| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Department of Otolaryngology, University of Mississippi Medical Center, Jackson, MS 39216, USA
2 Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA
Correspondence
Kong T. Chong
kchong{at}ent.umsmed.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). However, the use of mice has several drawbacks because mice are not natural hosts for the influenza virus infection. Furthermore, a recent report showed that mice lack 2,6-linked sialic acid, which is the preferred receptor for human IAV (Ibricevic et al., 2006). However, other investigators have shown that the presence of a major 2,6 sialic acid on N-linked glycoproteins is not essential for IAV infection in mice (Glaser et al., 2007). More importantly, mice are not suitable for the study of viral transmission because infected mice failed to transmit virus to other mice housed even within the same cages (Lowen et al., 2006). Unlike mice, ferrets are suitable for transmission and pathogenesis studies but they are not as readily available and their housing requirements are beyond the resource of many investigators (Zitzow et al., 2002). More recently, cotton rats are shown to be promising for IAV studies but it is not known if these animals are susceptible to transmission (Ottolini et al., 2005).
Guinea pigs (Cavia porcellus), which are not rodents based on phylogenetic analysis of amino acid sequences (Graur et al., 1991; D'Erchia et al., 1996), have recently been shown to be an excellent model for the study of IAV transmission (Lowen et al., 2006). Earlier studies in guinea pigs were limited to the analysis of IAV-induced lung changes by histology and electron microscopy (Azoulay-Dupuis et al., 1984) and immune responses such as delayed-type hypersensitivity (Wetherbee, 1973; Phair et al., 1979). However, despite the new found importance of guinea pigs for the study of environmental factors that affect IAV transmission (Lowen et al., 2007, 2008), very little is known about viral replication and histopathology in the respiratory tissues of this species. Here, we describe viral growth kinetics, target cells and histopathology in upper and lower airways of guinea pigs infected with the IAV strain A/Puerto Rico/8/34 (H1N1), designated PR8 (ATCC VR-95) and A/Hong Kong/8/68 (H3N2), designated HK/68 (ATCC VR-544). These IAV strains were grown in embryonated eggs and titrated in Madin-Darby canine kidney (MDCK) cells as previously reported (Chong et al., 2008). For these studies, we used juvenile out-bred Hartley guinea pigs of mixed sex at 190–220 g body weight (Charles River Laboratories Inc., Wilmington, Massachusetts, USA) that were housed individually in cages with food and water provided ad libitum. These animals were maintained and handled in accordance with the guidelines set by the Institutional Animal Care and Use Committee.
We initially inoculated non-anaesthetized guinea pigs by dosing them with 10 µl HK/68 virus per nostril to take advantage of the reported high susceptibility of the animals to droplet transmission. Animals infected with 2x105 p.f.u. showed little respiratory symptoms and there was no significant difference in body weight compared to mock-infected animals during the duration of infection. For the determination of virus titre, we collected tissue specimens from nasosinus mucosa, trachea and whole lungs of guinea pigs as previously reported (Chong et al., 2008). As shown in Fig. 1(a), infected animals showed a large increase in nasal titre between 4 and 8 h post-infection (p.i.) that led to a mean peak nasal titres of 
3x105 p.f.u. (g tissue)–1 at 1 day p.i. Following peak infection, there was a 1 log reduction in nasal viral levels by day 5 p.i. and a further 1 log reduction by day 7 p.i. Infected animals developed relatively low levels of virus in the lungs with mean peak titres of 103 p.f.u. (g tissue)–1 on day 3 p.i., which subsequently declined to undetectable levels (Fig. 1a). Viral growth in the trachea was significantly lower than in the nasal mucosa, but was more persistent than in the lungs.
|
). With this higher dose of virus, infected animals displayed ruffled fur and were noticeably less active, but no animal died during the experimental duration. Tissue dissection showed macroscopic gross pathology, including excessive mucus in the nasosinus tract and varying levels of lung congestion and inflammation on day 3 p.i. Most infected animals also showed areas of lung lobes that appeared normal and this may account for the mild disease symptoms in these animals. Unlike the lower dose group, animals infected with the higher dose were slower in gaining weight compared with mock-infected control animals on days 1 and 3 p.i., but this reduction in body weight was not statistically significant (P>0.05 Student's t-test).
