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Sample records for marburg virus vp40

  1. Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer.

    PubMed

    Bhattarai, Nisha; Gc, Jeevan B; Gerstman, Bernard S; Stahelin, Robert V; Chapagain, Prem P

    2017-04-26

    Filovirus infections cause hemorrhagic fever in humans and non-human primates that often results in high fatality rates. The Marburg virus is a lipid-enveloped virus from the Filoviridae family and is closely related to the Ebola virus. The viral matrix layer underneath the lipid envelope is formed by the matrix protein VP40 (VP40), which is also involved in other functions during the viral life-cycle. As in the Ebola virus VP40 (eVP40), the recently determined X-ray crystal structure of the Marburg virus VP40 (mVP40) features loops containing cationic residues that form a lipid binding basic patch. However, the mVP40 basic patch is significantly flatter with a more extended surface than in eVP40, suggesting the possibility of differences in the plasma membrane interactions and phospholipid specificity between the VP40 dimers. In this paper, we report on molecular dynamics simulations that investigate the roles of various residues and lipid types in PM association as well as the conformational changes of the mVP40 dimer facilitated by membrane association. We compared the structural changes of the mVP40 dimer with the mVP40 dimer in both lipid free and membrane associated conditions. Despite the significant structural differences in the crystal structure, the Marburg VP40 dimer is found to adopt a configuration very similar to the Ebola VP40 dimer after associating with the membrane. This conformational rearrangement upon lipid binding allows Marburg VP40 to localize and stabilize at the membrane surface in a manner similar to the Ebola VP40 dimer. Consideration of the structural information in its lipid-interacting condition may be important in targeting mVP40 for novel drugs to inhibit viral budding from the plasma membrane.

  2. Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40.

    PubMed

    Wijesinghe, Kaveesha J; Stahelin, Robert V

    2015-12-30

    Marburg virus (MARV), which belongs to the virus family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface. Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family Filoviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. The VP35 and VP40 proteins of filoviruses. Homology between Marburg and Ebola viruses.

    PubMed

    Bukreyev, A A; Volchkov, V E; Blinov, V M; Netesov, S V

    1993-05-03

    The fragments of genomic RNA sequences of Marburg (MBG) and Ebola (EBO) viruses are reported. These fragments were found to encode the VP35 and VP40 proteins. The canonic sequences were revealed before and after each open reading frame. It is suggested that these sequences are mRNA extremities and at the same time the regulatory elements for mRNA transcription. Homology between the MBG and EBO proteins was discovered.

  4. Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

    PubMed

    Oda, Shun-Ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J; Halfmann, Peter; Bornholdt, Zachary A; Abelson, Dafna M; Armbrust, Tammy; Stahelin, Robert V; Kawaoka, Yoshihiro; Saphire, Erica Ollmann

    2016-02-15

    Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. The VP40 protein of Marburg virus exhibits impaired budding and increased sensitivity to human tetherin following mouse adaptation.

    PubMed

    Feagins, Alicia R; Basler, Christopher F

    2014-12-01

    The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting Janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus [RAVV]) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by coprecipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells. Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens that cause severe disease in humans and nonhuman primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular

  6. Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress.

    PubMed

    Loughran, H Marie; Han, Ziying; Wrobel, Jay E; Decker, Sarah E; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N; Reitz, Allen B

    2016-08-01

    We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Crystal Structure of the Marburg Virus VP35 Oligomerization Domain.

    PubMed

    Bruhn, Jessica F; Kirchdoerfer, Robert N; Urata, Sarah M; Li, Sheng; Tickle, Ian J; Bricogne, Gérard; Saphire, Erica Ollmann

    2017-01-15

    Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics. Copyright © 2017 American Society for Microbiology.

  8. Interaction of Tsg101 with Marburg Virus VP40 Depends on the PPPY Motif, but Not the PT/SAP Motif as in the Case of Ebola Virus, and Tsg101 Plays a Critical Role in the Budding of Marburg Virus-Like Particles Induced by VP40, NP, and GP▿

    PubMed Central

    Urata, Shuzo; Noda, Takeshi; Kawaoka, Yoshihiro; Morikawa, Shigeru; Yokosawa, Hideyoshi; Yasuda, Jiro

    2007-01-01

    Marburg virus (MARV) VP40 is a matrix protein that can be released from mammalian cells in the form of virus-like particles (VLPs) and contains the PPPY sequence, which is an L-domain motif. Here, we demonstrate that the PPPY motif is important for VP40-induced VLP budding and that VLP production is significantly enhanced by coexpression of NP and GP. We show that Tsg101 interacts with VP40 depending on the presence of the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and plays an important role in VLP budding. These findings provide new insights into the mechanism of MARV budding. PMID:17301151

  9. Amino Acid Residue at Position 79 of Marburg Virus VP40 Confers Interferon Antagonism in Mouse Cells.

    PubMed

    Feagins, Alicia R; Basler, Christopher F

    2015-10-01

    Marburg viruses (MARVs) cause highly lethal infections in humans and nonhuman primates. Mice are not generally susceptible to MARV infection; however, if the strain is first adapted to mice through serial passaging, it becomes able to cause disease in this animal. A previous study correlated changes accrued during mouse adaptation in the VP40 gene of a MARV strain known as Ravn virus (RAVV) with an increased capacity to inhibit interferon (IFN) signaling in mouse cell lines. The MARV strain Ci67, which belongs to a different phylogenetic clade than RAVV, has also been adapted to mice and in the process the Ci67 VP40 acquired a different collection of genetic changes than did RAVV VP40. Here, we demonstrate that the mouse-adapted Ci67 VP40 more potently antagonizes IFN-α/β-induced STAT1 and STAT2 tyrosine phosphorylation, gene expression, and antiviral activity in both mouse and human cell lines, compared with the parental Ci67 VP40. Ci67 VP40 is also demonstrated to target the activation of kinase Jak1. A single change at VP40 residue 79 was found to be sufficient for the increased VP40 IFN antagonism. These data argue that VP40 IFN-antagonist activity plays a key role in MARV pathogenesis in mice. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

    SciTech Connect

    Bruhn, Jessica F.; Kirchdoerfer, Robert N.; Urata, Sarah M.

    ABSTRACT Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (generaMarburgvirusandEbolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOVmore » VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCEMarburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.« less

  11. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

    PubMed Central

    Wijesinghe, Kaveesha J.; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E.; Gerstman, Bernard S.; Chapagain, Prem P.; Li, Sheng; Stahelin, Robert V.

    2017-01-01

    Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. PMID:28167534

  12. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

    PubMed

    Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

    2017-04-14

    Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

    PubMed Central

    Liang, Jingjing; Sagum, Cari A.; Bedford, Mark T.; Sudol, Marius; Han, Ziying

    2017-01-01

    Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. PMID:28076420

  14. Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress.

    PubMed

    Liang, Jingjing; Sagum, Cari A; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Han, Ziying; Harty, Ronald N

    2017-01-01

    Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.

  15. A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner

    PubMed Central

    Koehler, Alexander; Kolesnikova, Larissa; Welzel, Ulla; Schudt, Gordian; Herwig, Astrid

    2015-01-01

    ABSTRACT Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling. IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding

  16. Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

    PubMed

    Johnson, Britney; Li, Jing; Adhikari, Jagat; Edwards, Megan R; Zhang, Hao; Schwarz, Toni; Leung, Daisy W; Basler, Christopher F; Gross, Michael L; Amarasinghe, Gaya K

    2016-08-28

    Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

    SciTech Connect

    Johnson, Britney; Li, Jing; Adhikari, Jagat

    Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulatemore » nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.« less

  18. Recombinant Marburg viruses containing mutations in the IID region of VP35 prevent inhibition of Host immune responses.

    PubMed

    Albariño, César G; Wiggleton Guerrero, Lisa; Spengler, Jessica R; Uebelhoer, Luke S; Chakrabarti, Ayan K; Nichol, Stuart T; Towner, Jonathan S

    2015-02-01

    Previous in vitro studies have demonstrated that Ebola and Marburg virus (EBOV and MARV) VP35 antagonize the host cell immune response. Moreover, specific mutations in the IFN inhibitory domain (IID) of EBOV and MARV VP35 that abrogate their interaction with virus-derived dsRNA, lack the ability to inhibit the host immune response. To investigate the role of MARV VP35 in the context of infectious virus, we used our reverse genetics system to generate two recombinant MARVs carrying specific mutations in the IID region of VP35. Our data show that wild-type and mutant viruses grow to similar titers in interferon deficient cells, but exhibit attenuated growth in interferon-competent cells. Furthermore, in contrast to wild-type virus, both MARV mutants were unable to inhibit expression of various antiviral genes. The MARV VP35 mutants exhibit similar phenotypes to those previously described for EBOV, suggesting the existence of a shared immune-modulatory strategy between filoviruses. Published by Elsevier Inc.

  19. Differential Regulation of Interferon Responses by Ebola and Marburg Virus VP35 Proteins

    SciTech Connect

    Edwards, Megan R.; Liu, Gai; Mire, Chad E.

    2016-02-11

    Suppression of innate immune responses during filoviral infection contributes to disease severity. Ebola (EBOV) and Marburg (MARV) viruses each encode a VP35 protein that suppresses RIG-I-like receptor signaling and interferon-α/β (IFN-α/β) production by several mechanisms, including direct binding to double stranded RNA (dsRNA). Here, we demonstrate that in cell culture, MARV infection results in a greater upregulation of IFN responses as compared to EBOV infection. This correlates with differences in the efficiencies by which EBOV and MARV VP35s antagonize RIG-I signaling. Furthermore, structural and biochemical studies suggest that differential recognition of RNA elements by the respective VP35 C-terminal IFN inhibitorymore » domain (IID) rather than affinity for RNA by the respective VP35s is critical for this observation. Our studies reveal functional differences in EBOV versus MARV VP35 RNA binding that result in unexpected differences in the host response to deadly viral pathogens.« less

  20. Structural Insight into Nucleoprotein Conformation Change Chaperoned by VP35 Peptide in Marburg Virus.

    PubMed

    Liu, Baocheng; Dong, Shishang; Li, Guobang; Wang, Wenming; Liu, Xiang; Wang, Yantong; Yang, Cheng; Rao, Zihe; Guo, Yu

    2017-08-15

    Marburg virus (MARV) encodes a nucleoprotein (NP) to encapsidate its genome by oligomerization and form a ribonucleoprotein complex (RNP). According to previous investigation on nonsegmented negative-sense RNA viruses (nsNSV), the newly synthesized NPs must be prevented from indiscriminately binding to noncognate RNAs. During the viral RNA synthesis process, the RNPs undergo a transition from an RNA-bound form to a template-free form, to open access for the interaction between the viral polymerase and the RNA template. In filoviruses, this transition is regulated by VP35 peptide and other viral components. To further understand the dynamic process of filovirus RNP formation, we report here the structure of MARV NP core , both in the apo form and in the VP35 peptide-chaperoned form. These structures reveal a typical bilobed structure, with a positive-charged RNA binding groove between two lobes. In the apo form, the MARV NP exists in an interesting hexameric state formed by the hydrophobic interaction within the long helix of the NP core C-terminal region, which shows high structural flexibility among filoviruses and may imply critical function during RNP formation. Moreover, the VP35 peptide-chaperoned NP core remains in a monomeric state and completely loses its affinity for single-stranded RNA (ssRNA). The structural comparison reveals that the RNA binding groove undergoes a transition from closed state to open state, chaperoned by VP35 peptide, thus preventing the interaction for viral RNA. Our investigation provides considerable structural insight into the filovirus RNP working mechanism and may support the development of antiviral therapies targeting the RNP formation of filovirus. IMPORTANCE Marburg virus is one of the most dangerous viruses, with high morbidity and mortality. A recent outbreak in Angola in 2005 caused the deaths of 272 persons. NP is one of the most essential proteins, as it encapsidates and protects the whole virus genome simultaneously

  1. Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

    SciTech Connect

    Feagins, Alicia R.; Basler, Christopher F., E-mail: chris.basler@mssm.edu

    Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, andmore » PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV. - Highlights: • Lloviu virus (LLOV) is a new member of the filovirus family. • LLOV VP35 blocks IRF3 phosphorylation, IFN-α/β production and PKR phosphorylation. • LLOV VP24 inhibits IFN responses by targeting phospho-STAT1 KPNA interaction. • Infection by LLOV may block innate immune responses in a manner similar to EBOV.« less

  2. Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

    PubMed Central

    Mühlberger, Elke; Lötfering, Beate; Klenk, Hans-Dieter; Becker, Stephan

    1998-01-01

    This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication. PMID:9765419

  3. Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

    PubMed Central

    Kamata, Teddy; Natesan, Mohan; Warfield, Kelly; Aman, M. Javad

    2014-01-01

    Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses. PMID:25230936

  4. Determination of specific antibody responses to the six species of ebola and Marburg viruses by multiplexed protein microarrays.

    PubMed

    Kamata, Teddy; Natesan, Mohan; Warfield, Kelly; Aman, M Javad; Ulrich, Robert G

    2014-12-01

    Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Evasion of interferon responses by Ebola and Marburg viruses.

