Co-infection of respiratory bacterium with severe acute respiratory syndrome coronavirus induces an exacerbated pneumonia in mice

Abstract

SARS-CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS-CoV infection in cultured cells, which suggests that SARS development may be due to elastase-mediated, enhanced SARS-CoV infection in the lungs. To explore this possibility, we examined whether co-infection of mice with SARS-CoV and Pp, a low-pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co-infected with SARS-CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33~90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co-infection with SARS-CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.

Figure 1

Elastase production in the lungs of mice after Pp infection or LPS administration. Mice inoculated i.n. with 1.5 × 10⁷ cfu of Pp suspended in 20 μl PBS (+) or mock‐infected mice (−) were killed one day after infection and elastase activity in BAL and lung homogenates were measured (a). Mice were inoculated i.n. with 2.3 × 10⁷ cfu of Pp or administered with 20 μl of LPS (1 mg/ml) dissolved in PBS and elastase activity in lung homogenates was determined one and two days after inoculation. Mock‐infected mice (No) were also examined for elastase activity (b). Mean values with standard deviation of three to five samples in each group are shown.

Figure 2

Body weights of mice infected with SARS‐CoV. Mice were infected i.n. with Pp (1.3 × 10⁷ cfu) and 1 day later with SARS‐CoV, either 1.1 × 10⁶ of p.f.u. Fr‐1 (a) or 0.8 × 10⁶ p.f.u. of Fr‐mo (b), and were weighed daily after Pp infection. Mice were administered i.n. with 20 μl of LPS (1 mg/ml), infected with 0.8 × 10⁶ p.f.u. of Fr‐mo 1 day later and weighed daily after LPS administration (c). Mean body weights are shown as a percentage compared with the mean weights of all mice measured just before Pp or LPS inoculation. Mice were inoculated with SARS‐CoV alone (○), Pp or LPS alone (▵) or Pp or LPS + SARS‐CoV (□). Two of six mice infected withPp + Fr‐mo died by day five p.i., and the body weight from days five to seven p.i. showed a significant difference when compared with those of Pp‐infected mice (*P < 0.001, **P < 0.005, ***P < 0.01 by Student's t test) (b). Three of six mice treated with LPS and infected with Fr‐mo died on day four, and body weights were significantly lower (*P < 0.001) on days three to four in comparison to those of mice treated with LPS alone (c).

Figure 3

Virus titers in the lungs of mice infected with Pp and/or SARS‐CoV: Mice infected (solid line) or mock‐infected (broken line) with Pp and one day later with Fr‐1 (a) or Fr‐mo (b), as described in the legend to Fig. 2, were killed on days two, four, six and eight, and virus titers in the lungs were determined by a plaque assay. Significant difference was shown (*P < 0.001, **P < 0.01 by Student's t test) (a and b). Mice were infected with Pp (2.0 × 10⁷ cfu) (black column) or mock‐infected (shaded column) and one day later further infected with 1 × 10⁴ (10⁴) or 1 × 10² (10²) p.f.u. of Fr‐mo. Virus titers in the lungs were examined on days two (2 d) and four (4 d) after Fr‐mo infection (c). Significant difference was shown (*P < 0.001, **P < 0.006 by Student's t test) (c). Virus titers are indicated in p.f.u. in log₁₀/50 mg tissue weight. Mean values of the titers with standard deviation are shown. Groups a and b consisted of four to five mice each and group c three mice.

Figure 4

Elastase activity in the lung of mice infected with Pp and SARS‐CoV. Lung homogenates from mice infected with Pp (black column) or mock‐ infected mice (shaded column) prepared on day two and four after SARS‐CoV (Fr‐1, Fr‐mo) or mock (no) infection were examined for elastase activity. Mean values of elastase activity with standard deviation are shown.

Figure 5

Comparison of cytokine concentrations in mice infected with Pp alone, Fr‐mo alone and those infected with Pp + Fr‐mo. Lung homogenates from Fr‐mo infected mice (shaded column) and Pp + Fr‐mo infected mice (black column) were examined for 20 different cytokines. Lung homogenates prepared from mice infected with Pp alone (white column) were also examined. The concentrations of three cytokines, IL‐1α (a), IP‐10 (b) and MIG (c) are illustrated, of which IP‐10 and MIG were significantly higher in the Pp + Fr‐mo infected mice than those infected with Fr‐mo alone, by Student's t test (IP‐10: P < 0.0025, MIG: P < 0.015, IL‐1α: P = 0.061).

Figure 6

Histopathological and immunohistochemical studies of lungs infected with Pp and/or SARS‐CoV. Exudation of hyaline substances into alveolar cavities, infiltration of macrophages and lymphocytes in alveolar walls, and hyperplastic changes of type 2 respiratory epithelial cells were observed in mice co‐infected withPp + Fr‐mo on day 4 (a), while the exudates in alveoli was not found in mice infected with Fr‐mo alone (b). Also, viral antigens (brown) were seen in the cytoplasm of respiratory epithelial cells (arrow) in mice infected withPp + Fr‐mo on day four (c). The lung lesion in the mouse that died on day four after infection byPp + Fr‐mo was similar to that seen in cases of diffuse alveolar damage, and was characterized by an exudative hyaline membrane (arrow) in the alveolar cavities (d,e). Antigens were detected with rabbit hyperimmune serum against SARS‐CoV using diaminobentizine for visualization, hematoxylin as a counterstain (c), and hematoxylin and eosin (the others) for routine stains. Original magnification, ×30 (a,b,c and e), ×6 (d).