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The clinical significance of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA in stool remains uncertain. We found that extrapulmonary dissemination of infection to the gastrointestinal tract, assessed by the presence of SARS-CoV-2 RNA in stool, is associated with decreased coronavirus disease 2019 (COVID-19) survival. Measurement of SARS-CoV-2 RNA in stool may have utility for clinical risk assessment.

We report cellular nanosponges as an effective medical countermeasure to the SARS-CoV-2 virus. Two types of cellular nanosponges are made of the plasma membranes derived from human lung epithelial type II cells or human macrophages. These nanosponges display the same protein receptors, both identified and unidentified, required by SARS-CoV-2 for cellular entry. It is shown that, following incubation with the nanosponges, SARS-CoV-2 is neutralized and unable to infect cells. Crucially, the nanosponge platform is agnostic to viral mutations and potentially viral species, as well. As long as the target of the virus remains the identified host cell, the nanosponges will be able to neutralize the virus.

Using an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent COVID-19 patients, we identified three human antibodies that when combined demonstrated both robust viral suppressive properties against all tested SARS-CoV-2 variants of concern in vitro and profound anti-viral efficacy in vivo. In this report, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant antibodies directed at non-overlapping surfaces on the Spike protein, each with particularly effective antiviral activity. One antibody has a composite epitope blocking ACE2 binding, one antibody bridges two Spike proteins, and one antibody neutralizes virus by binding to a conserved epitope outside of ACE2 binding site. These antibodies, when administered after viral infection, potently decreased viral load in lungs of infected Syrian golden hamsters in a dose-dependent manner, elicited broad anti-viral neutralizing activity against multiple SARS-CoV-2 variants, and induced a robust anti-viral effector function response, including phagocytosis, and activation of classical complement pathway. Our pre-clinical data demonstrate that the unique three antibody cocktail IMM-BCP-01 is a potent and dose-efficient approach to treat early viral infection and prevent SARS-CoV-2 in susceptible individuals. One sentence summaryWe describe three human antibodies that recognize unique non-overlapping epitopes on Spike protein and, when combined, exhibit highly potent in vitro activity against multiple SARS-CoV-2 variants of concern, including Delta, that translates into a striking dose-dependent efficacy against two SARS-CoV-2 isolates in vivo, augmented by a robust, rare epitope-driven Fc effector response.

The coronavirus disease 2019 (COVID‐19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID‐19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), the toll‐like receptor 4 (TLR‐4) agonist monophosphoryl lipid A (MPLA), and SARS‐CoV‐2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen‐presenting cells (APCs) for the generation of adaptive immunity. MSR‐based vaccines generate robust and durable cellular and humoral responses against SARS‐CoV‐2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime‐boost and a single‐dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS‐CoV‐2 variants or new pathogens. Together, the data show a promising COVID‐19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens., Mesoporous silica rods (MSRs) scaffolds are used as a platform to induce adaptive immunity against SARS‐CoV‐2 antigens. Sustained delivery of GM‐CSF, MPLA, and a variety of SARS‐CoV‐2 subunit immunogens from the MSR vaccine recruite immune cells into the scaffolds and consistently induce antigen‐specific long‐lasting antibodies, cytotoxic T lymphocyte responses (CTL), and viral neutralization in vitro.