    PubMed

    Basler, Christopher F; Amarasinghe, Gaya K

    2009-09-01

    The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause frequently lethal viral hemorrhagic fever. These infections induce potent cytokine production, yet these host responses fail to prevent systemic virus replication. Consistent with this, filoviruses have been found to encode proteins VP35 and VP24 that block host interferon (IFN)-alpha/beta production and inhibit signaling downstream of the IFN-alpha/beta and the IFN-gamma receptors, respectively. VP35, which is a component of the viral nucleocapsid complex and plays an essential role in viral RNA synthesis, acts as a pseudosubstrate for the cellular kinases IKK-epsilon and TBK-1, which phosphorylate and activate interferon regulatory factor 3 (IRF-3) and interferon regulatory factor 7 (IRF-7). VP35 also promotes SUMOylation of IRF-7, repressing IFN gene transcription. In addition, VP35 is a dsRNA-binding protein, and mutations that disrupt dsRNA binding impair VP35 IFN-antagonist activity while leaving its RNA replication functions intact. The phenotypes of recombinant EBOV bearing mutant VP35s unable to inhibit IFN-alpha/beta demonstrate that VP35 IFN-antagonist activity is critical for full virulence of these lethal pathogens. The structure of the VP35 dsRNA-binding domain, which has recently become available, is expected to provide insight into how VP35 IFN-antagonist and dsRNA-binding functions are related. The EBOV VP24 protein inhibits IFN signaling through an interaction with select host cell karyopherin-alpha proteins, preventing the nuclear import of otherwise activated STAT1. It remains to be determined to what extent VP24 may also modulate the nuclear import of other host cell factors and to what extent this may influence the outcome of infection. Notably, the Marburg virus VP24 protein does not detectably block STAT1 nuclear import, and, unlike EBOV, MARV infection inhibits STAT1 and STAT2 phosphorylation. Thus, despite their similarities, there are fundamental differences by which

  6. Human Survivors of Disease Outbreaks Caused by Ebola or Marburg Virus Exhibit Cross-Reactive and Long-Lived Antibody Responses.

    PubMed

    Natesan, Mohan; Jensen, Stig M; Keasey, Sarah L; Kamata, Teddy; Kuehne, Ana I; Stonier, Spencer W; Lutwama, Julius Julian; Lobel, Leslie; Dye, John M; Ulrich, Robert G

    2016-08-01

    A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Recombinant Marburg Virus Expressing EGFP Allows Rapid Screening of Virus Growth and Real-time Visualization of Virus Spread

    PubMed Central

    Schmidt, Kristina Maria; Schümann, Michael; Olejnik, Judith; Krähling, Verena

    2011-01-01

    The generation of recombinant enhanced green fluorescent protein (EGFP)--expressing viruses has significantly improved the study of their life cycle and opened up the possibility for the rapid screening of antiviral drugs. Here we report rescue of a recombinant Marburg virus (MARV) expressing EGFP from an additional transcription unit (ATU). The ATU was inserted between the second and third genes, encoding VP35 and VP40, respectively. Live-cell imaging was used to follow virus spread in real time. EGFP expression was detected at 32 hours postinfection (hpi), and infection of neighboring cells was monitored at 55 hpi. Compared to the parental virus, production of progeny rMARV-EGFP was reduced 4-fold and lower protein levels of VP40, but not nucleoprotein, were observed, indicating a decrease in downstream protein expression due to the insertion of an ATU. Interestingly, EGFP concentrated in viral inclusions in infected cells. This was reproduced by transient expression of both EGFP and other fluorescent proteins along with filovirus nucleocapsid proteins, and may suggest that a general increase in protein synthesis occurs at viral inclusion sites. In conclusion, the EGFP-expressing MARV will be a useful tool not only to monitor virus spread and screen for antiviral compounds, but also to investigate the biology of inclusion body formation. PMID:21987762

  8. Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

    PubMed

    Zhu, Tengfei; Song, Hao; Peng, Ruchao; Shi, Yi; Qi, Jianxun; Gao, George F

    2017-09-15

    Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs. IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and

  9. Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances

    PubMed Central

    Schudt, Gordian; Kolesnikova, Larissa; Dolnik, Olga; Sodeik, Beate; Becker, Stephan

    2013-01-01

    Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells. PMID:23940347

  10. Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances.

    PubMed

    Schudt, Gordian; Kolesnikova, Larissa; Dolnik, Olga; Sodeik, Beate; Becker, Stephan

    2013-08-27

    Transport of large viral nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, nucleocapsids were transported at ∼200 nm/s; (ii) nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.

  11. The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

    SciTech Connect

    Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.

    Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures ofmore » the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.« less

  12. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  13. Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

    PubMed Central

    Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

    2006-01-01

    The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

  14. The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes.

    PubMed

    Soni, Smita P; Stahelin, Robert V

    2014-11-28

    Ebola virus is from the Filoviridae family of viruses and is one of the most virulent pathogens known with ∼ 60% clinical fatality. The Ebola virus negative sense RNA genome encodes seven proteins including viral matrix protein 40 (VP40), which is the most abundant protein found in the virions. Within infected cells VP40 localizes at the inner leaflet of the plasma membrane (PM), binds lipids, and regulates formation of new virus particles. Expression of VP40 in mammalian cells is sufficient to form virus-like particles that are nearly indistinguishable from the authentic virions. However, how VP40 interacts with the PM and forms virus-like particles is for the most part unknown. To investigate VP40 lipid specificity in a model of viral egress we employed giant unilamellar vesicles with different lipid compositions. The results demonstrate VP40 selectively induces vesiculation from membranes containing phosphatidylserine (PS) at concentrations of PS that are representative of the PM inner leaflet content. The formation of intraluminal vesicles was not significantly detected in the presence of other important PM lipids including cholesterol and polyvalent phosphoinositides, further demonstrating PS selectivity. Taken together, these studies suggest that PM phosphatidylserine may be an important component of Ebola virus budding and that VP40 may be able to mediate PM scission. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

    PubMed

    Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

    2017-10-01

    The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Comparison of the Transcription and Replication Strategies of Marburg Virus and Ebola Virus by Using Artificial Replication Systems

    PubMed Central

    Mühlberger, Elke; Weik, Michael; Volchkov, Viktor E.; Klenk, Hans-Dieter; Becker, Stephan

    1999-01-01

    The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses. PMID:9971816

  17. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

    PubMed

    Dilley, Kari A; Voorhies, Alexander A; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B; Lorenzi, Hernan; Basler, Christopher F; Shabman, Reed S

    2017-01-01

    Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.

  18. The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

    PubMed Central

    Voorhies, Alexander A.; Luthra, Priya; Puri, Vinita; Stockwell, Timothy B.; Lorenzi, Hernan; Basler, Christopher F.; Shabman, Reed S.

    2017-01-01

    Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation. PMID:28636653

  19. Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress.

