key: cord-0327021-u3as3e4e
authors: Adney, Danielle R.; Lovaglio, Jamie; Schulz, Jonathan E.; Yinda, Claude Kwe; Avanzato, Victoria A.; Haddock, Elaine; Port, Julia R.; Holbrook, Myndi G.; Hanley, Patrick W.; Saturday, Greg; Scott, Dana; Spengler, Jessica R.; Tansey, Cassandra; Cossaboom, Caitlin M.; Wendling, Natalie M.; Martens, Craig; Easley, John; Yap, Seng Wai; Seifert, Stephanie N.; Munster, Vincent J.
title: Severe acute respiratory disease in American mink (Neovison vison) experimentally infected with SARS-CoV-2
date: 2022-01-24
journal: bioRxiv
DOI: 10.1101/2022.01.20.477164
sha: 33abb6f2a2da460c6024f75b2a83b00e1d81d96b
doc_id: 327021
cord_uid: u3as3e4e

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, a non-transgenic model that develops severe acute respiratory disease has not been described. 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 for nonstructural protein 6 in open reading frame 1a/1ab. Together, these data indicate that American mink develop clinical features characteristic of severe COVID19 and as such, are uniquely suited to test viral countermeasures. One Sentence Summary SARS-CoV-2 infected mink develop severe respiratory disease that recapitulates some components of severe acute respiratory disease, including ARDS.

To compare differences within the ACE2 interface with the SARS-CoV-2 spike receptor 94 binding domain (RBD), the residues participating in the interaction, as described by Lan, et al. 95 (17), were mapped onto an amino acid sequence alignment of ACE2 from American mink 96 (Neovison vison), European mink (Mustela lutreola biedermanni, GenBank QNC68911.1), and 97 humans (Homo sapiens, GenBank BAB40370.1)(18). The binding residues are 65% identical 98 between mink and human ACE2, with seven of the 20 total interface residues differing in mink 99 ( Figure 1A ). These residues are highlighted on the structure of SARS-CoV-2 RBD bound to 100 Residues that participate in the SARS-CoV-2 RBD -ACE2 interaction are noted below the alignment by a blue box. Residues that participate in intermolecular hydrogen bonding or salt bridges are marked with a black dot. ACE2 residues that differ between mink and human within the interface are outlined with a red box. The substitution at residue 90 affecting an N-linked glycosylation site is noted with a blue box. (B) Differences between mink and human ACE2 are highlighted on the structure of the complex of SARS-CoV-2 RBD in gray bound to human ACE2 in black. Sidechains of the ACE2 and RBD residues that participate in the binding interaction are shown as sticks. The mutated residues are indicated by red. (C) SARS-CoV-2 spike pseudotype assay showing relative entry compared to no spike control in BHK cells expressing human or mink ACE2. Bars depict standard deviation. end-point criteria on 3 DPI, and one animal recovered from severe disease and was euthanized 123 on the predetermined experimental endpoint of 28 DPI. 124

Marked weight loss (up to 15%) was observed in all animals by 3 DPI (Figure 2A) . In 125 the animal that survived infection, bodyweight returned to baseline values by 14 DPI 126 (Supplemental Figure 2A) . Clinical signs were first detectable on 1 DPI in 5 of 11 (45 %) 127 animals, with clinical signs observed in 9 of 11 (82 %) animals by 2 DPI and all remaining 128 animals by 3 DPI. Signs of clinical disease included dull mentation, shivering, hunched or balled 129 posture, lethargy, anorexia, increased respiratory effort, tachypnea, with occasional nasal 130 discharge that included both epistaxis and serous discharge ( Figure 2 ). Animals were examined 131 on 1,3, 5, 7, 10, 14, 17, 21, and 28 DPI under anesthesia; 10 animals were clinically dehydrated 132 by 3 DPI. 133

