Pathogenesis of Influenza A/H5N1 virus infection in ferrets differs between intranasal and intratracheal routes of inoculation

Abstract

Most patients infected with highly pathogenic avian influenza A/H5N1 virus develop severe pneumonia resulting in acute respiratory distress syndrome, with extrarespiratory disease as an uncommon complication. Intranasal inoculation of ferrets with influenza A/H5N1 virus causes lesions in both the respiratory tract and extrarespiratory organs (primarily brain). However, the route of spread to extrarespiratory organs and the relative contribution of extrarespiratory disease to pathogenicity are largely unknown. In the present study, we characterized lesions in the respiratory tract and central nervous system (CNS) of ferrets (n = 8) inoculated intranasally with influenza virus A/Indonesia/5/2005 (H5N1). By 7 days after inoculation, only 3 of 8 ferrets had a mild or moderate bronchointerstitial pneumonia. In contrast, all 8 ferrets had moderate or severe CNS lesions, characterized by meningoencephalitis, choroiditis, and ependymitis, and centered on tissues adjoining the cerebrospinal fluid. These findings indicate that influenza A/H5N1 virus spread directly from nasal cavity to brain, and that CNS lesions contributed more than pulmonary lesions to the pathogenicity of influenza A/H5N1 virus infection in ferrets. In comparison, intratracheal inoculation of ferrets with the same virus reproducibly caused severe bronchointerstitial pneumonia. The method of virus inoculation requires careful consideration in the design of ferret experiments as a model for influenza A/H5N1 in humans.

Figure 1

Comparison of histological lesions and viral antigen expression in lungs of ferrets inoculated with influenza virus A/Indonesia/5/2005 (H5N1) intranasally (A–C; present study) or intratracheally (D–F; Ref. 16). A and B: Absence of lesions in the lung of an intranasally inoculated ferret. C: Absence of cells expressing virus antigen in alveoli of an intranasally inoculated ferret. Inset: Rare type II pneumocyte expressing virus antigen. D: Bronchointerstitial pneumonia, with inflammatory exudate in bronchiolar and alveolar lumina, in the lung of an intratracheally inoculated ferret. E: Neutrophils, macrophages, cellular debris, and erythrocytes in the alveolus of an intratracheally inoculated ferret. F: Epithelial cells, mainly type II pneumocytes, expressing virus antigen in alveoli of an intratracheally inoculated ferret. Original magnification: ×200 (A and D); ×1000 (B, C, E, and F).

Figure 2

Histological lesions and viral antigen expression in the CNS of ferrets inoculated intranasally with influenza virus A/Indonesia/5/2005 (H5N1). A: Leptomeninges of the cerebrum infiltrated with many inflammatory cells, mainly macrophages. B and C: Influenza viral antigen in mesothelial cells of the leptomeninges. D: Infiltration of mainly macrophages in the connective tissue of the choroid plexus, and segmental dissociation of the epithelial layer. E and F: Influenza viral antigen in epithelial cells of the choroid plexus. G: Segmental loss of the epithelial layer of the ependyma, and infiltration of mainly macrophages. H and I: Influenza viral antigen in epithelial cells of the ependyma. J: Infiltration of inflammatory cells, vacuolation, and neuronal necrosis in the cerebrum. K and L: Influenza viral antigen in neurons and glial cells of the cerebrum. M: Infiltration of inflammatory cells, vacuolation, and neuronal necrosis in the olfactory bulb. N and O: Influenza viral antigen in glial cells and neuronal cells of the olfactory bulb. Original magnification: ×400 (A, B, D, E, G, H, J, K, M, and N); ×1000 (C, F, I, L, and O).