Eight-week-old female FVB/J mice weighing 20–25 g were obtained from Charles River Laboratories (L’Arbresle, France). Challenge studies were conducted under BioSafety Level 3 laboratory conditions and in facilities accredited by the Belgian Council for Laboratory Animal Science, under the guidance of the Institutional Animal Care and Use Committees of the Veterinary Agrochemical Research Center and University of Liège. The mice were housed in microisolator cages ventilated under negative pressure with HEPA-filtered air. The light/dark cycle was 12/12 h, and the animals were allowed free access to food and water. Before each inoculation or euthanasia procedure, the animals were anesthetized by intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (30 mg/kg).

Two influenza A virus strain subtypes that had low pathogenicity for laboratory mice were used in this study: a clade 1 avian influenza virus (H5N1) (A/crested_eagle/Belgium/1/2004), and a porcine influenza virus (H1N1) (A/swine/Iowa/4/76). Both viruses were first propagated in the allantoic cavity of 10-day-old embryonating hen eggs and then adapted to the mice by lung-to-lung passaging. At each passage, a set of mice were inoculated intranasally with 50 µL of either allantoic fluid or lung homogenate containing influenza A virus. At 5 days postinoculation (dpi), the mice were killed humanely by an overdose of pentobarbital, followed by exsanguination. The lungs were combined and homogenized in phosphate-buffered saline (PBS)–penicillin-streptomycin, the homogenates were centrifuged at 3,000 g for 10 min, and the supernatant was used for the next passage. The process was stopped when the mice showed a substantial loss of bodyweight on 4 dpi. This occurred after 5 (H5N1) or 31 (H1N1) passages. Lung homogenates from the last passage were homogenized and divided into aliquots for direct use in pathotyping studies, and their titers were determined by standard plaque (subtype H1N1) or median tissue culture infective dose assays (H5N1). Serial dilutions of each adapted virus stock were then injected into FVB/J mice, and the 50% mouse lethal dose (MLD₅₀) was calculated according to the method of Reed and Muench (5).

For assessment of virus-induced pathogenicity, 2 series of mice were inoculated intranasally with 10 MLD₅₀ of virus by instillation of 50 µL of diluted stock. Mice were monitored daily for changes in bodyweight to assess virus-induced illness. At selected intervals, 5 (virus titration or histopathology) or 10 (virus titration + dry/wet weight ratio) mice were given an overdose of sodium pentobarbital and exsanguinated by cutting the brachial artery. Lungs and pieces of heart, liver, spleen, pancreas, kidney, brain, and adipose tissue from 5 mice were fixed in 4% neutral-buffered, ice-cold paraformaldehyde, routinely processed, and embedded in paraffin for histopathologic evaluation. Five-micrometer sections were stained with hematoxylin and eosin (HE) or periodic acid–Schiff (PAS) for lesion detection. For virus detection, sections were stained by a streptavidin-biotin complex immunoperoxidase method. An in-house immunoglobulin (Ig) G–purified polyclonal rabbit antiserum raised against recombinant influenza virus nucleoprotein was used as the source of primary antibodies, and horseradish peroxidase (HRP)–conjugated anti–rabbit IgGs (Dako, Glostrup, Denmark) were used as secondary antibodies. Peroxidase was indicated by the bright red precipitate produced in the presence of 3-amino-9-ethyl-carbazole, and sections were counterstained with Mayer hematoxylin. For virus titrations, lungs from 5 mice were weighed, homogenized in 1 mL PBS, and clarified. The supernatants were used for virus titration by plaque or median tissue culture infectious dose assays. Because the appearance of a biphasic expiratory pattern has been shown to announce death within ≈24 h (6), this qualitative sign was chosen, for humane reasons, as the endpoint of the experimental disease. On this endpoint day, lungs from 5 mice were sampled and weighed, and homogenates thereof were desiccated for dry weight determination.

