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Address reprint requests to Thijs Kuiken, D.V.M., Ph.D., Department of Virology, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands
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.
Since the first human cases of infection with highly pathogenic avian influenza A/H5N1 virus were recognized in 1997, infection has been confirmed in more than 500 people, of whom approximately 60% have died (World Health Organization; http://www.who.int/csr/disease/avian_influenza/country/cases_table_2011_03_16/en/index.html, last accessed March 23, 2011). Typically, infected individuals develop a severe pneumonia, which often is fatal.
Writing Committee of the Second World Health Organization Consultation on Clinical Aspects of Human Infection with Avian Influenza A Virus Update on avian influenza A (H5N1) virus infection in humans.
In addition to the respiratory tract, influenza A/H5N1 virus has been detected in various extrarespiratory organs, particularly the central nervous system (CNS), both in a few human cases and in multiple experimental animal species.
The ferret (Mustela putorius furo) is considered a good animal model for influenza virus infection in humans and is frequently used to study the pathogenesis of influenza.
After intranasal inoculation, it is assumed that the virus spreads from the nose to the lower respiratory tract and, if pathogenic, causes pneumonia. From there, virus might spread systemically and cause extrarespiratory disease, including encephalitis. However, the route of spread to the CNS and the relative contribution of CNS disease to the pathogenicity of influenza A/H5N1 virus infection for ferrets are largely unknown. We therefore designed a study to determine the character and severity of lesions caused by influenza A/H5N1 virus in the respiratory tract and CNS after intranasal inoculation. To this end, ferrets were inoculated intranasally with highly pathogenic avian influenza virus A/Indonesia/5/2005 (H5N1) and multiple tissues were collected for macroscopic, microscopic, and immunohistochemical (IHC) evaluation after euthanasia at 7 days post inoculation (dpi). Results were compared with those of a previous study from our research group,
in which ferrets had been inoculated intratracheally with the same virus.
Materials and Methods
Virus Preparation
Influenza virus A/Indonesia/5/2005 (H5N1, clade 2.1) was propagated in confluent Madin-Darby canine kidney (MDCK) cells. After cytopathic changes were complete, culture supernatants were harvested, cleared by low-speed centrifugation, and stored at −80°C. The virus titer was determined in MDCK cells as described previously.
Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro.
Healthy outbred female ferrets between 6 and 12 months of age were purchased from a commercial breeder. All ferrets tested negative for the presence of antibodies against recent influenza A/H1N1 and A/H3N2 viruses, influenza virus A/Indonesia/5/2005, and Aleutian disease virus. During the experiment, ferrets were housed together and received food and water ad libitum. Eight ferrets were inoculated intranasally with 5 × 106 median tissue culture infectious dose (TCID50) influenza virus A/Indonesia/5/2005 in a total volume of 0.5 mL PBS under anesthesia with ketamine and medetomidine; sedation was reversed with atipamezole. Inoculation was performed by instilling 250 μL inoculum into each nostril while holding the ferret vertically with the head in line with the rest of its body. After instillation of the inoculum, the ferret was laid on its back with the head slightly raised to prevent the inoculum from dripping out of the nose. After inoculation, ferrets were checked daily for the presence of clinical signs. Before inoculation with influenza A/H5N1 virus and at 2, 4, 6, and 7 dpi, ferrets were weighed under anesthesia with ketamine. At 7 dpi, or earlier in case of ferrets that became moribund, animals were weighed and then euthanized by exsanguination under anesthesia with ketamine and medetomidine. Experimental procedures were approved by an independent animal ethics committee and were performed under biosafety level 3 conditions.
Pathological Examination and IHC
Necropsies were performed according to standard procedures. Area of affected lung was estimated visually. Samples of olfactory bulb, cerebrum, cerebellum, brainstem, lungs (all lobes of the right lung and the accessory lobe, after inflation with 10% neutral-buffered formalin), spleen, liver, and duodenum were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 4 μm. Tissue sections were stained with H&E for histological evaluation or with an immunoperoxidase method using a monoclonal antibody directed against the nucleoprotein of the influenza A virus for detection of virus-infected cells. An IgG2a isotype control for each tissue and a positive control tissue were included in each staining procedure.