Complete blood count (CBC) and complete chemistry panels were performed on blood 134 samples collected at least one week prior to infection and at 0, 1, 3, 5, 7, 10, 14, 17, 21, and 28 135 DPI. At all time-points post infection, the CBC was unremarkable apart from a decreased white 136 blood cell (WBC) count characterized by a mild lymphopenia that was most pronounced in the 9 137 remaining animals on 3 DPI ( Figure 2C ). The neutrophil-to-lymphocyte ratio was significantly 138 increased at the terminal endpoint for clinically ill animals ( Figure 2D ). The single surviving 139 animal had an elevated neutrophil-to-lymphocyte ratio (NLR) that peaked on 5 DPI as compared 140 to baseline then quickly decreased (Supplemental Figure 2B) . The blood chemistry panel was 141 clinically unremarkable for all values except for a mild hypoproteinemia and hypoalbuminemia 142 ( Figure 2E 

Radiographic scores on 1 and 3 DPI were increased as compared to baseline values 148 ( Figure 3A ) and indicated the presence of progressive pulmonary infiltrates consistent with viral 149 pneumonia likely with concurrent non-cardiogenic pulmonary edema secondary to acute 150 respiratory disease syndrome (ARDS)( Figure 3B ). On 1 DPI, radiographic changes consistent 151 with viral pneumonia were present in the thoracic radiographs of 5 (45%) of 11 mink. Of these 5, 152 4 had evidence of a mild-to-moderate ground glass/unstructured interstitial pattern and the 153 remaining animal had a moderate-to-marked alveolar pattern affecting multiple lung lobes. 154

Interestingly, this animal had progressive multifocal grade 3 and 4 pulmonary infiltrates at 2 DPI 155 prior to euthanasia (Figure 3 ). At 3 DPI, 8 of the 9 remaining animals displayed disease 156 weight ratio was assessed to estimate the extent of pulmonary edema, and the ratio was 180 significantly increased in infected animals compared to uninfected controls ( Figure 4A ). There 181 were varying degrees of gross pulmonary pathology evident in all 10 animals euthanized on 2 or 182 3 DPI, with 100% of some lungs affected ( Figure 4B ). Grossly, lungs were hyperemic, and 183 several animals had undergone pulmonary hepatization ( Figure 4B Table 1 ). The deep sequencing runs yielded an average of 88,686 reads mapped 278 for each sample (Supplemental Table 1 ). One sample for which fewer than 50,000 reads were 279 

We analyzed serum for the development of a neutralizing antibody response. All animals 307 had a titer of <20 prior to challenge and only the surviving animal developed a measurable 308 neutralizing response. This animal seroconverted by 14 DPI with a peak neutralizing titer of 960, 309 which decreased to a titer of 640 at euthanasia at 28 DPI. 310

The continued emergence of SARS-CoV-2 variants of interest and variants of concern 312 highlight the urgent need for animal models who consistently recapitulate the spectrum of 313 disease in COVID-19 patients. Overall, the pseudotype entry data from this study demonstrates 314 comparable spike entry between human and mink ACE2 confirming the suitability of mink for 315 modeling SARS-CoV-2 infection and COVID-19 diseases. ranges from asymptomatic infection to severe disease characterized by respiratory distress, 318 sepsis, or multiorgan failure. Currently, an animal model for severe COVID-19 disease is not 319 available (1). Most human infections are confined to the upper respiratory tract. Mild or early 320 disease manifests with nonspecific symptoms that can include fatigue, fever, headache, loss of 321 smell and taste, congestion, and fever (21). Progression into severe disease is typically presented 322 as worsening respiratory disease, hypoxemia, and radiographic lesions, with end-point markers 323 that can include coagulopathies, thromboembolism, acute kidney injury, and ARDS (21-24). 324

Infected mink displayed clinical disease consistent with worsening human COVID-19 disease. 325

Infected mink consistently demonstrated a greater degree of weight loss than that reported in 326 nonhuman primates or hamsters in the days following infection. Increased respiratory effort and 327 tachypnea in mink mark progression into severe COVID-19 disease. This study did not look at 328 odor discrimination; however, neutrophilic infiltrate in olfactory epithelium could suggest a loss 329 of smell that resulted in decreased appetite. Interestingly, while multiple field reports of fur farm 330 outbreaks commonly report nasal discharge, this was not a consistent finding in these mink. 331