Background and Objectives: Early warning of bacterial and viral infection, prior to the development of overt clinical symptoms, allows not only for improved patient care and outcomes but also enables faster implementation of public health measures (patient isolation and contact tracing). Our primary objectives in this effort are 3-fold. First, we seek to determine the upper limits of early warning detection through physiological measurements. Second, we investigate whether the detected physiological response is specific to the pathogen. Third, we explore the feasibility of extending early warning detection with wearable devices.Research Methods: For the first objective, we developed a supervised random forest algorithm to detect pathogen exposure in the asymptomatic period prior to overt symptoms (fever). We used high-resolution physiological telemetry data (aortic blood pressure, intrathoracic pressure, electrocardiograms, and core temperature) from non-human primate animal models exposed to two viral pathogens: Ebola and Marburg (N = 20). Second, to determine reusability across different pathogens, we evaluated our algorithm against three independent physiological datasets from non-human primate models (N = 13) exposed to three different pathogens: Lassa and Nipah viruses and Y. pestis. For the third objective, we evaluated performance degradation when the algorithm was restricted to features derived from electrocardiogram (ECG) waveforms to emulate data from a non-invasive wearable device.Results: First, our cross-validated random forest classifier provides a mean early warning of 51 ± 12 h, with an area under the receiver-operating characteristic curve (AUC) of 0.93 ± 0.01. Second, our algorithm achieved comparable performance when applied to datasets from different pathogen exposures – a mean early warning of 51 ± 14 h and AUC of 0.95 ± 0.01. Last, with a degraded feature set derived solely from ECG, we observed minimal degradation – a mean early warning of 46 ± 14 h and AUC of 0.91 ± 0.001.Conclusion: Under controlled experimental conditions, physiological measurements can provide over 2 days of early warning with high AUC. Deviations in physiological signals following exposure to a pathogen are due to the underlying host’s immunological response and are not specific to the pathogen. Pre-symptomatic detection is strong even when features are limited to ECG-derivatives, suggesting that this approach may translate to non-invasive wearable devices.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity—suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants., Graphical abstract, Unbiased charting of memory B cell receptor-encoded antibodies from COVID-19 convalescent subjects identifies seven major antibody competition groups recognizing epitopic regions with group assignment correlating with cross-CoV-reactivity breadth and neutralization potency. SARS-CoV-2 variants tend to escape antibodies from groups with the most potent neutralizers, but many retain affinity, showing that redundant components of a primary immune response establish durable protection from evolving pathogens.

SummaryThe SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross–SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection. CA extern

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 non-survivors 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.

When working with the novel coronavirus SARS-CoV-2 during a pandemic response, having a rapid, reproducible and reliable assay for infectious virus quantitation and utilization for evaluation of potential therapeutics is critical. Compared to traditional agarose overlay plaques visualized with neutral red, assays performed with Avicel R RC-591 semi-solid overlay provide a simplified format for rapid and easy detection and neutralization testing. The method is easily modified for higher throughput using dispensers or automated processing. Fixation using formalin provides flexibility when dealing with pathogenic agents such as SARS-CoV-2 where tissue culture plates might be removed from biocontainment for staining. Although plaque assays are considered straightforward in principle, having an easily reproducible, consistent plaque assay is an invaluable tool.

There are a variety of methods routinely used in the sterilization of medical devices using hydrogen peroxide (H2O2) including vaporization, plasma generation and ionization. Many of these systems are used for sterilization and are validated for bioburden reduction using bacterial spores.Here, we explored the benefits of using vaporized H2O2 (VHP) treatment of N95 respirators for emergency decontamination and reuse to alleviate PPE shortages for healthcare workers in the COVID-19 emergency. The factors that are considered for the effective reuse of these respirators are the fit, the filter efficiency and the decontamination/disinfection level for SARS-CoV-2, which is the causative virus for COVID-19 and other organisms of concern in the hospital environment such as methicillin-resistant Staphylococcus aureus or Clostridium difficile. WE showed that the method did not affect fit or filter efficiency at least for one cycle and resulted in a >6 log reduction in bacterial spores and >3.8 log reduction in the infectious SARS-CoV2 load on N95 respirators.

Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.

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.

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.

The recent epidemic of Ebola virus disease in West Africa resulted in an unprecedented number of cases and deaths. Due to the scope of the outbreak combined with the lack of available approved treatment options, there was strong motivation to investigate any potential drug which had existing data reporting anti-Ebola activity. Drugs with demonstrated antiviral activity in the nonhuman primate models already approved for another indication or for which there was existing safety data were considered to be priorities for evaluation by the World Health Organization. Sertraline hydrochloride was reported to have anti-Ebola activity in vitro alone and in combination with other approved drugs. Although the efficacy was less than 100% in the murine model, the established safety profile of this product, the potential benefit alone and in combination, as well as the lack of other available options prioritized this compound for testing in the Ebola virus intramuscular rhesus macaque challenge model. Using a blinded dosing strategy, we demonstrated that high dose sertraline monotherapy provided no benefit for the prevention of Ebola virus disease in rhesus macaques with regards to reduction of viral load, morbidity, or survival highlighting the challenges of translating results between in vitro and in vivo models.

