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Debby van Riel,Vincent J. Munster,Emmie de Wit,Guus F. Rimmelzwaan,Ron A. M. Fouchier,Ab D. M. E. Osterhaus,Thijs Kuiken*
Highly pathogenic avian influenza virus (H5N1) may cause severelower respiratory tract (LRT) disease in humans. However, theLRT cells to which the virus attaches are unknown for both humansand other mammals. We show here that H5N1 virus attached predominantlyto type II pneumocytes, alveolar macrophages, and nonciliatedbronchiolar cells in the human LRT, and this pattern was mostclosely mirrored in cat and ferret tissues. These findings mayexplain, at least in part, the localization and severity ofH5N1 viral pneumonia in humans. They also identify the cat andthe ferret as suitable experimental animals based on this criterion.
Department of Virology, Erasmus Medical Center, 3015 GE Rotterdam, Netherlands.
* To whom correspondence should be addressed. E-mail: t.kuiken{at}erasmusmc.nl
Highly pathogenic avian influenza virus of the subtype H5N1may cause infection of the lower respiratory tract (LRT) andsevere pneumonia in humans (1). However, the cell types in theLRT to which the virus attaches are unknown for both humansand experimental animals. Although attachment is not the onlyfactor required for virus replication, this information is importantboth to better understand the pathogenesis of H5N1 influenzaand to assess the suitability of animal models. Therefore, wecompared the pattern of H5N1 virus attachment to the LRT ofhumans and four animal species.
Influenza viruses attach to host cells by binding of the hemagglutininto sialosaccharides on the host cell surface. Human influenzaviruses prefer sialic acid (SA)–-2,6-Gal–terminatedsaccharides, whereas avian influenza viruses prefer those terminatingin SA--2,3-Gal (2). The use of lectins that specifically detect-2,6– and -2,3–linked sialosaccharides is an indirectmeasure of influenza virus attachment to host tissues and doesnot account for other variables that influence the binding avidity,such as type of SA, and glycosylation and sialylation of thehemagglutinin close to the receptor binding site (2). For amore direct method, which was modified from a previously usedtechnique (3), we incubated formalin-fixed, paraffin-embeddedtissue sections with formalin-inactivated fluorescein isothiocyanate(FITC)–labeled H5N1 virus (A/Vietnam/1194/04) and detectedvirus with a peroxidase-labeled rabbit antibody to FITC thatwas amplified with a tyramide signal amplification system. Tissuescomprised histologically normal LRT (including alveolus, bronchiole,and bronchus), as well as trachea from three individuals ofeach of the following species: human, mouse (C57BL/6), ferret,cynomolgus macaque, and domestic cat (4).
In the human LRT, H5N1 virus attached predominantly to typeII pneumocytes, alveolar macrophages, and nonciliated cuboidalepithelial cells in terminal bronchioles. Attachment becameprogressively rarer toward the trachea (Fig. 1 and table S1).The identity of type II pneumocytes was confirmed by doublestaining with antibody to human surfactant apoprotein A (fig.S1). These findings fit with the limited pathology data forH5N1 virus infection in humans, which show diffuse alveolardamage (1) and the presence of H5N1 virus antigen in type IIpneumocytes (5). However, they contrast with the idea that avianinfluenza viruses generally have little affinity for human respiratorytissues (2).
Fig. 1. Attachment of H5N1 virus to respiratory tissues of humans and four animal species. In the trachea, H5N1 virus—visible as red-brown staining—attached only to epithelial cells of mice. In the alveoli, H5N1 virus attached predominantly to type II pneumocytes (arrowheads) in humans and all animal species except the macaque, where attachment was predominantly to type I pneumocytes (arrows).
[View Larger Version of this Image (73K GIF file)]
The predilection of H5N1 virus for type II pneumocytes and alveolarmacrophages may contribute to the severity of the pulmonarylesion. Because type II pneumocytes are metabolically activeand are the most numerous cell type lining the alveoli, targetingof this cell type may lead to abundant virus production. Damageto type II pneumocytes may impair their functions, includingre-epithelialization after alveolar damage, ion transport, andsurfactant production, and so may inhibit tissue repair. Targetingof alveolar macrophages may be important because of their rolein limiting viral replication and in the immune response toviral infection.
The pattern of H5N1 virus attachment to cat LRT and, to a lesserextent, ferret LRT most closely resembled that in human tissues(Fig. 1 and table S1). Based on this criterion, we consideredthese two species as the most suitable models for H5N1 viralpneumonia in humans. However, other factors also need to beconsidered, such as the availability of reagents and immunologicsimilarity. In macaque alveoli, H5N1 virus attached predominantlyto type I pneumocytes instead of type II pneumocytes, as inhuman tissues. In mice, H5N1 virus attachment to cells was mostabundant in the trachea and became progressively rarer towardthe alveoli, whereas the opposite trend was observed in humantissues. The observed pattern of H5N1 virus attachment to theLRT is consistent with the respective pathology and immunohistochemistryresults of experimental H5N1 virus infection in mice (6), ferrets(7), macaques (8), and cats (9).
This study demonstrates the attachment of H5N1 virus to thehuman LRT in a pattern that corresponds with autopsy findings.It also identifies cat and ferret as the most suitable animalmodels for human H5N1 viral pneumonia, on the basis of the similarityof viral attachment pattern. This technique also could be appliedto further determine H5N1 virus attachment to the upper respiratorytract. Failure to attach to this site may be a limiting factorin human-to-human transmissibility of H5N1 virus.
References
1. J. H. Beigel et al., N. Eng. J. Med.353, 1374 (2005).[Free Full Text]
10. We thank W. Lim for providing the H5N1 virus isolate; M. den Bakker, H. Sharma, M. Vermeij, and J. van den Brand for providing tissues; and F. van der Panne for technical assistance.
Received for publication 30 January 2006. Accepted for publication 21 March 2006.
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