Features of complete blood counts of COVID-19 disease patients include leukopenia, 332 lymphopenia, thrombocytopenia, and an increased NLR (13,21,25,26). While we were unable to 333 rule out a stress leukogram resulting in increased NLR, this finding has been reported in ferrets Piscataway, NJ, USA). All DNA constructs were verified by Sanger sequencing (ACGT). 440

Pseudotype production was carried out as described previously (16 The animals used in the infection study consisted of 9 females and two males; intake female 465 body weight range 1.04 kg -1.47 kg, mean = 1.18 kg, male weights were 2.06 kg and 2.73. The 466 females were approximately two years of age, the males were approximately one year of age. 467

Upon arrival whole blood from all mink were screened for antibodies against SARS-CoV-2. 468

Animals were single-housed in a climate-controlled room with a fixed light-dark cycle (12-hour 469 light and 12-hour dark) for the duration of the experiment with access to food and water ad 470 libitum with enrichment that included human interaction, commercial toys, music, and treats. All 471 manipulations were done on anesthetized animals using Telazol (10-20 mg/kg administered 472 subcutaneously). 473

Eleven animals were inoculated intratracheally (1.7 mL) and intranasally (0.15 mL per 475 naris delivered using a MAD Nasal™ Mucosal Atomization Device (Teleflex, US) for a total 476 dose of 10 5 TCID50 delivered in 2 total mL. Animals were evaluated at least twice daily 477 throughout the study. Clinical exams (including thoracic radiographs) were performed on 0, 1, 3, 478 5, 7, 10, 14, 17, 21, 28 DPI on anesthetized animals, during which the following parameters were 479 assessed: bodyweight, body temperature, heart rate, respiratory rate, and radiographs. Clinical 480 samples collected included nasal, oral, rectal, and fur swabs, and blood. Fur swabs were 481 collected down the dorsal midline of the animal. Swabs were collected in 1mL of DMEM 482 supplemented with 2% FBS, 1 mM L-glutamine, 50 U/ml penicillin, and 50 g/ml streptomycin. 483

Ventrodorsal, left lateral, and right lateral thoracic radiographs were taken prior to 485 clinical exams on 0, 1, 3, 5, 7, 10, 14, 17, 21, and 28 DPI with 0 DPI being performed prior to 486 inoculation and serving as a baseline. Thoracic radiographs were taken immediately after 487 animals were anesthetized and each lung lobe was evaluated by a board-certified veterinary 488 radiologist as follows: 0 = normal lung, 1 = mild interstitial infiltrate, 2 = moderate to marked 489 unstructured interstitial pattern, 3 = <25% alveolar pattern, 4 = >25% alveolar pattern. 490

Hematology analysis was completed on a ProCyte Dx® (IDEXX Laboratories, 492

Westbrook, ME, USA) and the following parameters were evaluated: red blood cells (RBC); 493 hemoglobin (Hb); hematocrit (HCT); mean corpuscular volume (MCV); mean corpuscular 494 hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); red cell distribution 495 width (RDW); platelets; mean platelet volume (MPV); white blood cells (WBC); neutrophil 496 count (absolute and percentage); lymphocyte count (absolute and percentage); monocyte count 497 (absolute and percentage); eosinophil count (absolute and percentage); and basophil count 498 (absolute and percentage). Serum chemistry analysis was completed on a VetScan VS2® Chemistry Analyzer (Abaxis, Union City CA) and the following parameters were evaluated: 500 glucose; blood urea nitrogen (BUN); creatinine; calcium; albumin; total protein; alanine 501 aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP); total 502 bilirubin; globulin; sodium; potassium; chloride and total carbon dioxide. Clinical pathology 503 samples were evaluated by a board-certified clinical veterinarian. 504

Histopathology 505

Histopathology and immunohistochemistry were performed on mink tissues. Tissues 506 were fixed for a minimum of 7 days in 10% neutral-buffered formalin with 2 changes. Tissues 507 were placed in cassettes and processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated 508 schedule, using a graded series of ethanol, xylene, and PureAffin. Embedded tissues were 509 sectioned at 5um and dried overnight at 42 degrees C prior to staining. The skulls were placed in 510

