Persistent Crimean-Congo hemorrhagic fever virus infection in the testes and within granulomas of non-human primates with latent tuberculosis.

Crimean-Congo hemorrhagic fever (CCHF) is the most medically important tick-borne viral disease of humans and tuberculosis is the leading cause of death worldwide by a bacterial pathogen. These two diseases overlap geographically, however, concurrent infection of CCHF virus (CCHFV) with mycobacterial infection has not been assessed nor has the ability of virus to persist and cause long-term sequela in a primate model. In this study, we compared the disease progression of two diverse strains of CCHFV in the recently described cynomolgus macaque model. All animals demonstrated signs of clinical illness, viremia, significant changes in clinical chemistry and hematology values, and serum cytokine profiles consistent with CCHF in humans. The European and Asian CCHFV strains caused very similar disease profiles in monkeys, which demonstrates that medical countermeasures can be evaluated in this animal model against multiple CCHFV strains. We identified evidence of CCHFV persistence in the testes of three male monkeys that survived infection. Furthermore, the histopathology unexpectedly revealed that six additional animals had evidence of a latent mycobacterial infection with granulomatous lesions. Interestingly, CCHFV persisted within the granulomas of two animals. This study is the first to demonstrate the persistence of CCHFV in the testes and within the granulomas of non-human primates with concurrent latent tuberculosis. Our results have important public health implications in overlapping endemic regions for these emerging pathogens.

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.

Ebola virus persistence and disease recrudescence in the brains of antibody-treated nonhuman primate survivors.

Effective therapeutics have been developed against acute Ebola virus disease (EVD) in both humans and experimentally infected nonhuman primates. However, the risk of viral persistence and associated disease recrudescence in survivors receiving these therapeutics remains unclear. In contrast to rhesus macaques that survived Ebola virus (EBOV) exposure in the absence of treatment, we discovered that EBOV, despite being cleared from all other organs, persisted in the brain ventricular system of rhesus macaque survivors that had received monoclonal antibody (mAb) treatment. In mAb-treated macaque survivors, EBOV persisted in macrophages infiltrating the brain ventricular system, including the choroid plexuses. This macrophage infiltration was accompanied by severe tissue damage, including ventriculitis, choroid plexitis, and meningoencephalitis. Specifically, choroid plexus endothelium-derived EBOV infection led to viral persistence in the macaque brain ventricular system. This resulted in apoptosis of ependymal cells, which constitute the blood-cerebrospinal fluid barrier of the choroid plexuses. Fatal brain-confined recrudescence of EBOV infection manifested as severe inflammation, local pathology, and widespread infection of the ventricular system and adjacent neuropil in some of the mAb-treated macaque survivors. This study highlights organ-specific EBOV persistence and fatal recrudescent disease in rhesus macaque survivors after therapeutic treatment and has implications for the long-term follow-up of human survivors of EVD.

A Model for the Production of Regulatory Grade Viral Hemorrhagic Fever Exposure Stocks: From Field Surveillance to Advanced Characterization of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging human pathogen, endemic in areas of China, Japan, and the Korea (KOR). It is primarily transmitted through infected ticks and can cause a severe hemorrhagic fever disease with case fatality rates as high as 30%. Despite its high virulence and increasing prevalence, molecular and functional studies in situ are scarce due to the limited availability of high-titer SFTSV exposure stocks. During the course of field virologic surveillance in 2017, we detected SFTSV in ticks and in a symptomatic soldier in a KOR Army training area. SFTSV was isolated from the ticks producing a high-titer viral exposure stock. Through the use of advanced genomic tools, we present here a complete, in-depth characterization of this viral stock, including a comparison with both the virus in its arthropod source and in the human case, and an in vivo study of its pathogenicity. Thanks to this detailed characterization, this SFTSV viral exposure stock constitutes a quality biological tool for the study of this viral agent and for the development of medical countermeasures, fulfilling the requirements of the main regulatory agencies.

Nipah virus persists in the brains of nonhuman primate survivors.

Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes highly lethal henipavirus encephalitis in humans. Survivors develop various neurologic sequelae, including late-onset and relapsing encephalitis, several months up to several years following initial infection. However, the underlying pathology and disease mechanisms of persistent neurologic complications remain unknown. Here, we demonstrate persistent NiV infection in the brains of grivets that survived experimental exposure to NiV. Encephalitis affected the entire brains, with the majority of NiV detected in the neurons and microglia of the brainstems, cerebral cortices, and cerebella. We identified the vascular endothelium in the brain as an initial target of NiV infection during the acute phase of disease, indicating a primary path of entry for NiV into the brain. Notably, we were unable to detect NiV anywhere else except the brains in the examined survivors. Our findings indicate that late-onset and relapsing encephalitis of NiV in human survivors may be due to viral persistence in the brain and shed light on the pathogenesis of chronic henipavirus encephalitis.

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.

Persistent Marburg Virus Infection in the Testes of Nonhuman Primate Survivors.

Sexual transmission of filoviruses was first reported in 1968 after an outbreak of Marburg virus (MARV) disease and recently caused flare-ups of Ebola virus disease in the 2013-2016 outbreak. How filoviruses establish testicular persistence and are shed in semen remain unknown. We discovered that persistent MARV infection of seminiferous tubules, an immune-privileged site that harbors sperm production, is a relatively common event in crab-eating macaques that survived infection after antiviral treatment. Persistence triggers severe testicular damage, including spermatogenic cell depletion and inflammatory cell invasion. MARV mainly persists in Sertoli cells, leading to breakdown of the blood-testis barrier formed by inter-Sertoli cell tight junctions. This disruption is accompanied by local infiltration of immunosuppressive CD4+Foxp3+ regulatory T cells. Our study elucidates cellular events associated with testicular persistence that may promote sexual transmission of filoviruses and suggests that targeting immunosuppression may be warranted to clear filovirus persistence in damaged immune-privileged sites.