Similar to the low dose infection group, these animals also showed a rapid increase (100-fold) in titre within the nasal tract between 4 and 8 h p.i. (Fig. 1b
), but reached a much higher titre in the nasal tract with mean peak titre of 2x107 p.f.u. (g tissue)–1 on day 1 after infection. Nasal titre decreased rapidly to 3x104 p.f.u. g–1 by day 3 p.i. It was interesting to note that in the high dose infection groups animals displayed rapidly decreasing nasal titres, suggesting that infection was limited by the availability of susceptible epithelial cells. Moreover, the higher dose infection did not produce greater infection in the trachea or the lungs with lung titres peaking at 103 p.f.u. g–1 by day 3 p.i. and declining rapidly so that virus titres were below the assay limit of detection by day 5 p.i. In separate experiments using the PR8 strain of H1N1 virus, we noted that PR8 showed similar growth kinetics to the HK/68 strain except that titres were lower in all airway tissues, especially in nasal mucosa that showed a mean peak nasal titre of 2x106 p.f.u. g–1 (Fig. 1c). Unlike HK/68 strain, the PR8 virus that we used in this study may have been mouse-adapted and this would account for its poor replication in guinea pigs.
To correlate viral growth kinetics with histopathological analysis, we infected separate groups of guinea pigs with either HK/68 or PR8 strains by the high dose infection procedure as described above. Tissue specimens from infected animals on days 1, 3 and 7 p.i. were fixed in buffered formalin and processed for routine histology. In HK/68-infected animals, the conducting airway tissues including trachea, nasal, paranasal sinuses, bronchus and bronchioles showed varying levels of epithelial cell desquamation and inflammation beginning on day 1 p.i. Infected nasal mucosa showed a moderate increase in the number of goblet cells on days 1 (Fig. 2a and b) and 3 p.i. (Fig. 2d and e) and disorganized epithelial cells most likely due to epithelial tissue remodelling induced by viral cytopathic effect. To localize IAV antigen to target cells, we performed immunostaining on tissue sections that were treated for antigen retrieval by immersion in 0.01 M sodium citrate buffer (pH 6.0) and steam heated for 15 min. Subsequently, sections were stained using antibody preparations against influenza virions as previously reported (Chong et al., 2008). The staining results showed widespread viral antigens in nasal mucosa predominantly in ciliated epithelial cells on day 1 p.i. (Fig. 2c), and to a smaller extent on day 3 p.i. (Fig. 2f). Thereafter, the occurrence of virus-positive cells declined sharply so that only occasional foci of infection in the olfactory epithelia were demonstrable by day 7 p.i. (Fig. 2i). In contrast, excessive mucus secretion within the nasal tract was still evident by day 7 (Fig. 2g and h). For PR8-infected animals, we noted similar nasal tract histological changes (data not shown).
|
). By day 3 p.i., infected lungs showed severe bronchiolitis and alveolitis that were characterized by the presence of inflammatory cells within alveolar spaces (Fig. 3d and e). Viral antigens were readily demonstrated in the airway epithelial cells, including the bronchioles, alveolar epithelial cells and macrophages (Fig. 3c and f). Lung lesions persisted for 7 days p.i. as indicated by prominent alveolitis with extravasated red blood cells and oedema (Fig. 3g and h), but virus antigens were not detected in infected lung sections (Fig. 3i).
|
2x105 and 5x104 p.f.u. (g tissue)–1, respectively. In contrast, it was reported that guinea pigs inoculated with 103 p.f.u. led to nasal wash titres on days 1 and 3 p.i. of 3x105 and 3x106 p.f.u. ml–1, respectively (Lowen et al., 2006). One interpretation of this is that the nasopharyngeal washing procedure yields more virus than could be obtained in microdissected nasosinus mucosa. However, this is not likely to be a major factor since our animals also showed >1.5 log lower lung titres. A more important difference between the earlier transmission report and our findings is the nature of virus strains and their propagation methods. Whereas we have used an older non-mouse-adapted HK/68 isolate that was propagated in chick embryos, Lowen et al. (2006) had used a newer H3N2 virus human isolate (A/Panama/2007/99) that was cultured in MDCK cells. However, it is not known how virus strain differences can lead to differences in guinea pig susceptibility, since there have been very few studies of IAV infection in this animal species. Our findings suggest that it would be worthwhile to investigate if HK/68 and PR8 infections are also transmissible in guinea pigs, but we have not performed any transmission study.
