Longitudinal analyses using 18F-Fluorodeoxyglucose positron emission tomography with computed tomography as a measure of COVID-19 severity in the aged, young, and humanized ACE2 SARS-CoV-2 hamster models

Abstract

This study compared disease progression of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in three different models of golden hamsters: aged (≈60 weeks old) wild-type (WT), young (6 weeks old) WT, and adult (14-22 weeks old) hamsters expressing the human-angiotensin-converting enzyme 2 (hACE2) receptor. After intranasal (IN) exposure to the SARS-CoV-2 Washington isolate (WA01/2020), 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography with computed tomography (¹⁸F-FDG PET/CT) was used to monitor disease progression in near real time and animals were euthanized at pre-determined time points to directly compare imaging findings with other disease parameters associated with coronavirus disease 2019 (COVID-19). Consistent with histopathology, ¹⁸F-FDG-PET/CT demonstrated that aged WT hamsters exposed to 10⁵ plaque forming units (PFU) developed more severe and protracted pneumonia than young WT hamsters exposed to the same (or lower) dose or hACE2 hamsters exposed to a uniformly lethal dose of virus. Specifically, aged WT hamsters presented with a severe interstitial pneumonia through 8 d post-exposure (PE), while pulmonary regeneration was observed in young WT hamsters at that time. hACE2 hamsters exposed to 100 or 10 PFU virus presented with a minimal to mild hemorrhagic pneumonia but succumbed to SARS-CoV-2-related meningoencephalitis by 6 d PE, suggesting that this model might allow assessment of SARS-CoV-2 infection on the central nervous system (CNS). Our group is the first to use (¹⁸F-FDG) PET/CT to differentiate respiratory disease severity ranging from mild to severe in three COVID-19 hamster models. The non-invasive, serial measure of disease progression provided by PET/CT makes it a valuable tool for animal model characterization.

Keywords: 2-Deoxy-2-[fluorine-18]fluoro-D-glucose ((18)F-FDG) PET/CT; Animal models; COVID-19; Computed tomography (CT); Hamsters; SARS-CoV-2.

Fig. 1

Clinical signs of disease survival, body weight changes, and viral loads in all three hamster models. A. Survival of hamsters exposed to SARS-CoV-2 via intranasal inoculation. All WT hamsters survived virus exposure over the course of 8 d. Survival of hACE2 hamsters in a 10–14-d natural history study correlated with exposure dose. Significant differences were seen as p = 0.0013 (**). B. Individual relative body weight changes (weight loss in WT aged 10⁵ PFU in comparison to WT young 10³, 10⁵, hACE2 100, 10, 1, and 0.1 PFU; p < 0.0001***) over the duration of the disease. Data are presented as the mean percentage of the weight relative to 0 d (±SD). Statistical significance for average body weight was analyzed across the 8–14-d time course using a mixed-effects repeated measures model with Tukey's post-test multiple comparisons. C-F Virus loads were determined from C. RNA (per mg homogenized right cranial lung lobe) and infectious virus titers (per g homogenized right cranial lung lobe) (WT aged 10⁵ PFU in comparison to WT young 10⁵ PFU; p < 0.03*) and (D) nasal turbinates (E) Viral RNA from oropharyngeal samples collected by swabbing or lavage (per swab or per mL, respectively) to detect the virus shredding over the course of the experiment (F) Infectious virus titers in brain obtained at euthanasia. Horizontal lines show the mean. Points indicate data from individual hamsters. Solid symbols indicate viral RNA; open symbols indicate virus titers; dotted lines indicate the limit of detection; points indicate data from individual hamsters. One-way ANOVA with Tukey multiple comparison was performed in GraphPad Prism 9.3.1. p < 0.033 is *, p < 0.002 is **, and p < 0.001 is ***. SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; WT, wild-type; hACE2, human angiotensin-converting enzyme 2; SEM, standard error of the mean; SD, standard deviation; ANOVA, analysis of variance; LLOQ, lower limit of quantitation; PCR, polymerase chain reaction.

