Hemorrhagic Disease In Humans Research Articles

Autophagy facilitates antibody-enhanced dengue virus infection in human pre-basophil/mast cells.

BackgroundDengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.Methodology/Principal FindingsWe showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BC74A mutant.Conclusions/SignificanceOur results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

Ebola virus modulates transforming growth factor β signaling and cellular markers of mesenchyme-like transition in hepatocytes.

Ebola virus (EBOV) causes a severe hemorrhagic disease in humans and nonhuman primates, with a median case fatality rate of 78.4%. Although EBOV is considered a public health concern, there is a relative paucity of information regarding the modulation of the functional host response during infection. We employed temporal kinome analysis to investigate the relative early, intermediate, and late host kinome responses to EBOV infection in human hepatocytes. Pathway overrepresentation analysis and functional network analysis of kinome data revealed that transforming growth factor (TGF-β)-mediated signaling responses were temporally modulated in response to EBOV infection. Upregulation of TGF-β signaling in the kinome data sets correlated with the upregulation of TGF-β secretion from EBOV-infected cells. Kinase inhibitors targeting TGF-β signaling, or additional cell receptors and downstream signaling pathway intermediates identified from our kinome analysis, also inhibited EBOV replication. Further, the inhibition of select cell signaling intermediates identified from our kinome analysis provided partial protection in a lethal model of EBOV infection. To gain perspective on the cellular consequence of TGF-β signaling modulation during EBOV infection, we assessed cellular markers associated with upregulation of TGF-β signaling. We observed upregulation of matrix metalloproteinase 9, N-cadherin, and fibronectin expression with concomitant reductions in the expression of E-cadherin and claudin-1, responses that are standard characteristics of an epithelium-to-mesenchyme-like transition. Additionally, we identified phosphorylation events downstream of TGF-β that may contribute to this process. From these observations, we propose a model for a broader role of TGF-β-mediated signaling responses in the pathogenesis of Ebola virus disease. Ebola virus (EBOV), formerly Zaire ebolavirus, causes a severe hemorrhagic disease in humans and nonhuman primates and is the most lethal Ebola virus species, with case fatality rates of up to 90%. Although EBOV is considered a worldwide concern, many questions remain regarding EBOV molecular pathogenesis. As it is appreciated that many cellular processes are regulated through kinase-mediated phosphorylation events, we employed temporal kinome analysis to investigate the functional responses of human hepatocytes to EBOV infection. Administration of kinase inhibitors targeting signaling pathway intermediates identified in our kinome analysis inhibited viral replication in vitro and reduced EBOV pathogenesis in vivo. Further analysis of our data also demonstrated that EBOV infection modulated TGF-β-mediated signaling responses and promoted "mesenchyme-like" phenotypic changes. Taken together, these results demonstrated that EBOV infection specifically modulates TGF-β-mediated signaling responses in epithelial cells and may have broader implications in EBOV pathogenesis.

189: Differences in cytokine responses during infection with high and low pathogenic Ebola viruses

To date five Ebolavirus species have been identified, differing in pathogenicity. The highly pathogenic Zaire ebolavirus (EBOV) causes a hemorrhagic disease in humans with case fatality rates up to 90%. A hallmark of EBOV infection is a dysregulated immune response, including high levels of cytokines. In contrast, Reston ebolavirus (RESTV) seems to be nonpathogenic in humans, as only asymptomatic cases of RESTV infection in humans have been reported. The mechanisms of protection against RESTV in humans are not yet understood. As cytokines might play a role in the pathogenesis of EBOV infection, we analyzed the cytokine response in EBOV- and RESTV-infected human macrophages and dendritic cells. These cells not only orchestrate innate and adaptive immune responses but are also early targets of Ebolaviruses. We observed degradation of I κ Bα and nuclear translocation of p65 in EBOV-infected macrophages, indicating activation of NFκB signaling. qRT-PCR and Luminex assays revealed induction of selected cytokines in EBOV-infected macrophages. Most analyzed cytokines and chemokines were upregulated in EBOV infection at 6 hpi or later, whereas others, including IL10, did not show increased secretion levels. In contrast, p65 was not translocated into the nucleus in RESTV-infected macrophages and no significant changes in cytokine mRNA levels or secreted protein levels were observed. To investigate if this effect is due to inhibition of pro-inflammatory signaling pathways, RESTV-infected macrophages were treated with LPS at 1 dpi and cytokine production was determined. Similar levels of pro-inflammatory cytokines, including IL6 and TNFα, were detected in RESTV-infected and LPS-treated macrophages compared to non-infected LPS-treated cells, suggesting that RESTV is not able to inhibit LPS-mediated cytokine induction. Our data support the hypothesis that high pathogenicity correlates with high cytokine levels in Ebolavirus infection. Currently we are investigating which cellular signaling pathways are involved in the observed differential cytokine induction in EBOV versus RESTV infection.

Antibodies are necessary for rVSV/ZEBOV-GP–mediated protection against lethal Ebola virus challenge in nonhuman primates

Ebola viruses cause hemorrhagic disease in humans and nonhuman primates with high fatality rates. These viruses pose a significant health concern worldwide due to the lack of approved therapeutics and vaccines as well as their potential misuse as bioterrorism agents. Although not licensed for human use, recombinant vesicular stomatitis virus (rVSV) expressing the filovirus glycoprotein (GP) has been shown to protect macaques from Ebola virus and Marburg virus infections, both prophylactically and postexposure in a homologous challenge setting. However, the immune mechanisms of protection conferred by this vaccine platform remain poorly understood. In this study, we set out to investigate the role of humoral versus cellular immunity in rVSV vaccine-mediated protection against lethal Zaire ebolavirus (ZEBOV) challenge. Groups of cynomolgus macaques were depleted of CD4+ T, CD8+ T, or CD20+ B cells before and during vaccination with rVSV/ZEBOV-GP. Unfortunately, CD20-depleted animals generated a robust IgG response. Therefore, an additional group of vaccinated animals were depleted of CD4+ T cells during challenge. All animals were subsequently challenged with a lethal dose of ZEBOV. Animals depleted of CD8+ T cells survived, suggesting a minimal role for CD8+ T cells in vaccine-mediated protection. Depletion of CD4+ T cells during vaccination caused a complete loss of glycoprotein-specific antibodies and abrogated vaccine protection. In contrast, depletion of CD4+ T cells during challenge resulted in survival of the animals, indicating a minimal role for CD4+ T-cell immunity in rVSV-mediated protection. Our results suggest that antibodies play a critical role in rVSV-mediated protection against ZEBOV.

P014 Ebola virus induces a differential cytokine response and NFKB activation in infected cells

Introduction Zaire ebolavirus (EBOV) causes a hemorrhagic disease in humans with case fatality rates up to 90%. A hallmark of EBOV infection is a dysregulated immune response. Macrophages and dendritic cells not only orchestrate innate and adaptive immune responses but are also early target cells of EBOV. Using infected human macrophages, we analyzed the antiviral response to EBOV compared to a promising EBOV vaccine candidate, recombinant vesicular stomatitis virus expressing EBOV-GP (rVSV/EBOV-GP). rVSV/EBOV-GP conferred complete protection against EBOV in a NHP model (Jones et al., 2005). Methods Primary human macrophages or Vero cells were infected with EBOV or rVSV/EBOV-GP. Western blot analysis was performed to detect induction of antiviral signaling. To investigate if EBOV is able to block pro-inflammatory signaling pathways, EBOV-infected cells were treated with LPS and the cytokine/chemokine response was analyzed. Cytokine mRNA expression levels were analyzed by qRT-PCR and supernatants were used to detect secretion using 18-plex Luminex assay. Immunofluorescence analysis was used to analyze p65 translocation. Results rVSV/EBOV-GP infection of macrophages led to the activation of antiviral pathways, including dsRNA-dependent protein kinase R (PKR) activation, induction of apoptosis, degradation of IκBα, and increased expression of cytokines at RNA level with IFNβ and TNFα showing the highest expression levels. However, cytokine levels were not significantly elevated in supernatants of rVSV/EBOV-GP-infected macrophages. The observed lack of cytokine secretion is likely due to the inhibition of the nuclear export of cellular mRNA by VSV (Stojdl et al., 2003). As cytokines were present in supernatants of rVSV/EBOV-GP-infected PBMCs, we currently investigate which cell type secretes the cytokines. EBOV infection did not induce apoptosis in infected cells and activation of PKR was suppressed. Interestingly, EBOV was not able to block apoptosis induced by exogenous stimuli in Vero cells, including dsRNA-mediated apoptosis. Degradation of IκBα and nuclear translocation of p65 was observed in EBOV-infected macrophages, indicating that EBOV activates NFκB signaling. Cytokine expression was upregulated in EBOV-infected macrophages, and most of the cytokines/chemokines tested in the Luminex assay, including IL6 and TNFα, were secreted from EBOV-infected cells at 6 hpi or later. Treatment of EBOV-infected macrophages with LPS revealed an increase in IL6 expression, suggesting that LPS-mediated signaling is not blocked by EBOV. In contrast to non-infected cells treated with LPS, IL10 was not upregulated in EBOV-infected macrophages even after stimulation with LPS. Conclusion Significant antiviral responses were observed after infection of macrophages with rVSV/EBOV-GP whereas multiple antiviral responses including PKR activation and apoptosis signaling are not induced or blocked in EBOV-infection. rVSV/EBOV-GP induced an increase in cytokine gene expression, but secretion of the corresponding proteins from macrophages was not detected. Furthermore, we observed NFκB activation and a differential cytokine response in macrophages infected with EBOV. Our data suggest a complex regulation of cytokine expression by EBOV in infected primary cells. Further analysis are planned to reveal the underlying mechanisms.

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase–promoter complex

Arenaviruses form a noncytolytic infection in their rodent hosts, yet can elicit severe hemorrhagic disease in humans. How arenaviruses regulate gene expression remains unclear, and further understanding may provide insight into the dichotomy of these disparate infection processes. Here we reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified components and demonstrate a direct role of the viral Z protein in controlling RNA synthesis. Our data reveal that Z forms a species-specific complex with the viral polymerase (L) and inhibits RNA synthesis initiation by impairing L catalytic activity. This Z-L complex locks the viral polymerase in a promoter-bound, catalytically inactive state and may additionally ensure polymerase packaging during virion maturation. Z modulates host factors involved in cellular translation, proliferation, and antiviral signaling. Our data defines an additional role in governing viral RNA synthesis, revealing Z as the center of a network of host and viral connections that regulates viral gene expression.

A small nonhuman primate model for filovirus-induced disease

Ebolavirus and Marburgvirus are members of the filovirus family and induce a fatal hemorrhagic disease in humans and nonhuman primates with 90% case fatality. To develop a small nonhuman primate model for filovirus disease, common marmosets ( Callithrix jacchus) were intramuscularly inoculated with wild type Marburgvirus Musoke or Ebolavirus Zaire. The infection resulted in a systemic fatal disease with clinical and morphological features closely resembling human infection. Animals experienced weight loss, fever, high virus titers in tissue, thrombocytopenia, neutrophilia, high liver transaminases and phosphatases and disseminated intravascular coagulation. Evidence of a severe disseminated viral infection characterized principally by multifocal to coalescing hepatic necrosis was seen in EBOV animals. MARV-infected animals displayed only moderate fibrin deposition in the spleen. Lymphoid necrosis and lymphocytic depletion observed in spleen. These findings provide support for the use of the common marmoset as a small nonhuman primate model for filovirus induced hemorrhagic fever.

Intracellular Events and Cell Fate in Filovirus Infection

Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis.

Pathogenesis of lassa fever in cynomolgus macaques

BackgroundLassa virus (LASV) infection causes an acute and sometimes fatal hemorrhagic disease in humans and nonhuman primates; however, little is known about the development of Lassa fever. Here, we performed a pilot study to begin to understand the progression of LASV infection in nonhuman primates.MethodsSix cynomolgus monkeys were experimentally infected with LASV. Tissues from three animals were examined at an early- to mid-stage of disease and compared with tissues from three animals collected at terminal stages of disease.ResultsDendritic cells were identified as a prominent target of LASV infection in a variety of tissues in all animals at day 7 while Kupffer cells, hepatocytes, adrenal cortical cells, and endothelial cells were more frequently infected with LASV in tissues of terminal animals (days 13.5-17). Meningoencephalitis and neuronal necrosis were noteworthy findings in terminal animals. Evidence of coagulopathy was noted; however, the degree of fibrin deposition in tissues was less prominent than has been reported in other viral hemorrhagic fevers.ConclusionThe sequence of pathogenic events identified in this study begins to shed light on the development of disease processes during Lassa fever and also may provide new targets for rational prophylactic and chemotherapeutic interventions.

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. In this study, an infectious cDNA of OHFV was constructed to investigate the molecular mechanisms involved in OHFV pathogenesis for the first time. Our cDNA clone was capable of producing infectious virus which is genetically identical to the parental Guriev strain, and the recombinant virus showed similar biological properties to the parental virus including growth kinetics and virulence characteristics. While characterizing the cDNAs, fortuitous mutations at NS2A position 46 and NS5 position 836 were found to affect viral production. By using a viral replicon expressing luciferase, it was shown that both of the mutations produced a defect in RNA replication and that the NS5 mutation induced a temperature-sensitive phenotype, indicating the importance of these residues in RNA replication. This infectious cDNA will be a useful tool to study the replication and pathogenesis of OHFV.

Complement Protein C1q Reduces the Stoichiometric Threshold for Antibody-Mediated Neutralization of West Nile Virus

Virus neutralization is governed by the number of antibodies that bind a virion during the cellular entry process. Cellular and serum factors that interact with antibodies have the potential to modulate neutralization potency. Although the addition of serum complement can increase the neutralizing activity of antiviral antibodies in vitro, the mechanism and significance of this augmented potency in vivo remain uncertain. Herein, we show that the complement component C1q increases the potency of antibodies against West Nile virus by modulating the stoichiometric requirements for neutralization. The addition of C1q does not result in virolysis but instead reduces the number of antibodies that must bind the virion to neutralize infectivity. For IgG subclasses that bind C1q avidly, this reduced stoichiometric threshold falls below the minimal number of antibodies required for antibody-dependent enhancement (ADE) of infection of cells expressing Fc-gamma receptors (CD32) and explains how C1q restricts the ADE of flavivirus infection.

Sequence signatures in envelope protein may determine whether flaviviruses produce hemorrhagic or encephalitic syndromes

We analyzed the envelope proteins in pathogenic flaviviruses to determine whether there are sequence signatures associated with the tendency of viruses to produce hemorrhagic disease (H-viruses) or encephalitis (E-viruses). We found that, at the position corresponding to the glycosylated Asn-67 in dengue virus, asparagine (Asn) occurs in all seven viral species that cause hemorrhagic disease in humans. Furthermore, Asn was extremely rare at position 67 in six flaviviruses that cause encephalitis, being replaced by Asp in four of them. Of the 3,246 sequences from H- and E-viruses, we found that 2,916 sequences (90%) contained Asn in position 67 for H-viruses or Asp in position 67 for E-viruses. The change from Asn-67 that is prevalent in H-viruses to Asp-67 (common in E-viruses) contributes to a stronger electrostatically negative surface in the E-viruses as compared to the H-viruses. These findings should help predicting the disease potential of emerging and re-emerging flaviviruses and understanding the relationship between protein structure and disease outcome.

Sub-genomic replicon and virus-like particles of Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis serocomplex of flaviviruses, and causes hemorrhagic disease in humans. To investigate the molecular mechanisms involved in OHFV pathogenesis, we constructed several subgenomic OHFV replicons containing large deletions in the structural region. Replicon RNA was introduced into BHK cells by transfection and the production of viral proteins was monitored by IFA. GFP and luciferase genes were inserted into the OHFV replicon, and these reporter genes were expressed in cells harboring replicating replicon RNA. OHFV replicons were packaged into single-round infectious virus-like particles (VLPs) by sequential transfection with replicon RNA and a plasmid expressing the viral structural proteins. Reporter genes were expressed in cells infected with VLPs, and the infection was inhibited by neutralizing antibodies. These replicon and VLP systems will be useful tools for investigating the molecular mechanism of OHFV pathogenicity.

Cellular Immune Response to Marburg Virus Infection in Cynomolgus Macaques

Marburg virus (MARV) causes a severe and usually lethal hemorrhagic disease in humans and non-human primates. Here, 16 cynomolgus macaques were experimentally infected with the Ci67 strain of MARV. Blood and spleen samples were collected at various time points after infection to study the immunological response to MARV. Beginning at day 2 and continuing throughout the course of the infection there was a rise in antigen-presenting cells in both the blood and spleen expressing MARV glycoprotein. Natural killer (NK) cells declined in the blood after infection (from 15% on day 0 to 5% on day 6), but a small increase was seen in the spleen samples. Little or no change in CD4(+) or CD8(+) T cells was observed out to day 6 post-exposure in blood, while there was a continual decline in the percentage of CD8(+) T cells in spleen samples. Circulating B cells (defined as CD20(+)) increased during the course of the infection as did CD4(+) CD8(+) (double-positive) T cells. Intracellular cytokine staining indicated that by day 6 a large population of leukocytes in the spleen were producing IFN-alpha; analysis of surface markers indicated that these cells were plasmacytoid dendritic cells based on their expression of CD123(+), but these cells had decreased expression of class II MHC. IL-6 production was detected late in the infection in CD14(+) spleen cells. These results suggest a robust innate immune response to MARV; however, this response was delayed relative to the infection.

Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening

Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay.

Human Macrophage C-Type Lectin Specific for Galactose andN-Acetylgalactosamine Promotes Filovirus Entry

Filoviruses cause lethal hemorrhagic disease in humans and nonhuman primates. An initial target of filovirus infection is the mononuclear phagocytic cell. Calcium-dependent (C-type) lectins such as dendritic cell- or liver/lymph node-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as the hepatic asialoglycoprotein receptor, bind to Ebola or Marburg virus glycoprotein (GP) and enhance the infectivity of these viruses in vitro. Here, we demonstrate that a recently identified human macrophage galactose- and N-acetylgalactosamine-specific C-type lectin (hMGL), whose ligand specificity differs from DC-SIGN and L-SIGN, also enhances the infectivity of filoviruses. This enhancement was substantially weaker for the Reston and Marburg viruses than for the highly pathogenic Zaire virus. We also show that the heavily glycosylated, mucin-like domain on the filovirus GP is required for efficient interaction with this lectin. Furthermore, hMGL, like DC-SIGN and L-SIGN, is present on cells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggesting a role for these C-type lectins in viral replication in vivo. We propose that filoviruses use different C-type lectins to gain cellular entry, depending on the cell type, and promote efficient viral replication.

Pre-West Nile Virus Outbreak: Perceptions and Practices to Prevent Mosquito Bites and Viral Encephalitis in the United States

Mosquitoes can transmit over 100 of the viruses that can cause encephalitis, meningitis, and hemorrhagic disease in humans (Chin 2000; Gubler 1996; Monath 1989). While much is known about the ecology, epidemiology, and clinical manifestations of the arboviral encephalitides (Campbell et al. 2002; Centers for Disease Control and Prevention 1997; Gubler 1998; Hayes 1989; Hubálek and Halouzka 1999), little empirical research exists regarding the U.S. population's knowledge of mosquitoes and arboviral encephalitis, particularly prior to the U.S. outbreak of West Nile virus (WNV) in 1999. A nationally representative 55-item survey instrument was successfully administered to 1,500 adults in the United States and an additional 250 adults in six states in the Northeast (Connecticut, Delaware, New Jersey, New York, Pennsylvania, and Rhode Island) regarding mosquitoes and mosquito-borne viral encephalitis. A summary outcome measure for mosquito bite prevention was created. Analyses revealed that the following were statistically significant predictors of behaviors taken to prevent mosquito bites: being concerned about being bitten by mosquitoes, perceived effectiveness of staying indoors in late afternoon and early evening was protective, perceived effectiveness that mosquito repellent is not harmful to health, owning dogs and/or cats as pets, being married, and being > or = 18-44 years old. Being concerned about being bitten by mosquitoes was the most robust predictor of behavioral action to prevent mosquito bites (OR = 7.3; 95% CI = 4.3, 12.2). Observed misperceptions and inadequate knowledge regarding insect repellents suggest increased promotion of the safety and efficacy of DEET-containing insect repellents is warranted.

Pathogenesis of Ebola Hemorrhagic Fever in Primate Models: Evidence that Hemorrhage Is Not a Direct Effect of Virus-Induced Cytolysis of Endothelial Cells

Ebola virus (EBOV) infection causes a severe and often fatal hemorrhagic disease in humans and nonhuman primates. Whether infection of endothelial cells is central to the pathogenesis of EBOV hemorrhagic fever (HF) remains unknown. To clarify the role of endothelial cells in EBOV HF, we examined tissues of 21 EBOV-infected cynomolgus monkeys throughout time, and also evaluated EBOV infection of primary human umbilical vein endothelial cells and primary human lung-derived microvascular endothelial cells in vitro. Results showed that endothelial cells were not early cellular targets of EBOV in vivo, as viral replication was not consistently observed until day 5 after infection, a full day after the onset of disseminated intravascular coagulation. Moreover, the endothelium remained relatively intact even at terminal stages of disease. Although human umbilical vein endothelial cells and human lung-derived microvascular endothelial cells were highly permissive to EBOV replication, significant cytopathic effects were not observed. Analysis of host cell gene response at 24 to 144 hours after infection showed some evidence of endothelial cell activation, but changes were unremarkable considering the extent of viral replication. Together, these data suggest that coagulation abnormalities associated with EBOV HF are not the direct result of EBOV-induced cytolysis of endothelial cells, and are likely triggered by immune-mediated mechanisms.

Analysis of the complete genome of the tick-borne flavivirus Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHF) is a tick-borne flavivirus endemic to Western Siberia. This virus is the only known tick-borne flavivirus to cause hemorrhagic disease in humans in the absence of encephalitis. OHF virus circulates within a small, defined niche in which other tick-borne complex flaviviruses are also present. The objectives of this study were to genetically classify OHF virus based on its complete genome and to identify genetic determinants that might be involved in tissue tropism and viral replication leading to the disease state caused by this virus. The OHF virus genome was sequenced and phylogenetic analysis demonstrated that OHF virus falls within the tick-borne encephalitis serocomplex of flaviviruses, yet is distinct from other members of the complex, including those closely associated geographically. OHF is also distinct from Alkhurma (ALK) and Kyasanur forest disease (KFD) viruses, both of which cause disease that includes hemorrhagic and encephalitic manifestations. Several amino acid residues were found to be distinct among OHF, KFD, and ALK viruses; these residues include E-76, which is closely associated with the viral envelope protein fusion peptide. In addition, variation between the viral 5′-untranslated region of OHF and other tick-borne flaviviruses suggests potential variability in viral replication. These data demonstrate that OHF is a unique virus among the tick-borne flaviviruses and also provide insight to viral biodiversity and tropism.

Antibody-dependent enhancement of Ebola virus infection.

Most strains of Ebola virus cause a rapidly fatal hemorrhagic disease in humans, yet there are still no biologic explanations that adequately account for the extreme virulence of these emerging pathogens. Here we show that Ebola Zaire virus infection in humans induces antibodies that enhance viral infectivity. Plasma or serum from convalescing patients enhanced the infection of primate kidney cells by the Zaire virus, and this enhancement was mediated by antibodies to the viral glycoprotein and by complement component C1q. Our results suggest a novel mechanism of antibody-dependent enhancement of Ebola virus infection, one that would account for the dire outcome of Ebola outbreaks in human populations.