To measure the extent to which IFN-mediated antiviral responses control the virulence of CCHFV in mice, we evaluated CCHFV infection in mice lacking the STAT1 protein, a central component of the IFN signaling pathways. In the absence of the IFN response, CCHFV infection of STAT129 mice leads rapidly to disease and death. A low virus dose of 10 PFU results in 100% mortality, indicating the importance of the IFN response in controlling virus replication and dissemination in these animals. A more limited study using IFN receptor knockout mice demonstrated that mice succumb to CCHFV infection with a similar GMD, providing supporting evidence that the IFN response is crucial for disease manifestation (5
). In this study, high viral replication in CCHFV-infected STAT129 mice was present despite the high plasma levels of IFN-α and IFN-β, indicating that IFN is produced but unable to induce an IFN-mediated antiviral state due to the STAT1 knockout. We hypothesize that the ability of CCHFV to disable or evade components of the IFN response may be human specific. CCHFV infection of animals with a functional IFN antiviral response is, therefore, attenuated and does not cause disease in the animals.
CCHFV-infected STAT129 mice undergo an early burst of viral replication in the blood on day 1 postinfection and in liver and spleen on day 2 before virus disseminates systemically into multiple organs. The resulting high viremia levels in the blood on days 2 and 3 postinfection are comparable to severe disease in human cases (7
). In STAT129 mice, the liver and spleen are the major sites of replication and the only sites where prominent histopathologic changes were noticed. Interestingly, high CCHFV titers in spleen and low titers, late, in the brain were detected in some samples. This differs from the infant mouse model, which found high virus titers in the brain and none in the spleen (33
). CCHFV in the model presented here exhibits hepatotropism, and the virus detected in the brain late in the infection is from blood-borne systemic spread rather than local replication. A subcutaneous inoculation would be a more relevant route when mimicking transmission by tick bite. A recent pilot study in our lab indicated that the subcutaneous route is still 100% lethal. However, the GMD was delayed by 1 day compared to the results for the same virus dose given by the i.p. route. Future studies will utilize this route of infection and compare the response to that seen with the i.p. route.
Clinical studies have demonstrated that thrombocytopenia, leukopenia, and raised levels of liver transaminases are hallmarks of CCHFV infection and can be used to predict fatal outcome in 90% of patients (20
). Similar changes in clinical chemistry and hematology can be found in the STAT129 mouse model, where white blood cell and platelet counts dropped on day 1 postinfection and reached their lowest values on day 2, while increasing slightly on day 3. At this point, further experiments are needed to determine if the leukopenia and thrombocytopenia are due to the migration of circulating white blood cells and platelets to infected organs or diminished production in and mobilization from the bone marrow. The more than 10-fold increase in ALT serum levels of CCHFV-infected animals by 3 dpi compared to the levels in mock-infected animals is a clear indicator of severe liver damage.
Histopathologic findings in CCHF cases are limited because histopathologic studies have been limited to a small number of human cases (6
). A spectrum of severity of hepatic damage was identified, ranging from mild necrosis with occasional Councilman bodies to more severe necrosis with extensive damage to hepatic lobules (6
). The main findings in other tissues included prominent splenic lymphoid apoptosis and depletion and interstitial pneumonia. Except for the interstitial pneumonia, the histopathologic findings in this mouse model, particularly the association of CCHFV with Kupffer cells and hepatocytes, as well as prominent lymphocyte depletion in the spleen, reflect the findings in human cases. Hemorrhages, especially intestinal hemorrhaging, are often seen in human cases but were not found in this mouse model. This might be due to the rapid disease progression or differences in the murine coagulation cascade compared to that in humans.