Our results showed that pathological lesions that developed in the upper and lower airways of guinea pigs corresponded with the time course of acute IAV infection. Since both HK/68 and PR8 have been widely used in animal model studies, our findings allow comparison of IAV-induced airway disease in guinea pigs with published results on airway lesions in mice and ferrets (Mbawuike et al., 2007; Sanford & Ramsay, 1987). For these IAV strains, infected guinea pigs showed somewhat more severe nasal and lung disease than have been previously reported for mice (Iwasaki et al., 1999; Chong et al., 2008). For instance, guinea pigs produced excessive amounts of mucus in the nasosinus tract by day 3 p.i., which were much greater than we previously observed in infected mice (Chong et al., 2008). This suggests that infection in guinea pigs produced acute airway symptoms more similar to upper airway infection in humans. This is perhaps not surprising since guinea pigs are the preferred species for the study of upper airway responses such as antigen-induced rhinitis that involved the measurement of nasal secretion and sneezing responses (Fujita et al., 1999). As early as 1 day after infection, we noted widespread localization of IAV in nasal mucosa, which suggested that ciliated nasal epithelial cells in guinea pigs are highly susceptible to IAV infection. Therefore, the ease with which nasal epithelial cells supported IAV growth along with the excessive nasal mucus secretions might contribute to the susceptibility of guinea pigs to droplet spread.
Our results suggested that IAV-inoculated guinea pigs produced a predominantly upper airway infection with localized infection foci in the lungs. Infection in the lungs induced marked lung inflammatory responses, resulting in severe bronchopneumonia and alveolitis. Unlike IAV infection in mice, infected guinea pigs developed pronounced nasal tract mucus secretion, which resembled rhinitis in IAV-infected persons. In addition, elevated tissue eosinophils were suggestive of the induction of airway hypersensitivity.
Therefore, in addition to their use for viral transmission studies, guinea pigs should be further explored for their potential contributions in the understanding of virus–host interactions in IAV infection and airway allergic responses such as asthma. In this respect, the guinea pig is unique because its airway innervation is very similar to that of humans (Wang et al., 2005). Moreover, guinea pigs are widely used in pulmonary pharmacology because their responses to various mediators and drugs are more similar to that of the human airway (Ressmeyer et al., 2006). For instance, leukotrienes readily cause bronchoconstriction in humans and guinea pigs, but not in mice or rats (Dahlèn et al., 1980; Drazen et al., 1980; Hedqvist et al., 1980; Held et al., 1999).
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Chong, K. T., Thangavel, R. R. & Tang, X. (2008). Enhanced expression of murine β-defensins (MBD-1, -2,- 3, and -4) in upper and lower airway mucosa of influenza virus infected mice. Virology 380, 136–143.
Dahlèn, S. E., Hedqvist, P., Hammarstrom, S. & Samuelsson, B. (1980). Leukotrienes are potent constrictors of human bronchi. Nature 288, 484–486.[CrossRef][Medline]
D'Erchia, A. M., Gissi, C., Pesole, G., Saccone, C. & Arnason, U. (1996). The guinea-pig is not a rodent. Nature 381, 597–600.[CrossRef][Medline]
Drazen, J. M., Austen, K. F. & Lewis, R. A. (1980). Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro. Proc Natl Acad Sci U S A 77, 4354–4358.
