoV-2 to mink or ferret do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs, and farmed mink. Since the start of the 2019 pandemic, several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all three mink adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.