Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus)

Infection with respiratory syncytial virus (RSV) generally presents as a mild, upper airway disease in human patients but may cause severe lower airway disease in the very young and very old. Progress toward understanding the mechanisms of RSV pathogenesis has been hampered by a lack of relevant rodent models. Mice, the species most commonly used in RSV research, are resistant to upper respiratory infection and do not recapitulate the pattern of virus spread in the human host. To address the need for better rodent models of RSV infection, we have characterized the acute and chronic pathology of RSV infection of a relatively permissive host, cotton rats (Sigmodon hispidus). We demonstrate that virus delivered to the upper airway results in widespread RSV replication in the ciliated respiratory epithelial cells of the nasal cavity and, to a lesser extent, of the lung. Although acute inflammation is relatively mild and rapidly eliminated after viral clearance, chronic, eosinophilic lung pathology persists. These data support the use of cotton rats as a robust rodent model of human RSV disease, including the association between RSV pneumonia and subsequent development of allergic asthma.Abbreviations: BAL, bronchoalveolar lavage; RSV, human respiratory syncytial virusHuman respiratory syncytial virus (RSV) is the primary cause of lower airway disease in infants and children worldwide.³⁹ Although generally limited to the upper airway, RSV infection can also manifest as bronchiolitis and pneumonia in young children and the elderly and has been implicated as a major cause of middle ear infections (otitis media).^3,55 In addition to the problems associated with acute illness, children who experience severe RSV disease in infancy are at increased risk for development of asthma and recurrent wheezing later in childhood.^35,53,56RSV is ubiquitous worldwide, with most children infected in infancy, and essentially all children are infected by 3 y of age.²⁰ Although reinfection throughout life has been documented, the true incidence of upper airway infection is difficult to quantify, given that treatment for such infections typically is not sought. In comparison, lower airway infection is more likely to come to medical attention, and it is currently estimated that at least 33.8 million episodes of RSV-induced acute pulmonary infection in children younger than 5 y occur yearly and that as many as 200,000 of those episodes are fatal.⁴⁰ Although supportive treatment for severe RSV disease is highly effective in infants, access to such treatment is generally only available in industrialized countries. More than 90% of fatal RSV cases occur in the developing world.⁴⁰Mice have been used extensively to study RSV infection, yet there are key limitations of the mouse model for the study of human RSV disease and immunity. The most important of these is the poor permissiveness of the mouse for human RSV. RSV replicates to a limited extent in the mouse lung, and large viral loads delivered in a relatively large volume are generally used with this model. Even with intranasal inoculation, primarily lower, and not upper, airway infection is achieved in WT mice. This scenario is unlike the human disease, in which the upper airway is the primary target of RSV replication.^24,25 Mice are essentially resistant to upper airway infection, with little to no virus detected in nasal washes evaluated by plaque assay, and only rare RSV-antigen–positive cells are detected by immunohistochemistry.^13,18,21 In addition to this altered route of entry, the pattern of lung infection is markedly divergent between humans and mice. In humans, RSV primarily infects ciliated bronchiolar epithelial cells and, to a much lesser extent, alveolar cells.³² In contrast, bronchiolar epithelial cells are infected only rarely in the mouse lung; instead RSV targets the pneumocytes.³⁷Unlike mice, cotton rats are susceptible to RSV infection of the upper airway, and the pattern of lower airway infection mirrors that seen in human patients. The cotton rat was established as a model of RSV infection more than 3 decades ago and has since emerged as the preferred rodent model in which to evaluate RSV therapeutics and vaccine candidates.^{19,23,33,41,49,57,60} In addition, as the availability of species-specific reagents has improved, cotton rats have become an increasingly useful model in which to study RSV pathogenesis.⁶ However, whereas the susceptibility of cotton rats to RSV infection has been established firmly, the pathology of RSV infection in this species has not yet been extensively characterized.Asthma clearly is a multifactorial disease, dependent on both genetic and environmental factors (see references 28 and 30 for recent reviews), and many studies have pointed to a role for respiratory virus infection in the induction of asthma. In one study,¹⁷ the combination of atopy and the presence of virus in nasal secretions synergistically increased the odds ratio for wheezing in children 25-fold, and another study²⁹ showed that repeated rhinovirus infections in the first 3 y of life increased by 26-fold the risk of developing asthma by the age of 6 y. This relationship is obviously a complex one, influenced both by the nature and the timing of the viral infection. Nonetheless, as many as 80% of acute asthma exacerbations in children and approximately 50% in adults are associated with viral infection.^33,42 The majority of these infections are attributed to rhinoviruses, but other respiratory viruses, including influenza virus, RSV, and coronaviruses, can provoke these attacks.^33,42 Beyond the exacerbation of established asthma, evidence that severe RSV disease in infancy is correlated with development of asthma and recurrent wheezing in later childhood is mounting. An association between lower airway RSV infection and subsequent development of recurrent wheezing and asthma was demonstrated more than 30 y ago,¹⁶ and several recent prospective studies have strengthened this correlation.^27,53In the current study, we expand upon existing knowledge of the cotton rat model of RSV infection by characterizing the spread of virus after droplet inhalation and the early and late inflammatory response to viral challenge. We confirm the observation that cotton rats are relatively permissive in regard to RSV infection as compared with mice, and we demonstrate that the infection of cotton rats, like that in the human host, is primarily an upper airway phenomenon. In addition, we show that primary RSV infection of the cotton rat lung, even at the low levels in this study, induces chronic changes consistent with those in cases of human allergic asthma. This finding is of interest because of the substantial literature associating early, severe respiratory infection with the development of asthma later in life. The studies of RSV infection in cotton rats that we describe here suggest that this species may be predisposed to atopy and that, long after virus clearance, changes associated with allergic inflammation persist in human hosts. Therefore, in addition to the usefulness of cotton rats for testing RSV treatments and vaccine candidates, we suggest that this species may serve as a model system for determining the involvement of virus infection in the development of allergic asthma.