Fig. 2

Micro-PET/CT imaging of the lungs from three models of virus-exposed hamsters over an 8–10-d period. A. Representative chest micro-PET/CT images in the coronal plane. Columns from left to right show lung images from groups of aged WT hamsters (exposed to mock inoculum and 10⁵ PFU virus), young WT hamsters (exposed to mock inoculum, 10⁵ PFU virus, and 10³ PFU virus), and adult hACE2 hamsters (exposed to mock inoculum, 100 PFU virus, and 1 PFU virus). All hamsters were exposed to mock inoculum or SARS-CoV-2 intranasally. Rows from top to bottom are images show disease progress over time (baseline to 8 d). The scale bar indicates radiodensity value (in HU). B. Semi-quantitative average of the whole lung (sum of lobes) CT consolidation score (±SD) from three hamster models. C. Quantitative lung CT: average of PCLH (±SD) from three hamster models. D.¹⁸F-FDG PET images show ¹⁸F-FDG-avid areas, which generally correlated with CT lung consolidation. (PET images show the same lungs from panels in A.) The color bar indicates SUV. E. Quantitation of ¹⁸F-FDG uptake in lungs. Normalized SUVmax was calculated to reflect the longitudinal change of the ¹⁸F-FDG uptake in lungs at the different days post-exposure. Data are presented as the average of the normalized lung SUVmax (±SD). Imaging data were analyzed using GraphPad Prism 9.3.1. Two-way ANOVA with Tukey multiple comparison test was used to determine statistical significance. Micro-PET, micro-positron emission tomography; CT, computed tomography; WT, wild-type; hACE2, human angiotensin-converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; HU, Hounsfield Unit; SD, standard deviation; PCLH, percent change in lung hyperdense volume; ¹⁸F-FDG, 2-deoxy-2-[fluorine-18]fluoro-D-glucose; SUV, standardized uptake value; GGOs, ground-glass opacities; ANOVA, analysis of variance; BL, baseline; D2, day 2; D5, day 5; D8, day 8; LN, lymph node.

Fig. 3

¹⁸F-FDG uptake determined by tracking of the posterior nasal soft tissues using micro-PET/CT images. A. Representative ¹⁸F-FDG micro-PET/CT images from groups of WT aged hamsters (10⁵ PFU), young hamsters (mock, 10⁵ PFU, and 10³ PFU) and adult hACE2 hamsters (100 and 1 PFU) exposed to SARS-CoV-2 intranasally with different doses (columns from left to right) in the sagittal plane show metabolic activities located in the regions of interests (nasal tissues; white arrow) for each indicated study day (rows from top to bottom). B. Quantitation of ¹⁸F-FDG uptake in nasal cavities. Normalized nasal SUVmean was calculated to reflect the longitudinal change of the ¹⁸F-FDG uptake in the nasal and nasopharyngeal structures at the different days post-exposure. Data are presented as the mean percentage of the normalized nasal SUVmean (±SD). Imaging data were analyzed using GraphPad Prism 9.3.1. Two-way ANOVA with Tukey multiple comparison test was used to determine statistical significance. ¹⁸F-FDG, 2-deoxy-2-[fluorine-18]fluoro-D-glucose; micro-PET, micro-positron emission tomography; CT, computed tomography; WT, wild-type; hACE2, human angiotensin-converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; SUV, standardized uptake value; SD, standard deviation; ANOVA, analysis of variance; BL, baseline; D2, day 2; D5, day 5; D8, day 8.

Fig. 4

Histopathologic changes in the lungs and nasal turbinates. A. Histopathology (row 1: lungs; row 3: nasal turbinates) and immunochemistry (row 2: lungs; row 4: nasal turbinates) from aged WT, young WT, and hACE2 hamsters at 5 d (WT 10⁵ PFU and hACE2 100 PFU, respectively). At 5 d PE, aged WT hamsters showed a hemorrhagic interstitial pneumonia (upper-left panel) that lacked the robust, adenomatous, type II pneumocyte hyperplasia (upper-middle panel) seen in young hamster's lungs, while hACE2 hamsters exposed to 100 PFU of virus showed only a mild interstitial pneumonia at the same post-exposure time point. B. A heat map of histopathology scores in the lungs and nasal turbinates by day post-exposure and SARS-CoV-2 exposure dose in the three hamster models. An “X” indicates insufficient sample collection for comparison. Data were analyzed using GraphPad Prism 9.3.1, Two-way ANOVA with Tukey multiple comparison test. (WT aged 10⁵ PFU in comparison to hACE2 100 PFU, degeneration necrosis and interstitial pneumonia p < 0.05*). WT, wild-type; hACE2, human angiotensin-converting enzyme 2; PE, post-exposure; IHC, immunohistochemistry; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; NP, nucleocapsid protein; ANOVA, analysis of variance; D5, day 5; H&E, hematoxylin and eosin.

Fig. 5

Brain histopathology of hACE2 hamsters. Histopathology (A–C) and IHC (D–F) of the brain and olfactory bulb from one hACE2 hamster that sucumbed to disease at 5 d. A. Moderate, multifocal, neuronal necrosis of brainstem neurons (black arrows). B. Dense, perivascular lymphoplasmacytic cuffing. C. Lymphoplasmacytic meningitis. D. Strong cytoplasmic positivity in brainstem neurons for SARS-CoV-2 NP antigen. E. Most neurons in the basal ganglia showed strong cytoplasmic positivity for SARS-CoV-2 NP. F. Scattered juxtaglomerular cells within the olfactory bulb showed SARS-CoV-2 NP antigen positivity. G. Correlation heat map of brain histopathology and IHC scores among hACE2 hamsters. Data were analyzed using GraphPad Prism 9.3.1, Two-way ANOVA with Tukey multiple comparison test (at 5 d post-exposure, hACE2 100 PFU versus hACE2 0.1 PFU; p < 0.001 is ***). hACE2, human angiotensin-converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; NP, nucleoprotein; ANOVA, analysis of variance; H&E, hematoxylin and eosin; IHC, immunohistochemistry; T (5–6), terminal.

Fig. 6

Humoral systemic immune response and SARS-CoV-2 acute local immune gene activation. A. Median of endpoint SARS-CoV-2-specific IgG antibody in serum obtained at the indicated study days and measured by ELISA. (IgG significantly increased in WT young 10⁵ PFU and 10³ PFU with respect to WT aged 10⁵ PFU, p < 0.001*** and p < 0.05*). B. Reciprocal live virus neutralization titers in serum obtained at the indicated days post-exposure. Horizontal bars show the median. Points indicate data from individual hamsters. (WT aged 10⁵ PFU in comparison to hACE2 100; p < 0.05*) C. Heat map of cytokine and chemokine protein expression levels in lung homogenates collected from the three hamster models from different SARS-CoV-2 dose groups at the indicated time points. Heatmap are a representation of the log2 fold change of selected hamster chemokine/cytokine RNA levels as determined by RT-qPCR on lung extracts and normalized against RPL18 mRNA levels. (IL2 reduced significantly in WT aged and young 10⁵ PFU in comparison to hACE2 100 and 10 PFU; p = 0.019 is * and p = 0.0003 is ***. IL10 significantly increased WT aged and young; p < 0.0063 is * and p < 0.001 is ***.) SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; IgG, immunoglobulin G; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; hACE2, human angiotensin-converting enzyme 2; RT-qPCR, real-time reverse transcription polymerase chain reaction; mRNA, messenger RNA; IL, interleukin; WA01, Washington isolate; IFNG, interferon gamma; TNFA, tumor necrosis factor alpha; TGFB1, transformation growth factor beta 1; CCL, C–C motif ligand; IP-10, IFNG-induced protein 10 kDa; T (5–6), terminal.

Fig. 7

Correlation heatmap of imaging, pathology, and clinical data. Spearman correlations of lung imaging (CT consolidation score, PCLH, and normalized SUVmax), histopathology scores (interstitial pneumonia), clinical signs of disease (weight loss, viral shedding, lung titers) and neutralizing antibody titers at 5 d (Spearman correlation analysis, p < 0.05; significant correlations are shown in red). CT, computed tomography; PCLH, percent change in lung hyperdense volume; SUV, standardized uptake value; PET, positron emission tomography; Ab, antibody.