    PubMed

    Han, Ziying; Sagum, Cari A; Takizawa, Fumio; Ruthel, Gordon; Berry, Corbett T; Kong, Jing; Sunyer, J Oriol; Freedman, Bruce D; Bedford, Mark T; Sidhu, Sachdev S; Sudol, Marius; Harty, Ronald N

    2017-10-15

    Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress. IMPORTANCE Ebola

  20. The Role of Exosomal VP40 in Ebola Virus Disease.

    PubMed

    Pleet, Michelle L; DeMarino, Catherine; Lepene, Benjamin; Aman, M Javad; Kashanchi, Fatah

    2017-04-01

    Ebola virus (EBOV) can cause a devastating hemorrhagic disease, leading to death in a short period of time. After infection, the resulting EBOV disease results in high levels of circulating cytokines, endothelial dysfunction, coagulopathy, and bystander lymphocyte apoptosis in humans and nonhuman primates. The VP40 matrix protein of EBOV is essential for viral assembly and budding from the host cell. Recent data have shown that VP40 exists in the extracellular environment, including in exosomes, and exosomal VP40 can impact the viability of recipient immune cells, including myeloid and T cells, through the regulation of the RNAi and endosomal sorting complexes required for transport pathways. In this study, we discuss the latest findings of the impact of exosomal VP40 on immune cells in vitro and its potential implications for pathogenesis in vivo.

  1. Marburg Virus VP24 Protein Relieves Suppression of the NF-κB Pathway Through Interaction With Kelch-like ECH-Associated Protein 1.

    PubMed

    Edwards, Megan R; Basler, Christopher F

    2015-10-01

    Marburg virus (MARV) is an emerging zoonotic pathogen that causes hemorrhagic fever. MARV VP24 (mVP24) protein interacts with the host cell protein Kelch-like-ECH-associated protein 1 (Keap1). Keap1 interacts with and promotes the degradation of IκB kinase β (IKKβ), a component of the IκB kinase (IKK) complex that regulates nuclear factor-κB (NF-κB) activity. We studied whether mVP24 could relieve Keap1 repression of the NF-κB pathway. Coimmunoprecipitation assays were used to examine the interaction between Keap1 and IKKβ in the presence of wild-type mVP24 and mutants of mVP24 defective for binding to Keap1. Western blotting was used to determine levels of IKKβ expression in the presence of Keap1 and mVP24. NF-κB promoter-luciferase assays were used to determine the effect of mVP24 on Keap1-induced repression of activity. Expression of wild-type mVP24 disrupted the interaction of IKKβ and Keap1, whereas weakly interacting and noninteracting mVP24 mutants did not disrupt the interaction between Keap1 and IKKβ. The interaction of mVP24 with Keap1 enhanced IKKβ levels in the presence of Keap1. The interaction of mVP24 with Keap1 also relieved Keap1 repression of NF-κB reporter activity. mVP24 can relieve Keap1 repression of the NF-κB pathway through its interaction with Keap1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. [Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

    PubMed

    Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

    2012-08-01

    Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

  3. Molecular docking based screening of compounds against VP40 from Ebola virus.

    PubMed

    M Alam El-Din, Hanaa; A Loutfy, Samah; Fathy, Nasra; H Elberry, Mostafa; M Mayla, Ahmed; Kassem, Sara; Naqvi, Asif

    2016-01-01

    Ebola virus causes severe and often fatal hemorrhagic fevers in humans. The 2014 Ebola epidemic affected multiple countries. The virus matrix protein (VP40) plays a central role in virus assembly and budding. Since there is no FDA-approved vaccine or medicine against Ebola viral infection, discovering new compounds with different binding patterns against it is required. Therefore, we aim to identify small molecules that target the Arg 134 RNA binding and active site of VP40 protein. 1800 molecules were retrieved from PubChem compound database based on Structure Similarity and Conformers of pyrimidine-2, 4-dione. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find the potent inhibitors for VP40 based on calculated ligand-protein pairwise interaction energies. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Ten independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ RMSD criteria. The post-docking analysis showed that binding energies ranged from -8.87 to 0.6 Kcal/mol. We report 7 molecules, which showed promising ADMET results, LD-50, as well as H-bond interaction in the binding pocket. The small molecules discovered could act as potential inhibitors for VP40 and could interfere with virus assembly and budding process.

  4. Molecular docking based screening of compounds against VP40 from Ebola virus

    PubMed Central

    M Alam El-Din, Hanaa; A. Loutfy, Samah; Fathy, Nasra; H Elberry, Mostafa; M Mayla, Ahmed; Kassem, Sara; Naqvi, Asif

    2016-01-01

    Ebola virus causes severe and often fatal hemorrhagic fevers in humans. The 2014 Ebola epidemic affected multiple countries. The virus matrix protein (VP40) plays a central role in virus assembly and budding. Since there is no FDA-approved vaccine or medicine against Ebola viral infection, discovering new compounds with different binding patterns against it is required. Therefore, we aim to identify small molecules that target the Arg 134 RNA binding and active site of VP40 protein. 1800 molecules were retrieved from PubChem compound database based on Structure Similarity and Conformers of pyrimidine-2, 4-dione. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find the potent inhibitors for VP40 based on calculated ligand-protein pairwise interaction energies. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Ten independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ RMSD criteria. The post-docking analysis showed that binding energies ranged from -8.87 to 0.6 Kcal/mol. We report 7 molecules, which showed promising ADMET results, LD-50, as well as H-bond interaction in the binding pocket. The small molecules discovered could act as potential inhibitors for VP40 and could interfere with virus assembly and budding process. PMID:28149054

  5. Characteristics of Filoviridae: Marburg and Ebola Viruses

    NASA Astrophysics Data System (ADS)

    Beer, Brigitte; Kurth, Reinhard; Bukreyev, Alexander

    Filoviruses are enveloped, nonsegmented negative-stranded RNA viruses. The two species, Marburg and Ebola virus, are serologically, biochemically, and genetically distinct. Marburg virus was first isolated during an outbreak in Europe in 1967, and Ebola virus emerged in 1976 as the causative agent of two simultaneous outbreaks in southern Sudan and northern Zaire. Although the main route of infection is known to be person-to-person transmission by intimate contact, the natural reservoir for filoviruses still remains a mystery.

  6. Identification of Continuous Human B-Cell Epitopes in the VP35, VP40, Nucleoprotein and Glycoprotein of Ebola Virus

    PubMed Central

    Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M.

    2014-01-01

    Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7–12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods. PMID:24914933

  7. Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

    PubMed

    Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M

    2014-01-01

    Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

  8. Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections.

    PubMed

    Iversen, Patrick L; Warren, Travis K; Wells, Jay B; Garza, Nicole L; Mourich, Dan V; Welch, Lisa S; Panchal, Rekha G; Bavari, Sina

    2012-11-06

    There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

  9. Forty-five years of Marburg virus research.

    PubMed

    Brauburger, Kristina; Hume, Adam J; Mühlberger, Elke; Olejnik, Judith

    2012-10-01

    In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of our current knowledge about Marburg virus disease ranging from ecology to pathogenesis and molecular biology.

