New Insights Into Marburg Virus Disease Pathogenesis in the Rhesus Macaque Model.

Previously, several studies have been performed to delineate the development and progression of Marburg virus infection in nonhuman primates (NHPs), primarily to clarify the mechanisms of severe (fatal) disease. After the 2013-2016 Ebola virus disease (EVD) epidemic in Western Africa, there has been a reassessment of the available filovirus animal models and the utility of these to faithfully recapitulate human disease. The high lethality of the NHP models has raised doubts as to their ability to provide meaningful data for the full spectrum of disease observed in humans. Of particular interest are the etiologic and pathophysiologic mechanisms underlying postconvalescent sequelae observed in human survivors of EVD and Marburg virus disease (MVD). In the current study, we evaluated the lesions of MVD in NHPs; however, in contrast to previous studies, we focused on the potential for development of sequelae similar to those reported in human survivors of MVD and EVD. We found that during acute MVD in the macaque model, there is frequent inflammation of peripheral nerves, autonomic ganglia, and the iris of the eye. Furthermore, we demonstrate viral infection of the ocular ciliary body and retina, testis, epididymis, ovary, oviduct, uterine endometrium, prostate, and mammary gland. These findings are relevant for both development of postconvalescent sequelae and the natural transmission of virus.

Comparative Transcriptomics in Ebola Makona-Infected Ferrets, Nonhuman Primates, and Humans.

The domestic ferret is a uniformly lethal model of infection for 3 species of Ebolavirus known to be pathogenic in humans. Reagents to systematically analyze the ferret host response to infection are lacking; however, the recent publication of a draft ferret genome has opened the potential for transcriptional analysis of ferret models of disease. In this work, we present comparative analysis of longitudinally sampled blood taken from ferrets and nonhuman primates infected with lethal doses of the Makona variant of Zaire ebolavirus. Strong induction of proinflammatory and prothrombotic signaling programs were present in both ferrets and nonhuman primates, and both transcriptomes were similar to previously published datasets of fatal cases of human Ebola virus infection.

In Vitro and In Vivo Activity of Amiodarone Against Ebola Virus.

At the onset of the 2013-2016 epidemic of Ebola virus disease (EVD), no vaccine or antiviral medication was approved for treatment. Therefore, considerable efforts were directed towards the concept of drug repurposing or repositioning. Amiodarone, an approved multi-ion channel blocker for the treatment of cardiac arrhythmia, was reported to inhibit filovirus entry in vitro. Compassionate use of amiodarone in EVD patients indicated a possible survival benefit. In support of further clinical testing, we confirmed anti-Ebola virus activity of amiodarone in different cell types. Despite promising in vitro results, amiodarone failed to protect guinea pigs from a lethal dose of Ebola virus.

Within-Host Evolution of Simian Arteriviruses in Crab-Eating Macaques.

Simian arteriviruses are a diverse clade of viruses infecting captive and wild nonhuman primates. We recently reported that Kibale red colobus virus 1 (KRCV-1) causes a mild and self-limiting disease in experimentally infected crab-eating macaques, while simian hemorrhagic fever virus (SHFV) causes lethal viral hemorrhagic fever. Here we characterize how these viruses evolved during replication in cell culture and in experimentally infected macaques. During passage in cell culture, 68 substitutions that were localized in open reading frames (ORFs) likely associated with host cell entry and exit became fixed in the KRCV-1 genome. However, we did not detect any strong signatures of selection during replication in macaques. We uncovered patterns of evolution that were distinct from those observed in surveys of wild red colobus monkeys, suggesting that these species may exert different adaptive challenges for KRCV-1. During SHFV infection, we detected signatures of selection on ORF 5a and on a small subset of sites in the genome. Overall, our data suggest that patterns of evolution differ markedly among simian arteriviruses and among host species. IMPORTANCE: Certain RNA viruses can cross species barriers and cause disease in new hosts. Simian arteriviruses are a diverse group of related viruses that infect captive and wild nonhuman primates, with associated disease severity ranging from apparently asymptomatic infections to severe, viral hemorrhagic fevers. We infected nonhuman primate cell cultures and then crab-eating macaques with either simian hemorrhagic fever virus (SHFV) or Kibale red colobus virus 1 (KRCV-1) and assessed within-host viral evolution. We found that KRCV-1 quickly acquired a large number of substitutions in its genome during replication in cell culture but that evolution in macaques was limited. In contrast, we detected selection focused on SHFV ORFs 5a and 5, which encode putative membrane proteins. These patterns suggest that in addition to diverse pathogenic phenotypes, these viruses may also exhibit distinct patterns of within-host evolution both in vitro and in vivo.

Necrotizing Scleritis, Conjunctivitis, and Other Pathologic Findings in the Left Eye and Brain of an Ebola Virus-Infected Rhesus Macaque (Macaca mulatta) With Apparent Recovery and a Delayed Time of Death.