Fujita, M., Yonetomi, Y., Shimouchi, K., Takeda, H., Aze, Y., Kawabata, K. & Ohno, H. (1999). Involvement of cysteinyl leukotrienes in biphasic increase of nasal airway resistance of antigen-induced rhinitis in guinea pigs. Eur J Pharmacol 369, 349–356.[CrossRef][Medline]
Glaser, L., Conenello, G., Paulson, J. & Palese, P. (2007). Effective replication of human influenza viruses in mice lacking a major 
2,6 sialyltransferase. Virus Res 126, 9–18.[CrossRef][Medline]
Graur, D., Hide, W. A. & Li, W. H. (1991). Is the guinea-pig a rodent? Nature 351, 649–652.[CrossRef][Medline]
Gubareva, L. V., McCullers, J. A., Bethell, R. C. & Webster, R. G. (1998). Characterization of influenza A/HongKong/156/97 (H5N1) virus in a mouse model and protective effect of zanamivir on H5N1 infection in mice. J Infect Dis 178, 1592–1596.[CrossRef][Medline]
Hedqvist, P., Dahlèn, S. E., Gustafsson, L., Hammarstrom, S. & Samuelsson, B. (1980). Biological profile of leukotrienes C4 and D4. Acta Physiol Scand 110, 331–333.[Medline]
Held, H. D., Martin, C. & Uhlig, S. (1999). Characterization of airway and vascular responses in murine lungs. Br J Pharmacol 126, 1191–1199.[CrossRef][Medline]
Ibricevic, A., Pekosz, A., Walter, M. J., Newby, C., Battaile, J. T., Brown, E. G., Holtzman, M. J. & Brody, S. L. (2006). Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells. J Virol 80, 7469–7480.
Iwasaki, T., Tamura, S., Kumasaka, T., Sato, Y., Hasegawa, H., Asanuma, H., Aizawa, S., Yanagihara, R. & Kurata, T. (1999). Exacerbation of influenza virus pneumonia by intranasal administration of surfactant in a mouse model. Arch Virol 144, 675–685.[CrossRef][Medline]
Lowen, A. C., Mubareka, S., Tumpey, T. M., García-Sastre, A. & Palese, P. (2006). The guinea pig as a transmission model for human influenza viruses. Proc Natl Acad Sci U S A 103, 9988–9992.
Lowen, A. C., Mubareka, S., Steel, J. & Palese, P. (2007). Influenza virus transmission is dependent on relative humidity and temperature. PLoS Pathog 3, 1470–1476.[Medline]
Lowen, A. C., Steel, J., Mubareka, S. & Palese, P. (2008). High temperature (30 °C) blocks aerosol but not contact transmission of influenza virus. J Virol 82, 5650–5652.
Mbawuike, I. N., Zhang, Y. & Couch, R. B. (2007). Control of mucosal virus infection by influenza nucleoprotein-specific CD8+ cytotoxic T lymphocytes. Respir Res 8, 44[Medline]
Ottolini, M. G., Blanco, J. C. G., Eichelberger, M. C., Pletneva, L., Richardson, J. Y. & Porter, D. D. (2005). The cotton rat provides a useful small animal model for the study of influenza virus pathogenesis. J Gen Virol 86, 2823–2830.
Phair, J. P., Kauffman, C. A., Jennings, R. & Potter, C. W. (1979). Influenza virus infection of the guinea pig: immune response and resistance. Med Microbiol Immunol 165, 241–254.[CrossRef][Medline]
Ressmeyer, A. R., Larsson, A. K., Vollmer, E., Dahlen, S. E., Uhlig, S. & Martin, C. (2006). Characterization of guinea pig precision-cut lung slices: comparison with human tissues. Eur Respir J 28, 603–611.
Sanford, B. A. & Ramsay, M. A. (1987). Bacterial adherence to the upper respiratory tract of ferrets infected with influenza A virus. Proc Soc Exp Biol Med 185, 120–128.[CrossRef][Medline]
Wang, L., Pasquale, C. & Thomas, M. M. (2005). Length oscillation induces force potentiation in infant guinea pig airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 289, L909–L915.
Wetherbee, R. E. (1973). Induction of systemic delayed hypersensitivity during experimental viral infection of the respiratory tract with a myxovirus or paramyxovirus. J Immunol 111, 157–163.
Zitzow, L. A., Rowe, T., Morken, T., Shieh, W. J., Zaki, S. & Katz, J. M. (2002). Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J Virol 76, 4420–4429.
Received 3 September 2008;
accepted 21 October 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
