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

Abstract

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.

Keywords: COVID-19; Molecular genetics.

Figure 1. Functional SARS-CoV-2 entry analysis with human and mink ACE2.

(A) An aa sequence alignment of ACE2 from mink and humans. 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 humans 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. Side chains 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 with no-spike control in BHK cells expressing human or mink ACE2. Bars depict SD, no significant difference detected between groups by 2-tailed t test.

Figure 2. Features of acute respiratory disease in mink.

(A) Percent of original BW was collected during clinical exams on 1 and 3 dpi. Mink lost a significant amount of BW at both 1 and 3 dpi (1-way ANOVA with Tukey’s multiple comparisons test). (B) Mink were assessed at least twice daily and evaluated for hunched posture, respiratory effort, mentation, nasal discharge, and appetite. (C) CBC values collected after infection. The medians with the 95% CI are depicted. (D) Increased neutrophil/lymphocyte ratio as determined from the CBC. Mean with SD depicted, 2-way ANOVA with Tukey’s multiple comparisons test. (E) Selected blood chemistry values, median with 95% CI depicted.

Figure 3. Severe radiological changes after infection with SARS-CoV-2.

(A) Compiled radiographic scores. Bar graph depicts the mean with SD and individuals, ordinary 1-way ANOVA with Tukey’s multiple comparisons test. Radiographs demonstrate multifocal pulmonary infiltrates, most severe in the left and right caudal lung lobes depicted in the (B) dorsoventral, (C) left lateral, and (D) right lateral radiographs on evening at 2 dpi. Arrows depict grade 4 pulmonary disease in the left and right caudal lung lobes with grade 3 pulmonary disease in the right middle lung lobe and cranial subsegment of the left cranial lung lobe.

Figure 4. Pulmonary histopathology and IHC.

(A) Lung to BW ratio, Mann-Whitney test. Graph depicts median with individuals. (B) Diffusely consolidated dark-mottled red lungs. (C) Multifocal pulmonary congestion (box). (D) Enlarged section of image C, vascular thrombi (arrows) and alveolar edema (asterisks). (E) Alveolar fibrin (arrows). (F) Lymphoplasmacytic perivascular cuffing (arrows). (G) Pulmonary PTAH staining (black). (H) Enlarged section of G, microthrombi (arrows). Scale bar expressed in μm in lower right corner of each image.

Figure 5. Nasal turbinate pathology and histopathology.

(A) Sagittal section of skull, H&E. (B) Respiratory epithelium neutrophilic infiltrates (small arrows), necrotic epithelium (long arrow), and cellular exudate (asterisk). (C) Olfactory epithelium with cellular exudate. (D) Respiratory epithelium SARS-CoV-2 IHC immunoreactivity (brown) and immunoreactive cellular exudate (asterisk). (E) Olfactory epithelium SARS-CoV-2 IHC immunoreactivity and cellular exudate (brown). Scale bars expressed in μm unless indicated.

Figure 6. Viral shedding in infected mink.

Oral (A), nasal (B), rectal (C), and fur (D) swabs were collected at 1 and 3 dpi and analyzed for gRNA, sgRNA, and infectious virus. Bar graphs depict the mean, SD, and individuals. Dotted line indicates the limit of detection.

Figure 7. High levels of viral replication in the respiratory tract of infected mink.

Tissues from animal euthanized at 3 dpi were analyzed for genomic (A), subgenomic (B), and infectious virus (C). Bar graphs depict individuals, mean, and SD. The dotted line depicts the limit of detection.

Figure 8. Enrichment of a nonsynonymous mutation (L260F) in the gene encoding nsp6 in ORF1a of SARS-CoV-2 in oral swabs and lung tissue samples of 5 experimentally challenged mink.

Deep sequencing of SARS-CoV-2-positive samples show rapid enrichment for L260F in nsp6 in lung tissue samples but not the oral swabs for 5 of 10 experimentally inoculated mink with SARS-CoV-2 genomic material detected in the lungs. Oral swab samples indicated by blue and lung tissue samples indicated by red, the line indicates group mean. Allele frequency for the L260F mutation is plotted on the y axis.