A 3.5-year-old adult female rhesus macaque (Macaca mulatta) manifested swelling of the left upper eyelid and conjunctiva and a decline in clinical condition 18 days following intramuscular challenge with Ebola virus (EBOV; Kikwit-1995), after apparent clinical recovery. Histologic lesions with strong EBOV antigen staining were noted in the left eye (scleritis, conjunctivitis, and peri-optic neuritis), brain (choriomeningoencephalitis), stomach, proximal duodenum, and pancreas. Spleen, liver, and adrenal glands, common targets for acute infection, appeared histologically normal with no evidence of EBOV immunoreactivity. These findings may provide important insight for understanding sequelae seen in West African survivors of Ebola virus disease.

Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques.

UNLABELLED: Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model. IMPORTANCE: In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure.

Ebola virus exploits a monocyte differentiation program to promote its entry.

Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo. However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.

An atlas of gross and histologic lesions and immunohistochemical immunoreactivity during the temporal progression of aerosolized Lassa virus induced hemorrhagic fever in cynomolgus macaques.

Lassa virus (LASV) causes an acute multisystemic hemorrhagic fever in humans known as Lassa fever, which is endemic in several African countries. This manuscript focuses on the progression of disease in cynomolgus macaques challenged with aerosolized LASV and serially sampled for the development and progression of gross and histopathologic lesions. Gross lesions were first noted in tissues on day 6 and persisted throughout day 12. Viremia and histologic lesions were first noted on day 6 commencing with the pulmonary system and hemolymphatic system and progressing at later time points to include all systems. Immunoreactivity to LASV antigen was first observed in the lungs of one macaque on day 3 and appeared localized to macrophages with an increase at later time points to include immunoreactivity in all organ systems. Additionally, this manuscript will serve as a detailed atlas of histopathologic lesions and disease progression for comparison to other animal models of aerosolized Arenaviral disease.

A Single-shot ChAd3 Vaccine Provides Protection from Intramuscular and Aerosol Sudan Virus Exposure.

Infection with Sudan virus (SUDV) is characterized by an aggressive disease course with case fatality rates between 40-100% and no approved vaccines or therapeutics. SUDV causes sporadic outbreaks in sub-Saharan Africa, including a recent outbreak in Uganda which has resulted in over 100 confirmed cases in one month. Prior vaccine and therapeutic efforts have historically prioritized Ebola virus (EBOV), leading to a significant gap in available treatments. Two vaccines, Erbevo ® and Zabdeno ® /Mvabea ® , are licensed for use against EBOV but are ineffective against SUDV. Recombinant adenovirus vector vaccines have been shown to be safe and effective against filoviruses, but efficacy depends on having low seroprevalence to the vector in the target human population. For this reason, and because of an excellent safety and immunogenicity profile, ChAd3 was selected as a superior vaccine vector. Here, a ChAd3 vaccine expressing the SUDV glycoprotein (GP) was evaluated for immunogenicity and efficacy in nonhuman primates. We demonstrate that a single dose of ChAd3-SUDV confers acute and durable protection against lethal SUDV challenge with a strong correlation between the SUDV GP-specific antibody titers and survival outcome. Additionally, we show that a bivalent ChAd3 vaccine encoding the GP from both EBOV and SUDV protects against both parenteral and aerosol lethal SUDV challenge. Our data indicate that the ChAd3-SUDV vaccine is a suitable candidate for a prophylactic vaccination strategy in regions at high risk of filovirus outbreaks. One Sentence Summary: A single-dose of ChAd3 vaccine protected macaques from lethal challenge with Sudan virus (SUDV) by parenteral and aerosol routes of exposure.

Computed Tomography Imaging for Monitoring of Marburg Virus Disease: a Nonhuman Primate Proof-Of-Concept Study.

Marburg virus (MARV) is a highly virulent zoonotic filovirid that causes Marburg virus disease (MVD) in humans. The pathogenesis of MVD remains poorly understood, partially due to the low number of cases that can be studied, the absence of state-of-the-art medical equipment in areas where cases are reported, and limitations on the number of animals that can be safely used in experimental studies under maximum containment animal biosafety level 4 conditions. Medical imaging modalities, such as whole-body computed tomography (CT), may help to describe disease progression in vivo, potentially replacing ethically contentious and logistically challenging serial euthanasia studies. Towards this vision, we performed a pilot study, during which we acquired whole-body CT images of 6 rhesus monkeys before and 7 to 9 days after intramuscular MARV exposure. We identified imaging abnormalities in the liver, spleen, and axillary lymph nodes that corresponded to clinical, virological, and gross pathological hallmarks of MVD in this animal model. Quantitative image analysis indicated hepatomegaly with a significant reduction in organ density (indicating fatty infiltration of the liver), splenomegaly, and edema that corresponded with gross pathological and histopathological findings. Our results indicated that CT imaging could be used to verify and quantify typical MVD pathogenesis versus altered, diminished, or absent disease severity or progression in the presence of candidate medical countermeasures, thus possibly reducing the number of animals needed and eliminating serial euthanasia. IMPORTANCE Marburg virus (MARV) is a highly virulent zoonotic filovirid that causes Marburg virus disease (MVD) in humans. Much is unknown about disease progression and, thus, prevention and treatment options are limited. Medical imaging modalities, such as whole-body computed tomography (CT), have the potential to improve understanding of MVD pathogenesis. Our study used CT to identify abnormalities in the liver, spleen, and axillary lymph nodes that corresponded to known clinical signs of MVD in this animal model. Our results indicated that CT imaging and analyses could be used to elucidate pathogenesis and possibly assess the efficacy of candidate treatments.