Nasal swabs were collected from ferrets at 2, 4, 6, and 7 dpi and were stored at −70°C in Hank's balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/mL penicillin, 200 μg/mL streptomycin, 100 U/mL polymyxin B sulfate, 250 μg/mL gentamicin, and 50 U/mL nystatin (ICN Pharmaceuticals, Zoetermeer, The Netherlands). Samples from cerebrum (temporal lobe), lung (left cranial, medial, and caudal lobes), spleen, and duodenum were collected from ferrets at necropsy, snap-frozen on dry ice with ethanol, and stored at −70°C until further processing. Tissue samples were weighed and subsequently homogenized with a FastPrep-24 homogenizer (MP Biomedicals, Eindhoven, The Netherlands) in the same medium as used for nasal swabs and then were centrifuged briefly. Quadruplicate 10-fold serial dilutions of nasal samples and organ samples were used to infect MDCK cells, and hemagglutination activity of the culture supernatants collected 5 dpi was used as indicator of infection, as described previously.
Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro.
that were inoculated intratracheally with exactly the same batch of highly pathogenic avian influenza virus A/Indonesia/5/2005 (H5N1). In that study, 3 mL of inoculum containing 1 × 105 TCID50 virus was instilled into the trachea by catheter. The higher volume of inoculum was used to facilitate spread into the lungs. The lower dose of virus was used because intratracheal inoculation of this virus is known to be highly lethal to ferrets.
Severity of pneumonia due to new H1N1 influenza virus in ferrets is intermediate between that due to seasonal H1N1 virus and highly pathogenic avian influenza H5N1 virus.
Even so, all six ferrets were moribund by 4 dpi and either had died or were euthanized on humane grounds at that time point. Although the volumes used for intranasal (0.5 mL) and intratracheal (3 mL) inoculation differed, they are representative of the currently used methods in experimental influenza virus infections of ferrets.
Presence of gross lung lesions, area of affected lung tissue, relative lung weight, histopathological changes in the lungs, and virus titers in the lungs were measured according to the same methods as used for the intranasally inoculated ferrets. Semiquantitative comparison of pulmonary lesions in both intranasally and intratracheally inoculated ferrets was performed without knowledge of the identity of the ferrets. Four lung sections per ferret (a longitudinal section and a cross-section of the right cranial lung lobe and a longitudinal section and a cross section of the right caudal lung lobe) were scored for size and severity of inflammatory foci as described previously,
with the following modification. Five arbitrarily chosen fields in each lung section were examined for the presence of inflammation by light microscopy using a 10× objective. Each field was scored for size of inflamed area (1 = smaller than or equal to the area of a 10× objective, 2 = larger than the area of a 10× objective and smaller than or equal to the area of a 2.5× objective, and 3 = larger than the area of a 2.5× objective) and for severity of inflammation (1 = mild, 2 = moderate, and 3 = marked). The average cumulative value of size and severity of inflammation per field provided the histopathology score per ferret.
Results
Clinical Signs
From 1 dpi onwards, ferrets developed clinical signs including anorexia, diarrhea, and lethargy, as well as neurological signs. Neurological signs consisted of unsteady gait and uncontrolled movements. All eight ferrets had lost between 13% and 24% of their original body weight by 7 dpi. Two ferrets were found dead, at 5 dpi and at 7 dpi. One ferret was euthanized at 6 dpi because of the presence of severe clinical signs, in accordance with animal welfare regulations.
Macroscopic and Microscopic Observations
At necropsy, three of the eight ferrets had multifocal dark red areas of consolidation in the lungs. The estimated area of affected lung tissue per ferret was 8 ± 14% (mean ± SD) and the relative lung weight was 0.9 ± 0.48% (mean ± SD) (Table 1). No macroscopic lesions were seen in other organs, except for diffuse hepatic pallor in all eight ferrets.
Table 1Comparison of Pulmonary Disease between Ferrets Inoculated Intranasally or Intratracheally with Influenza Virus A/Indonesia/5/2005 (H5N1)
On histological examination, three of the eight ferrets had a mild or moderate multifocal bronchointerstitial pneumonia; the remaining five ferrets had no pulmonary lesions (Figure 1, A and B; Table 1, Table 2). In the affected areas, alveolar lumina were filled with cellular debris, edema fluid, erythrocytes, fibrin, and inflammatory cells (many macrophages and some neutrophils); alveolar walls showed epithelial necrosis, moderate hypertrophy and hyperplasia of type II pneumocytes, and infiltration with moderate numbers of macrophages and neutrophils. Bronchiolar lumina contained cellular debris, macrophages, and neutrophils; bronchiolar walls had loss of epithelium and infiltration with moderate numbers of macrophages and neutrophils. Bronchial lumina contained a few inflammatory cells and fibrin; bronchial walls contained a few inflammatory cells.
Figure 1Comparison 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.
). 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).
Table 2Presence of Histological Lesions and Viral Antigen Expression in Tissues of Ferrets (n = 8) Inoculated Intranasally with Influenza Virus A/Indonesia/5/2005 (H5N1)
Tissue
Histological lesion (no. of ferrets positive)
Virus antigen expression (no. of ferrets positive)
All eight ferrets had a moderate or severe diffuse nonsuppurative leptomeningitis in cerebrum, cerebellum, brainstem, and olfactory bulb (Figure 2A). The condition was characterized by an infiltrate in the subarachnoid space ranging from 4 to 20 cells in thickness and consisting of many large mononuclear cells (macrophages), a few lymphocytes, and rare neutrophils. Multifocally, there was dissociation of the superficial mesothelial cells of the arachnoid membrane and endothelial hypertrophy of the meningeal blood vessels.
Figure 2Histological 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).
Five of seven ferrets had a moderate or severe, necrotizing choroiditis (tissue was not collected from one of the eight ferrets) (Figure 2D).The connective tissue of affected choroid plexi had necrosis of cells and infiltration, with many macrophages; cellular debris, inflammatory cells, fibrin, and erythrocytes were present on the epithelial surface.
Six of seven ferrets had a moderate or severe necrotizing ependymitis (tissue was not collected from one of the eight ferrets). This was characterized by segmental loss of the ependymal cell lining and infiltration by many macrophages, although at some locations higher proportions of lymphocytes or neutrophils were present (Figure 2G).
All eight ferrets had a multifocal necrotizing encephalitis, which was moderate in cerebrum and olfactory bulb and mild in the brainstem. Affected areas were characterized by infiltration with neutrophils, necrosis of neurons, gliosis, and vacuolization of neuropil (Figure 2, J and M). Affected areas in the cerebrum were superficial and localized adjacent to the inflammatory lesions present in leptomeninges, ependyma, and olfactory bulb. Affected areas in the olfactory bulb were present throughout the tissue.
All eight ferrets had mild diffuse hepatic lipidosis, consistent with anorexia. None of the eight ferrets had microscopic lesions in spleen or duodenum.
Expression of Viral Antigen
Virus antigen expression was detected in the CNS of all eight ferrets and in the respiratory tract of three of the eight ferrets (Table 2). In the CNS, moderate or abundant virus antigen expression was detected in mesothelial cells of cerebral leptomeninges overlying the frontal lobe (Figure 2, B and C), in epithelial cells of choroid plexus (Figure 2, E and F) and ependyma (Figure 2, H and I), and in neurons and glial cells of cerebrum (Figure 2, K and L), brainstem, and olfactory bulb (Figure 2, N and O). Of note, virus-positive cells in cerebrum and brainstem were located superficially, adjacent to meninges, ependyma, and olfactory bulb, whereas virus-positive cells in the olfactory bulb were located in the central part of this tissue.
In the respiratory tract, scant virus antigen expression was detected in the lungs, mainly in type II pneumocytes (Figure 1C). None of the eight ferrets showed virus antigen expression in spleen, liver, or duodenum.
Virus Titers in Nasal Swabs and Organs
Influenza virus was isolated from nasal swabs of all eight ferrets at 2, 4, and 6 dpi and in three out of seven ferrets at 7 dpi. Mean virus titers were 4.9 (SD = 0.7) at 2 dpi, 4.7 (SD = 0.6) at 4 dpi, 3.1 (SD = 1.4) at 6 dpi, and 0.6 (SD = 0.2) at 7 dpi. Influenza virus was isolated from lung from two of seven ferrets at 7 dpi (Table 1), but not from cerebrum, spleen, or duodenum of any ferrets. The discrepancy between virological results for the cerebrum and IHC results for this tissue (see Table 2, expression of viral antigen) may be because samples for virus isolation were collected from the parietal lobe, in which virus antigen expression was not observed.
Comparison of Pulmonary Disease in Intranasally and Intratracheally Inoculated Ferrets
From 3 dpi onward, intratracheally inoculated ferrets showed severe clinical signs, including breathing difficulties, lethargy, decreased appetite, and weight loss, but no specific neurological signs.
At necropsy, all six intratracheally inoculated ferrets had multifocal or diffuse dark red areas of consolidation in the lungs, affecting a larger area of lung tissue and associated with a higher relative lung weight than in intranasally inoculated ferrets (Table 1). On histological examination, all six intratracheally inoculated ferrets had severe multifocal or extensive bronchointerstitial pneumonia (Figure 1, D and E), with abundant virus antigen expression in alveolar epithelial cells, mainly type II pneumocytes (Figure 1F). The histological lesions were similar in character to those in intranasally inoculated ferrets, except that there were more inflammatory cells in alveolar, bronchiolar, and bronchial lumina and walls, and more extensive epithelial necrosis in alveolar, bronchiolar, and bronchial walls. From semiquantitative analysis, the mean histopathology score for the intratracheally inoculated ferrets was more than four times higher than that for the intranasally inoculated ferrets (Table 1). From virological analysis, infectious virus was detected in all six intratracheally inoculated ferrets at a mean virus titer approximately five times higher than that in the intranasally inoculated ferrets (Table 1).
Discussion
In the present study. all the ferrets inoculated intranasally with this influenza A/H5N1 virus developed widespread and often fatal CNS disease, characterized by nonsuppurative meningoencephalitis, choroiditis, and ependymitis. Surprisingly for a highly pathogenic respiratory virus inoculated into the respiratory tract, bronchointerstitial pneumonia developed in only a minor proportion of ferrets, and was milder than the CNS disease.
Comparison of our results with those from other influenza A/H5N1 virus infections in experimental animals points to the importance of route of inoculation for primary disease presentation and character of CNS lesions.
First, route of inoculation is important for primary disease presentation. Intranasal inoculation in our ferrets resulted in primary disease of the CNS rather than of the respiratory tract. These findings are consistent with those of previous studies,
in which intranasal inoculation of influenza A/Vietnam/1203/04 (H5N1) in ferrets resulted in death from severe neurological disease but caused only mild lung lesions. The authors suggested that these results were likely due to the low virus inoculation dose used [10 or 102 median egg infectious dose (EID50) per ferret], and indicated that higher doses (ie, 106 or 107 EID50) led to more severe pulmonary lesions,
However, in the present study, we used a relatively high dose (5 × 106 TCID50) without inducing severe pulmonary lesions in any ferrets. A possible explanation is that intranasal inoculation results in virus deposition in the lower respiratory tract of some ferrets but not others. In contrast, intratracheal inoculation of a 50-fold lower dose (1 × 105 TCID50) of exactly the same virus stock
resulted in severe bronchointerstitial pneumonia associated with influenza virus infection in all ferrets (Figure 1, D–F; Table 1), which had to be euthanized at 4 dpi because of severe respiratory distress. CNS disease was not evaluated pathologically, but was not considered significant at that time point, based on lack of specific neurological signs.
Second, route of inoculation is important for the character of CNS lesions. In our intranasally inoculated ferrets, CNS lesions were centered on tissues adjacent to cerebrospinal fluid in the subarachnoidal space and ventricular system (ie, leptomeninges, choroid plexi, and ependyma) and were characterized by prominent infiltration of mononuclear cells; there was lesser involvement of adjacent brain parenchyma. Other studies also have indentified the infiltration of mononuclear cells in the meninges of intranasally inoculated ferrets.
In contrast, intratracheal inoculation of influenza A/H5N1 virus in cats resulted in randomly distributed foci of necrosis and inflammation in the brain parenchyma, with minor involvement of leptomeninges, choroid plexi, and ependyma.
A possible explanation for these disparities in pathological changes between routes of inoculation is a different route of virus entry into the CNS. As has been suggested previously, intranasal inoculation of influenza A/H5N1 virus in ferrets may lead to direct spread of virus from the nasal cavity, via olfactory nerves through the ethmoidal plate, to the olfactory bulb.
virus would have easy and rapid access to tissues around the subarachnoid space and ventricular system. This route of entry would explain the prominent involvement of the olfactory bulb, leptomeninges, choroid plexi, and ependyma in our intranasally inoculated ferrets. In contrast, there was evidence of bloodborne spread of influenza A/H5N1 virus in intratracheally inoculated cats,
which might explain the random distribution of lesions in the brain parenchyma and the lesser involvement of tissues around the subarachnoid space and ventricular system in those animals.
In conclusion, we have shown that choice of route of inoculation is critical in designing ferret models to study the pathogenesis of influenza A/H5N1 virus infection in humans. Based on our results with different routes of inoculation of influenza virus A/Indonesia/5/2005 (H5N1) in ferrets, intranasal inoculation would be the route of choice to study influenza A/H5N1-virus-induced CNS disease, whereas intratracheal inoculation may be more appropriate to study influenza A/H5N1-virus-induced lower respiratory tract disease.
Acknowledgments
We thank Peter van Run and animal care takers of the Dutch Vaccine Institute for excellent technical assistance and Debby van Riel and Judith van den Brand for stimulating discussions.
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Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro.
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Supported in part by the VIRGO consortium (T.K.), an Innovative Cluster approved by the Netherlands Genomics Initiative, and in part by the Government of The Netherlands (BSIK 03012).
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