Expression of interferon-induced antiviral genes is delayed in a STAT1 knockout mouse model of Crimean-Congo hemorrhagic fever
© Bowick et al.; licensee BioMed Central Ltd. 2012
Received: 23 November 2011
Accepted: 19 June 2012
Published: 19 June 2012
Crimean Congo hemorrhagic fever (CCHF) is a tick-borne hemorrhagic zoonosis associated with high mortality. Pathogenesis studies and the development of vaccines and antivirals against CCHF have been severely hampered by the lack of suitable animal model. We recently developed and characterized a mature mouse model for CCHF using mice carrying STAT1 knockout (KO).
Given the importance of interferons in controlling viral infections, we investigated the expression of interferon pathway-associated genes in KO and wild-type (WT) mice challenged with CCHF virus. We expected that the absence of the STAT1 protein would result in minimal expression of IFN-related genes. Surprisingly, the KO mice showed high levels of IFN-stimulated gene expression, beginning on day 2 post-infection, while in WT mice challenged with virus the same genes were expressed at similar levels on day 1.
We conclude that CCHF virus induces similar type I IFN responses in STAT1 KO and WT mice, but the delayed response in the KO mice permits rapid viral dissemination and fatal illness.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a Nairovirus of the Bunyaviridae and is the etiological agent of Crimean-Congo hemorrhagic fever (CCHF). The virus is associated with mortality rates from 5 to 70% . The pathogenesis of the disease is largely not understood due the lack of a suitable animal model. We recently described the establishment of an adult mouse model for CCHF using STAT1 knockout (KO) mice. STAT1 KO mice are highly susceptible to the virus and the mouse model exhibits key features seen in human cases of CCHF. Mice lacking interferon responses have been increasingly used as models for other hemorrhagic fever viruses, including the families flaviviridae, filoviridae, arenaviridae, and bunyaviridae [2–8].
Using the STAT1 KO model, we noted the production of high levels of interferon α and β in plasma of infected mice . We hypothesized that, despite the lack of STAT1, this interferon may lead to activation of certain interferon-regulated genes, possibly by signaling through alternative pathways. We used interferon α/β response PCR arrays (SA Biosciences, Frederick, MD) to investigate gene expression changes in the spleen and the liver of KO and wild-type (WT) mice infected with CCHFV at 1, 2 and 3 days post-infection. Experiments were conducted as previously described . Briefly, KO and WT mice were challenged intraperitoneally with 100 PFU of CCHFV strain IbAr 10200. Livers and spleens were harvested on day 1, 2 and 3 post-infection. Gene expression of infected KO and WT mice was compared to mock infected KO and WT mice. Liver and spleen were chosen as they were the tissues that showed histopathological changes and had the highest viral titers . All animal studies were performed in accordance with institutional animal care and use protocols. An overview of all data is provided in Additional file 1: Tables S1 and Additional file 2: Tables S2. We noted modest induction of STAT1 in the KO mice; we attribute this to the incomplete knockout of all exons of the STAT1 gene which leads to production of a non-functional STAT1 protein .
We next characterized the significance of specific cellular functions and pathways during infection (Additional file 3: Figure S1). At 1 day post infection, both groups of mice showed similar significance of antigen presentation and immune cell trafficking in the spleen. However, WT mice showed a greater transcriptional response of genes involved in the inflammatory response, antimicrobial response, gene expression, post-translational modification and protein folding. Analysis of gene expression associated with canonical pathways revealed increased numbers of differentially expressed genes associated with the roles of pattern recognition receptors (PRRs), interferon signaling, interferon response factor activation by PRRs, dendritic cell maturation and PKR induction of the antiviral response in the WT mice at 1 day post-infection. However, by 2 days post-infection, there were no striking differences between the pathway significance profiles between the two groups, suggesting that, by 2 days post-infection, KO mice are responding qualitatively similarly to WT mice.
In this report, we have shown that infection of STAT KO mice with CCHFV leads to induction of many interferon-induced genes associated with the antiviral response, but that this response is delayed compared to infection of WT mice. STAT1 KO mice have been used in a number of studies using a range of pathogens [16–22]. Similar to what we demonstrated here, some of these studies reported STAT1-independent IFN pathway gene expression patterns. A recent study demonstrated that much of the STAT1-independent activity is dependent on STAT2, since mice lacking both STAT1 and STAT2 show susceptibility to dengue virus . Our report is consistent with the observations of STAT1-independent type-I IFN signaling described in this study, with STAT1 being required for an early activation of type-I IFN production and antiviral gene expression. Consistent with our mouse model, several studies showed an increased replication in target tissues most likely due to lack of antiviral state of cells allowing the virus to replicate in more cells compared to the wildtype [17, 18, 22]. Interestingly, we also demonstrated residual STAT1 activity in this study, a phenomenon also described by another group . At this point, it is unclear whether the IFN-dependent gene expression seen in this study arose from STAT1-independent pathways, might have resulted, at least in part, from a small amount of residual STAT1 activity, or if they are induced by another mechanism, e.g., the virus itself or by other cytokines. Further studies in our laboratory will focus on elucidating this mechanism.
Many of the findings described are consistent with our previous report  and provide a basis for these observations. Interestingly, in human cells, the type-I interferon response and the MxA protein in particular have been shown to inhibit the replication of CCHFV [25, 26]. Others have demonstrated with Dugbe virus, closely related to CCHFV, that certain IFN genes such as PKR and MxA are not sufficient for protection in a mouse model . Our data shows that induction of these genes is insufficient for protection against CCHFV. This may reveal species-specific differences, in vitro versus in vivo differences, or could be further evidence that the delay in induction of antiviral genes is critical.
In summary, we have further characterized the STAT1 KO mouse model for CCHFV infection. Given our finding of significant antiviral gene expression, we postulate that, as discussed in our previous characterization of the model, that, while the IFN response is critical in the control of CCHFV, it appears the kinetics of the response, combined with the activation of multiple pathways, is the most important factor in determining the outcome of infection.
We thank Dr. Mike Bray for critical reading of the manuscript. This work was funded by the Public Health Agency of Canada; DAB received funding from the Natural Sciences and Engineering Research Council of Canada; GCB is funded by the University of Texas Medical Branch Department of Microbiology & Immunology.
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