Real-time monitoring of cardiovascular function in rhesus macaques infected with Zaire ebolavirus.

Nine rhesus macaques were implanted with multisensor telemetry devices and internal jugular vein catheters before being infected with Zaire ebolavirus. All animals developed viremia, fever, a hemorrhagic rash, and typical changes of Ebola hemorrhagic fever in clinical laboratory tests. Three macaques unexpectedly survived this usually lethal disease, making it possible to compare physiological parameters in lethally challenged animals and survivors. After the onset of fever, lethal illness was characterized by a decline in mean arterial blood pressure, an increase in pulse and respiratory rate, lactic acidosis, and renal failure. Survivors showed less pronounced change in these parameters. Four macaques were randomized to receive supplemental volumes of intravenous normal saline when they became hypotensive. Although those animals had less severe renal compromise, no apparent survival benefit was observed. This is the first report of continuous physiologic monitoring in filovirus-infected nonhuman primates and the first to attempt cardiovascular support with intravenous fluids.

Detailed analysis of the pathologic hallmarks of Nipah virus (Malaysia) disease in the African green monkey infected by the intratracheal route.

Disease associated with Nipah virus infection causes a devastating and often fatal spectrum of syndromes predominated by both respiratory and neurologic conditions. Additionally, neurologic sequelae may manifest months to years later after virus exposure or apparent recovery. In the two decades since this disease emerged, much work has been completed in an attempt to understand the pathogenesis and facilitate development of medical countermeasures. Here we provide detailed organ system-specific pathologic findings following exposure of four African green monkeys to 2.41×105 pfu of the Malaysian strain of Nipah virus. Our results further substantiate the African green monkey as a model of human Nipah virus disease, by demonstrating both the respiratory and neurologic components of disease. Additionally, we demonstrate that a chronic phase of disease exists in this model, that may provide an important opportunity to study the enigmatic late onset and relapse encephalitis as it is described in human disease.

Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study.

BACKGROUND: We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. METHODS: A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV. FINDINGS: Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. INTERPRETATION: This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections. FUNDING: Defense Threat Reduction Agency.

Natural History of Aerosol-Induced Ebola Virus Disease in Rhesus Macaques.

Ebola virus disease (EVD) is a serious global health concern because case fatality rates are approximately 50% due to recent widespread outbreaks in Africa. Well-defined nonhuman primate (NHP) models for different routes of Ebola virus exposure are needed to test the efficacy of candidate countermeasures. In this natural history study, four rhesus macaques were challenged via aerosol with a target titer of 1000 plaque-forming units per milliliter of Ebola virus. The course of disease was split into the following stages for descriptive purposes: subclinical, clinical, and decompensated. During the subclinical stage, high levels of venous partial pressure of carbon dioxide led to respiratory acidemia in three of four of the NHPs, and all developed lymphopenia. During the clinical stage, all animals had fever, viremia, and respiratory alkalosis. The decompensatory stage involved coagulopathy, cytokine storm, and liver and renal injury. These events were followed by hypotension, elevated lactate, metabolic acidemia, shock and mortality similar to historic intramuscular challenge studies. Viral loads in the lungs of aerosol-exposed animals were not distinctly different compared to previous intramuscularly challenged studies. Differences in the aerosol model, compared to intramuscular model, include an extended subclinical stage, shortened clinical stage, and general decompensated stage. Therefore, the shortened timeframe for clinical detection of the aerosol-induced disease can impair timely therapeutic administration. In summary, this nonhuman primate model of aerosol-induced EVD characterizes early disease markers and additional details to enable countermeasure development.

Postexposure treatment of Marburg virus infection.

Rhesus monkeys are protected from disease when a recombinant vesicular stomatitis virus-based vaccine is administered 20-30 min after infection with Marburg virus. We protected 5/6 monkeys when this vaccine was given 24 h after challenge; 2/6 animals were protected when the vaccine was administered 48 h postinfection.

Previremic Identification of Ebola or Marburg Virus Infection Using Integrated Host-Transcriptome and Viral Genome Detection.

Outbreaks of filoviruses, such as those caused by the Ebola (EBOV) and Marburg (MARV) virus, are difficult to detect and control. The initial clinical symptoms of these diseases are nonspecific and can mimic other endemic pathogens. This makes confident diagnosis based on clinical symptoms alone impossible. Molecular diagnostics for these diseases that rely on the detection of viral RNA in the blood are only effective after significant disease progression. As an approach to identify these infections earlier in the disease course, we tested the effectiveness of viral RNA detection combined with an assessment of sentinel host mRNAs that are upregulated following filovirus infection. RNAseq analysis of EBOV-infected nonhuman primates identified host RNAs that are upregulated at early stages of infection. NanoString probes that recognized these host-response RNAs were combined with probes that recognized viral RNA and were used to classify viral infection both prior to viremia and postviremia. This approach was highly successful at identifying samples from nonhuman primate subjects and correctly distinguished the causative agent in a previremic stage in 10 EBOV and 5 MARV samples. This work suggests that unified host response/viral fingerprint assays can enable diagnosis of disease earlier than testing for viral nucleic acid alone, which could decrease transmission events and increase therapeutic effectiveness.IMPORTANCE Current molecular tests that identify infection with high-consequence viruses such as Ebola virus and Marburg virus are based on the detection of virus material in the blood. These viruses do not undergo significant early replication in the blood and, instead, replicate in organs such as the liver and spleen. Thus, virus begins to accumulate in the blood only after significant replication has already occurred in those organs, making viremia an indicator of infection only after initial stages have become established. Here, we show that a multianalyte assay can correctly identify the infectious agent in nonhuman primates (NHPs) prior to viremia through tracking host infection response transcripts. This illustrates that a single-tube, sample-to-answer format assay could be used to advance the time at which the type of infection can be determined and thereby improve outcomes.

Discovery of Lanama Virus, a Distinct Member of Species Kunsagivirus C (Picornavirales: Picornaviridae), in Wild Vervet Monkeys (Chlorocebus pygerythrus).

We report the discovery and sequence-based molecular characterization of a novel virus, lanama virus (LNMV), in blood samples obtained from two wild vervet monkeys (Chlorocebus pygerythrus), sampled near Lake Nabugabo, Masaka District, Uganda. Sequencing of the complete viral genomes and subsequent phylogenetic analysis identified LNMV as a distinct member of species Kunsagivirus C, in the undercharacterized picornavirid genus Kunsagivirus.

Natural History of Aerosol Induced Lassa Fever in Non­Human Primates.

Lassa virus (LASV), an arenavirus causing Lassa fever, is endemic to West Africa with up to 300,000 cases and between 5000 and 10,000 deaths per year. Rarely seen in the United States, Lassa virus is a CDC category A biological agent inasmuch deliberate aerosol exposure can have high mortality rates compared to naturally acquired infection. With the need for an animal model, specific countermeasures remain elusive as there is no FDA-approved vaccine. This natural history of aerosolized Lassa virus exposure in Macaca fascicularis was studied under continuous telemetric surveillance. The macaque response to challenge was largely analogous to severe human disease with fever, tachycardia, hypotension, and tachypnea. During initial observations, an increase trend of activated monocytes positive for viral glycoprotein was accompanied by lymphocytopenia. Disease uniformly progressed to high viremia followed by low anion gap, alkalosis, anemia, and thrombocytopenia. Hypoproteinemia occurred late in infection followed by increased levels of white blood cells, cytokines, chemokines, and biochemical markers of liver injury. Viral nucleic acids were detected in tissues of three non­survivors at endpoint, but not in the lone survivor. This study provides useful details to benchmark a pivotal model of Lassa fever in support of medical countermeasure development for both endemic disease and traditional biodefense purposes.

Author Correction: Histology, immunohistochemistry, and in situ hybridization reveal overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In Vivo Activity of Amodiaquine against Ebola Virus Infection.

During the Ebola virus disease (EVD) epidemic in Western Africa (2013‒2016), antimalarial treatment was administered to EVD patients due to the high coexisting malaria burden in accordance with World Health Organization guidelines. In an Ebola treatment center in Liberia, EVD patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. As artemether and artesunate are derivatives of artemisinin, the beneficial anti-Ebola virus (EBOV) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. Amodiaquine is a widely used antimalarial in the countries that experience outbreaks of EVD and, therefore, holds promise as an approved drug that could be repurposed for treating EBOV infections. We investigated the potential anti-EBOV effect of amodiaquine in a well-characterized nonhuman primate model of EVD. Using a similar 3-day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. However, the treatment regimen did not result in a survival benefit or decrease of disease signs in EBOV-infected animals. While amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral.