The rapid introduction of swine origin H1N1 viruses into human populations in 2009 necessitated the detailed study of these viruses to better understand their capacity to cause disease in mammalian species. Because of the similarity in the HA proteins of the 2009 H1N1 viruses and classical swine H1N1, including the 1918 pandemic virus, a side-by-side comparison was made among these viruses representing a span of approximately 91 years. This animal model has proven a useful tool for the study of influenza viruses with pandemic potential, due to its utility in measuring infectivity, pathogenesis, and subsequent application for the evaluation of vaccine and antiviral candidates. In this study, we used this model to assess the relative virulence of 2009 H1N1 viruses isolated from human patients compared with both swine lineage influenza viruses previously associated with human infection and highly pathogenic H1N1 and H5N1 viruses. Importantly, these studies revealed that 2009 H1N1 viruses exhibited mild to moderate virulence in mice compared with highly pathogenic avian H5N1 or 1918 viruses, as defined by morbidity, mortality, hemostatic assessments, lung histopathology, and lung cytokine production. Viruses isolated from severe or fatal human cases did not possess heightened virulence in this model. As the 2009 H1N1 pandemic continues to cause human infection and disease, this information will contribute toward a more comprehensive understanding of these viruses in the context of previous human infections with swine lineage influenza viruses and provide a well-characterized model for the study of antiviral and other therapeutic strategies against this emergent pandemic strain.
Viruses isolated from 2009 pandemic H1N1 patients replicated efficiently in the lungs of mice and sporadically and to lower titers in the noses and were restricted to the murine respiratory tract. This pattern of virus replication is similar to that observed with the 1918 reconstructed virus in mice (40
). A recent study by Itoh et al., which assessed 2009 H1N1 virus replication in the mouse, confirmed this lack of systemic spread but detected virus at high titers in nasal turbinates (18
). This study also reported that select 2009 H1N1 viruses were lethal to mice at high inoculum doses, a finding not recapitulated here. Differences between laboratories in anesthesia agents used, in addition to laboratory-specific conditions, may have contributed to the discordance in severity of disease observed between studies. In agreement with previous work, the 2009 H1N1 viruses tested here produced pulmonary pathology similar to those of other influenza A viruses and included mild to moderate bronchiolitis and alveolitis (18
). Necrosis was not a common feature, and neutrophilic responses were typically mild. In contrast, the 1918 and VN/1203 viruses produced moderate to severe inflammation, and necrosis of bronchiolar and alveolar epithelium was frequent, as was alveolar and hilar interstitial edema.
Gastrointestinal symptoms were reported in approximately 40% of 2009 H1N1 virus-infected patients, and virus was recovered from the intestinal tract of ferrets following inoculation with 2009 H1N1 viruses; however, the 2009 H1N1 viruses tested here were not recovered from intestinal mouse tissue (7
). We also assessed the ability of 2009 H1N1 viruses to cause disease following ocular inoculation, as some influenza virus subtypes can use this portal of entry to initiate virus infection (3
). The inability of 2009 H1N1 viruses to mount a productive infection in mice following ocular inoculation recapitulates the absence of conjunctivitis reported following 2009 H1N1 human infection and further suggests that the primary mode of 2009 H1N1 virus transmission is via the respiratory tract (7
Collection of blood from mice during the acute phase of infection was performed to assess the influence of 2009 H1N1 virus infection on lymphocyte populations and other hemostatic parameters. Severe influenza virus infection in humans with H5N1 and 2009 H1N1 viruses has been associated with leukopenia, lymphopenia, and thrombocytopenia; these clinical characteristics have not been consistently observed among mild cases of 2009 H1N1 virus infection in humans (4
). Highly virulent viruses, including the reconstructed 1918 virus and the H5N1 virus used in this study, have previously been shown to result in peripheral blood lymphopenia in the mouse model (24
). While transient lymphopenia was observed in mice following 2009 H1N1 virus infection, there was significantly less overall lymphocyte depletion compared with highly virulent viruses or the triple-reassortant OH/2 virus (Table ; Fig. ). Further clinical data from 2009 H1N1 virus-infected patients will contribute toward a comprehensive understanding of the change in hemostatic parameters following infection with this pandemic virus. There is currently a paucity of information pertaining to the consequence of influenza virus infection on complete blood counts in the mouse model as presented in this study in Table . By comparing viruses of different subtypes and degrees of virulence, we are better able to place in context the results obtained from 2009 H1N1 virus-infected mice.