  10. Forty-Five Years of Marburg Virus Research

    PubMed Central

    Brauburger, Kristina; Hume, Adam J.; Mühlberger, Elke; Olejnik, Judith

    2012-01-01

    In 1967, the first reported filovirus hemorrhagic fever outbreak took place in Germany and the former Yugoslavia. The causative agent that was identified during this outbreak, Marburg virus, is one of the most deadly human pathogens. This article provides a comprehensive overview of our current knowledge about Marburg virus disease ranging from ecology to pathogenesis and molecular biology. PMID:23202446

  11. Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections

    PubMed Central

    Iversen, Patrick L.; Warren, Travis K.; Wells, Jay B.; Garza, Nicole L.; Mourich, Dan V.; Welch, Lisa S.; Panchal, Rekha G.; Bavari, Sina

    2012-01-01

    There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates. PMID:23202506

  12. [Ebola and Marburg viruses: the humans strike back].

    PubMed

    Alazard-Dany, Nathalie; Ottmann Terrangle, Michèle; Volchkov, Viktor

    2006-04-01

    Ebola and Marburg viruses are the causative agents of rapidly progressive hemorrhagic fevers with high mortality rates. Pre- or post-exposure treatments against the diseases are currently not available for human use. In the field, establishment of strict quarantine measures preventing further virus transmission are still the only way to fight the infections. However, our knowledge of Ebola and Marburg viruses has markedly increased as a result of two recent discoveries discussed in this review. Chandran et al. have elucidated the mechanism by which Ebola GP is converted to a fusion-active form. Infectivity of Ebola virus was shown to be dependent on the cleavage of GP by cellular endosomal proteases, cathepsin B and L, thus opening new therapeutic approaches options. As for Jones SM et al., they have successfully vaccinated monkeys with recombinant vesicular stomatitis virus expressing Ebola or Marburg virus surface glycoprotein GP, a promising vaccine approach.

  13. Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections.

    PubMed

    Geisbert, Thomas W; Feldmann, Heinz

    2011-11-01

    The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections.

  14. The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

    PubMed

    Adu-Gyamfi, Emmanuel; Soni, Smita P; Xue, Yi; Digman, Michelle A; Gratton, Enrico; Stahelin, Robert V

    2013-02-22

    Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

  15. Studies of Reservoir Hosts for Marburg Virus

    PubMed Central

    Smit, Sheilagh B.; Rollin, Pierre E.; Formenty, Pierre; Leman, Patricia A.; Kemp, Alan; Burt, Felicity J.; Grobbelaar, Antoinette A.; Croft, Janice; Bausch, Daniel G.; Zeller, Hervé; Leirs, Herwig; Braack, L.E.O.; Libande, Modeste L.; Zaki, Sherif; Nichol, Stuart T.; Ksiazek, Thomas G.; Paweska, Janusz T.

    2007-01-01

    To determine reservoir hosts for Marburg virus (MARV), we examined the fauna of a mine in northeastern Democratic Republic of the Congo. The mine was associated with a protracted outbreak of Marburg hemorrhagic fever during 1998–2000. We found MARV nucleic acid in 12 bats, comprising 3.0%–3.6% of 2 species of insectivorous bat and 1 species of fruit bat. We found antibody to the virus in the serum of 9.7% of 1 of the insectivorous species and in 20.5% of the fruit bat species, but attempts to isolate virus were unsuccessful. PMID:18258034

  16. Isolated Case of Marburg Virus Disease, Kampala, Uganda, 2014.

    PubMed

    Nyakarahuka, Luke; Ojwang, Joseph; Tumusiime, Alex; Balinandi, Stephen; Whitmer, Shannon; Kyazze, Simon; Kasozi, Sam; Wetaka, Milton; Makumbi, Issa; Dahlke, Melissa; Borchert, Jeff; Lutwama, Julius; Ströher, Ute; Rollin, Pierre E; Nichol, Stuart T; Shoemaker, Trevor R

    2017-06-01

    In September 2014, a single fatal case of Marburg virus was identified in a healthcare worker in Kampala, Uganda. The source of infection was not identified, and no secondary cases were identified. We describe the rapid identification, laboratory diagnosis, and case investigation of the third Marburg virus outbreak in Uganda.

  17. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats.

    PubMed

    Towner, Jonathan S; Amman, Brian R; Sealy, Tara K; Carroll, Serena A Reeder; Comer, James A; Kemp, Alan; Swanepoel, Robert; Paddock, Christopher D; Balinandi, Stephen; Khristova, Marina L; Formenty, Pierre B H; Albarino, Cesar G; Miller, David M; Reed, Zachary D; Kayiwa, John T; Mills, James N; Cannon, Deborah L; Greer, Patricia W; Byaruhanga, Emmanuel; Farnon, Eileen C; Atimnedi, Patrick; Okware, Samuel; Katongole-Mbidde, Edward; Downing, Robert; Tappero, Jordan W; Zaki, Sherif R; Ksiazek, Thomas G; Nichol, Stuart T; Rollin, Pierre E

    2009-07-01

    In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

  18. Isolation of Genetically Diverse Marburg Viruses from Egyptian Fruit Bats

    PubMed Central

    Towner, Jonathan S.; Amman, Brian R.; Sealy, Tara K.; Carroll, Serena A. Reeder; Comer, James A.; Kemp, Alan; Swanepoel, Robert; Paddock, Christopher D.; Balinandi, Stephen; Khristova, Marina L.; Formenty, Pierre B. H.; Albarino, Cesar G.; Miller, David M.; Reed, Zachary D.; Kayiwa, John T.; Mills, James N.; Cannon, Deborah L.; Greer, Patricia W.; Byaruhanga, Emmanuel; Farnon, Eileen C.; Atimnedi, Patrick; Okware, Samuel; Katongole-Mbidde, Edward; Downing, Robert; Tappero, Jordan W.; Zaki, Sherif R.; Ksiazek, Thomas G.; Nichol, Stuart T.; Rollin, Pierre E.

