The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis

Martin G. Ottolini1, Jorge C. G. Blanco2, Maryna C. Eichelberger2, David D. Porter2, Lioubov Pletneva2, Joann Y. Richardson1 and Gregory A. Prince2

1 Department of Pediatrics, F. Edward Hébert School of Medicine, The Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA
2 Virion Systems, Inc., Rockville, MD, USA

Correspondence
Martin G. Ottolini
mottolini{at}usuhs.mil


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Influenza A virus continues to cause annual epidemics. The emergence of avian viruses in the human population poses a pandemic threat, and has highlighted the need for more effective influenza vaccines and antivirals. Development of such therapeutics would be enhanced by the use of a small-animal model that is permissive for replication of human influenza virus, and for which reagents are available to dissect the host response. A model is presented of nasal and pulmonary infection in adult inbred cotton rats (Sigmodon hispidus) that does not require viral ‘adaptation’. It was previously demonstrated that animals infected intranasally with 107 TCID50 of a recent H3N2 influenza, A/Wuhan/359/95, have increased breathing rates. In this report it is shown that this is accompanied by weight loss and decreased temperature. Virus replication peaked within 24 h in the lung, with peak titres proportional to the infecting dose, clearing by day 3. Replication was more permissive in nasal tissues, and persisted for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia, which persisted for nearly 3 weeks. Interleukin 1 alpha (IL1{alpha}), alpha interferon (IFN-{alpha}), IL6, tumour necrosis factor alpha (TNF-{alpha}), GRO{alpha} and MIP-1{beta} mRNA were elevated soon after infection, and expression coincided with virus replication. A biphasic response was observed for RANTES, IFN-{gamma}, IL4, IL10 and IL12-p40, with increased mRNA levels early during virus replication followed by a later increase that coincided with pulmonary inflammation. These results indicate that cotton rats will be useful for further studies of influenza pathogenesis and immunity.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human disease caused by influenza viruses has been recognized since ancient times. While much is known about the structure and function of many individual viral proteins, further studies of pathogenesis and immunity are needed to facilitate the identification of new strategies toward protection from infection and treatment of disease.

The animal models presently available for the study of the pathogenesis of influenza virus disease have limitations (reviewed by Renegar, 1992). Influenza A virus will experimentally infect a number of Old World and New World primates. The gibbon and baboon develop clinical illness with nasal application of the virus, and the squirrel, cynomolgus and rhesus monkeys develop illness when the virus is inoculated intratracheally (Berendt & Scott, 1977; Scott et al., 1978). Primate models suffer a number of disadvantages, including the limited availability of expensive animals. In addition, these animals are outbred and the models lack many of the reagents necessary to characterize the host response in detail. Mammals such as horses and pigs that are natural hosts for influenza have also been used experimentally. However, their large size and the limited number of reagents available precludes their use in the laboratory.

Small-animal models that have been used to study influenza virus pathogenesis include the ferret, in which human influenza virus was originally isolated (Smith et al., 1933). Adult ferrets become ill after infection with unadapted influenza A viruses, exhibiting fever, lethargy and weight loss (reviewed by Smith & Sweet, 1988). The ferret model has been used in recent studies of H5N1 viruses (Zitzow et al., 2002), the transmission of influenza (Herlocher et al., 2001) and the development of resistance to antiviral therapy (Herlocher et al., 2003). Unfortunately, ferrets are outbred and reagents are not available for dissecting the correlates of protective immunity. In contrast, laboratory strains of mice are inbred and there is an abundance of reagents to characterize both innate and adaptive immune responses. However, mice are not naturally infected with influenza viruses, and therefore most studies are performed using strains that have been adapted by extensive serial passage to replicate efficiently in this mammal. Laboratory mouse strains lack expression of Mx (Lindenmann et al., 1963), an interferon (IFN)-induced antiviral response that contributes to the inhibition of virus replication (Haller et al., 1979; Krug et al., 1985). Consequently there is prolonged influenza virus replication in the lungs of mice compared to ferrets, primates or even man, with clearance of the virus in mice depending more on the cellular immune response than the innate immune response.

