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Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.

The ability to appropriately mimic human disease is critical for using animal models as a tool for understanding virus pathogenesis. In the case of Nipah virus (NiV), infection of humans appears to occur either through inhalation, contact with or consumption of infected material. In two of these circumstances, respiratory or sinusoidal exposure represents a likely route of infection. In this study, intermediate-size aerosol particles (~7 μm) of NiV-Malaysia were used to mimic potential routes of exposure by focusing viral deposition in the upper respiratory tract. Our previous report showed this route of exposure extended the disease course and a single animal survived the infection. Here, analysis of the peripheral immune response found minimal evidence of systemic inflammation and depletion of B cells during acute disease. However, the animal that survived infection developed an early IgM response with rapid development of neutralizing antibodies that likely afforded protection. The increase in NiV-specific antibodies correlated with an expansion of the B cell population in the survivor. Cell-mediated immunity was not clearly apparent in animals that succumbed during the acute phase of disease. However, CD4+ and CD8+ effector memory cells increased in the survivor with correlating increases in cytokines and chemokines associated with cell-mediated immunity. Interestingly, kinetic changes of the CD4+ and CD8bright T cell populations over the course of acute disease were opposite from animals that succumbed to infection. In addition, increases in NK cells and basophils during convalescence of the surviving animal were also evident, with viral antigen found in NK cells. These data suggest that a systemic inflammatory response and "cytokine storm" are not major contributors to NiV-Malaysia pathogenesis in the AGM model using this exposure route. Further, these data demonstrate that regulation of cell-mediated immunity, in addition to rapid production of NiV specific antibodies, may be critical for surviving NiV infection.

Antibody titers against a viral pathogen are typically measured using an antigen binding assay, such as an enzyme-linked immunosorbent assay (ELISA), which only measures the ability of antibodies to identify a viral antigen of interest. Neutralization assays measure the presence of virus-neutralizing antibodies in a sample. Traditional neutralization assays, such as the plaque reduction neutralization test (PRNT), are often difficult to use on a large scale due to being both labor and resource intensive. Here we describe an Ebola virus fluorescence reduction neutralization assay (FRNA), which tests for neutralizing antibodies, that requires only a small volume of sample in a 96-well format and is easy to automate. The readout of the FRNA is the percentage of Ebola virus-infected cells measured with an optical reader or overall chemiluminescence that can be generated by multiple reading platforms. Using blinded human clinical samples (EVD survivors or contacts) obtained in Liberia during the 2013-2016 Ebola virus disease outbreak, we demonstrate there was a high degree of agreement between the FRNA-measured antibody titers and the Filovirus Animal Non-clinical Group (FANG) ELISA titers with the FRNA providing information on the neutralizing capabilities of the antibodies.

Nipah virus (NiV) infection can lead to severe respiratory or neurological disease in humans. Transmission of NiV has been shown to occur through contact with virus contaminated fomites or consumption of contaminated food. Previous results using the African green monkey (AGM) model of NiV infection identified aspects of infection that, while similar to humans, don't fully recapitulate disease. Previous studies also demonstrate near uniform lethality that is not consistent with human NiV infection. In these studies, aerosol exposure using an intermediate particle size (7μm) was used to mimic potential human exposure by facilitating virus deposition in the upper respiratory tract. Computed tomography evaluation found some animals developed pulmonary parenchymal disease including consolidations, ground-glass opacities, and reactive adenopathy. Despite the lack of neurological signs, magnetic resonance imaging identified distinct brain lesions in three animals, similar to those previously reported in NiV-infected patients. Immunological characterization of tissues collected at necropsy suggested a local pulmonary inflammatory response with increased levels of macrophages in the lung, but a limited neurologic response. These data provide the first clear evidence of neurological involvement in the AGM that recapitulates human disease. With the development of a disease model that is more representative of human disease, these data suggest that NiV infection in the AGM may be appropriate for evaluating therapeutic countermeasures directed at virus-induced neuropathogenesis.

