Exploring Crimean-Congo Hemorrhagic Fever Virus-Induced Hepatic Injury Using Antibody-Mediated Type I Interferon Blockade in Mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hepatic injury in humans. However, the mechanism(s) causing this damage is poorly characterized. CCHFV produces an acute disease, including liver damage, in mice lacking type I interferon (IFN-I) signaling due to either STAT-1 gene deletion or disruption of the IFN-I receptor 1 gene. Here, we explored CCHFV-induced liver pathogenesis in mice using an antibody to disrupt IFN-I signaling. When IFN-I blockade was induced within 24 h postexposure to CCHFV, mice developed severe disease with greater than 95% mortality by 6 days postexposure. In addition, we observed increased proinflammatory cytokines, chemoattractants, and liver enzymes in these mice. Extensive liver damage was evident by 4 days postexposure and was characterized by hepatocyte necrosis and the loss of CLEC4F-positive Kupffer cells. Similar experiments in CCHFV-exposed NOD-SCID-γ (NSG), Rag2-deficient, and perforin-deficient mice also demonstrated liver injury, suggesting that cytotoxic immune cells are dispensable for hepatic damage. Some apoptotic liver cells contained viral RNA, while other apoptotic liver cells were negative, suggesting that cell death occurred by both intrinsic and extrinsic mechanisms. Protein and transcriptional analysis of livers revealed that activation of tumor necrosis factor superfamily members occurred by day 4 postexposure, implicating these molecules as factors in liver cell death. These data provide insights into CCHFV-induced hepatic injury and demonstrate the utility of antibody-mediated IFN-I blockade in the study of CCHFV pathogenesis in mice.IMPORTANCE CCHFV is an important human pathogen that is both endemic and emerging throughout Asia, Africa, and Europe. A common feature of acute disease is liver injury ranging from mild to fulminant hepatic failure. The processes through which CCHFV induces severe liver injury are unclear, mostly due to the limitations of existing small-animal systems. The only small-animal model in which CCHFV consistently produces severe liver damage is mice lacking IFN-I signaling. In this study, we used antibody-mediated blockade of IFN-I signaling in mice to study CCHFV liver pathogenesis in various transgenic mouse systems. We found that liver injury did not depend on cytotoxic immune cells and observed extensive activation of death receptor signaling pathways in the liver during acute disease. Furthermore, acute CCHFV infection resulted in a nearly complete loss of Kupffer cells. Our model system provides insight into both the molecular and the cellular features of CCHFV hepatic injury.

B Cells and Antibodies in Kawasaki Disease.

The etiology of Kawasaki disease (KD), the leading cause of acquired heart disease in children, is currently unknown. Epidemiology supports a relationship of KD to an infectious disease. Several pathological mechanisms are being considered, including a superantigen response, direct invasion by an infectious etiology or an autoimmune phenomenon. Treating affected patients with intravenous immunoglobulin is effective at reducing the rates of coronary aneurysms. However, the role of B cells and antibodies in KD pathogenesis remains unclear. Murine models are not clear on the role for B cells and antibodies in pathogenesis. Studies on rare aneurysm specimens reveal plasma cell infiltrates. Antibodies generated from these aneurysmal plasma cell infiltrates showed cross-reaction to intracellular inclusions in the bronchial epithelium of a number of pathologic specimens from children with KD. These antibodies have not defined an etiology. Notably, a number of autoantibody responses have been reported in children with KD. Recent studies show acute B cell responses are similar in children with KD compared to children with infections, lending further support of an infectious disease cause of KD. Here, we will review and discuss the inconsistencies in the literature in relation to B cell responses, specific antibodies, and a potential role for humoral immunity in KD pathogenesis or diagnosis.

Single molecule-sensitive probes for imaging RNA in live cells.

To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.

Respiratory syncytial virus induces host RNA stress granules to facilitate viral replication.

Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.

Respiratory syncytial virus uses a Vps4-independent budding mechanism controlled by Rab11-FIP2.

Respiratory syncytial virus (RSV) infects polarized epithelia, which have tightly regulated trafficking because of the separation and maintenance of the apical and basolateral membranes. Previously we established a link between the apical recycling endosome (ARE) and the assembly of RSV. The current studies tested the role of a major ARE-associated protein, Rab11 family interacting protein 2 (FIP2) in the virus life cycle. A dominant-negative form of FIP2 lacking its N-terminal C2 domain reduced the supernatant-associated RSV titer 1,000-fold and also caused the cell-associated virus titer to increase. These data suggested that the FIP2 C2 mutant caused a failure at the final budding step in the virus life cycle. Additionally, truncation of the Rab-binding domain from FIP2 caused its accumulation into mature filamentous virions. RSV budding was independent of the ESCRT machinery, the only well-defined budding mechanism for enveloped RNA viruses. Therefore, RSV uses a virus budding mechanism that is controlled by FIP2.

GP38-targeting monoclonal antibodies protect adult mice against lethal Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. Limited evidence suggests that antibodies can protect humans against lethal CCHFV disease but the protective efficacy of antibodies has never been evaluated in adult animal models. Here, we used adult mice to investigate the protection provided against CCHFV infection by glycoprotein-targeting neutralizing and non-neutralizing monoclonal antibodies (mAbs). We identified a single non-neutralizing antibody (mAb-13G8) that protected adult type I interferon-deficient mice >90% when treatment was initiated before virus exposure and >60% when administered after virus exposure. Neutralizing antibodies known to protect neonatal mice from lethal CCHFV infection failed to confer protection regardless of immunoglobulin G subclass. The target of mAb-13G8 was identified as GP38, one of multiple proteolytically cleaved glycoproteins derived from the CCHFV glycoprotein precursor polyprotein. This study reveals GP38 as an important antibody target for limiting CCHFV pathogenesis and lays the foundation to develop immunotherapeutics against CCHFV in humans.

Intracellular Detection of Viral Transcription and Replication Using RNA FISH.

Many hemorrhagic fever viruses require BSL-3 or BSL-4 laboratory containment for study. The necessary safety precautions associated with this work often contribute to longer assay times and lengthy decontamination procedures. Here we will discuss recent advances in RNA fluorescence in situ hybridization (FISH) that not only allow entirely new investigations into the replication of these viruses but also demonstrate how this method can be applied to any virus with a known sequence and how it can be rapidly performed to minimize time spent in high containment. We have adapted existing protocols for mRNA detection with appropriate changes for examining viruses in a variety of containment laboratories (Shaffer et al., PLoS One 8:e75120, 2013; Raj et al., Nat Methods 5:877-879, 2008).

A Multicomponent Animal Virus Isolated from Mosquitoes.

RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

Activation of protein kinase R is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication.

We performed experiments to determine the effect of PKR activation on respiratory syncytial virus (RSV) replication. We first determined that RSV infection activates PKR which induces the phosphorylation of eIF2α, resulting in the formation of host stress granules. We used RNA interference to decrease endogenous PKR levels. RSV replication was not altered in cells deficient for PKR expression. However, RSV-mediated stress granule formation was significantly reduced in PKR-knockdown cells. As an alternative method to block PKR activation, we used treatment with the kinase inhibitor 2-aminopurine (2-AP). We observed that 2-AP treatment significantly reduced viral replication. We also treated PKR-knockdown cells with 2-AP and inoculated with RSV. Under these conditions, 2-AP treatment diminished viral replication in the absence of PKR expression. These results suggest that PKR activation has a minimal effect on RSV replication and that the antiviral effect of 2-AP during RSV infection likely occurs via a PKR-independent mechanism.