The influenza A virus strains (subtypes H1N1 and H5N1) used in this study were isolated, respectively, from a diseased pig in the United States in 1976 and from a crested eagle smuggled from Thailand in 2003 (7). Both were nonpathogenic for FVB/J mice (MLD₅₀ >10⁶ PFU/50% tissue culture infective dose [TCID₅₀]). After adaptation, the strains showed a similar pathogenic outcome in FVB/J mice, i.e., close MLD₅₀ values: 3.2 PFUs for the subtype H1N1 strain and 6.4 TCID₅₀ for the subtype H5N1 strain. These results allowed a relevant comparison of their respective pathologic signatures. Overall, virus-associated illness, bodyweight loss, and gross lesions caused by inoculation of 10 MLD₅₀ were similar for both viruses, except that body condition and respiratory function deteriorated far more rapidly after subtype H5N1 inoculation, the endpoint being reached on 4 dpi for subtype H5N1–induced disease and 8 dpi for subtype H1N1–induced disease. The pathologic processes caused no symptoms for the first 2 (H5N1) or 3 (H1N1) days and then gave rise to general signs such as gradually slower, less frequent, and more erratic spontaneous displacements and a ruffled coat. By 3 dpi (H5N1) or 5 dpi (H1N1), all mice became lethargic and abruptly showed clinical signs of respiratory disease, including respiratory distress, labored breathing, and forced expiration. Mice inoculated with subtype H5N1 lost 10% of their bodyweight during the last 48 hours before the endpoint day. In mice that were inoculated with subtype H1N1, weight loss was acute and biphasic: a 10% loss occurred between virus inoculation and the appearance of respiratory symptoms, and an additional 20% was lost during ARDS (Figure 1). Autopsies performed on the endpoint day of subtype H1N1 disease consistently showed dark, purplish, bulky, noncrepitant, liverlike lungs, findings compatible with a diagnosis of massive pulmonary congestion and consolidation. In subtype H5N1–inoculated mice, the lungs at endpoint were bulky, noncrepitant, and diffusely pinkish gray, which suggests a diagnosis of congestion with massive pulmonary edema. Mice inoculated with either virus had a lung wet weight at endpoint approximately double that of controls, but this weight gain was achieved during the last ≈24 hours in mice inoculated with subtype H5N1, whereas mice inoculated with subtype H1N1 showed a progressive lung weight increase over 96 hours, from 4 dpi to the endpoint day (Figure 2). At the endpoint, the dry/wet weight ratio of the lungs was ≈22% lower for subtype H5N1-infected mice (17.6% ± 1.1%) than for subtype H1N1-infected mice (21.4% ± 1.4%). No obvious gross lesions were observed in the heart, liver, spleen, kidney, brain, or perivisceral fat. The lung virus loads measured on 2, 4, 6, and 8 dpi are shown in Figure 3. The time required to reach the peak virus titer was the same for both virus strains. Death occurred at the peak lung virus concentration for subtype H5N1, but subtype H1N1–associated disease did not become fatal until 4 days after this peak, when virus clearance was already substantial (Figure 3).

An exhaustive list of the histopathologic lesions caused by the 2 viruses is given in Table A1. Some changes in lung morphology were identical for both viruses. First, a clear topographic extension of the lesions was perceptible between the first and the last day of infection, with centrifugal spreading from the terminal bronchioles or the alveoli adjacent to the airways. Qualitatively, all alterations characterizing the exudative phase of the histopathologic condition termed diffuse alveolar damage were identifiable, with intense congestion of the alveolar capillaries, marginated intracapillary neutrophils, necrosis of the alveolar epithelium, interstitial and alveolar edema, hyaline membranes, and invasion of the alveoli by (mostly) mononucleate cells. On the other hand, we did not observe cuboidalization of the alveoli (hyperplasia of type II pneumocytes) or hyperplasia or squamous metaplasia of the airway epithelia. These results indicate extremely rapid disease progression, nearly complete elimination of type II pneumocytes, or both. Despite these similarities, when sections of lung tissue samples taken on the last day from infected mice were pooled by subtype, an examiner unaware of which infection he was looking could easily distinguish one from the other (Figures 4, 5). The criteria for attributing lung lesions to the subtype H1N1 strain were the following: 1) earlier and much more extensive degeneration, necrosis, and desquamation of the airway epithelium; 2) a much higher cell density of the peribronchial, peribronchiolar, interstitial, and intra-alveolar infiltrates; 3) the presence of dense cuffs of mononucleate cells around the arterioles; 4) far less extensive alveolar edemas; and 5) the rarity of alveolar hemorrhages. The lesions caused by the subtype H5N1 strain were distinguishable by the late and mild regressive alterations of the airway epithelium, the extent of alveolar edema, the low cell density of the inflammatory infiltrates, the high number of alveolar hemorrhage foci, and the unusual appearance of the pulmonary arterioles (which seemed to have been dissected from the surrounding tissues because of the magnitude of the perivascular edema).

On the other hand, no arteriole showed any cuff of infiltrated mononucleate cells. Some blood-vessel walls also showed hemorrhage inside the muscle layer. No other organ examined was found to carry any histopathologic lesions except, notably, the liver in subtype H5N1–infected mice (Figure 5). These livers displayed multifocal necrosis, with necrotic foci consisting of aggregates of hypereosinophilic, pyknotic, and caryorhectic hepatocytes, admixed with a few neutrophils and lymphocytes. Such foci were also seen in the spleen in some animals. Strikingly, numerous PAS-positive islets were detected throughout the livers of subtype H5N1–infected animals, each overlapping with a necrotic focus. Patterns of centrolobular, hydropic, granular (2 dpi), centrolobular (3 dpi), and panlobular (4 dpi) microvesicular fatty degeneration were also observed in the livers of all subtype H5N1-infected animals. Interstitial hemorrhages were seen in the renal medulla.

The results of immunohistochemical tests were homogeneous for mice infected with the same strain. Overall, they showed that the subtype H1N1 strain swarmed centrifugally from the bronchioles throughout the lungs over 4–5 days, but remained strictly confined to the lungs. The subtype H5N1 virus, in contrast, conquered the whole lung over 24–48 hours; infected some bronchioles only later; and spread to the liver, pancreas, kidneys, spleen, brain, and perivisceral fat.

The virus was first detectable in the epithelium of the bronchi and bronchioles on 3 dpi. By 5 dpi, the stain was more conspicuous and appeared also in the alveolar epithelium of the areas adjacent to the airways. By 7 dpi, the virus was detectable in the epithelia of almost all bronchi and bronchioles and in the alveolar epithelium in extensive areas of the lungs. In the alveolar structures, staining showed the virus in type I and type II pneumocytes and in alveolar macrophages (Figure 6, panels A, C, and E). Nonrespiratory organs sampled on 3, 5, or 7 dpi remained strictly virus negative.

The virus was detectable from 2 dpi in some type II pneumocytes in peribronchiolar alveoli, some interstitial/alveolar macrophages, and some endothelial cells in the vicinity of the positive alveoli. In contrast, no nonrespiratory organ examined showed any virus-positive cells. By 3 dpi, staining of the airway epithelium was still discrete and limited, whereas the alveolar epithelium showed more pronounced staining, diffusely distributed throughout the lung. In the liver, multiple nests of positive hepatocytes were detectable, corresponding exactly with the above-mentioned necrotic PAS-positive foci. A few renal tubular epithelial cells were also positive. On 4 dpi, the alveolar epithelium was still diffusely stained, but more intensely than on 3 dpi. For the first time, staining of the bronchiolar epithelium was also visible, but not all bronchioles—far from all, in fact—showed this staining. Type II pneumocytes and alveolar macrophages were more often positive than type I pneumocytes (Figure 6, panels B, D, and F). The appearance of the kidneys and liver was the same as on 3 dpi, with more conspicuous staining. Additionally, virus-positive glial cells, splenic macrophages, cardiomyocytes, islets of Langerhans cells, and peritoneal adipocytes were also detected (Figure 7).