TRS H1N1 influenza viruses circulating in North America have caused only sporadic and isolated human infection since 2005, in contrast to the 2009 H1N1 virus, which was responsible for the first pandemic of the 21st century. In this study, we utilized the ferret model to assess the relative virulence and transmissibility of two TRS H1N1 influenza viruses associated with human illness compared with those of both seasonal and 2009 H1N1 virus infection. Overall, TRS H1N1 viruses isolated from humans in 2007 and 2008 exhibited pathogenicities in ferrets similar to that of the 2009 H1N1 viruses but displayed a limited capacity for transmission by respiratory droplets compared with that of either seasonal or 2009 H1N1 viruses. To further expand the utility of the ferret model in the study of influenza virus pathogenesis, we analyzed complete blood counts and serum chemistries to identify detrimental lymphohematopoietic effects of H1N1 and H5N1 virus infection in this mammalian model. By providing comprehensive analyses of both baseline and virus-infected ferrets, we describe for the first time the potentiation of the ferret model to recapitulate many of these parameters not previously studied in an outbred laboratory mammalian model following influenza virus infection.
Virus was detected throughout respiratory and intestinal tissues of ferrets infected with TRS or 2009 H1N1 viruses, unlike seasonal influenza viruses, which are typically not detected in the lungs or intestinal tract of infected ferrets (17
). Both swH1β and swH1λ lineage TRS viruses replicated to generally similar titers in respiratory tract tissues; however, Tx/14 virus was detected in the intestinal tissue of 50% of infected ferrets (3/6) compared to only 17% (1/6) following OH/2 virus infection. Both TRS viruses elicited lung pathology that was more pronounced than that elicited by seasonal H1N1 infection and generally similar to that observed following 2009 H1N1 infection in ferrets (10
). Histological features characteristic of pandemic 2009 H1N1 infection, including necrosis of the bronchial epithelium and alveolar edema with inflammatory cell infiltrate, were also observed in TRS H1N1-infected lungs. A recent study of fatal 2009 H1N1 human cases identified a range of histopathological changes in autopsy tissues indicative of diffuse alveolar damage following infection of the lower respiratory tract (27
). The detection of high titers of virus on day 3 p.i. in ferret lung tissue following 2009 H1N1 and TRS virus infection further establishes the ability of these swine origin H1N1 viruses to cause extensive damage to the mammalian lung (17
Unlike seasonal influenza virus infection in ferrets, clinical signs of respiratory symptoms, including sneezing and nasal secretions, were rarely observed among TRS H1N1-infected ferrets. Moreover, TRS H1N1 viruses did not possess the capacity for efficient aerosolized respiratory droplets, which is consistent with the limited, nonsustained transmission of TRS H1N1 viruses observed in humans (17
). It is important to note that, like 2009 H1N1 viruses which possess the capacity to spread efficiently between cohoused ferrets (17
), TRS H1N1 viruses were readily transmitted by direct contact in this animal model. It is reasonable to speculate that such a transmission phenotype could possess an advantage over viruses that transmit poorly or not at all, even in the presence of direct contact (e.g., H5N1 virus [17
]). Thus, virus passage from animal to animal by direct contact may ultimately result in the generation of mutant viruses conferring enhanced transmissibility. Experimentally, such natural virus variants have been isolated from contact ferrets following a rare transmission event in which an H2N2 mutant influenza strain demonstrated improved respiratory droplet transmission to naïve ferrets (22
). Moreover, Sorrell et al. demonstrated that after 10 passages of an H9N2 virus in ferrets, a mutant influenza virus emerged that conferred efficient transmission via respiratory droplets (29
), further demonstrating that minimal molecular changes, often those affecting receptor binding, will result in enhanced transmissibility of some influenza strains. An avian/swine reassortant H2N3 virus isolated from pigs exhibiting respiratory disease was found to possess molecular changes associated with increased affinity for α2-6-linked sialic acid receptors and was capable of direct contact transmission in the ferret model (15
). As such, the capacity for direct contact transmission of TRS H1N1 viruses underscores the public health threat posed by this subtype despite the absence of documented human-to-human transmission of these viruses to date (28
). Identification of the genetic determinants that govern airborne transmission and the receptor binding properties of influenza viruses isolated from swine is needed to understand the factors that may lead to the emergence of future swine origin pandemic influenza viruses.
There is a need to establish additional markers of virulence associated with virus-host interactions that may help identify viruses which possess the capacity to cause severe disease. The use of blood chemistry analysis has been employed to assess ferret health following severe acute respiratory syndrome coronavirus (SARS-CoV) infection; however, investigation of serum chemistries following influenza virus infection in the ferret model has not been previously reported (4
). Several parameters associated with influenza virus infection in humans were also present in ferrets, notably decreased levels of serum albumin following infection with any H1N1 virus tested here. Serum albumin levels were even further reduced from baseline levels following H5N1 infection in ferrets, at a magnitude in accordance with reports of hypoalbuminemia among H5N1 virus-infected patients (12
). Atypical levels of alanine aminotransferase and amylase, detected in some ferrets exhibiting severe H1N1 and H5N1 disease, have also been reported following severe human cases of H5N1, 2009 H1N1, and TRS H1N1 influenza virus infection in humans (1
). The abnormal levels of these and other analytes observed in our hands are indicative of numerous systemic disruptions in severely infected ferrets, including liver and kidney disease and metabolic and nutritional disorders, indicating that severe H5N1 or H1N1 virus infection in this model can result in dysfunction of multiple organ systems in the absence of detectable infectious virus in these tissues. Elevated levels of creatinine and total bilirubin, reported among H5N1 and hospitalized H1N1 virus-infected patients, respectively, were not observed here (11
). Examination of serum chemistries from ferrets infected with an expanded range of influenza viruses, such as additional highly pathogenic avian influenza viruses, will provide a greater understanding of the biological relevance of these findings as they pertain to ferret health following severe infection. Furthermore, increased use of these analyses will allow for a more thorough assessment of ferret health prior to experimental use, potentially identifying ferrets with congenital abnormalities which otherwise would not be detected until postmortem necropsy.
With the exception of transient lymphopenia, there were no significant changes in white blood cell or erythrocyte populations detected among ferrets surviving infection with any H1N1 viruses tested here, similar to what was observed in BALB/c mice following 2009 H1N1 and TRS virus infection (2
). However, complete blood counts performed on ferrets with severe disease requiring euthanasia revealed several abnormalities, including leukopenia, leukocytosis, and lymphopenia, all of which have been observed following severe human infection with H5N1, H1N1, and TRS viruses (1
). While there is currently no established absolute lymphocyte cutoff value associated with lymphopenia in the ferret model, these lymphocytic abnormalities are associated with a poor prognosis in both human infection and mammalian models (19
). Anemia has been reported among H1N1 and H5N1 virus-infected patients, but consistent levels of hemoglobin and hematocrit levels observed in ferrets exhibiting mild to severe disease suggest that the ferret model may not be an ideal model to study erythrocyte parameters following influenza virus infection (11
). Despite the altered lymphohematopoietic parameters discussed here, systemic tissues collected from ferrets euthanatized during the course of the observational period due to severe disease did not possess more extensive histopathological changes than infected ferrets exhibiting milder disease (data not shown).
While the 2009 H1N1 pandemic virus has caused severe and fatal disease in previously healthy individuals, the majority of cases worldwide have resolved without the need for hospitalization or medical treatment. Likewise, 2009 H1N1 and TRS virus infection in ferrets generally resulted in moderate illness, with only few ferrets exhibiting severe disease. By differentiating between mild to moderately ill ferrets and those animals exhibiting severe disease, a more comprehensive evaluation of ferret health following infection with these H1N1 viruses was possible and provides a greater understanding of lymphohematopoietic involvement during influenza virus infection in this model. Determination of TRS H1N1 virus pathogenicity and transmissibility further places in context those features shared by swine lineage influenza viruses associated with disease in humans. Given the ability of swine lineage influenza viruses to jump the species barrier to infect humans, continuous surveillance and monitoring of influenza viruses that circulate among swine remain of critical importance to public health.