    2009-01-01

    In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans. PMID:19649327

  19. Marburg virus-like particles produced in insect cells induce neutralizing antibodies in rhesus macaques.

    PubMed

    Weiwei, Gai; Xuexing, Zheng; Chong, Wang; Yongkun, Zhao; Qi, Wang; Hualei, Wang; Gary, Wong; Ying, Xie; Haijun, Wang; Zengguo, Cao; Na, Feng; Hang, Chi; Tiecheng, Wang; Yuwei, Gao; Junjie, Shan; Songtao, Yang; Xianzhu, Xia

    2017-12-01

    Marburg virus (MARV), which is one of the most virulent agents in the world, causes lethal haemorrhagic fever in humans and nonhuman primates (NHPs) with a mortality rate of up to 90%. Currently, there is no effective treatment or approved vaccine for MARV for human use to control disease outbreak and spread. Virus-like particles (VLPs), which are morphologically identical to the native infectious virus particle, are efficacious as vaccines against many viruses, including human papilloma virus (HPV), porcine circovirus (PCV) type 2 and hepatitis B virus (HBV). In this study, we generated MARV virus-like particles (VLPs) by co-expressing a glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. Rhesus macaques vaccinated with MARV VLPs mixed with adjuvant Poria cocos polysaccharides (PCP-II) produced a GP-specific IgG titer of up to 1:1280 and virus-neutralizing antibody titers that reached 1:320. MARV VLPs also elicited interferon-γ (IFN-γ) and interleukin-4 (IL-4) secretion associated with T-helper 1 cell (Th1)- and T-helper 2 cell (Th2)-mediated immunity, as detected using enzyme-linked immunospot (ELISpot) assays. These data indicate that MARV VLPs mixed with adjuvant PCP-II have excellent immunogenicity in rhesus macaques and may be a promising candidate vaccine against MARV. © 2017 Wiley Periodicals, Inc.

  20. Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells

    PubMed Central

    Kolesnikova, Larissa; Welsch, Sonja; Krähling, Verena; Davey, Norman; Parsy, Marie-Laure; Becker, Stephan; Briggs, John A. G.

    2011-01-01

    Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps. PMID:22110401

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

    SciTech Connect

    Leung, Daisy W.; Shabman, Reed S.; Farahbakhsh, Mina

    2010-09-21

    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 thismore » 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-{angstrom} 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 {angstrom}, 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

  2. Structural and functional characterization of Reston Ebola virus VP35 interferon inhibitory domain.

    PubMed

    Leung, Daisy W; Shabman, Reed S; Farahbakhsh, Mina; Prins, Kathleen C; Borek, Dominika M; Wang, Tianjiao; Mühlberger, Elke; Basler, Christopher F; Amarasinghe, Gaya K

    2010-06-11

    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-A 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 A, 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

  3. Transcriptional Profiling of the Immune Response to Marburg Virus Infection.

    PubMed

    Connor, John H; Yen, Judy; Caballero, Ignacio S; Garamszegi, Sara; Malhotra, Shikha; Lin, Kenny; Hensley, Lisa; Goff, Arthur J

    2015-10-01

    Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptomes of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early infection, 1 to 3 days postexposure; midinfection, 5 days postexposure; late infection, 7 to 9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg virus was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus and further suggested that the early immune response is skewed toward a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the upregulation of coagulation-associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich data set for identification of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy. Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study, we investigated how the immune cells

  4. Distribution of Marburg virus in Africa: An evolutionary approach.

    PubMed

    Zehender, Gianguglielmo; Sorrentino, Chiara; Veo, Carla; Fiaschi, Lisa; Gioffrè, Sonia; Ebranati, Erika; Tanzi, Elisabetta; Ciccozzi, Massimo; Lai, Alessia; Galli, Massimo

    2016-10-01

    The aim of this study was to investigate the origin and geographical dispersion of Marburg virus, the first member of the Filoviridae family to be discovered. Seventy-three complete genome sequences of Marburg virus isolated from animals and humans were retrieved from public databases and analysed using a Bayesian phylogeographical framework. The phylogenetic tree of the Marburg virus data set showed two significant evolutionary lineages: Ravn virus (RAVV) and Marburg virus (MARV). MARV divided into two main clades; clade A included isolates from Uganda (five from the European epidemic in 1967), Kenya (1980) and Angola (from the epidemic of 2004-2005); clade B included most of the isolates obtained during the 1999-2000 epidemic in the Democratic Republic of the Congo (DRC) and a group of Ugandan isolates obtained in 2007-2009. The estimated mean evolutionary rate of the whole genome was 3.3×10(-4) substitutions/site/year (credibility interval 2.0-4.8). The MARV strain had a mean root time of the most recent common ancestor of 177.9years ago (YA) (95% highest posterior density 87-284), thus indicating that it probably originated in the mid-XIX century, whereas the RAVV strain had a later origin dating back to a mean 33.8 YA. The most probable location of the MARV ancestor was Uganda (state posterior probability, spp=0.41), whereas that of the RAVV ancestor was Kenya (spp=0.71). There were significant migration rates from Uganda to the DRC (Bayes Factor, BF=42.0) and in the opposite direction (BF=5.7). Our data suggest that Uganda may have been the cradle of Marburg virus in Africa. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine.

    PubMed

    Del Vecchio, Kathryn; Frick, Cary T; Gc, Jeevan B; Oda, Shun-Ichiro; Gerstman, Bernard S; Saphire, Erica Ollmann; Chapagain, Prem P; Stahelin, Robert V

    2018-03-02

    Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures ( i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys 224, 225 and Lys 274, 275 ). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Marburg Virus Reverse Genetics Systems.

    PubMed

    Schmidt, Kristina Maria; Mühlberger, Elke

    2016-06-22

    The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems.

  7. VP40 of the Ebola Virus as a Target for EboV Therapy: Comprehensive Conformational and Inhibitor Binding Landscape from Accelerated Molecular Dynamics.

    PubMed

    Balmith, Marissa; Soliman, Mahmoud E S

    2017-03-01

    The first account of the dynamic features of the loop region of VP40 of the Ebola virus was studied using accelerated molecular dynamics simulations and reported herein. Among the proteins of the Ebola virus, the matrix protein (VP40) plays a significant role in the virus lifecycle thereby making it a promising therapeutic target. Of interest is the newly elucidated N-terminal domain loop region of VP40 comprising residues K127, T129, and N130 which when mutated to alanine have demonstrated an unrecognized role for N-terminal domain-plasma membrane interaction for efficient VP40-plasma membrane localization, oligomerization, matrix assembly, and egress. The molecular understanding of the conformational features of VP40 in complex with a known inhibitor still remains elusive. Using accelerated molecular dynamics approaches, we conducted a comparative study on VP40 apo and bound systems to understand the conformational features of VP40 at the molecular level and to determine the effect of inhibitor binding with the aid of a number of post-dynamic analytical tools. Significant features were seen in the presence of an inhibitor as per molecular mechanics/generalized born surface area binding free energy calculations. Results revealed that inhibitor binding to VP40 reduces the flexibility and mobility of the protein as supported by root mean square fluctuation and root mean square deviation calculations. The study revealed a characteristic "twisting" motion and coiling of the loop region of VP40 accompanied by conformational changes in the dimer interface upon inhibitor binding. We believe that results presented in this study will ultimately provide useful insight into the binding landscape of VP40 which could assist researchers in the discovery of potent Ebola virus inhibitors for anti-Ebola therapies.

  8. Marburg Virus Reverse Genetics Systems

    PubMed Central

    Schmidt, Kristina Maria; Mühlberger, Elke

    2016-01-01

    The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems. PMID:27338448

  9. Recombinant Vesicular Stomatitis Virus–Based Vaccines Against Ebola and Marburg Virus Infections

    PubMed Central

    Feldmann, Heinz

    2011-01-01

    The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections. PMID:21987744

  10. Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

    PubMed

    Lázaro-Frías, Adrián; Gómez-Medina, Sergio; Sánchez-Sampedro, Lucas; Ljungberg, Karl; Ustav, Mart; Liljeström, Peter; Muñoz-Fontela, César; Esteban, Mariano; García-Arriaza, Juan

    2018-06-01

    Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV. IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths

  11. ALIX Rescues Budding of a Double PTAP/PPEY L-Domain Deletion Mutant of Ebola VP40: A Role for ALIX in Ebola Virus Egress.

    PubMed

    Han, Ziying; Madara, Jonathan J; Liu, Yuliang; Liu, Wenbo; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N

    2015-10-01

    Ebola (EBOV) is an enveloped, negative-sense RNA virus belonging to the family Filoviridae that causes hemorrhagic fever syndromes with high-mortality rates. To date, there are no licensed vaccines or therapeutics to control EBOV infection and prevent transmission. Consequently, the need to better understand the mechanisms that regulate virus transmission is critical to developing countermeasures. The EBOV VP40 matrix protein plays a central role in late stages of virion assembly and egress, and independent expression of VP40 leads to the production of virus-like particles (VLPs) by a mechanism that accurately mimics budding of live virus. VP40 late (L) budding domains mediate efficient virus-cell separation by recruiting host ESCRT and ESCRT-associated proteins to complete the membrane fission process. L-domains consist of core consensus amino acid motifs including PPxY, P(T/S)AP, and YPx(n)L/I, and EBOV VP40 contains overlapping PPxY and PTAP motifs whose interactions with Nedd4 and Tsg101, respectively, have been characterized extensively. Here, we present data demonstrating for the first time that EBOV VP40 possesses a third L-domain YPx(n)L/I consensus motif that interacts with the ESCRT-III protein Alix. We show that the YPx(n)L/I motif mapping to amino acids 18-26 of EBOV VP40 interacts with the Alix Bro1-V fragment, and that siRNA knockdown of endogenous Alix expression inhibits EBOV VP40 VLP egress. Furthermore, overexpression of Alix Bro1-V rescues VLP production of the budding deficient EBOV VP40 double PTAP/PPEY L-domain deletion mutant to wild-type levels. Together, these findings demonstrate that EBOV VP40 recruits host Alix via a YPx(n)L/I motif that can function as an alternative L-domain to promote virus egress. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. A hamster model for Marburg virus infection accurately recapitulates Marburg hemorrhagic fever

    PubMed Central

    Marzi, Andrea; Banadyga, Logan; Haddock, Elaine; Thomas, Tina; Shen, Kui; Horne, Eva J.; Scott, Dana P.; Feldmann, Heinz; Ebihara, Hideki

    2016-01-01

    Marburg virus (MARV), a close relative of Ebola virus, is the causative agent of a severe human disease known as Marburg hemorrhagic fever (MHF). No licensed vaccine or therapeutic exists to treat MHF, and MARV is therefore classified as a Tier 1 select agent and a category A bioterrorism agent. In order to develop countermeasures against this severe disease, animal models that accurately recapitulate human disease are required. Here we describe the development of a novel, uniformly lethal Syrian golden hamster model of MHF using a hamster-adapted MARV variant Angola. Remarkably, this model displayed almost all of the clinical features of MHF seen in humans and non-human primates, including coagulation abnormalities, hemorrhagic manifestations, petechial rash, and a severely dysregulated immune response. This MHF hamster model represents a powerful tool for further dissecting MARV pathogenesis and accelerating the development of effective medical countermeasures against human MHF. PMID:27976688

  13. A hamster model for Marburg virus infection accurately recapitulates Marburg hemorrhagic fever.

    PubMed

    Marzi, Andrea; Banadyga, Logan; Haddock, Elaine; Thomas, Tina; Shen, Kui; Horne, Eva J; Scott, Dana P; Feldmann, Heinz; Ebihara, Hideki

    2016-12-15

    Marburg virus (MARV), a close relative of Ebola virus, is the causative agent of a severe human disease known as Marburg hemorrhagic fever (MHF). No licensed vaccine or therapeutic exists to treat MHF, and MARV is therefore classified as a Tier 1 select agent and a category A bioterrorism agent. In order to develop countermeasures against this severe disease, animal models that accurately recapitulate human disease are required. Here we describe the development of a novel, uniformly lethal Syrian golden hamster model of MHF using a hamster-adapted MARV variant Angola. Remarkably, this model displayed almost all of the clinical features of MHF seen in humans and non-human primates, including coagulation abnormalities, hemorrhagic manifestations, petechial rash, and a severely dysregulated immune response. This MHF hamster model represents a powerful tool for further dissecting MARV pathogenesis and accelerating the development of effective medical countermeasures against human MHF.

  14. Sequence analysis of the GP, NP, VP40 and VP24 genes of Ebola virus isolated from deceased, surviving and asymptomatically infected individuals during the 1996 outbreak in Gabon: comparative studies and phylogenetic characterization.

    PubMed

    Leroy, Eric M; Baize, Sylvain; Mavoungou, Elie; Apetrei, Cristian

    2002-01-01

    The aims of this study were to determine if the clinical outcome of Ebola virus (EBOV) infection is associated with virus genetic structure and to document the genetic changes in the Gabon strains of EBOV by sequencing the GP, NP, VP40 and VP24 genes from deceased and surviving symptomatic and asymptomatic individuals. GP and NP sequences were identical in the three groups of patients and only one silent substitution occurred in the VP40 and VP24 genes in asymptomatic individuals. A strain from an asymptomatic individual had a reverse substitution to the Gabon-94 sequence, indicating that minor virus variants may cocirculate during an outbreak. These results suggest that the different clinical outcomes of EBOV infection do not result from virus mutations. Phylogenetic analysis confirmed that Gabon-96 belonged to the Zaire subtype of EBOV and revealed that synonymous substitution rates were higher than nonsynonymous substitution rates in the GP, VP40 and VP24 genes. In contrast, nonsynonymous substitutions predominated over synonymous substitutions in the NP gene of the two Gabon strains, pointing to divergent evolution of these strains and to selective pressures on this gene.

