Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans

Abstract

Advanced age has repeatedly been identified as an independent correlate of adverse outcome and a predictor of mortality in cases of severe acute respiratory syndrome (SARS). SARS-associated mortality may exceed 50% for persons aged 60 years or older. Heightened susceptibility of the elderly to severe SARS and the ability of SARS coronavirus to replicate in mice led us to examine whether aged mice might be susceptible to disease. We report here that viral replication in aged mice was associated with clinical illness and pneumonia, demonstrating an age-related susceptibility to SARS disease in animals that parallels the human experience.

FIG. 1.

Clinical illness in aged BALB/c mice during SARS-CoV infection. (A) Mean percentages of change in weight for mock-infected (open squares) (n = 5) or SARS-CoV-infected (filled squares) (n = 6) mice. Error bars indicate standard errors. The results of one of four replicates (mean weight loss of 6 to 10%) are presented. Asterisks indicate statistically significant weight loss (P < 0.045). The mean initial weight of mice (at day 0) was 26.9 ± 0.5 g. Various findings for the lungs of mice infected with SARS-CoV are indicated below the graph. Plus signs indicate the presence of a finding, minus signs indicate the absence of a finding, and blanks indicate that no evaluation was made at that time point. (B) Replication of SARS-CoV in various tissues following intranasal inoculation. The mean virus titer is expressed as the log₁₀ TCID₅₀ per gram of tissue (n = 4). Filled bars, lungs; gray bars, nasal turbinates; open bars, liver; hatched bars, spleen. The limit of detection was 10^1.5 TCID₅₀/g of tissue (dashed line). Error bars indicate standard errors. All animal experiments were approved by the National Institutes of Health Animal Care and Use Committee, all work with infectious virus was performed inside a biosafety cabinet in a biosafety containment level 3 facility, and personnel wore powered air-purifying respirators (HEPA AirMate; 3M, Saint Paul, Minn.).

FIG. 2.

Histopathological and immunohistochemical findings for aged BALB/c mice 2 to 3 days following SARS-CoV infection. (A) SARS-CoV antigens (arrowheads) in ciliated columnar epithelium of nasal turbinates (day 2 p.i.); (B) SARS-CoV antigens (arrowhead) in the cytoplasm of bronchiolar epithelium and alveolar pneumocytes (day 2 p.i.); (C) necrosis and sloughing of respiratory epithelial cells in a bronchiole and adjacent, predominantly lymphohistiocytic, inflammatory cell infiltrates (day 2 p.i.); (D-F) extensive cellular debris in airway lumen comprised of necrotic epithelium and inflammatory cells (arrow) with abundant staining of SARS-CoV antigens (arrowheads) (day 3 p.i.). Antigens were detected with hyperimmune mouse anti-SARS CoV ascitic fluid at a 1:1,000 dilution. Stains used were immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain (A, B, E, and F) and hematoxylin and eosin stain (C and D). Original magnifications, ×50 (A-E) and ×100 (F).

FIG. 3.

Histopathological and immunohistochemical findings in aged BALB/c mice 5 to 13 days following SARS-CoV infection. (A) SARS-CoV antigens (arrowhead) in alveolar pneumocytes (day 5 p.i.); (B) focus of early alveolar damage showing intraalveolar edema (large arrows) and interstitial, predominantly lymphohistiocytic, inflammatory cell infiltrates (day 5 p.i.); (C) lymphohistiocytic inflammatory cell foci accompanied by fibroblast proliferation (small arrows) in pulmonary parenchyma (day 9 p.i.); (D) focus of residual alveolar damage in predominantly normal lung (day 13 p.i.). Antigens were detected with hyperimmune mouse anti-SARS-CoV ascitic fluid at a 1:1,000 dilution. Stains used were immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counterstain (A) and hematoxylin and eosin stain (B-D). Original magnifications, ×100 (A), ×25 (B, C), and ×12.5 (D).

FIG. 4.

Pulmonary cytokines detected by enzyme-linked immunosorbent assay in SARS-CoV-infected aged BALB/c mice. SARS-CoV-infected mice (shaded bars) (four mice per group) demonstrated elevated levels of cytokines IFN-α (A), IFN-γ (B), and TNF-α (C) compared to mock-infected, age-matched controls (open bars) (two mice per group) at days 2 and 3 p.i. Consistent rises in cytokine levels, defined as a >2-fold increase over mock levels for more than a single time point, were not observed for IL-10 (D), IL-12 (E), and IL-4 (F). Y-axes indicate pg of measured cytokine per g lung tissue. X-axes indicate days after inoculation with SARS- CoV. Bars indicate geometric means within each group. Samples were assayed in duplicate, and open circles represent mean values of duplicates. Error bars indicate standard errors of geometric means.