Ebola Virus Disease in Pregnancy: Clinical, Histopathologic, and Immunohistochemical Findings.

Here we describe clinicopathologic features of Ebola virus disease in pregnancy. One woman infected with Sudan virus in Gulu, Uganda, in 2000 had a stillbirth and survived, and another woman infected with Bundibugyo virus had a live birth with maternal and infant death in Isiro, the Democratic Republic of the Congo in 2012. Ebolavirus antigen was seen in the syncytiotrophoblast and placental maternal mononuclear cells by immunohistochemical analysis, and no antigen was seen in fetal placental stromal cells or fetal organs. In the Gulu case, ebolavirus antigen localized to malarial parasite pigment-laden macrophages. These data suggest that trophoblast infection may be a mechanism of transplacental ebolavirus transmission.

Implementation of a National Semen Testing and Counseling Program for Male Ebola Survivors - Liberia, 2015-2016.

According to World Health Organization (WHO) data, the Ebola virus disease (Ebola) outbreak that began in West Africa in 2014 has resulted in 28,603 cases and 11,301 deaths (1). In March 2015, epidemiologic investigation and genetic sequencing in Liberia implicated sexual transmission from a male Ebola survivor, with Ebola virus detected by reverse transcription-polymerase chain reaction (RT-PCR) 199 days after symptom onset (2,3), far exceeding the 101 days reported from an earlier Ebola outbreak (4). In response, WHO released interim guidelines recommending that all male survivors, in addition to receiving condoms and sexual risk reduction counseling at discharge from an Ebola treatment unit (ETU), be offered semen testing for Ebola virus RNA by RT-PCR 3 months after disease onset, and every month thereafter until two consecutive semen specimens collected at least 1 week apart test negative for Ebola virus RNA (5). Male Ebola survivors should also receive counseling to promote safe sexual practices until their semen twice tests negative. When these recommendations were released, testing of semen was not widely available in Liberia. Challenges in establishing and operating the first nationwide semen testing and counseling program for male Ebola survivors included securing sufficient resources for the program, managing a public health semen testing program in the context of ongoing research studies that were also collecting and screening semen, identification of adequate numbers of trained counselors and appropriate health communication messages for the program, overcoming Ebola survivor-associated stigma, identification and recruitment of male Ebola survivors, and operation of mobile teams.

Use of Postexposure Prophylaxis After Occupational Exposure to Zaire ebolavirus.

From September 2014 to April 2015, 6 persons who had occupational exposures to Zaire ebolavirus in West Africa received investigational agent rVSV-ZEBOV or TKM-100802 for postexposure prophylaxis and were monitored in the United States. All patients experienced self-limited symptoms after postexposure prophylaxis; none developed Ebola virus disease.

Human Ebola virus infection results in substantial immune activation.

Four Ebola patients received care at Emory University Hospital, presenting a unique opportunity to examine the cellular immune responses during acute Ebola virus infection. We found striking activation of both B and T cells in all four patients. Plasmablast frequencies were 10-50% of B cells, compared with less than 1% in healthy individuals. Many of these proliferating plasmablasts were IgG-positive, and this finding coincided with the presence of Ebola virus-specific IgG in the serum. Activated CD4 T cells ranged from 5 to 30%, compared with 1-2% in healthy controls. The most pronounced responses were seen in CD8 T cells, with over 50% of the CD8 T cells expressing markers of activation and proliferation. Taken together, these results suggest that all four patients developed robust immune responses during the acute phase of Ebola virus infection, a finding that would not have been predicted based on our current assumptions about the highly immunosuppressive nature of Ebola virus. Also, quite surprisingly, we found sustained immune activation after the virus was cleared from the plasma, observed most strikingly in the persistence of activated CD8 T cells, even 1 mo after the patients' discharge from the hospital. These results suggest continued antigen stimulation after resolution of the disease. From these convalescent time points, we identified CD4 and CD8 T-cell responses to several Ebola virus proteins, most notably the viral nucleoprotein. Knowledge of the viral proteins targeted by T cells during natural infection should be useful in designing vaccines against Ebola virus.

Identification of cell surface molecules involved in dystroglycan-independent Lassa virus cell entry.

Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.

Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies.

Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.

An antibody directed against the fusion peptide of Junin virus envelope glycoprotein GPC inhibits pH-induced membrane fusion.

The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.

Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication.

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.

Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1)-antitrypsin variants.

BACKGROUND: Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1)-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1)-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1)-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1)-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. CONCLUSIONS/SIGNIFICANCE: Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

Breakdown of paraendothelial barrier function during Marburg virus infection is associated with early tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1.

Marburg virus (MARV) infection often causes fulminant shock due to pathologic immune responses and alterations of the vascular system. Cytokines released from virus-infected monocytes/macrophages provoke endothelial activation and vascular hyperpermeability and contribute to the development of shock. Tyrosine phosphorylation of cell-junction proteins is important for the regulation of paraendothelial barrier function. We showed that mediators released from MARV-infected monocytes/macrophages, as well as recombinant tumor necrosis factor (TNF)- alpha /H2O2 and interferon (IFN)- gamma , caused tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) but not of the vascular endothelial (VE) cadherin/catenin complex proteins. Tyrosine phosphorylation of PECAM-1 was associated with delayed opening of interendothelial junctions. Interestingly, we observed an early increase in water permeability in response to TNF- alpha /H2O2 that was not due to an opening of the interendothelial junctions. These data indicate 2 distinct mechanisms for the TNF- alpha /H2O2-mediated decrease in endothelial barrier function involving tyrosine phosphorylation of PECAM-1 but not requiring tyrosine phosphorylation of VE-cadherin or catenin proteins.

Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function.

Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP(1,2) has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP(1,2) were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and delta-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-alpha), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not delta-peptide, induced a recovery of endothelial barrier function following treatment with TNF-alpha. Our results demonstrate that Ebola virus GP(1,2) in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.

Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages.

Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Delta-peptide), GP(1), and GP(1,2Delta). While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP(1,2) in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.

Ectodomain shedding of the glycoprotein GP of Ebola virus.

In this study, release of abundant amounts of the Ebola virus (EBOV) surface glycoprotein GP in a soluble form from virus-infected cells was investigated. We demonstrate that the mechanism responsible for the release of GP is ectodomain shedding mediated by cellular sheddases. Proteolytic cleavage taking place at amino-acid position D637 removes the transmembrane anchor and liberates complexes consisting of GP1 and truncated GP2 (GP(2delta)) subunits from the cell surface. We show that tumor necrosis factor alpha-converting enzyme (TACE), a member of the ADAM family of zinc-dependent metalloproteases, is involved in EBOV GP shedding. This finding shows for the first time that virus-encoded surface glycoproteins are substrates for ADAMs. Furthermore, we provide evidence that shed GP is present in significant amounts in the blood of virus-infected animals and that it may play an important role in the pathogenesis of infection by efficiently blocking the activity of virus-neutralizing antibodies.

The Genome sequence of the SARS-associated coronavirus.

We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.