Cancer Diagnostic acid free EDTA for 4 weeks and the solution was changed weekly. and SARS-CoV-2 genome copy (equivalent/mL) numbers were determined by absolute 566 quantitation method. Next generation libraries were generated using the TruSeq DNA PCR Free 567 Nano kit (Illumina, Inc., San Diego, CA, USA) and the ARTIC multiplex PCR genome 568 amplification protocol with the V3 primer scheme (www.protocols.io/view/ncov-2019-569 sequencing-protocol-bbmuik6w) and libraries were sequenced on an Illumina MiSeq at 2 x 250 570 paired-end reads. The ARTIC multiplex PCR SARS-CoV-2 genome amplification protocol has 571 been widely used in viral genome sequencing during the COVID-19 pandemic and to study 572 within-host dynamics of SARS-CoV-2 (21). 573

To determine reproducibility of our assay, a subset of 12 samples determined to have 574 high (10 4 ), medium (10 3 ), and low (10 2 ) SARS-CoV-2 genome copy (equivalent/mL) numbers 575 by NSP5 qRT-PCR were selected as technical replicates. ARTIC primers and Illumina adapters 576 were trimmed, low quality bases and duplicate reads were filtered out, and mapping and variant 577 calling were completed as described in the iVar and PrimalSeq pipeline described by Grubaugh 578 et al.(47) . Intrahost single nucleotide variants (iSNVs) were included in further analysis if they 579 passed the Fisher's exact test for variation above the mean error rate at that locus and had a depth 580 of coverage at or above 100X. iSNVs were called with minor allele frequency (MAF) thresholds 581 at 3% and 5% and compared against technical replicates (Supplemental SNS2). iSNVs detected 582 at 3% MAF were plotted against the SARS-CoV-2 genome copy number for each sample 583 (Supplemental SNS3A) and the number of reads mapped for each sample (Supplemental 584 SNS3B). 585

To compare variation arising in the experimentally challenged mink to variation 586 previously detected in SARS-CoV-2 circulating at mink farms, all available mink-associated 587 SARS-CoV-2 genomes were downloaded from GISAID from 01-Jan-2020 through 22-Nov-588 2021. The resulting alignment of 1002 SARS-CoV-2 genome sequences included 999 with Neovison vison as the host species and 3 SARS-CoV-2 sequences from the genus Mustela 590 (GISAID Acknowledgements, Supplemental Table SNS4) . 591

Serology 592

Sera were heat-inactivated (30 min, 56°C). After an initial 1:10 dilution of the sera, two-593 fold serial dilutions were prepared in DMEM. 100 TCID50 of SARS-CoV02 variant B.1.1.7 was 594 added to the diluted sera. After a 1-hour incubation at 37°C and 5% CO2, the virus-serum 595 mixture was added to VeroE6 cells. The cells were incubated for 6 days at 37°C and 5% CO2 at 596 which time they were evaluated for CPE. The virus neutralization titer was expressed at the 597 reciprocal value of the highest dilution of the serum that still inhibited virus replication. 598

Statistical analysis was performed using GraphPad Version 8.4.3. Significance tests were 600 performed as indicated where appropriate with reported p-values. 601 well as the mink ACE2 sequencing. We also thank Randy Elkins, Steve Denny, and Alphie Cisar 736 for additional support. The surviving animal was monitored for change in relative weight (A). Weight loss was most severe on 5 DPI, after which the animal began recovery. Neutrophil-to-lymphocyte ratio was monitored over time (B), with the most severe change appreciated on 5 DPI. Nasal and oral swabs were evaluated for resolution of viral shedding through genomic and subgenomic RT-PCR (C). All swabs on 14 DPI were below the limit of detectable virus. Table 1 . Samples from mink experimentally challenged with SARS-CoV-2 that were deep sequenced for within-host evolutionary analyses.