There has been one report, published in Polish and largely overlooked, of the use of outbred cotton rats (Sigmodon hispidus) for pathogenesis experiments with influenza viruses (Sadowski et al., 1987). Nasal administration of virus in lightly anaesthetized cotton rats resulted in virus replication, the production of pulmonary lesions and a strong immune response. In this study we confirm the report using additional viral strains, describe the histopathology, and characterize the kinetics of virus replication and induction of chemokine and cytokines in the lungs of inbred cotton rats.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Animals.
Young adult (6–12 weeks old) cotton rats (S. hispidus) of both genders were obtained from an inbred colony maintained at Virion Systems. The animals were housed in large polycarbonate cages, and fed a diet of standard rodent chow and water. The animals were seronegative for adventitious respiratory viruses and other common rodent pathogens. All experiments were performed using protocols that followed federal guidelines and were approved by the Institutional Animal Care and Use Committee. Animals were sacrificed by carbon dioxide inhalation.

Infection and clinical evaluation of disease.
Cotton rats were infected intranasally with the influenza viruses described below while under isoflurane anaesthesia by the application of 0·1 ml virus solution per 100 g body weight (an 8-week-old cotton rat weighs approx. 100 g). To evaluate the ability of different viral strains to replicate in cotton rats, approximately 106 TCID50 of each virus was administered to each cotton rat intranasally. The pathogenesis of A/Wuhan/395/95 was studied using a 107 TCID50 inoculum since at this dose increased breathing rates and severe lung pathology had previously been reported (Eichelberger et al., 2004). Core body temperature was measured using a digital rectal thermometer (Becton-Dickinson). To determine serological responses, cotton rats were bled from the retro-orbital venous plexus under anaesthesia. Haemagglutination inhibition (HAI) titres were measured in these serum samples using the WHO standard assay (WHO, 2004).

Viruses.
The following influenza viruses were used in this study: influenza A viruses with H1N1 subtype – mouse-adapted A/PR/8/34, tissue culture-adapted A/PR/8/34 and A/Malaya/302/54; influenza A viruses with H3N2 subtype – X-31 and A/Wuhan/359/95; influenza B viruses – B/HK/73, B/HK/330/01 and B/Sichuan/379/99. Viruses that were not available in our laboratory were either purchased from the ATCC or obtained from the Centers for Disease Control, Atlanta, GA, USA. Stocks of all viruses were obtained by harvesting the supernatants of MDCK cells that had been inoculated 3 days previously at a low m.o.i. A large stock of A/Wuhan/359/95 was prepared by Novavax.

Virus titration.
MDCK cells were grown to confluence in 96-well plates. The cells were washed with serum-free medium prior to inoculation of quadruplicate wells with serial 10-fold dilutions of sample. After 1 h incubation at 37 °C, an equal volume of tissue culture medium that contained 1 µg TPCK-trypsin (Worthington) was added to each well. After a further 4 days of culture, the cells were fixed and stained with 0·06 % crystal violet in 1 % glutaraldehyde. Wells that were not stained demonstrated the cytopathic effect (CPE) of the virus. The titre of the virus was recorded as the TCID50, i.e. the inverse of the dilution that resulted in the CPE in 50 % of wells, (ml virus stock solution)–1 or (g tissue)–1. These tissues included lung, nose and larynx. After dissection of these tissues, they were homogenized in 10 parts (w/v) Earle's modified Eagle's medium supplemented with 0·218 M sucrose, 4·4 mM glutamate, 3·8 mM KH2HPO4 and 7·2 mM K2HPO4 (to stabilize the virus during freeze–thawing). After centrifugation, supernatants were removed and stored at –70 °C until assayed. The lowest level of detection of this assay was 102·5 TCID50 influenza (g lung tissue)–1.

Histopathology of the lung, larynx and nasal tissues.
Lungs were dissected with the lower one-third of the trachea left attached. They were inflated with 10 % neutral buffered formalin to their normal volume, and then immersed in the same fixative solution. In some experiments the left lung was first tied off, and divided for viral titration and cytokine analysis; only the right lung was inflated and fixed for histology. Laryngeal tissue included the upper two-thirds of the trachea together with the epiglottis and larynx. Noses were prepared by removal of the skull, with later disarticulation and separation of the jaw. Following formalin fixation, the anterior two-thirds of the head, containing the nasal and paranasal sinus structures, were demineralized with neutral 10 % EDTA before coronal sections were prepared for examination. Sections of lung, larynx and nose were stained with haematoxylin and eosin (H & E), and evaluated for inflammation and epithelial cell damage. Lungs were evaluated for three indices of pulmonary inflammatory changes: peribronchiolitis (inflammatory cells clustered around the periphery of small airways), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls) and alveolitis (cells within the alveolar spaces). Slides were scored blind, with validation of scoring by two pathologists experienced in respiratory viral pathogenesis, on a 0–4 severity scale, as previously reported (Prince et al., 2001).

RT-PCR analysis.
The expression of selected cytokines and chemokines was determined by the semi-quantitative measurement of mRNA in pulmonary tissue using RT-PCR, as previously described (Blanco et al., 2002). In brief, lung tissue was homogenized and total RNA was isolated by the use of an RNeasy purification kit (Qiagen) according to the manufacturer's instructions. The total amount of mRNA isolated was quantified by UV spectrophotometry at 260 nm. RNA (1 µg) from each sample was used for the preparation of cDNA using oligo (dT) primers and SuperScript II RT enzyme (Invitrogen). Specific PCR reactions for cotton rat interleukin 1 beta (IL1{beta}), IFN-{alpha}, IL6, tumour necrosis factor alpha (TNF-{alpha}), GRO{alpha}, MIP-1{beta}, RANTES, IFN-{gamma}, IL4, IL10 and IL12-p40 were performed using the primers and conditions previously described (Blanco et al., 2002, 2004). Amplified products were electrophoresed and transferred to Hybond-N+ membranes (Amersham) by Southern blotting. DNA was cross-linked by exposure to UV light, and hybridized with specific internal oligonucleotide probes. Labelling of the probe and subsequent detection of bound probe was carried out with an enhanced chemiluminescence system (Amersham) and autoradiography on Kodak X-Omat AR film. The signals were quantified with a scanner using National Institutes of Health image software. The signal was normalized to the {beta}-actin level expressed in the lung of the individual animal.

Statistical analysis.
Viral titres were calculated as geometric means±SE for all animals in a group at a given time. Pulmonary lesion scores are expressed as the arithmetic mean±SEM for all animals in a group.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection with various strains of human influenza
Cotton rats were infected with human influenza A and B viruses that were isolated between 1934 and 2001. Virus replication was determined by the titration of lung and nose on day 2 post-infection (p.i.) (Table 1). All influenza B viruses (B/HK/73, B/HK/330/01 and B/Sichuan/379/99) that were used to inoculate cotton rats replicated in both the nose and lungs. A/Malaya/302/54 (H1N1) and tissue-culture-adapted A/PR/8/34 (H1N1) were also able to infect the lungs and nose. However, poor replication of mouse-adapted A/PR/8/34 in the lower respiratory tract was observed. The dose used to inoculate cotton rats is lethal to mice, showing that the adaptation to mice does not facilitate replication in these rats. Both influenza A viruses with the H3N2 subtype (X-31 and A/Wuhan/359/95) replicated in the lungs and nose. To characterize influenza pathogenicity in cotton rats, further studies of the kinetics of replication and induction of the host response were carried out using A/Wuhan/359/95. All subsequent studies described in this report use this H3N2 virus to inoculate animals intranasally with a dose of 107 TCID50 per 100 g.


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Table 1. Replication of influenza viruses in cotton rats

Groups of cotton rats were inoculated intranasally with 0·1 ml of a virus preparation containing approximately 107 TCID50 ml–1. Two days after infection the cotton rats were sacrificed and their lungs homogenized for virus titration.

 
Clinical signs of influenza disease
Although mortality due to influenza infection was low (<5 %), the animals appeared ill, with visible respiratory distress. Infected cotton rats showed significant weight loss for several days, which normalized 1 week after infection (Fig. 1a). Body temperature dropped at 1–3 days p.i., then returned to normal baseline measures (Fig. 1b). Respiratory rates nearly doubled from 1–3 days p.i., an observation we have published separately (Eichelberger et al., 2004). At 3 weeks p.i., all animals had HAI titres of 16, while control animals had no detectable influenza-specific antibodies.



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Fig. 1. Cotton rats infected with influenza virus experience weight loss and decreased body temperature. Serial measurement of weight (a) and temperature (b) were made on days 0–10 after intranasal infection with A/Wuhan/395/95 ({square}), and in age-matched uninfected control animals ({blacksquare}).