Previous studies have demonstrated little efficacy of interferons (IFNs) in animal models of Ebola virus disease. However, these studies were limited to a small number of type I IFNs and, during the most recent outbreak of Ebola virus, questions regarding the suitability of the animal models to evaluate IFNs were raised. To address the potential that anti-Ebola virus activity was overlooked, type I and type II IFNs (α-2a, α-2b, -β, -γ, and -universal) were tested in a variety of cell types (Vero E6, Huh 7 cells, and human macrophages). IFNs are weak inhibitors of Ebola virus Makona in these cell lines.

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.

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.

Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

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.

Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention., Author summary Lassa fever virus (LASV) is endemic in West Africa and can cause fatal infection. Currently, there is no vaccine or effective post-exposure treatment. Here we identify 3.3, a small molecule that inhibits LASV infection by targeting lysosome-associated membrane protein 1 (LAMP1), which binds to the LASV glycoprotein (GP) and promotes virus membrane fusion and infection. Our analysis reveals that cholesterol is an important cofactor for LAMP1 binding to LASV GP and that 3.3 inhibits infection by displacing cholesterol from LAMP1. Thus, 3.3 is a candidate for further development as a LASV inhibitor.

Identifying effective antivirals for treating Ebola virus disease (EVD) and minimizing transmission of such disease is critical. A variety of cell-based assays have been developed for evaluating compounds for activity against Ebola virus. However, very few reports discuss the variable assay conditions that can affect the results obtained from these drug screens. Here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-Ebola virus activity using established cell lines and human primary cells. The effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for 50% and/ or 90% inhibition (EC50, EC90) was evaluated using the FDA-approved compound, toremifene citrate. In these studies, we show that altering cell-based assay conditions can have an impact on apparent drug potency as measured by the EC50. These results further support the importance of developing standard operating procedures for generating reliable and reproducible in vitro data sets for potential antivirals.

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.

The limited data available for long-term Ebola virus disease health outcomes suggest that sequelae persist for longer than 1 year after infection. The magnitude of the present outbreak in west Africa necessitates a more complete understanding of the health effects and future medical needs of these patients.We invited adult survivors of the 2007 Bundibugyo Ebola virus outbreak in Uganda and their contacts to take part in an observational study roughly 29 months after the outbreak. We collected information about health status, functional limitations, and demographics. We collected blood samples for clinical chemistry, haematology, and filovirus antibodies using ELISA. Analyses were restricted to probable and confirmed survivors and their seronegative contacts.We recruited 70 survivors of the 2007 Bundibugyo Ebola virus and 223 contacts. We did analyses for 49 probable and confirmed survivors and 157 seronegative contacts. Survivors of the Bundibugyo Ebola virus were at significantly increased risk of ocular deficits (retro-orbital pain [RR 4·3, 95% CI 1·9-9·6; p0·0001], blurred vision [1·9, 1·1-3·2; p=0·018]), hearing loss (2·3, 1·2-4·5; p=0·010), difficulty swallowing (2·1, 1·1-3·9; p=0·017), difficulty sleeping (1·9, 1·3-2·8; p=0·001), arthralgias (2·0, 1·1-3·6; p=0·020), and various constitutional symptoms controlling for age and sex. Chronic health problems (prevalence ratio [PR] 2·1, 95% CI 1·2-3·6; p=0·008) and limitations due to memory loss or confusion (PR 5·8, 1·5-22·4; p=0·010) were also reported more frequently by survivors of Bundibugyo Ebola virus.Long-term sequelae persist for more than 2 years after Ebola virus disease. Definition of health consequences related to Ebola virus disease could improve patient care for survivors and contribute to understanding of disease pathogenesis.Chemical Biological Technologies Directorate, Defense Threat Reduction Agency.