Middle East respiratory syndrome coronavirus (MERS-CoV) presents an emerging threat to public health worldwide by causing severe respiratory disease in humans with high virulence and case fatality rate (about 35%) since 2012. Little is known about the pathogenesis and innate antiviral response in primary human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) upon MERS-CoV infection. In this study, we assessed MERS-CoV replication as well as induction of inflammatory cytokines and chemokines in MDMs and immature and mature MDDCs. Immature MDDCs and MDMs were permissive for MERS-CoV infection, while mature MDDCs were not, with stimulation of proinflammatory cytokine and chemokine upregulation in MDMs, but not in MDDCs. To further evaluate the antiviral activity of well-defined drugs in primary antigen presenting cells (APCs), three compounds (chloroquine, chlorpromazine and toremifine), each with broad-spectrum antiviral activity in immortalized cell lines, were evaluated in MDMs and MDDCs to determine their antiviral effect on MERS-CoV infection. While chloroquine was not active in these primary cells, chlorpromazine showed strong anti-MERS-CoV activity, but it was associated with high cytotoxicity narrowing the potential window for drug utilization. Unlike in established cells, toremifene had marginal activity when tested in antigen presenting cells, with high apparent cytotoxicity, also limiting its potential as a therapeutic option. These results demonstrate the value of testing drugs in primary cells, in addition to established cell lines, before initiating preclinical or clinical studies for MERS treatment and the importance of carefully assessing cytotoxicity in drug screen assays. Furthermore, these studies also highlight the role of APCs in stimulating a robust protective immune response to MERS-CoV infection.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that can result in severe pulmonary disease and fatal encephalitis in humans and is responsible for outbreaks in Bangladesh, Malaysia, Singapore, India and possibly the Philippines. NiV has a negative-sense RNA genome that contains six genes and serves as a template for production of viral mRNA transcripts. NiV mRNA transcripts are subsequently translated into viral proteins. Traditionally, NiV quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays have relied on using primer sets that amplify a target (N that encodes the nucleocapsid) within the coding region of the viral gene that also amplifies viral mRNA. Here we describe a novel one-step qRT-PCR assay targeting the intergenic region separating the viral F and G proteins, thereby eliminating amplification of the viral mRNA. This assay is more accurate than the traditional qRT-PCR in quantifying concentrations of viral genomic RNA.

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.

Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8-10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition.

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease.

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.

Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers.

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells. The combined effort resulted in successful virus isolation; however, no single approach was successful for all 4 simarteriviruses. We describe several approaches that might be used to isolate additional simarteriviruses for phenotypic characterization. Our results will expedite laboratory studies of simarteriviruses to elucidate virus-host interactions, assess zoonotic risk, and develop medical countermeasures.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in Western Asia, Middle East respiratory syndrome. Attempts to identify the natural reservoirs of MERS-CoV have focused in part on dromedaries. Bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. For this study, ten distinct cell lines derived from bats of divergent species were exposed to MERS-CoV. Plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. We found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed CD26/DPP4, the functional human receptor for MERS-CoV. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. Overexpression of human CD26/DPP4 receptor conferred MERS-CoV susceptibility to resistant bat cell lines. Finally, sequential passage of MERS-CoV in permissive bat cells established persistent infection with concomitant downregulation of CD26/DPP4 surface expression. Together, these results imply that bats indeed could be among the MERS-CoV host spectrum, and that cellular restriction of MERS-CoV is determined by CD26/DPP4 expression rather than by downstream restriction factors.

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1-2×10(7) PFU) or CPXV (>10(2) PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.

To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.

A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVDeltaG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVDeltaG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.

Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.

Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine.To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10) particles, two logs lower than that used previously.Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10) rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.

The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV.In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain.SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children.

BackgroundRecent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge.Methods and findingsWe developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection.ConclusionOur data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease.