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

Abstract

Following emergence in late 2019, SARS-CoV-2 rapidly became pandemic and is presently responsible for millions of infections and hundreds of thousands of deaths worldwide. There is currently no approved vaccine to halt the spread of SARS-CoV-2 and only very few treatment options are available to manage COVID-19 patients. For development of preclinical countermeasures, reliable and well-characterized small animal disease models will be of paramount importance. Here we show that intranasal inoculation of SARS-CoV-2 into Syrian hamsters consistently caused moderate broncho-interstitial pneumonia, with high viral lung loads and extensive virus shedding, but animals only displayed transient mild disease. We determined the infectious dose 50 to be only five infectious particles, making the Syrian hamster a highly susceptible model for SARS-CoV-2 infection. Neither hamster age nor sex had any impact on the severity of disease or course of infection. Finally, prolonged viral persistence in interleukin 2 receptor gamma chain knockout hamsters revealed susceptibility of SARS-CoV-2 to adaptive immune control. In conclusion, the Syrian hamster is highly susceptible to SARS-CoV-2 making it a very suitable infection model for COVID-19 countermeasure development.

Figure 1:. SARS-CoV-2 spike receptor binding data.

A VSV pseudotype assay was used to assess the binding affinity of the SARS-CoV-2 RBD. BHK cells expressing either the human or Syrian hamster or no ACE2 receptor were infected with VSV-pseudotyped particles carrying either the SARS-CoV-1 spike (S) protein or a chimeric SARS-CoV-1 spike with the SARS-CoV-2 receptor binding domain (RBD). Note: red circles, SARS-CoV-1 S; yellow circles with red outline, SARS-CoV-1 S with SARS-CoV-2 RBD; black circle, no ACE-2; S, spike protein; RBD, receptor binding domain.

Figure 2:. Susceptibility of Syrian hamsters to SARS-CoV-2.

Syrian hamsters were inoculated intranasally with 10-fold limiting dilutions of SARS-CoV-2 beginning at 10³ TCID₅₀. Weights were collected daily and shedding was assessed via swab samples (nasal and rectal) collected at 3dpi and 5dpi. Viral loads were determined as genome copies and infectious virus. (A) Daily weights. (B) Shedding at 3dpi. (C) Shedding at 5dpi. (D) Viral genome load in the lungs at 5dpi. (E) Infectious lung titers at 5dpi. A statistical significance was found between the groups presented in (A), with the group receiving the highest dose of 10³ TCID₅₀ losing the most weight. The group receiving the second highest infectious dose (10² TCID₅₀) lost statistically less than the 10³ TCID₅₀ group but statistically more weight than the 2 groups receiving the two lowest infectious doses. (B-E) A statistically significance difference was found between the group receiving the lowest dose (10⁰ TCID₅₀) and all other groups. Multiple t tests comparing groups directly were used to analyze significance. Note: blue circles, 10⁰ TCID₅₀ dose; red square, 10¹ TCID₅₀ dose; green triangle, 10² TCID₅₀ dose; purple triangle, 10³ TCID₅₀ dose.

Figure 3:. Increased infectious dose does not affect shedding or disease severity.

Syrian hamsters were infected intranasally with either 500 ID₅₀ (10³ TCID₅₀) or 5×10⁴ ID₅₀ (10⁵ TCID₅₀) of SARS-CoV-2. Samples were collected at the time points noted. Weight were collected daily, shedding from mucosal membranes and viral genome load and infectivity in the lungs were measured. (A) Daily weights. (B) Viral genome load recovered from nasal swabs. (C) Viral genome load recovered from rectal swabs. (D) Viral genome load in the lungs. (E) Infectious titers in the lungs. T-tests were used to compare the two groups at each time where samples were collected. A significant difference was observed at 10dpi in the lung titers (E), but no other significant differences were observed in this study. Note: blue circles, 10⁵ TCID₅₀ dose; red square, 10³ TCID₅₀ dose.

Figure 4:. SARS-CoV-2 infection of Syrian hamsters results in broncho-interstitial pneumonia.

Syrian hamsters were infected intranasally with 500 ID₅₀ (10³ TCID₅₀) of SARS-CoV-2. Lungs were fixed in 10% formalin, cut and stained with Hematoxylin and Eosin (HE) to examine pulmonary pathology at 3, 5 and 10dpi. (A-C), 3dpi. (A) Inflammation initiates within interstitial spaces in and around terminal airways with a minimal cellular exudate into the airway spaces (100x, size bar is 50um). (B) Bronchiolar epithelial necrosis with influx of neutrophils into the mucosa and airway lumen (400x, size bar is 20um). (C) Attenuation of the tracheal mucosa with loss of apical cilia accompanied with an influx of moderate numbers of degenerate and non-degenerate neutrophils (400x, size bar is 20um). (D-F), 5dpi. (D) Locally extensive inflammation is noted (100x, size bar is 50um). (E) Progressive bronchiolitis with degenerate and non-degenerate neutrophils and exudate within the lumen and prominent epithelial syncytial cells (arrows; 400x, size bar is 20um). (F) Alveolar spaces contain macrophages and neutrophils. Alveolar septa are thickened and expanded by fibrin, edema fluid and infiltrating leukocytes and are lined by prominent type II pneumocytes (arrowhead) and rare syncytial cells (arrow; 400x, size bar is 20um). (G-I), 10dpi. (G) Resolving inflammation is largely limited to bronchioles and the adjacent alveolar spaces (100x, size bar is 50um) (H) Alveolar septa are thickened by collagen with lymphocytes and lined by numerous plump type II pneumocytes that surround low numbers of foamy alveolar macrophages (400x, size bar is 20um). (I) Multifocal pleural fibrosis is evident with mild subpleural inflammation (200x, size bar is 20um).

Figure 5:. SARS-CoV-2 viral antigen in the lungs over the course of infection.

Syrian hamsters were infected intranasally with 500 ID₅₀ (10³ TCID₅₀) of SARS-CoV-2. Histopathology (HE) and immunohistochemistry (IHC) was used to assess pathology with the presence of SARS-CoV-2 antigen in pulmonary sections at 3, 5 and 10dpi. (A-C), 3dpi. (A) Histopathology is largely limited to bronchioles and terminal airway spaces and is not readily apparent at a low magnification (H&E, 20x, size bar is 200um). (B) Immunohistochemical reaction highlights antigen distribution in bronchioles and terminal airway spaces (20x, size bar is 200um). (C) Bronchiolar epithelial cell immunoreactivity with limited antigen detection in alveolar spaces (100x, size bar is 50um). (D-F), 5dpi. (D) Extension of cellular exudate from bronchioles into alveolar spaces (H&E, 20x, size bar is 200um). (E) Immunoreactivity is detected along the periphery of regions of pathology and has largely been cleared from bronchiolar epithelium (20x, size bar is 200um). (F) Immunoreactivity is noted in type I and type II pneumocytes and few alveolar macrophages (200x, size bar is 20um). (G-I), 10dpi (G) Resolving inflammation is limited to bronchioles and adjacent terminal airways (H&E, 20x, size bar is 200um). (H) SARS-CoV-2 immunoreactivity is not observed in regions of resolving inflammation (20x, size bar is 200um). (I) No immunoreactivity is observed (200x, size bar is 20um).

Figure 6:. Neither age nor sex affects shedding or disease following infection with SARS-CoV-2.

To compare the effects of aging and sex on disease following SARS-CoV-2 infection, young female and male (4–6weeks) and aged female and male (>6months) Syrian hamsters were infected intranasally with 500 ID₅₀ (10³ TCID₅₀) of SARS-CoV-2. Samples were collected at the time points noted. Weights were collected daily, shedding and viral loads in the lungs were measured. (A) Daily weights. (B) Viral genome load recovered from oral swabs at each time point. (C) Viral genome load recovered from rectal swabs at each time point. (D) Viral genome load recovered from lungs at each terminal point. (E) Infectious titers in the lungs. ANOVA was used to compare groups at each time where samples were collected. No significant differences were observed between groups at any time point collected in in this study. Note: blue circles, aged females; red circle, aged male; green circle, young female; purple circle, young male.

Figure 7:. SARS-CoV-2 infection of Interleukin-2 receptor subunit gamma knockout hamsters (IL2RG^−/−) results in persistent infection and pneumonia.

IL2RG KO hamsters lacking mature B-cells, T-cells and NK cells, were infected with 5×10⁴ ID₅₀ (10^5 TCID₅₀) and followed for 24 days to determine if disease developed. Weights were collected daily and shedding from mucosal membranes and viral infectivity in the lungs were measured at the time points noted. (A) Daily weights. (B) Viral genome load recovered from oral and rectal swabs at each time point. (C) Infectious titers in the lungs. (D) Alveoli frequently contain macrophages, neutrophils and sloughed epithelial cells, and are lined by numerous hyperplastic type II pneumocytes (H&E, 200x). (E) Immunoreactivity is observed in hyperplastic type II pneumocytes and macrophages (Anti-SARS-CoV-2 nuclear protein, 200x). Note (B): blue circles, oral swabs; red circle, rectal swabs. Size bar is 100um.