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Early pathogen exposure detection allows better patient care and faster implementation of public health measures (patient isolation, contact tracing). Existing exposure detection most frequently relies on overt clinical symptoms, namely fever, during the infectious prodromal period. We have developed a robust machine learning based method to better detect asymptomatic states during the incubation period using subtle, sub-clinical physiological markers. Starting with high-resolution physiological waveform data from non-human primate studies of viral (Ebola, Marburg, Lassa, and Nipah viruses) and bacterial (Y. pestis) exposure, we processed the data to reduce short-term variability and normalize diurnal variations, then provided these to a supervised random forest classification algorithm and post-classifier declaration logic step to reduce false alarms. In most subjects detection is achieved well before the onset of fever; subject cross-validation across exposure studies (varying viruses, exposure routes, animal species, and target dose) lead to 51h mean early detection (at 0.93 area under the receiver-operating characteristic curve [AUCROC]). Evaluating the algorithm against entirely independent datasets for Lassa, Nipah, andY. pestisexposures un-used in algorithm training and development yields a mean 51h early warning time (at AUCROC=0.95). We discuss which physiological indicators are most informative for early detection and options for extending this capability to limited datasets such as those available from wearable, non-invasive, ECG-based sensors.

Multiple products are being developed for use against filoviral infections. Efficacy for these products will likely be demonstrated in nonhuman primate models of filoviral disease to satisfy licensure requirements under the Animal Rule, or to supplement human data. Typically, the endpoint for efficacy assessment will be survival following challenge, however, there exists no standardized approach for assessing the health or euthanasia criteria for filovirus-exposed nonhuman primates. Consideration of objective criteria is important to (a) ensure test subjects are euthanized without unnecessary distress, (b) enhance the likelihood that animals exhibiting mild or moderate signs of disease are not prematurely euthanized, (c) minimize the occurrence of spontaneous deaths and loss of end-stage samples, (d) enhance the reproducibility of experiments between different researchers, and (e) provide a defensible rationale for euthanasia decisions that withstands regulatory scrutiny. Historic records were compiled for 58 surviving and non-surviving monkeys exposed to Ebola virus at the US Army Medical Research Institute of Infectious Diseases. Clinical pathology parameters were statistically analyzed and those exhibiting predicative value for survival are reported. These findings may be useful for standardization of objective euthanasia assessments in rhesus monkeys exposed to Ebola virus and may serve as a useful approach for other standardization efforts.

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences.

Antigen presenting cells (APCs), including macrophages and dendritic cells, are early and sustained targets of Ebola virus (EBOV) infection in vivo. Because EBOV activates mitogen-activated protein kinase (MAPK) signaling upon infection of APCs, we evaluated the effect of pyridinyl imidazole inhibitors of p38 MAPK on EBOV infection of human APCs and EBOV mediated cytokine production from human DCs. The p38 MAPK inhibitors reduced viral replication in PMA-differentiated macrophage-like human THP-1 cells with an IC50 of 4.73 μM (SB202190), 8.26 μM (p38kinhIII) and 8.21 μM (SB203580) and primary human monocyte-derived dendritic cells (MDDCs) with an IC50 of 2.67 μM (SB202190). Furthermore, cytokine production from EBOV-treated MDDCs was inhibited in a dose-dependent manner. A control pyridinyl imidazole compound failed to inhibit either EBOV infection or cytokine induction. Using an established EBOV virus-like particle (VLP) entry assay, we demonstrate that inhibitor pretreatment blocked VLP entry suggesting that the inhibitors blocked infection and replication at least in part by blocking EBOV entry. Taken together, our results indicate that pyridinyl imidazole p38 MAPK inhibitors may serve as leads for the development of therapeutics to treat EBOV infection.

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.

Background Ebola virus is the causative agent of a severe syndrome in humans with a fatality rate that can approach 90 %. During infection, the host immune response is thought to become dysregulated, but the mechanisms through which this happens are not entirely understood. In this study, we analyze RNA sequencing data to determine the host response to Ebola virus infection in circulating immune cells. Results Approximately half of the 100 genes with the strongest early increases in expression were interferon-stimulated genes, such as ISG15, OAS1, IFIT2, HERC5, MX1 and DHX58. Other highly upregulated genes included cytokines CXCL11, CCL7, IL2RA, IL2R1, IL15RA, and CSF2RB, which have not been previously reported to change during Ebola virus infection. Comparing this response in two different models of exposure (intramuscular and aerosol) revealed a similar signature of infection. The strong innate response in the aerosol model was seen not only in circulating cells, but also in primary and secondary target tissues. Conversely, the innate immune response of vaccinated macaques was almost non-existent. This suggests that the innate response is a major aspect of the cellular response to Ebola virus infection in multiple tissues. Conclusions Ebola virus causes a severe infection in humans that is associated with high mortality. The host immune response to virus infection is thought to be an important aspect leading to severe pathology, but the components of this overactive response are not well characterized. Here, we analyzed how circulating immune cells respond to the virus and found that there is a strong innate response dependent on active virus replication. This finding is in stark contrast to in vitro evidence showing a suppression of innate immune signaling, and it suggests that the strong innate response we observe in infected animals may be an important contributor to pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3060-0) contains supplementary material, which is available to authorized users.

Early detection of Ebola virus (EBOV) infection is essential to halting transmission and adjudicating appropriate treatment. However, current methods rely on viral identification, and this approach can misdiagnose presymptomatic and asymptomatic individuals. In contrast, disease-driven alterations in the host transcriptome can be exploited for pathogen-specific diagnostic biomarkers. Here, we present for the first time EBOV-induced changes in circulating miRNA populations of nonhuman primates (NHPs) and humans. We retrospectively profiled longitudinally-collected plasma samples from rhesus macaques challenged via intramuscular and aerosol routes and found 36 miRNAs differentially present in both groups. Comparison of miRNA abundances to viral loads uncovered 15 highly correlated miRNAs common to EBOV-infected NHPs and humans. As proof of principle, we developed an eight-miRNA classifier that correctly categorized infection status in 64/74 (86%) human and NHP samples. The classifier identified acute infections in 27/29 (93.1%) samples and in 6/12 (50%) presymptomatic NHPs. These findings showed applicability of NHP-derived miRNAs to a human cohort, and with additional research the resulting classifiers could impact the current capability to diagnose presymptomatic and asymptomatic EBOV infections.

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.

Viruses of the family Filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. While many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. Current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. Contemporary methods of supportive care and previous treatment approaches for human patients are also discussed.

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.

Detailed studies describing the pathogenesis of Rift Valley fever (RVF) virus (RVFV) in the mouse model are lacking. A fully characterized small animal model of RVF is needed to evaluate potential vaccines and therapeutics. In this study, we characterized the pathogenesis of RVFV throughout the disease course in mice. Infection produced high-titer viremia and demonstrated RVFV tropism for a variety of tissue and individual cell types. Overwhelming infection of hepatocytes, accompanied by apoptosis, was a major consequence of infection. The majority of mice died or were euthanatized between days 3 and 6 postinfection with severe hepatitis. The remaining mice effectively cleared virus from the liver and blood, but exhibited neuroinvasion and developed panencephalitis. In addition, we characterized a number of other virological, clinicopathological, and histopathological features of RVFV infection in mice. The mouse model therefore mimics both the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF.

We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus–cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure–activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus–cell but not cell–cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus–cell fusion.

We evaluated the ability of flagellin, a highly effective mucosal adjuvant in mice and non-human primates, to promote mucosal innate and adaptive immunity in aged mice. We found that intratracheal instillation of flagellin induced a stronger respiratory innate response in aged mice than in young mice, and that intranasal instillation of flagellin was equally effective at triggering recruitment of T and B lymphocytes to the draining lymph nodes of young and aged mice. Intranasal immunization of aged mice with flagellin and the Yersinia pestis protein F1 promoted specific IgG and IgA production, but at lower levels and lower avidities than in young mice. Although intranasal instillation of flagellin and F1 antigen increased germinal center formation and size in young mice, it did not do so in aged mice. Our findings are consistent with the conclusion that flagellin can promote adaptive immune responses in aged mice, but at a less robust level than in young mice.

Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein’s poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed, however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations.