Acute respiratory distress syndrome induced by a swine 2009 H1N1 variant in mice

Abstract

Background: Acute respiratory distress syndrome (ARDS) induced by pandemic 2009 H1N1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 H1N1 infection. However, no animal model has been developed for ARDS caused by infection with 2009 H1N1 virus. Here, we present a mouse model of ARDS induced by 2009 H1N1 virus.

Methodology principal findings: Mice were inoculated with A/swine/Shandong/731/2009 (SD/09), which was a 2009 H1N1 influenza variant with a G222D mutation in the hemagglutinin. Clinical symptoms were recorded every day. Lung injury was assessed by lung water content and histopathological observation. Arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. Mice infected with SD/09 virus showed typical ARDS symptoms characterized by 60% lethality on days 8-10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines.

Conclusions/significance: These results suggested that we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ARDS caused by 2009 H1N1 virus.

Figure 1. Virus titers in lung (A), wet:dry weight ratio (B) and wet weight∶body weight ratios (C) of SD/09-infected mice.

Mice were inoculated i.n. with 10^2.5 pfu SD/09 viruses; tissues were collected at indicated times p.i. and viruses were titrated in MDCK cells. Body weight, lung wet and dry weight were determined and recorded. The lung wet weight∶body weight ratio and lung wet∶dry weight ratio were calculated and used as an indicator of lung edema. *p<0.05, **p<0.01, ***p<0.001, comparison between ratios obtained from the virus-infected and control groups. Bars represent means ± SD of data from three mice.

Figure 2. Hematoxylin-eosin stained sections from lung tissues of SD/09-infected mice.

Normal mouse lungs (A, B). On day 4 p.i., dropout of mucous epithelium and inflammatory cells adhered to the surface of bronchioles (C, D, solid arrows). On day 6 p.i., severe edema around blood vessels (E, open arrows); dropout of alveolar epithelial, erythrocytes, and inflammatory cells within alveolar spaces (E, F, Δ). On day 8 p.i., the virus caused more severe interstitial pneumonia (G, H, Δ), bronchiolitis (G, H, solid arrows) and peribronchiolitis (G, H, open arrows). On day 14 p.i., the interstitial tissue was thickened with fibrin exudation and organization (I, Δ). Lymphocytes and alveolar macrophages were the predominant inflammatory cells observed (J, solid arrows). Magnification: C, E, G, and I, ×100; A, D and H, ×200; B, F and J, ×400.

Figure 3. Masson's trichrome stained sections (A–C) and immunohistochemical staining for influenza A nucleoprotein (D–F) of the SD/09-infected mouse lung.

Alveoli were collapsed and filled with collagen fibers (A, B, open arrows); normal mouse lungs (C). Influenza A nucleoprotein (brown) in the epithelial cells of the bronchioles (D, solid arrows); viral nucleoprotein in abundant terminal bronchioles epithelial cells and few alveolar epithelial cells (E, solid arrows); normal mouse lungs (F). Magnification: A, ×100; B, C, D, E and F, ×400.

Figure 4. Kinetic analysis of circulating leukocytes (A) and lymphocyte (B) following SD/09 virus infection.

Peripheral blood samples were collected at indicated times p.i. The total number of leukocytes and differential blood counts for three individual mice were analyzed. *p<0.05 **, p<0.01, ***p<0.001, comparison between virus-infected and control groups.

Figure 5. Cytokine levels in virus-infected mouse lungs.

Mice were euthanized at indicated times p.i. The collected lungs were weighed, and 10% homogenates were prepared in cold PBS. Cytokine levels from lung homogenates were measured using the Cytokine Bead Array system. *p<0.05, **p<0.01, ***p<0.001, comparison between virus-infected and control groups.