Acute respiratory distress syndrome induced by H9N2 virus in mice

Abstract

H9N2 avian influenza viruses have repeatedly caused infections in swine and humans in some countries. The purpose of the present study was to evaluate the pulmonary pathology caused by H9N2 viral infection in mice. Six- to eight-week-old BALB/c mice were infected intranasally with 1 x 10(4) MID(50) of A/Chicken/Hebei/4/2008(H9N2) virus. Clinical signs, pathological changes and viral replication in lungs, arterial blood gas, and cytokines in bronchoalveolar lavage fluid (BALF) were observed at different time points after infection. A control group was infected intranasally with noninfectious allantoic fluid. H9N2-infected mice exhibited severe respiratory syndrome, with a mortality rate of 60%. Gross observations showed that infected lungs were highly edematous. Major histopathological changes in infected lungs included diffuse pneumonia and alveolar damage, with neutrophil-dominant inflammatory cellular infiltration, interstitial and alveolar edema, hemorrhage, and severe bronchiolitis/peribronchiolitis. In addition, H9N2 viral infection resulted in severe progressive hypoxemia, lymphopenia, and a significant increase in neutrophils, tumor necrosis factor-alpha and interleukin-6 in BALF. The features described above satisfy the criteria for acute respiratory distress syndrome (ARDS). Our data show that H9N2 viral infection resulted in ARDS in mice, and this may facilitate studies of the pathogenesis of future potential H9N2 disease in humans.

Fig. 1

Gross pathology and histopathology of H9N2-virus-infected lung. a shows severe edema, congestion and hemorrhage in the infected lung (left) on day 4 p.i. (right, control lung). Histopathology with hematoxylin-eosin staining in infected lung is shown in b–h. On day 3 p.i., thickening of the alveolar wall, interstitial edema around small blood vessels (c, d, solid arrows) and dropout of mucous epithelium adhering to the surface of bronchioles (c, d, open arrows) were observed. On days 4–5 p.i., diffuse pneumonia (b, e, f) with severe edema and neutrophil-dominant inflammatory cellular infiltration around small blood vessels (e, f, solid arrows) and severe alveolar injury (e, f, open arrows) were observed. By day 6 p.i., more severe diffuse pneumonia with alveolar lumens flooded with edema fluid mixed with fibrin, erythrocytes and inflammatory cells was observed (g, h, open arrows), as well as severe peribronchiolitis (g, h, solid arrows). Original magnification: b, c, e, g ×10; d, f, h ×40

Fig. 2

Replication of H9N2 virus in mouse lungs. Mice were infected with 10⁴ MID₅₀ of Ck/HB/4/08 virus, tissues were collected on the indicated days p.i., and the virus was titrated in embryonated eggs. Mean viral titers based on three mice per group are expressed as log₁₀ EID₅₀ per milliliter ± SD. The limit of virus detection was 10^1.2 EID₅₀/ml for lungs

Fig. 3

Lung wet:dry weight ratios (a) and lung wet weight:body weight ratios (b) of H9N2-virus-infected mice. The means ± SD (n = 4) at each time point are shown. Open bars control group, dotted bars infection group. *p < 0.05 and **p < 0.01 compared with those in control mice

Fig. 4

Kinetic changes in circulating leukocytes (a) and blood differential counts (b) after H9N2 virus infection. The means ± SD (n = 4) at each time point are shown. a Solid triangles leukocytes of control mice, open squares leukocytes of infected mice, b Open bars monocytes, striped bars neutrophils, solid bars lymphocytes. *p < 0.05 and **p < 0.01 compared with those in control mice

Fig. 5

Changes in leukocyte (a) and neutrophil counts (b) in BALF after H9N2 virus infection. The means ± SD (n = 4) at each time point are shown. Open bars control group, solid bars infection group. *p < 0.05 and **p < 0.01 compared with those in control mice

Fig. 6

TNF-α and IL-6 levels in BALF and serum of mice infected with H9N2 viruses. TNF-α in BALF (a) and in serum (b); IL-6 in BALF (c) and in serum (d). Open bars control group, solid bars infection group. *p < 0.05 and **p < 0.01 compared with those in control mice