Animal models for COVID-19

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Defining the Syrian hamster as a highly susceptible preclinical model 609 for SARS-CoV-2 infection

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SARS-coronavirus

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SARS-CoV-2 is transmitted via contact and via the air between ferrets

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SARS-CoV-2 Exposure in Escaped Mink

First Description of SARS-CoV-2 Infection in Two Feral

Neovison vison) Caught in the Wild. Animals : an open access journal 632 from MDPI 11

Clinical and Pathological Findings in SARS-CoV-2 Disease

Outbreaks in Farmed Mink (Neovison vison)

In vitro characterization of fitness and convalescent antibody 637 neutralization of SARS-CoV-2 Cluster 5 variant emerging in mink at Danish farms

Coronavirus rips through Dutch mink farms, triggering culls

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Structure of the SARS-CoV-2 spike receptor-binding domain bound to the 645 ACE2 receptor

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SARS-CoV-2 within-host diversity and transmission

Patterns of within-host genetic diversity in SARS-CoV-2

Pathological Findings in COVID-19 as a Tool to Define SARS-CoV

Severe Covid-19

Acute Respiratory Distress Syndrome: The Berlin Definition

Diffuse alveolar damage (DAD) resulting from coronavirus disease 661 2019 Infection is Morphologically Indistinguishable from Other Causes of DAD

Neutrophil-to-lymphocyte ratio predicts critical illness patients with 2019 664 coronavirus disease in the early stage

Neutrophil-to-lymphocyte ratio as an independent risk factor for mortality 667 in hospitalized patients with COVID-19

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COVID-19 patients in

Laboratory parameters in patients with COVID-19 on first emergency 675 admission is different in non-survivors: albumin and lactate dehydrogenase as risk 676 factors

Syndrome and COVID-19 Lung Injury

Western diet increases COVID-19 disease severity in the Syrian hamster

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Immune 686 mechanisms of pulmonary intravascular coagulopathy in COVID-19 pneumonia

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Ebola Virus Genome From Ecological and Clinical Samples

S304

A Novel Field-Deployable Method for Sequencing and Analyses of

Henipavirus Genomes From Complex Samples on the MinION Platform

SPAdes: a new genome assembly algorithm and its applications to 695 single-cell sequencing

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ENDscript server

The EMBL-EBI search and sequence analysis tools APIs in

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Protease-mediated entry via the 709 endosome of human coronavirus 229E

A system for functional analysis of Ebola virus glycoprotein

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Transmission of 2019-nCoV Infection from an Asymptomatic Contact in

An amplicon-based sequencing framework for accurately 718 measuring intrahost virus diversity using PrimalSeq and iVar

Acknowledgements: We would like to thank Emmie de Wit

Anzick 726 for their assistance with the SARS-CoV-2 isolate. The following reagent was obtained through 727 BEI Resources, NIAID NIH: Severe Acute Respiratory Syndrome-Related Coronavirus 2, Isolate 728 hCoV-19/England/204820464/20200, NR-54000, contributed by Bassam Hallis. We are grateful 729 to Dr. High Hildebrant for providing our Aleution Lateral Flow test

and Taylor Saturday for technical and data assistance

We thank Kimmo Virtaneva and Dan Bruno for their assistance with extractions, the 734 ARTIC protocol, and sequencing. Stacy Ricklefs assisted with the original isolate sequencing as Author contributions: 746 Conceptualization: DRA

Competing interests: The authors declare no competing interests

SARS-CoV-2 Pulmonary immunohistochemistry. (A) Lung: Bar=200um (B) Alveolar macrophage immunoreactivity (C) Type I & II pneumocyte immunoreactivity (D) Bronchiolar epithelium immunoreactivity (brown=immunoreactive cells) C-E Bar=20um

Emmanuelle Munger; Irina Chestakova; Marion Koopmans; Marjan Boter; OH consortium; Reina Sikkema

EPI_ISL_1265367 see above Dutch COVID-19 response team Wageningen Bioveterinary Research OH consortium EPI_ISL_8144460, EPI_ISL_8144461, EPI_ISL_8144462

Laboratorija Latvian Biomedical Research and Study Centre Alise Jakovele

Davids Fridmanis; Dmitrijs Perminovs

Janis Pjalkovskis; Jurijs Perevoscikovs

Lauma Freimane; Laura Ansone; Liga Birzniece; Mikus Gavars; Monta Briviba; Nikita Zrelovs

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