Cytokines are major players in the pathogenesis of viral hemorrhagic fevers. In this study, we demonstrate that STAT129 mice respond to CCHFV infection with high levels of IFN-γ, IL-1β, IL-6, IL-10, CCL-2, and TNF on days 2 and 3 postinfection. IL-1β is a major mediator of innate immune reactions and is largely responsible for the acute-phase response, including fever and anorexia (9
). CCL-2 is a marker of severe disease in murine and primate models of Ebola virus (17
). In this model, IL-1β and CCL-2 peak on day 2 postinfection, with high levels on day 3, and correlate with viremia levels. We hypothesize that CCL-2 and IL-1β expression could enhance CCHFV infection by recruiting monocytes/macrophage cells to inflammatory sites early on and therefore increase virus replication. IFN-γ is critical for innate and adaptive immunity against viral infections and is produced by NK cells and activated memory CTL cells (28
). Therefore, high levels of IFN-γ on days 2 and 3 postinfection suggest a substantial activation of those cells. Recent clinical studies detected elevated levels of TNF, IL-6, and IL-10 in patients with CCHF (13
). Significantly higher levels of IL-6 and TNF were seen in CCHF patients with a fatal outcome than in nonfatal cases, whereas the IL-10 levels were not significantly different (13
). In another study, high TNF levels correlated with the severity of CCHF disease, and the results suggested that IL-6 can be found in both mild and severe cases of CCHF (25
). The results of these studies clearly signify the relevance of TNF, IL-6, and IL-10 in human CCHF cases, and the results presented here imply that they also play a critical role in the pathogenesis of our mouse model. IL-6 is a major cytokine of the acute-phase response and has been shown to raise body temperature, and TNF was shown to cause hypothermia in mice (21
). Strikingly, IL-6 peaks on day 2 postinfection and TNF peaks on day 3, clearly coinciding with body temperature changes observed in animals. TNF production also contributes to macrophage activation, with resulting hemophagocytosis, and is correlated with the severity of other viral hemorrhagic fevers (16
The changes in peripheral lymphocytes affected by CCHFV and their association with the severity of disease and mortality are very limited. However, evidence from human infections suggests immunological correlations with death. Two studies have looked at T, B, and NK cells in CCHF cases with contradicting results (1
). The first study correlated an increase in total NK numbers with severe cases of CCHF, where the highest NK cell counts occurred in fatal cases. In the second study, the NK cell counts were unchanged, while higher CTL counts correlated with both fatal cases and a higher viral load. However, this study looked at percentages and not absolute counts (38
). In our study, the lymphocyte and APC populations in the spleen were activated within 3 dpi. The numbers of NK, B and T cells initially increased on day 1 postinfection, possibly influenced by early cytokine/chemokine production or other factors resulting from early viral replication in the spleen. However, the increases in absolute numbers were not sustained, and this was confirmed by the histopathological evidence of lymphocyte depletion in the spleen and leukopenia in the blood. This suggests that both the innate and adaptive immune systems may be sabotaged by the loss of leukocytes in the blood and spleen. However, since the STAT129 mice died by day 4 and there were no virus-specific IgM antibodies by 3 dpi in CCHFV-infected WT129 mice, clearly the innate immune system was unable to control the infection long enough for the adaptive immune response to ramp up. The percentage of activated NK cells increased up to day 3, but the absolute numbers showed the reverse trend, suggesting a loss of NK cells regardless of activation. This severely undermined the ability of the NK cells to control early viral replication. This may be due to CCHFV infecting primarily macrophages and DC (26
). In addition to the loss of leukocytes, there is also some indication of a defective activation of immune cells. Our study demonstrates an insufficient upregulation of MHC-II in macrophages and DC that would be necessary for the correct activation of the adaptive immune response. Adequate levels of MHC-II are important for priming of naïve T cells; therefore, low MHC-II levels may contribute to the elimination of lymphocytes and hinder the ability to control the CCHFV infection.
Ribavirin is a synthetic purine nucleotide analog with a broad-spectrum antiviral activity that may be due to the alteration of the cellular nucleotide pools and inhibition of viral mRNA synthesis (19
). The effectiveness of ribavirin in CCHF cases has been described in a few observational studies (12
) that have been criticized for a lack of randomization and small sample size. Ribavirin significantly reduced mortality and extended GMD in a neonate mouse model (33
). Here, we show that ribavirin protected 100% of STAT129 mice when challenged with 2.5 times the LD50
of CCHFV. Partial protection and extended GMD was achieved when animals were challenged with 250 times the LD50
and ribavirin was given early. Survivors showed delayed elevation in temperature compared to the results for nonsurvivors and untreated animals, suggesting delayed or lower viremia. When ribavirin was given 24 h postinfection, none of the animals survived, although GMD was delayed in 40% of the animals. Thus, the viremia levels at 24 h postinfection might already be too high to be significantly reduced by ribavirin. As a result, we hypothesize that the initial phase following CCHFV infection is crucial in controlling virus replication. Ribavirin may reach most of the primary cells infected in the abdominal cavity before systemic spread of the virus occurs, therefore reducing the initial virus load significantly. We did not measure viremia and virus titer in organs of infected STAT129 mice treated with ribavirin. Future work will show to what degree viremia and virus titers in organs are reduced after ribavirin treatment.
In conclusion, we have developed and characterized a new mature mouse model for CCHFV infection, allowing us to study the pathogenesis of this reemerging arbovirus. This study presents the first in-depth in vivo analysis of CCHFV pathophysiology and offers a validated small-animal model that exhibits key features of fatal human CCHF. This model may prove useful to study pathogenesis, test therapeutic strategies, and study virus attenuation.