Influenza A Strain-Dependent Pathogenesis in Fatal H1N1 and H5N1 Subtype Infections of Mice

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Volume 16, Number 4–April 2010
Research
Influenza A Strain-Dependent Pathogenesis in Fatal H1N1 and H5N1 Subtype Infections of Mice
Mutien-Marie Garigliany, Adélite Habyarimana, Bénédicte Lambrecht, Els Van de Paar, Anne Cornet, Thierry van den Berg, and Daniel Desmecht
Author affiliations: University of Liège, Liège, Belgium (M.-M. Garigliany, E. Van de Paar, A. Cornet, D. Desmecht); and Veterinary Agrochemical Center, Brussels, Belgium (A. Habyarimana, B. Lambrecht, T. van den Berg)

Suggested citation for this article

Abstract
To determine if fatal infections caused by different highly virulent influenza A viruses share the same pathogenesis, we compared 2 different influenza A virus subtypes, H1N1 and H5N1. The subtypes, which had shown no pathogenicity in laboratory mice, were forced to evolve by serial passaging. Although both adapted viruses evoked diffuse alveolar damage and showed a similar 50% mouse lethal dose and the same peak lung concentration, each had a distinct pathologic signature and caused a different course of acute respiratory distress syndrome. In the absence of any virus labeling, a histologist could readily distinguish infections caused by these 2 viruses. The different histologic features described in this study here refute the hypothesis of a single, universal cytokine storm underlying all fatal influenza diseases. Research is thus crucially needed to identify sets of virulence markers and to examine whether treatment should be tailored to the influenza virus pathotype.

According to the World Health Organization, influenza annually infects 5%–15% of the global population, causing 3–5 million cases of severe illness and ≈500,000 reported deaths. The persistence of influenza A virus (H5N1) in poultry populations over the past 6 years and the ability of those viruses to cause fatal infections in humans, along with the recent pandemic (H1N1) 2009 outbreaks, have raised fears of a renewed catastrophic influenza outbreak comparable to that of 1918, which caused death in 0.2%–8% of those infected in various countries and ≈50 million deaths worldwide (1). Standard influenza symptoms include fever, cough, headache, sore throat, and dehydration, with some reports of diarrhea, vomiting, and bleeding from the mouth or throat. In benign cases, not all of these symptoms are exhibited. In severe cases, additional signs typical of either secondary bacterial pneumonia or acute respiratory distress syndrome (ARDS) occur. Notably, these 2 manifestations are those that cause death in patients with influenza, whether seasonal or pandemic or caused by the 1918 subtype H1N1 strain or by recent subtype H5N1 strains.

The catastrophic lethality of the 1918 pandemic makes it paramount that we understand the disease pathogenesis of both severe forms of influenza. Because most secondary bacterial pneumonias can be controlled with antimicrobial agents, prevention and treatment of influenza-associated ARDS are the major medical challenges that must be addressed to reduce the influenza-related death rate. This requires more knowledge about the pathogenesis of ARDS. Alterations in human and mouse lungs have been described for fatal virus infections with pandemic virus strains (subtypes H1N1, H2N2, and H3N2 strains of 1918, 1957, and 1968, respectively) or subtype H5N1 strains. They are all characterized by similar lung dysfunctions and lesions (2,3). The lung becomes flooded as its alveolocapillary membranes leak, and the alveoli fill with body fluids. Consequently, the exchange of carbon dioxide and oxygen is reduced, and fatal acute lung failure ensues. The histologic findings depend on the stage of the disease. Edema, epithelial necrosis, fibrin, and hyaline membranes are found during the early exudative phase, and fibroblast and type II cell hyperplasia are found during the proliferative phase. This array of morphologic alterations is known as diffuse alveolar damage. Moreover, mice infected with the 1918 influenza virus or with a recent subtype H5N1 human isolate also show considerable similarities in overall lung cellularity, composition of lung immune cell subpopulation, and cellular immune temporal dynamics (4). On the basis of these mostly retrospective studies, the pathogenesis of influenza-associated ARDS is widely viewed as being the same whatever the infecting strain.

In this study, we closely monitored ARDS in mice, caused by inoculation of identical doses of 2 different influenza strains rendered highly pathogenic toward mice by adaptation. The 2 strains elicited dramatically different disease courses and histopathologic signatures, although both strains caused death in 100% of those infected, evoked the expected diffuse alveolar damage, and led to comparable virus titers in the lungs. The pathogenesis underlying influenza-associated fatal ARDS thus depended on the infecting strain.

Materials and Methods
Animals
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).

Viruses
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).

Pathotyping Studies
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.

Suggested Citation for this Article
Garigliany M-M, Habyarimana A, Lambrecht B, Van de Paar E, Cornet A, van den Berg T, et al. Influenza A strain-dependent pathogenesis in fatal H1N1 and H5N1 subtype infections of mice. Emerg Infect Dis [serial on the Internet]. 2010 April [date cited]. http://www.cdc.gov/EID/content/16/4/595.htm

DOI: 10.3201/eid1604.091061

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http://www.cdc.gov/eid/content/16/4/595.htm