Absence of COVID-19-associated changes in plasma coagulation proteins and pulmonary thrombosis in the ferret model

Abstract

Background: Many patients who are diagnosed with coronavirus disease 2019 (COVID-19) suffer from venous thromboembolic complications despite the use of stringent anticoagulant prophylaxis. Studies on the exact mechanism(s) underlying thrombosis in COVID-19 are limited as animal models commonly used to study venous thrombosis pathophysiology (i.e. rats and mice) are naturally not susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ferrets are susceptible to SARS-CoV-2 infection, successfully used to study virus transmission, and have been previously used to study activation of coagulation and thrombosis during influenza virus infection.

Objectives: This study aimed to explore the use of (heat-inactivated) plasma and lung material from SARS-CoV-2-inoculated ferrets studying COVID-19-associated changes in coagulation and thrombosis.

Material and methods: Histology and longitudinal plasma profiling using mass spectrometry-based proteomics approach was performed.

Results: Lungs of ferrets inoculated intranasally with SARS-CoV-2 demonstrated alveolar septa that were mildly expanded by macrophages, and diffuse interstitial histiocytic pneumonia. However, no macroscopical or microscopical evidence of vascular thrombosis in the lungs of SARS-CoV-2-inoculated ferrets was found. Longitudinal plasma profiling revealed minor differences in plasma protein profiles in SARS-CoV-2-inoculated ferrets up to 2 weeks post-infection. The majority of plasma coagulation factors were stable and demonstrated a low coefficient of variation.

Conclusions: We conclude that while ferrets are an essential and well-suited animal model to study SARS-CoV-2 transmission, their use to study SARS-CoV-2-related changes relevant to thrombotic disease is limited.

Keywords: COVID-19; Ferrets; Mass spectrometry; Proteomics; Thrombosis.

Fig. 1

Virus replication and lung histology after SARS-CoV-2 inoculation of ferrets. (A) Ferrets (n = 4) were inoculated intranasally with 5.4 × 10⁵ median Tissue Culture Infectious Dose (TCID)50/ml of SARS-CoV-2. At the indicated days post inoculation (DPI) nose and throat swabs were taken for the detection of SARS-CoV-2 genomes. In addition, at the days indicated, citrated blood samples were taken for plasma proteomic profiling and detection of virus neutralizing antibodies. At 20 DPI the ferrets were sacrificed and the right lung lobules were processed for histological and immunohistochemical analysis. Ferret image Healthy Pets, Healthy People: Ferrets. [Internet]. (B) Virus genomes in throat swabs (blue circles) was determined by RT-qPCR. When the cycle threshold (Ct) was not surpassed after 40 cycles, the genome load was considered to be zero. Viral genomes detected in nose swabs showed comparable kinetics (not shown). Due to overlap in the datapoint not all are visible, values are below <LOD = limit of detection. Presence of virus neutralizing antibodies was determined (red squares), end-point serum dilution factor that block SARS-CoV-2 infection in vitro are displayed (VNtiter). (C) Images of representative hematoxylin and eosin staining of a 5 μm thick paraffin lung section obtained from a negative control ferret (left) and a SARS-CoV-2 inoculated ferret (right). Visible (left) is the honeycomb pattern of the alveoli and optical empty spaces typical for well-functioning lungs). Alveolar septa of SARS-CoV-2-inoculated ferrets were mildly expanded (right, white asterisks), mostly by macrophages, fewer lymphocytes, and showing an diffuse interstitial histiocytic pneumonia (not visible for the magnification presented here). (D) Fibrin(ogen) staining of slides serial to the HE slides presented in panel C. Black arrow heads; small positive spots from capillary vessels likely containing fibrinogen rather than pathological fibrin-rich microthrombi. Black bars represent 50 μm. Note: Screening on the specificity of the polyclonal anti-human fibrin(ogen) antibody used showed that this specifically bound to ferret fibrin(ogen), since no antibody-binding was observed in the IgG isotype controls (see methods, data not shown). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Plasma profiling mass spectrometry approach on ferrets with CDV- and SARS-CoV-2-inoculated ferrets. (A) Label-free Mass-spectrometry analysis shows impact of heat-inactivation on healthy individual (human) citrated plasma samples by decreased levels of fibrinogens (FGA, FGG, FGB), ficolin3 (FCN3) and carboxypeptidase N catalytic chain 1 (CPN1). (HI: heat inactivation plasma, no-HI: non heat inactivated plasma) (B) Direct comparison of heat-inactivated and non-heat-inactivated citrated ferret plasma depicts the limited impact of heat inactivation on the ferret plasma proteome. (C) Number of significantly altered proteins between day 0 and 10 for CDV- and SARS-CoV-2-inoculated ferrets (D) Hierarchical clustering of z-scored median LFQ-intensities for 40 significant proteins following over time for inoculation with CDV. (E) Hierarchical clustering of z-scored median LFQ intensities for significant 10 proteins over time following SARS-CoV-2 inoculation. Including serum amyloid A (SAA), haptoglobin (HP), ceruloplasmin (CP), fibrinogen gamma (FGG), apolipoprotein B (APOB), lipase A (LIPA), lipocalin (LCN). (F) The inter-individual and intra-individual variation of protein levels calculated as the means coefficient of variation (CV) for each protein within each SARS-CoV-2-inoculated ferret and across all analysed SARS-CoV-2-inoculated ferrets. Zoom-in highlights coagulation proteins within a CV of 2%.