The dysregulation of cytokines and chemokines during infection with H5N1 avian influenza viruses has been proposed to contribute to the severity of disease in human patients (25
). Currently, there is little information on the proinflammatory response induced by 2009 H1N1 viruses and its relevance to disease severity in humans. A recent study described the increased production of several cytokines in mice infected with the 2009 H1N1 virus CA/4 compared with a seasonal H1N1 virus (18
). As these experiments were performed with only one 2009 H1N1 isolate and did not offer a side-by-side comparison with viruses known to elicit hypercytokinemia following infection, selected analytes were chosen for further study here to more broadly assess the induction of cytokines and chemokines following infection with multiple human 2009 H1N1 isolates. We did not observe substantial differences in mouse cytokine production among 2009 H1N1 viruses isolated from patients in the early stages of the pandemic. Levels of IL-12 and MCP-1 following 2009 H1N1 virus infection were significantly higher compared with NJ/8 virus, similar to the results in the study by Itoh et al., which demonstrated elevated levels of cytokines from CA/4 virus compared with a less virulent seasonal H1N1 virus (18
). Conversely, cytokine production following OH/2 virus infection was elevated compared with 2009 H1N1 viruses, which correlates with the heightened pathogenicity observed with the triple-reassortant virus in mice. Furthermore, production of IL-12 and MCP-1 by H1N1 viruses in this study was significantly reduced compared with the H5N1 virus VN/1203, similarly to a previous report demonstrating excessive release of cytokines by H5N1 viruses compared with H1N1 viruses, including the 1918 reconstructed virus (33
). Our results agree with a recent study which found weak cytokine production following 2009 H1N1 virus infection in human macrophages and dendritic cells (31
). These findings suggest that hypercytokinemia is not a general feature of 2009 H1N1 viruses, including those viruses isolated from fatal cases.
Similarly to previously published studies, we did not observe molecular features among 2009 H1N1 viruses which are known to correlate with virus pathogenicity in the mouse model. The E627K substitution in PB2 has been associated with enhanced virulence of HPAI viruses and is also present in the reconstructed 1918 virus (17
). The presence of this amino acid in NJ/8 virus indicates that not all H1N1 viruses which bear this substitution possess high virulence in the mouse model. The accessory protein PB1-F2 has known roles in virus pathogenicity and inflammation following influenza virus infection (27
). All viruses in this study which possessed a full-length PB1-F2 protein (VN/1203, 1918, and OH/2), and only these viruses, were capable of mounting a lethal infection in the mouse model. Future study addressing the potential for enhanced pathogenicity of 2009 H1N1 viruses following the introduction of a full-length PB1-F2 is warranted. The triple-reassortant H1N1 virus OH/2 demonstrated enhanced morbidity and mortality in mice compared with 2009 H1N1 viruses, along with elevated levels of proinflammatory cytokines and more pronounced dysregulation of hemostatic and lymphostatic parameters. The genetic composition of OH/2 virus, a member of the swH1-gamma lineage, generally aligns with 2009 H1N1 viruses, with the exception of Eurasian and not classical swine lineage NA and M genes present in 2009 isolates (11
). Triple-reassortant H1N1 viruses, including OH/2 virus, remain antigenically similar to the 2009 H1N1 isolates (11
). Detailed study of the contribution of individual genes to the heightened pathogenicity of OH/2 virus found in this study compared with 2009 isolates will allow for a greater understanding of the potential for these viruses to cause severe disease.
Well-characterized mammalian models have afforded the opportunity for a more comprehensive understanding of previous pandemic viruses and those viruses considered to hold pandemic potential (24
). Establishing similar models for 2009 H1N1 viruses, such as the mouse model presented here, will accelerate the identification of those molecular correlates which contribute to the unique features of this newest pandemic strain and support efforts to limit the spread and severity of disease via the use of vaccines and antivirals. Direct comparison in vivo
between 2009 H1N1 viruses and previous viruses of swine origin associated with human infection additionally presents the opportunity to more closely examine viruses within this lineage as they evolved from causing sporadic human infections to acquiring properties which resulted in the first pandemic of the 21st century.