 
Kinetics of influenza virus replication in cotton rat lung, nose and larynx
Cotton rats were infected intranasally with A/Wuhan/359/95 (107 TCID50 per 100 g body weight). Four animals were sacrificed for virus titration at time points starting at 5 min p.i. Four uninfected animals served as controls. An initial virus titre of 106 TCID50 g–1 was recovered from lung tissues 5 min after infection (Fig. 2), which dropped to 104 TCID50 g–1 at 4 h p.i. (indicating that this was the time of viral ‘eclipse’) before rising to a peak of 107 TCID50 g–1 by 8 h. Nasal tissue eclipse of virus replication occurred slightly later, and lasted a few hours longer. Virus replication in the lung peaked within the first day of infection, and rapidly dropped to undetectable levels by 3 days p.i. Nasal virus replication continued for 6 days (Fig. 2). The experiment was repeated with similar results. Laryngeal virus titres paralleled lung titres, although they never exceeded 103·5 TCID50 g–1 (results not shown).



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Fig. 2. Kinetics of virus replication in the nose and lung of cotton rats. Animals were inoculated intranasally with A/Wuhan/359/95 and sacrificed at 5 min, then hourly for the first 8 h and on days 1, 2, 3, 4, 6 and 8 p.i. Virus titres for lungs ({blacksquare}) and noses ({square}) were measured as described in Methods. Each point represents the geometric mean±SEM titre for >=8 total animals. m, minutes; h, hours; d, days.

 
Groups of cotton rats were infected with different doses (107, 105, 103 and 101 TCID50 per 100 g body weight) of A/Wuhan/359/95. Four animals from each group were sacrificed on different days after infection for determination of pulmonary and nasal virus titres. Virus titres in the lungs were proportional to the input dose (Fig. 3), indicative of semi-permissive replication, while all input doses resulted in high levels of replication (106–107 TCID50 g–1) in the nose (results not shown). There was a small delay in achieving peak titres as well as clearance when animals were inoculated with low doses of virus. No virus was isolated from animals infected with 101 TCID50 per 100 g body weight, demonstrating that the infectious dose required to infect 50 % of cotton rats in this experiment was 102 TCID50 per 100 g body weight.



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Fig. 3. Dose-dependent replication of influenza virus in the lungs of cotton rats. Animals were inoculated intranasally with 107 ({blacksquare}), 105 ({square}) or 103 ({blacktriangleup}) TCID50 A/Wuhan/359/95. At each time point shown, four animals in each group were sacrificed and the virus titre determined in lung homogenates. The geometric mean titre is shown in the graph.

 
Pathology in influenza-infected cotton rat lung, nose and larynx
Cotton rats that had been infected intranasally with 107 TCID50 A/Wuhan/359/95 were sacrificed on days 1, 2, 3, 4, 6, 8, 10, 14, 21 and 28 p.i. for histopathological evaluation of the lungs, nose and larynx. Infected animals had few nasal lesions, with only a slight decrease in the number of ciliated cells. There was no nasal inflammation at any time point studied, despite the high viral titres. There were very mild laryngeal lesions, with some loss of ciliated cells, and loss of a few columnar cells. No inflammation was present in the larynx at any time point. In contrast, regions of the columnar epithelium of the larger airways in the lung had sloughed off at 2 days p.i. (Fig. 4a). The columnar epithelium, including a full complement of ciliated cells, regenerated by 5 days p.i. (Fig. 4b).



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Fig. 4. Histopathology evident in the lungs of influenza A virus infected cotton rats. Formalin-fixed lung sections were stained with H & E. (a) Bronchus at 2 days p.i. shows widespread epithelial cell death and sloughing of the columnar surface epithelium. (b) Bronchus at 5 days p.i. shows that the columnar epithelium has regenerated. The tissue is indistinguishable from an uninfected control. (c) Uninfected cotton rat lung for comparison with d–f. (d) Lung at 2 days p.i. shows marked alveolar inflammation with infiltration of numerous neutrophils. (e) Lung at 5 days p.i. shows marked alveolar, peribronchiolar and interstitial inflammation with a predominantly mononuclear cell infiltrate. (f) Lung at day 10 p.i. shows partial but slow resolution of the pneumonic process. Sections in (a)–(b) and (c)–(f) are shown at x320 and x80 magnification, respectively.

