Intensive Care Unit-Like Care of Nonhuman Primates with Ebola Virus Disease.

BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (n = 7), intravenous fluids plus levofloxacin (n = 2), or a control group (n = 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; P = .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.

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.

Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters.

UNLABELLED: Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1α and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS. IMPORTANCE: Hantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract, alveolar macrophages are poised to defend against hantavirus infection, but those antiviral responses may also contribute to hantavirus disease. Here, we demonstrate that, like in our prior T and B cell studies, alveolar macrophages neither prevent hantavirus infection nor cause hantavirus disease. While these studies reflect pathogenesis in the hamster model, they should help us rule out specific cell types and prompt us to consider other potential mechanisms of disease in an effort to improve the outcome of human HPS.

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.

Ebola Virus Causes Intestinal Tract Architectural Disruption and Bacterial Invasion in Non-Human Primates.

In the 2014⁻2016 West Africa Ebola Virus (EBOV) outbreak, there was a significant concern raised about the potential for secondary bacterial infection originating from the gastrointestinal tract, which led to the empiric treatment of many patients with antibiotics. This retrospective pathology case series summarizes the gastrointestinal pathology observed in control animals in the rhesus EBOV-Kikwit intramuscular 1000 plaque forming unit infection model. All 31 Non-human primates (NHPs) exhibited lymphoid depletion of gut-associated lymphoid tissue (GALT) but the severity and the specific location of the depletion varied. Mesenteric lymphoid depletion and necrosis were present in 87% (27/31) of NHPs. There was mucosal barrier disruption of the intestinal tract with mucosal necrosis and/or ulceration most notably in the duodenum (16%), cecum (16%), and colon (29%). In the intestinal tract, hemorrhage was noted most frequently in the duodenum (52%) and colon (45%). There were focal areas of bacterial submucosal invasion in the gastrointestinal (GI) tract in 9/31 (29%) of NHPs. Only 2/31 (6%) had evidence of pancreatic necrosis. One NHP (3%) experienced jejunal intussusception which may have been directly related to EBOV. Immunofluorescence assays demonstrated EBOV antigen in CD68+ macrophage/monocytes and endothelial cells in areas of GI vascular injury or necrosis.

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.

Zika Virus Infection in Syrian Golden Hamsters and Strain 13 Guinea Pigs.

To evaluate potential immunocompetent small animal models of Zika virus (ZIKV) infection, we inoculated Syrian golden hamsters (subcutaneously or intraperitoneally) and strain 13 guinea pigs (intraperitoneally) with Senegalese ZIKV strain ArD 41525 or Philippines ZIKV strain CPC-0740. We did not detect viremia in hamsters inoculated subcutaneously with either virus strain, although some hamsters developed virus neutralizing antibodies. However, we detected statistically significant higher viremias (P = 0.0285) and a higher median neutralization titer (P = 0.0163) in hamsters inoculated intraperitoneally with strain ArD 41525 compared with strain CPC-0740. Furthermore, some hamsters inoculated with strain ArD 41525 displayed mild signs of disease. By contrast, strain 13 guinea pigs inoculated intraperitoneally with either strain did not have detectable viremias and less than half developed virus neutralizing antibodies. Our results support the use of the Syrian golden hamster intraperitoneal model to explore phenotypic variation between ZIKV strains.

Disease progression in mice exposed to low-doses of aerosolized clinical isolates of Burkholderia pseudomallei.

Mouse models have been essential to generate supporting data for the research of infectious diseases. Burkholderia pseudomallei, the etiological agent of melioidosis, has been studied using mouse models to investigate pathogenesis and efficacy of novel medical countermeasures to include both vaccines and therapeutics. Previous characterization of mouse models of melioidosis have demonstrated that BALB/c mice present with an acute infection, whereas C57BL/6 mice have shown a tendency to be more resistant to infection and may model chronic disease. In this study, either BALB/c or C57BL/6 mice were exposed to aerosolized human clinical isolates of B. pseudomallei. The bacterial strains included HBPUB10134a (virulent isolate from Thailand), MSHR5855 (virulent isolate from Australia), and 1106a (relatively attenuated isolate from Thailand). The LD50 values were calculated and serial sample collections were performed in order to examine the bacterial burdens in tissues, histopathological features of disease, and the immune response mounted by the mice after exposure to aerosolized B. pseudomallei. These data will be important when utilizing these models for testing novel medical countermeasures. Additionally, by comparing highly virulent strains with attenuated isolates, we hope to better understand the complex disease pathogenesis associated with this bacterium.

Low potential for mechanical transmission of Ebola virus via house flies (Musca domestica).

BACKGROUND: Ebola virus (EBOV) infection results in high morbidity and mortality and is primarily transmitted in communities by contact with infectious bodily fluids. While clinical and experimental evidence indicates that EBOV is transmitted via mucosal exposure, the ability of non-biting muscid flies to mechanically transmit EBOV following exposure to the face had not been assessed. RESULTS: To investigate this transmission route, house flies (Musca domestica Linnaeus) were used to deliver an EBOV/blood mixture to the ocular/nasal/oral facial mucosa of four cynomolgus macaques (Macaca fascicularis Raffles). Following exposure, macaques were monitored for evidence of infection through the conclusion of the study, days 57 and 58. We found no evidence of systemic infection in any of the exposed macaques. CONCLUSIONS: The results of this study indicate that there is a low potential for the mechanical transmission of EBOV via house flies - the conditions in this study were not sufficient to initiate infection.

Identification and pathological characterization of persistent asymptomatic Ebola virus infection in rhesus monkeys.

Ebola virus (EBOV) persistence in asymptomatic humans and Ebola virus disease (EVD) sequelae have emerged as significant public health concerns since the 2013-2016 EVD outbreak in Western Africa. Until now, studying how EBOV disseminates into and persists in immune-privileged sites was impossible due to the absence of a suitable animal model. Here, we detect persistent EBOV replication coinciding with systematic inflammatory responses in otherwise asymptomatic rhesus monkeys that had survived infection in the absence of or after treatment with candidate medical countermeasures. We document progressive EBOV dissemination into the eyes, brain and testes through vascular structures, similar to observations in humans. We identify CD68+ cells (macrophages/monocytes) as the cryptic EBOV reservoir cells in the vitreous humour and its immediately adjacent tissue, in the tubular lumina of the epididymides, and in foci of histiocytic inflammation in the brain, but not in organs typically affected during acute infection. In conclusion, our data suggest that persistent EBOV infection in rhesus monkeys could serve as a model for persistent EBOV infection in humans, and we demonstrate that promising candidate medical countermeasures may not completely clear EBOV infection. A rhesus monkey model may lay the foundation to study EVD sequelae and to develop therapies to abolish EBOV persistence.