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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 freezethawing). 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 04 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
), IFN-
, IL6, tumour necrosis factor alpha (TNF-
), GRO
, MIP-1
, RANTES, IFN-
, 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
-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.
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RESULTS |
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DISCUSSION |
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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
, GRO
), as well as late (RANTES) after infection. In addition, proinflammatory cytokines such as IL1
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-
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- plays a critical role in the induction of antiviral genes such as Mx. In the cotton rat, the levels of IFN-
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
).
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ACKNOWLEDGEMENTS |
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Received 27 April 2005;
accepted 17 July 2005.
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