 
Pathology scores for epithelial injury, alveolitis, interstitial pneumonia and bronchiolitis are presented in Fig. 5. While the peak of epithelial cell damage was evident on day 2 p.i., alveolitis and interstitial pneumonia were most prominent on day 4 p.i. Inflammatory cells were observed around bronchioles soon after infection, but were most abundant on day 6 p.i. The early inflammatory response included the accumulation of neutrophils. The later inflammatory response that included a lymphocytic infiltrate resolved slowly over 4 weeks (Figs 4 and 5).



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Fig. 5. Arithmetic mean pathology scores±SEM for epithelial injury, alveolitis, interstitial pneumonia and bronchiolitis in the lungs of influenza A infected cotton rats. Each bar represents at least seven animals.

 
Chemokine and cytokine responses in the lungs of cotton rats infected with influenza virus
Chemokine and cytokine mRNA was amplified from RNA isolated from the lungs of groups of seven cotton rats infected for different time periods with A/Wuhan/359/95. Baseline values were obtained using RNA isolated from control groups of animals that had been inoculated with a ‘mock’ preparation of viral culture media to rule out the possibility of non-specific inflammatory gene induction. These baseline values did not change with time p.i., and the mean value is presented as the zero time point in the graphs in Fig. 6. During RNA purification, the yield of RNA increased fourfold, being maximal on days 4 and 6 p.i., likely reflecting the degree of cellular infiltration. mRNA for IL1{beta}, IFN-{alpha}, IL6, TNF-{alpha}, GRO{alpha}, MIP-1{beta}, RANTES, IFN-{gamma}, IL4, IL10 and IL12-p40 was quantified at different time points p.i. (Fig. 6). With the exception of IFN-{alpha}, RANTES, IFN-{gamma} and IL10, all genes examined showed detectable increases in their expression as early as 6 h p.i. mRNA induction could be clustered into two different groups. In the first group, mRNA levels coincided with virus replication in the lung, and showed reduced expression after 3 days. This included mRNAs for IL1{beta}, IFN-{alpha}, IL6, TNF-{alpha}, GRO{alpha} and MIP-1{beta}. In the second group, mRNA induction was biphasic, with one phase overlapping with the peak of virus replication, and a second peak coinciding with the cellular inflammatory response. This is the case for RANTES, IFN-{gamma}, IL4, IL10 and IL12-p40. RANTES, IFN-{gamma} and IL12-p40 continued to be expressed at elevated levels 28 days p.i. This finding was consistent with a cellular infiltration that persisted in the lung for at least 28 days.



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Fig. 6. Cytokine and chemokine gene expression in the lungs of cotton rats during A/Wuhan/359/95 infection. The upper left graph represents the total amount of RNA isolated from half of the left lung. All other graphs represent the amount of cytokine/chemokine mRNA relative to {beta}-actin mRNA at the indicated time points. The dotted line at the top of each graph indicates the period during which virus replication is observed. The amounts shown are the mean±SD of at least seven individual samples.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our results suggest that the cotton rat may serve as a useful model for the study of influenza pathogenesis. The animals are small, inbred, easy to handle, and relatively inexpensive to purchase and maintain. We demonstrate that adult cotton rats can be infected with human influenza viruses, resulting in virus replication in the nose, larynx and lung. Evidence of virus replication includes early epithelial cell destruction and alveolitis that is followed by long-lasting peribronchiolitis. Clinical illness in this model includes increased respiratory rate, hypothermia and temporary weight loss. Each of these clinical signs is a characteristic observed in many severely ill human infants (Olshaker, 2003), suggesting that observations reported in cotton rats may have clinical relevance. The severity of respiratory symptoms seen in influenza-infected cotton rats has already led us to use this marker of disease to study the effect of a variety of therapeutic approaches (Eichelberger et al., 2004; Ottolini et al., 2003).

The greatest advantage that this model has over that of the mouse is that the virus does not need to be adapted to replicate in the lower or upper respiratory tract of adult animals. As in infected cotton rats, in other species, including monkeys, the disease signs are often dependent on the inoculum dose used (Murphy et al., 1980). This is not a concern in animal models of influenza as fever and signs of upper respiratory tract infection in humans experimentally infected with influenza virus correlate with the amount of virus present in nasal washes (Murphy et al., 1973).

