The Roborovski Dwarf Hamster Is A Highly Susceptible Model for a Rapid and Fatal Course of SARS-CoV-2 Infection

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated an unprecedented and yet-unresolved health crisis worldwide. Different mammals are susceptible to SARS-CoV-2; however, few species examined so far develop robust clinical disease that mirrors severe human cases or allows testing of vaccines and drugs under conditions of severe disease. Here, we compare the susceptibilities of three dwarf hamster species (Phodopus spp.) to SARS-CoV-2 and introduce the Roborovski dwarf hamster (P. roborovskii) as a highly susceptible COVID-19 model with consistent and fulminant clinical signs. Particularly, only this species shows SARS-CoV-2-induced severe acute diffuse alveolar damage and hyaline microthrombi in the lungs, changes described in patients who succumbed to the infection but not reproduced in any experimentally infected animal. Based on our findings, we propose the Roborovski dwarf hamster as a valuable model to examine the efficacy and safety of vaccine candidates and therapeutics, particularly for use in highly susceptible individuals.

Keywords: COVID-19; Campbell’s dwarf hamster (Phodopus campbelli); Djungarian hamster (Phodopus sungorus); Roborovski dwarf hamster (Phodopus roborovskii); animal model; coronavirus; histopathology; pneumonia; thrombosis.

Graphical abstract

Figure 1

Changes in Body Temperature and Body Weight Following SARS-CoV-2 Infection in Phodopus Hamsters (A–C) Temperature changes of (A) Roborovski dwarf hamsters, (B) Campbell’s dwarf hamsters, and (C) Djungarian hamsters over the course of infection (shown as means with SD of at least 12 [2 and 3 dpi], 7 [5 dpi], and 6 animals [after 5 dpi] per group). (D–F) Corresponding individual relative body weights of (D) Roborovski dwarf hamsters (n = 22), (E) Campbell’s dwarf hamsters (n = 24), and (F) Djungarian hamsters (n = 24) are given. The color code represents mock-infected animals (green) or Phodopus hamsters infected with the low dose (blue) or standard dose (red) (Wichmann et al., 2020). The dotted lines at 85% in (D)–(F) refer to 15% body weight losses (first defined humane endpoint). Mann-Whitney U tests (B, C, E, and F) and Kruskal-Wallis tests (A and D) for each time point revealed significant differences in body temperatures and body weights of infected versus non-infected Roborovski dwarf hamsters at 2 and 3 dpi (^∗∗∗p ≤ 0.0001, ^∗∗p ≤ 0.001).

Figure 2

Virus Loads in the Respiratory Tract and Whole-Blood Samples (A and B) Virus titers in 50-mg lung tissues were determined by plaque assay in Vero E6 cells (A) and virus genome copy numbers per 2.5-mg lung tissue as determined by qRT-PCR at different time points after infection (B). (C and D) Virus loads were also determined by qRT-PCR in (C) bucco-laryngeal swabs and (D) 2.5 μl of whole-blood samples. Kruskal-Wallis tests were employed to determine significant differences between the Phodopus hamsters. Shown are results for Roborovski dwarf hamsters infected with the standard dose (black circles, n = 2 at 2 dpi, n = 8 at 3 dpi) and low dose infection (white circles, n = 2 at 4 dpi, n = 1 at 5 dpi), Campbell’s dwarf hamsters (dark gray circles, n = 3 at all time points), and Djungarian hamsters (light gray circles, n = 3 at all time points). Significant differences are marked with asterisks (^∗) and indicate a p value of ≤ 0.05.

Figure 3

Virus Loads in Intestinal and Kidney Samples of Infected Roborovski Dwarf Hamsters Virus loads in 2.5 mg of (A) jejunal and (B) kidney tissues of animals infected with the standard dose (black circles) or low dose (white circles). Viral RNA copies were determined by qRT-PCR at different time points after infection and revealed systemic virus spread.

Figure 4

Lung Histopathology and In Situ Hybridization of SARS-CoV-2-Infected Roborovski Dwarf Hamsters (A–D) Following infection with 1 × 105 plaque-forming units (pfu, standard dose; A–D) P. roborovskii developed devastating diffuse lung damage at 3 dpi (A) with only mild necrosis of bronchial epithelium (BE) and bronchitis (B) but severe diffuse alveolar damage, including necrosis of alveolar epithelial cells (AECs; C, arrows), hyaline membranes and fibrin extravasation (C, arrowhead), hemorrhage and alveolar edema, as well as multiple hyaline thrombi in alveolar capillaries (C, inset, arrowheads; PAS reaction). Consistent with the homogeneous distribution of lesions, in situ hybridization localized viral RNA throughout the entire lungs predominantly within AEC-II (D, inset, arrowheads) but also in bronchi (D, hash) and macrophages, while AEC-I were completely spared. (E–H) Infection with 20-times-less virus (5 × 10³ pfu, low dose; E–H) still resulted in extensive pneumonia at 4 dpi (E) with inflammation and regeneration of BE (F, arrows), necrosis of AEC and inflammatory cells (G, arrow), as well as mainly neutrophilic and heterophilic infiltration (G, arrowhead). Viral RNA was detected with a distribution similar to the standard dose group; however, areas of dense inflammation had obviously cleared the virus (H, hash). Red, signals for viral RNA; blue, hemalaun counterstain. (I–L) Mock-infected animals developed none of the pathologic findings of animals infected with SARS-CoV-2. Bars: 1 mm (A, E, and I), 50 μm (B, C, F, G, J, and K), 200 μm (D, H, and L), and 20 μm (insets of C, H, and K).

Figure 5

Comparison of Genomic ACE-2 Sequences ACE-2 residues that interact with the receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein (S) are almost completely conserved among different species of the family Cricetidae (the only exception is ACE-2 residue 354G/E). The interacting residues of the two proteins are highlighted in grey shading. Amino acids are colored according to their physico-chemical properties: red, hydrophobic; blue, negatively charged; pink, positively charged; purple, small nonpolar; green, polar residue. Asterisk, fully conserved residue; colon, residue with very similar properties.