Human macrophage C-type lectin specific for galactose and N-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.     

Serological evidence of Ebola virus infection in Indonesian orangutans

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae and cause severe hemorrhagic fever in humans and nonhuman primates. Despite the discovery of EBOV (Reston virus) in nonhuman primates and domestic pigs in the Philippines and the serological evidence for its infection of humans and fruit bats, information on the reservoirs and potential amplifying hosts for filoviruses in Asia is lacking. In this study, serum samples collected from 353 healthy Bornean orangutans (Pongo pygmaeus) in Kalimantan Island, Indonesia, during the period from December 2005 to December 2006 were screened for filovirus-specific IgG antibodies using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with recombinant viral surface glycoprotein (GP) antigens derived from multiple species of filoviruses (5 EBOV and 1 MARV species). Here we show that 18.4% (65/353) and 1.7% (6/353) of the samples were seropositive for EBOV and MARV, respectively, with little cross-reactivity among EBOV and MARV antigens. In these positive samples, IgG antibodies to viral internal proteins were also detected by immunoblotting. Interestingly, while the specificity for Reston virus, which has been recognized as an Asian filovirus, was the highest in only 1.4% (5/353) of the serum samples, the majority of EBOV-positive sera showed specificity to Zaire, Sudan, Cote d'Ivoire, or Bundibugyo viruses, all of which have been found so far only in Africa. These results suggest the existence of multiple species of filoviruses or unknown filovirus-related viruses in Indonesia, some of which are serologically similar to African EBOVs, and transmission of the viruses from yet unidentified reservoir hosts into the orangutan populations. Our findings point to the need for risk assessment and continued surveillance of filovirus infection of human and nonhuman primates, as well as wild and domestic animals, in Asia.     

Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.     

Structural and Functional Characterization of Reston Ebola Virus VP35 Interferon Inhibitory Domain

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-Å crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 Å, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.     

Association of the caveola vesicular system with cellular entry by filoviruses

The filoviruses Ebola Zaire virus and Marburg virus are believed to infect target cells through endocytic vesicles, but the details of this pathway are unknown. We used a pseudotyping strategy to investigate the cell biology of filovirus entry. We observed that specific inhibitors of the caveola system, including cholesterol-sequestering drugs and phorbol esters, inhibited the entry of filovirus pseudotypes into human cells. We also measured slower cell entry kinetics for both filovirus pseudotypes than for pseudotypes of vesicular stomatitis virus (VSV), which has been recognized to exploit the clathrin-mediated entry pathway. Finally, visualization by immunofluorescence and confocal microscopy revealed that the filovirus pseudotypes colocalized with the caveola protein marker caveolin-1 but that VSV pseudotypes did not. Collectively, these results provide evidence suggesting that filoviruses use caveolae to gain entry into cells.     

Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence

The Ebola virus envelope glycoprotein (GP) derived from the pathogenic Zaire subtype mediates cell rounding and detachment from the extracellular matrix in 293T cells. In this study we provide evidence that GPs from the other pathogenic subtypes, Sudan and C?te d'Ivoire, as well as from Reston, a strain thought to be nonpathogenic in humans, also induced cell rounding, albeit at lower levels than Zaire GP. Sequential removal of regions of potential O-linked glycosylation at the C terminus of GP1 led to a step-wise reduction in cell detachment without obviously affecting GP function, suggesting that such modifications are involved in inducing the detachment phenotype. While causing cell rounding and detachment in 293T cells, Ebola virus GP did not cause an increase in cell death. Indeed, following transient expression of GP, cells were able to readhere and continue to divide. Also, the rounding effect was not limited to 293T cells. Replication-deficient adenovirus vectors expressing Ebola virus GP induced the loss of cell adhesion in a range of cell lines and primary cell types, including those with proposed relevance to Ebola virus infection in vivo, such as endothelial cells and macrophages. In both transfected 293T and adenovirus-infected Vero cells, a reduction in cell surface expression of adhesion molecules such as integrin beta1 concurrent with the loss of cell adhesion was observed. A number of other cell surface molecules, however, including major histocompatibility complex class I and the epidermal growth factor receptor, were also down-modulated, suggesting a global mechanism for surface molecule down-regulation.     

Recombinant vesicular stomatitis virus vector mediates postexposure protection against Sudan Ebola hemorrhagic fever in nonhuman primates

Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Infectivity-enhancing antibodies to Ebola virus glycoprotein

Ebola virus causes severe hemorrhagic fever in primates, resulting in mortality rates of up to 100%, yet there are no satisfactory biologic explanations for this extreme virulence. Here we show that antisera produced by DNA immunization with a plasmid encoding the surface glycoprotein (GP) of the Zaire strain of Ebola virus enhances the infectivity of vesicular stomatitis virus pseudotyped with the GP. Substantially weaker enhancement was observed with antiserum to the GP of the Reston strain, which is much less pathogenic in humans than the Ebola Zaire and Sudan viruses. The enhancing activity was abolished by heat but was increased in the presence of complement system inhibitors, suggesting that heat-labile factors other than the complement system are required for this effect. We also generated an anti-Zaire GP monoclonal antibody that enhanced viral infectivity and another that neutralized it, indicating the presence of distinct epitopes for these properties. Our findings suggest that antibody-dependent enhancement of infectivity may account for the extreme virulence of the virus. They also raise issues about the development of Ebola virus vaccines and the use of passive prophylaxis or therapy with Ebola virus GP antibodies.     

Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway.

Monocytes and macrophages play a significant role in host's defense system, since they produce a number of cytokines in response to microbial infections. We have studied IL-1 beta, IL-18, IFN-alpha/beta, and TNF-alpha gene expression and protein production in human primary monocytes and GM-CSF-differentiated macrophages during influenza A and Sendai virus infections. Virus-infected monocytes released only small amounts of IL-1 beta or IL-18 protein, whereas 7- and 14-day-old GM-CSF-differentiated macrophages readily produced these cytokines. Constitutive expression of proIL-18 was seen in monocytes and macrophages, and the expression of it was enhanced during monocyte/macrophage differentiation. Expression of IL-18 mRNA was clearly induced only by Sendai virus, whereas both influenza A and Sendai viruses induced IL-1 beta mRNA expression. Since caspase-1 is known to cleave proIL-1 beta and proIL-18 into their mature, active forms, we analyzed the effect of a specific caspase-1 inhibitor on virus-induced IL-1 beta and IL-18 production. The release of IL-1 beta and IL-18, but not that of IFN-alpha/beta or TNF-alpha, was clearly blocked by the inhibitor. Our results suggest that the cellular differentiation is a crucial factor that affects the capacity of monocytes/macrophages to produce IL-1 beta and IL-18 in response to virus infections. Furthermore, the virus-induced activation of caspase-1 is required for the efficient production of biologically active IL-1 beta and IL-18.     

Evaluation of perceived threat differences posed by filovirus variants

In the United States, filoviruses (ebolaviruses and marburgviruses) are listed as National Institute of Allergy and Infectious Diseases (NIAID) Category A Priority Pathogens, Select Agents, and Centers for Disease Control and Prevention (CDC) Category A Bioterrorism Agents. In recent months, U.S. biodefense professionals and policy experts have initiated discussions on how to optimize filovirus research in regard to medical countermeasure (ie, diagnostics, antiviral, and vaccine) development. Standardized procedures and reagents could accelerate the independent verification of research results across government agencies and establish baselines for the development of animal models acceptable to regulatory entities, such as the Food and Drug Administration (FDA), while being fiscally responsible. At the root of standardization lies the question of which filovirus strains, variants, or isolates ought to be the prototypes for product development, evaluation, and validation. Here we discuss a rationale for their selection. We conclude that, based on currently available data, filovirus biodefense research ought to focus on the classical taxonomic filovirus prototypes: Marburg virus Musoke in the case of marburgviruses and Ebola virus Mayinga in the case of Zaire ebolaviruses. Arguments have been made in various committees in favor of other variants, such as Marburg virus Angola, Ci67 or Popp, or Ebola virus Kikwit, but these rationales seem to be largely based on anecdotal or unpublished and unverified data, or they may reflect a lack of awareness of important facts about the variants' isolation history and genomic properties.     

Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha

The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by beta-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.     

Interaction between Ebola Virus Glycoprotein and Host Toll-Like Receptor 4 Leads to Induction of Proinflammatory Cytokines and SOCS1

Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection.Ebola virus and Marburg virus comprise the Filoviridae family and represent important human pathogens and potential agents of bioterrorism. Currently there are no approved vaccines or specific treatments available to prevent or treat filovirus infections. The filoviruses are the cause of severe hemorrhagic disease in humans (7). Ebola virus initially targets monocytes/macrophages and dendritic cells (DCs), which can lead to the release of proinflammatory cytokines and chemokines (3, 7). A better understanding of the physical and functional interactions between Ebola virus proteins and cellular factors regulating the host innate immune response may reveal novel insights into the pathogenesis of Ebola virus and offer new strategies to inhibit Ebola virus replication.The VP40 matrix protein of Ebola virus is a key structural protein critical for budding virus-like particles (VLPs) and virion egress. Interactions between late budding domains of VP40 and specific host proteins facilitate efficient release of VLPs and infectious virus. Viral proteins other than VP40 also contribute to efficient budding of VLPs. Ebola virus glycoprotein (GP), when coexpressed with VP40, is incorporated into budding VLPs and enhances VLP egress (15), possibly by antagonizing the function of host proteins (12).Several studies have reported the induction of an innate immune response following infection or stimulation of macrophages/monocytes and DCs with Ebola virus or VLPs, respectively (2, 31). For example, incubation of Ebola virus VP40+GP VLPs with DCs led to the induction of interleukin-6 (IL-6), IL-8, NF-κB and ERK1/2 (18, 31). The triggering mechanism by which Ebola virus VLPs stimulate cytokine production is unknown. Here, we present evidence that Ebola virus VLPs stimulate induction of proinflammatory cytokines as well as SOCS1 (a ubiquitin ligase and negative feedback regulator of cytokine production) by interacting with host Toll-like receptor 4 (TLR4). Importantly, Ebola virus VP40+GP VLPs, but not VP40 VLPs, induced cytokine and SOCS1 expression in a TLR4/MD2 dependent manner both in a human monocytic cell line (THP-1 cells) and in 293T cells expressing a functional TLR4/MD2 receptor. These results indicate that the stimulation of TLR4 by Ebola virus envelope GP results in an innate host response, induction of SOCS1 protein, and potential enhancement of virus egress.     

Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin alpha proteins with activated STAT1

The Zaire ebolavirus protein VP24 was previously demonstrated to inhibit alpha/beta interferon (IFN-α/β)- and IFN-γ-induced nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1) and to inhibit IFN-α/β- and IFN-γ-induced gene expression. These properties correlated with the ability of VP24 to interact with the nuclear localization signal receptor for PY-STAT1, karyopherin α1. Here, VP24 is demonstrated to interact not only with overexpressed but also with endogenous karyopherin α1. Mutational analysis demonstrated that VP24 binds within the PY-STAT1 binding region located in the C terminus of karyopherin α1. In addition, VP24 was found to inhibit PY-STAT1 binding to both overexpressed and endogenous karyopherin α1. We assessed the binding of both PY-STAT1 and the VP24 proteins from Zaire, mouse-adapted Zaire, and Reston Ebola viruses for interaction with all six members of the human karyopherin α family. We found, in contrast to previous studies, that PY-STAT1 can interact not only with karyopherin α1 but also with karyopherins α5 and α6, which together comprise the NPI-1 subfamily of karyopherin αs. Similarly, all three VP24s bound and inhibited PY-STAT1 interaction with karyopherins α1, α5, and α6. Consistent with their ability to inhibit the karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN-β-induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with karyopherins α1, α5, or α6 by binding within the PY-STAT1 binding region of the karyopherins and that this function is conserved among the VP24 proteins of different Ebola virus species.     

Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species

Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.     

Molecular Evolution of Viruses of the Family Filoviridae Based on 97 Whole-Genome Sequences

Viruses in the Ebolavirus and Marburgvirus genera (family Filoviridae) have been associated with large outbreaks of hemorrhagic fever in human and nonhuman primates. The first documented cases occurred in primates over 45 years ago, but the amount of virus genetic diversity detected within bat populations, which have recently been identified as potential reservoir hosts, suggests that the filoviruses are much older. Here, detailed Bayesian coalescent phylogenetic analyses are performed on 97 whole-genome sequences, 55 of which are newly reported, to comprehensively examine molecular evolutionary rates and estimate dates of common ancestry for viruses within the family Filoviridae. Molecular evolutionary rates for viruses belonging to different species range from 0.46 × 10⁻⁴ nucleotide substitutions/site/year for Sudan ebolavirus to 8.21 × 10⁻⁴ nucleotide substitutions/site/year for Reston ebolavirus. Most recent common ancestry can be traced back only within the last 50 years for Reston ebolavirus and Zaire ebolavirus species and suggests that viruses within these species may have undergone recent genetic bottlenecks. Viruses within Marburg marburgvirus and Sudan ebolavirus species can be traced back further and share most recent common ancestors approximately 700 and 850 years before the present, respectively. Examination of the whole family suggests that members of the Filoviridae, including the recently described Lloviu virus, shared a most recent common ancestor approximately 10,000 years ago. These data will be valuable for understanding the evolution of filoviruses in the context of natural history as new reservoir hosts are identified and, further, for determining mechanisms of emergence, pathogenicity, and the ongoing threat to public health.     

Histopathology of natural Ebola virus subtype Reston infection in cynomolgus macaques during the Philippine outbreak in 1996.

We investigated the livers, spleens, kidneys and lungs collected from 24 cynomolgus macaques (Macaca fascicularis) naturally infected with Ebola virus subtype Reston (EBO-R) during the Philippine outbreak in 1996, in order to reveal the histopathologic findings. These macaques showed necrotic hepatocytes with inclusions, slight to massive fibrin deposition in splenic cords, depletion of lymphoid cells in the white pulp of the spleen, and fibrin thrombi in some organs. Immunohistochemical analysis using anti-leukocyte antigen L1 antibody revealed an increase in blood-derived macrophages/monocytes in the livers, kidneys and lungs of EBO-R infected macaques. EBO-R NP antigens were detected in the macrophages/monocytes, endothelial cells and fibroblasts in the liver, spleen, kidney and lung. These results indicate that EBO-R infection is characterized by systemic coagulopathy and an increase in blood-derived macrophages/monocytes in accordance with the EBO-R propagation in macrophages/monocytes.