Similar to humans, ferrets and pigs usually have fevers after inoculation with high doses of influenza virus(es). Instead of an increased body temperature, decreased temperatures are measured in infected cotton rats. The molecular mechanisms responsible for thermoregulation, which include the regulation of PGE2 synthesis and signalling through adrenoreceptors, are still under investigation (Blatteis et al., 2005). However, hypothermia is not unique to influenza-infected cotton rats – paediatricians have long recognized the occurrence of hypothermia in up to 15 % of infants during both systemic viral and bacterial infections (Klein, 2001). The predisposition of neonates to hypothermia is thought to be partly due to their large surface area to mass ratio, lack of a ‘shivering’ mechanism, and possibly metabolic disturbances. Body temperature also decreases in rats injected with high doses of endotoxin. However, hypothermia is dependent on the ambient temperature – when it is below the thermal ‘neutral’ zone for that particular species, body temperature decreases, but when the ambient temperature is neutral, body temperature increases in response to endotoxin (Romanovsky, 2000; Romanovsky et al., 2002). It is likely that, like other small rodents with high basal metabolic rates, the thermal neutral zone of the cotton rat is above normal room temperature at which our experiments were performed. Further investigation is required to determine whether the hypothermic response in influenza-infected cotton rats is related to the ambient temperature, the physical features of this small animal with a proportionally large surface area, or metabolic disturbances due to the inflammatory response.

In cotton rats, virus replication in the lung coincides with the induction of cytokines characteristic of the innate immune response, providing a small-animal model that will be useful to delineate the importance of early antiviral mechanisms. The ferret, as well as some primates, are clearly very good models for influenza virus disease; however, these animals are not inbred and species-specific reagents are needed before host responses can be easily characterized. The cotton rat has a distinct advantage in that the methods and reagents are now available to characterize the innate and adaptive immune responses to influenza in large groups of inbred cotton rats. Over 70 cotton-rat-specific reagents are now available commercially (R & D Systems) that can support such studies, providing a relevant model that may prove to be a highly flexible and relatively inexpensive tool for the study of influenza pathogenesis.

These studies are believed to be the first to show a detailed pattern of cytokines induced following influenza infection in the cotton rat. Both the kinetics of virus replication in the nose, and the pattern of cytokines induced are similar to those observed when human volunteers were inoculated with an H1N1 influenza A (Hayden et al., 1998). The cytokine profile, including the biphasic response of certain cytokines in cotton rats, is similar to that observed in mice infected with a mouse-adapted human virus (Conn et al., 1995; Hennet et al., 1992), and in pigs infected with a swine influenza virus (Van Reeth et al., 2002). This includes the production of chemotactic factors early (MIP-1{beta}, GRO{alpha}), as well as late (RANTES) after infection. In addition, proinflammatory cytokines such as IL1{beta} and IL6 were expressed immediately upon infection. Those cytokines that are expressed in a biphasic pattern are more than likely induced in different cell subsets. For example, the early induction of IFN-{gamma} may reflect the presence of activated natural killer cells, while the later response may be the product of antigen-specific T cells. While the cytokines that are expressed after viral clearance may not contribute to viral clearance during primary infection, they reflect the activation of the adaptive immune response that will prevent or control reinfection.

IFN-{alpha} plays a critical role in the induction of antiviral genes such as Mx. In the cotton rat, the levels of IFN-{alpha} mRNA coincided with virus replication, and suggest that this early host response is effective in controlling influenza virus replication in this model. This is distinct from the control of primary influenza virus infection in laboratory mice, where the adaptive response plays a critical role in viral clearance. This occurs because the innate response to influenza is impaired in mice due to the absence of some IFN-induced antiviral gene expression. The cotton rat can therefore be used to evaluate the importance of the host's early innate response. This is particularly relevant to the study of new emerging strains such as the H5N1 avian influenza viruses that have evolved mechanisms to overcome these early antiviral responses (Seo et al., 2002).


   ACKNOWLEDGEMENTS
 
We thank Sally Hensen and Lorraine Ward for their help with virological assays, and animal care and handling, respectively. This work was supported by USUHS research grants RO86DM and HO86DM, and by Virion Systems, Inc. corporate funds. The opinions and assertions contained herein are those of the author(s) alone and do not reflect the views of the Uniformed Services University of the Health Sciences, The United States Air Force or The Department of Defense.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 27 April 2005; accepted 17 July 2005.



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