Figure 1.
Survival rate, body weight and viral titers in lungs of PR8-infected mice.
Mice were intranasally inoculated with PR8 virus. (A) Survival rates of PR8-infected mice (n = 10). (B) Body weight of PR8-infected mice (0–5 days postinfection, n = 10; 6 days postinfection, n = 5). Percentages of mean body weight ± SD based on day 0 are shown. (C) Viral titers in lungs of PR8-infected mice (n = 5, each day) was measured by plaque forming assay using MDCK cells at 2, 4, and 6 days postinfection. Mean viral titers ± SD are shown. Representative results are shown.
Figure 2.
Macroscopic appearance, weight of lungs and water content in lungs of PR8-infected mice.
(A) Gross pathology of mouse lung. Large, edematous and dusky red in color in infected lung at 6 days postinfection (right panel). Lung of non-infected control (left panel). dpi, days postinfection. (B) Weight of lungs excised from PR8-infected mice (n = 5, each day) sacrificed at indicated days. Significant differences were observed on days 2, 4 and 6 when compared with day 0 as a control. (C) Percentages of lung water content in PR8-infected mice (n = 5, each day) determined by dry-wet ratio assay modified by incubation at 50°C for 24 h. Significant differences were observed on days 4 and 6 when compared with day 0 as a control. Differences in means ± SD and p values are shown. *p<0.05, ***p<0.001. Data represent three independent experiments.
Figure 3.
Serial histopathological changes in lungs infected with PR8 virus.
(A–D) Non-infected mice were sacrificed and excised the lungs as a control. (E–P) Mice (n = 6, each day) were infected with PR8 influenza virus and sacrificed at the indicated days after infection. (Q–T) Dead infected mice were performed postmortem examination immediately after their death. (A, E, I, M and Q) Magnification is ×40. (B–D, F–H, J–L, N–P and R–T) Magnification is ×400. (A, B, E, F, I, J, M, N, Q and R) HE staining. (C, G, K, O and S) Immunohistochemical staining using anti-influenza virus polyclonal antibody. (D, H, L, P and T) elastica van Gieson (EVG) staining. (E–H) Infected lung at 2 days postinfection. (E) Focal inflammation in the lung and invagination of the pulmonary pleura (arrowheads). (F) Congestion in thick alveolar septa and mild collapse. (G) Antigen-positive cells are stained red in alveoli around bronchioles. (H) Mild alveolar collapse. (I–L) Infected lung at 4 days postinfection. (I) Expansion of inflammation and invagination of the pulmonary pleura (arrowheads). (J) Alveolar collapse and cell debris (arrowheads) in bronchiolar lumen. (K) Antigen-positive cell debris in bronchiolar lumen (arrowheads). (L) Increased alveolar collapse. (M–P) Infected lung from live mice at 6 days postinfection. (M) Lung deformation with enlargement of alveolar duct and alveolar collapse. (N) Cell debris in bronchiolar lumen (arrowhead). (O) Antigen-positive cell debris in bronchiolar lumen (arrowheads). (P) Alveolar collapse and enlargement of alveolar ducts. (Q–T) Infected lung from dead mice at 6 days postinfection. (Q) Severe collapse throughout entire lung. (R) Hyaline membrane formation lining alveolar ducts (arrowhead) and collapse. (S) Antigen-positive cells in collapsed area. (T) Severe alveolar collapse. HE, hematoxyline-eosin staining. IHC, immunohistochemistry. EVG, elastica van Gieson staining.
Figure 4.
DAD with hyaline membrane formation demonstrated by Masson's trichrome staining.
Postmortem examinations of dead infected mice were performed immediately after their death. (A and B) Hyaline membrane stained pastel purple (arrowheads) in enlarged alveolar ducts and alveolar spaces throughout collapsed lungs. (A) Magnification, ×200. (B) Magnification, ×400. (C) Elastica van Gieson staining shows alveolar collapse throughout entire lungs. Magnification, ×200 (D) Influenza virus antigen-positive cells in collapsed area beside hyaline membrane. Magnification, ×400 (E–J) Hyaline membrane was not found in any lungs from the live infected mice at 2–6 days postinfection. (E, G, and I) Magnification, ×200. (F, H, and J) Magnification, ×400.
Figure 5.
Levels of SP-A and -D in sera from PR8-infected mice.
Upper and lower panels show SP-A and SP-D respectively, determined by Western blotting using the respective antibodies. Each lane was loaded with 10 µg of mouse serum without albumin and immunoglobulin.
Figure 6.
Hyaline Membrane Formation with a Low Dose of Virus.
(A–F) Postmortem examinations of dead mice infected with 1× MLD50 of PR8 virus were performed at the indicated days. (A, C, and E) Magnification, ×40. (B, D, and F) Magnification, ×400. (A) Alveolar collapse and expanded alveolar ducts on 10 days postinfection. (B) Hyaline membrane lining alveolar ducts (arrowheads). (C) Alveolar collapse and expanded alveolar ducts at 12 days postinfection. (D) Hyaline membrane lining alveolar ducts (arrowheads). (E) Alveolar collapse and expanded alveolar ducts at 14 days postinfection. (F) Hyaline membrane lining alveolar ducts (arrowheads). (G and H) surviving mice infected with 1× MLD50 of PR8 virus were sacrificed at 20 days postinfection for histopathological examination. (F) Alveolar collapse, expanded alveolar ducts, glandular metaplasia, and thick alveolar septa were observed without hyaline membrane formation. Magnification, ×40. (G) Remarkable glandular metaplasia. Magnification, ×400.