An immunosuppressed Syrian golden hamster model for SARS-CoV infection

Abstract

Several small animal models have been developed for the study of severe acute respiratory syndrome coronavirus (SARS-CoV) replication and pathogenesis. Syrian golden hamsters are among the best small animal models, though little clinical illness and no mortality are observed after virus infection. Cyclophosphamide was used to immunosuppress hamsters leading to a prolonged disease course and higher mortality after SARS-CoV infection. In addition, there was a significant weight loss, expanded tissue tropism, and increased viral pathology in the lung, heart, kidney, and nasal turbinate tissues. Infection with recombinant SARS-CoV viruses bearing disruptions in the gene 7 coding region showed no significant change in replication kinetics, tissue tropism, morbidity, or mortality suggesting that the ORF7a (7a) and ORF7b (7b) proteins are not required for virus replication in immunosuppressed hamsters. This modified hamster model may provide a useful tool for SARS-CoV pathogenesis studies, evaluation of antiviral therapy, and analysis of additional SARS-CoV mutants.

Fig. 1

Cyclophosphamide treatment results in immunosuppression of Syrian golden hamsters. (A) White blood cell (WBC) counts per mm³ from saline or CP-A treated hamsters were determined in uninfected (mock) or rSARS-CoV infected animals. Data points are from n = 1 mock infected, and n = 2–6 for all other samples. The WBC counts in saline-treated, mock infected animals did not change significantly over the course of the experiment. The average and standard error are graphed. (B) Complete WBC counts per mm³ at days-1 and 19 pi from saline or CP-D treated hamsters infected with rSARS-CoV. The average and standard error are graphed. (C) Serum neutralizing antibody titers from saline or CP-treated hamsters either mock infected or infected with the indicated recombinant SARS-CoV. Serum was collected from animals at days 19 and 42 pi, heat inactivated, and serial dilutions were assayed for neutralizing capacity against 100 TCID₅₀ of SARS-CoV. Data represent mean neutralizing titers.

Fig. 2

Daily mean weight and survival of saline and cyclophosphamide treated hamsters infected with rSARS-CoV. Groups of 3 golden Syrian hamsters were dosed with CP per the regimen specified in Table 1 and inoculated intranasally with either saline (A,B) or 10³ TCID₅₀ of rSARS-CoV (C,D), rSARS-CoV GFPΔORF7ab (E,F), or rSARS-CoV ΔORF7ab (G,H). Individual animals were weighed and data was normalized to animal weight on day 0 (A,C,E,G). Data represent average normalized weight and standard error. Percent survival of mock or CP-treated animals infected with each virus is shown (B,D,F,H). Black line, saline treatment; yellow line, CP-A; green line, CP-B; blue line, CP-C; red line, CP-D.

Fig. 3

SARS-CoV titers in tissues from immunosuppressed hamsters. (A) CP-B treated hamsters were infected intranasally with the indicated rSARS-CoV, sacrificed at the indicated times pi, and virus titers were measured in designated tissues. Open bars represent saline-treated animals; hashed bars represent CP-B treated hamsters. (B) CP-D treated hamsters were infected intranasally with either rSARS-CoV or rSARS-CoV ΔORF7ab virus. Animals were sacrificed at day 19 pi and virus titers were determined in the indicated tissues. Open bars represent saline-treated animals; hashed bars represent CP-D treated animals. (C) CP-B, CP-C, or CP-D treated hamsters were infected with rSARS-CoV or rSARS-CoV GFPΔORF7ab. Moribund animals were sacrificed and virus titers were measured in the indicated tissues. Mean days to moribundity for rSARS-CoV infected animals treated with CP-B, CP-C, and CP-D were 26.0 (n = 2), 31.3 (n = 3), and 25.3 (n = 3), respectively. Mean days to death for rSARS-CoV GFPΔORF7ab infected animals treated with CP-B, CP-C, and CP-D were 26.5 (n = 2), 31.0 (n = 3), and 27.0 (n = 3), respectively. TCID₅₀ limit of detection = 4.64 × 10² TCID₅₀/mL (D) CP-D treated hamsters were infected with rSARS-CoV or treated with saline. Moribund animals were sacrificed along with uninfected CP-D and age-matched saline-treated animals. Virus titer was determined in heart tissue. In all panels, virus titers from n = 3 animals were determined by TCID₅₀.assay, and presented as means and standard error.

Fig. 4

Histopathology of SARS-CoV infected hamsters. Hematoxylin and eosin (H and E) stain of pulmonary and extrapulmonary tissues. (A) Histopathologic lesions in the lungs of rSARS-CoV infected hamsters treated with CP-D. (i) Bronchoalveolar junction of control animal lung. (ii, iii, iv) Temporal changes in the lung of hamsters at 5, 19, and 23 days pi, respectively. In animals sacrificed moribund (n = 1 at day 23 pi and n = 2 at day 24 pi) (iv), changes were severe, widespread and frequently associated with hemorrhage. (B) Myocardium sections of (i) uninfected and (ii) rSARS-CoV infected, CP-D treated hamsters at 19 days pi. Subacute inflammation in the myocardium was present in rSARS-CoV infected animals, as well as in those animals sacrificed moribund. (C) Nasal turbinates of (i) uninfected and (ii) rSARS-CoV infected, CP-D treated hamsters 19 days pi.