  15. Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

    PubMed Central

    Watanabe, Marika; Phamduong, Ellen; Huang, Chu-Han; Itoh, Noriko; Bernal, Janie; Nakanishi, Akira; Rundell, Kathleen; Gjoerup, Ole

    2013-01-01

    The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1ΔC, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1ΔC in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1ΔC. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins. PMID:23427157

  16. Knowledge and attitude towards Ebola and Marburg virus diseases in Uganda using quantitative and participatory epidemiology techniques

    PubMed Central

    Skjerve, Eystein; Nabadda, Daisy; Sitali, Doreen Chilolo; Mumba, Chisoni; Mwiine, Frank N.; Lutwama, Julius J.; Balinandi, Stephen; Shoemaker, Trevor; Kankya, Clovice

    2017-01-01

    Background Uganda has reported five (5) Ebola virus disease outbreaks and three (3) Marburg virus disease outbreaks from 2000 to 2016. Peoples’ knowledge and attitude towards Ebola and Marburg virus disease impact on control and prevention measures especially during outbreaks. We describe knowledge and attitude towards Ebola and Marburg virus outbreaks in two affected communities in Uganda to inform future outbreak responses and help in the design of health education and communication messages. Methods The study was a community survey done in Luweero, Ibanda and Kamwenge districts that have experienced outbreaks of Ebola and Marburg virus diseases. Quantitative data were collected using a structured questionnaire and triangulated with qualitative participatory epidemiology techniques to gain a communities’ knowledge and attitude towards Ebola and Marburg virus disease. Results Out of 740 respondents, 48.5% (359/740) were categorized as being knowledgeable about Ebola and Marburg virus diseases, whereas 60.5% (448/740) were having a positive attitude towards control and prevention of Ebola and Marburg virus diseases. The mean knowledge and attitude percentage scores were 54.3 (SD = 23.5, 95%CI = 52.6–56.0) and 69.9 (SD = 16.9, 95%CI = 68.9–71.1) respectively. People educated beyond primary school were more likely to be knowledgeable about Ebola and Marburg virus disease than those who did not attain any formal education (OR = 3.6, 95%CI = 2.1–6.1). Qualitative data revealed that communities describe Ebola and Marburg virus diseases as very severe diseases with no cure and they believe the diseases spread so fast. Respondents reported fear and stigma suffered by survivors, their families and the broader community due to these diseases. Conclusion Communities in Uganda affected by filovirus outbreaks have moderate knowledge about these diseases and have a positive attitude towards practices to prevent and control Ebola and Marburg viral diseases. The public

  17. Knowledge and attitude towards Ebola and Marburg virus diseases in Uganda using quantitative and participatory epidemiology techniques.

    PubMed

    Nyakarahuka, Luke; Skjerve, Eystein; Nabadda, Daisy; Sitali, Doreen Chilolo; Mumba, Chisoni; Mwiine, Frank N; Lutwama, Julius J; Balinandi, Stephen; Shoemaker, Trevor; Kankya, Clovice

    2017-09-01

    Uganda has reported five (5) Ebola virus disease outbreaks and three (3) Marburg virus disease outbreaks from 2000 to 2016. Peoples' knowledge and attitude towards Ebola and Marburg virus disease impact on control and prevention measures especially during outbreaks. We describe knowledge and attitude towards Ebola and Marburg virus outbreaks in two affected communities in Uganda to inform future outbreak responses and help in the design of health education and communication messages. The study was a community survey done in Luweero, Ibanda and Kamwenge districts that have experienced outbreaks of Ebola and Marburg virus diseases. Quantitative data were collected using a structured questionnaire and triangulated with qualitative participatory epidemiology techniques to gain a communities' knowledge and attitude towards Ebola and Marburg virus disease. Out of 740 respondents, 48.5% (359/740) were categorized as being knowledgeable about Ebola and Marburg virus diseases, whereas 60.5% (448/740) were having a positive attitude towards control and prevention of Ebola and Marburg virus diseases. The mean knowledge and attitude percentage scores were 54.3 (SD = 23.5, 95%CI = 52.6-56.0) and 69.9 (SD = 16.9, 95%CI = 68.9-71.1) respectively. People educated beyond primary school were more likely to be knowledgeable about Ebola and Marburg virus disease than those who did not attain any formal education (OR = 3.6, 95%CI = 2.1-6.1). Qualitative data revealed that communities describe Ebola and Marburg virus diseases as very severe diseases with no cure and they believe the diseases spread so fast. Respondents reported fear and stigma suffered by survivors, their families and the broader community due to these diseases. Communities in Uganda affected by filovirus outbreaks have moderate knowledge about these diseases and have a positive attitude towards practices to prevent and control Ebola and Marburg viral diseases. The public health sector should enhance this community

  18. Structural basis for Marburg virus VP35-mediated immune evasion mechanisms

    SciTech Connect

    Ramanan, Parameshwaran; Edwards, Megan R.; Shabman, Reed S.

    2013-07-22

    Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen–associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFNmore » production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.« less

  19. Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

    PubMed Central

    Sänger, Christian; Mühlberger, Elke; Ryabchikova, Elena; Kolesnikova, Larissa; Klenk, Hans-Dieter; Becker, Stephan

    2001-01-01

    Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein. PMID:11152500

  20. Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    SciTech Connect

    Pleet, Michelle L.; Mathiesen, Allison; DeMarino, Catherine

    Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80–90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, wemore » examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. In addition, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the

  1. Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    DOE PAGES

    Pleet, Michelle L.; Mathiesen, Allison; DeMarino, Catherine; ...

    2016-11-07

    Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80–90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, wemore » examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. In addition, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the

  2. Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.

    PubMed

    Pleet, Michelle L; Mathiesen, Allison; DeMarino, Catherine; Akpamagbo, Yao A; Barclay, Robert A; Schwab, Angela; Iordanskiy, Sergey; Sampey, Gavin C; Lepene, Benjamin; Nekhai, Sergei; Aman, M J; Kashanchi, Fatah

    2016-01-01

    Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable of causing severe hemorrhagic fever with up to 80-90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, we examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. Additionally, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the deregulation

  3. [Ebola and Marburg hemorrhagic fever viruses: update on filoviruses].

    PubMed

    Leroy, E; Baize, S; Gonzalez, J P

    2011-04-01

    The Ebola and Marburg viruses are the sole members of the Filoviridae family of viruses. They are characterized by a long filamentous form that is unique in the viral world. Filoviruses are among the most virulent pathogens currently known to infect humans. They cause fulminating disease characterized by acute fever followed by generalized hemorrhagic syndrome that is associated with 90% mortality in the most severe forms. Epidemic outbreaks of Marburg and Ebola viruses have taken a heavy toll on human life in Central Africa and devastated large ape populations in Gabon and Republic of Congo. Since their discovery in 1967 (Marburg) and 1976 (Ebola), more than 2,300 cases and 1,670 deaths have been reported. These numbers pale in comparison with the burden caused by malnutrition or other infectious disease scourges in Africa such as malaria, cholera, AIDS, dengue or tuberculosis. However, due to their extremely high lethality, association with multifocal hemorrhaging and specificity to the African continent, these hemorrhagic fever viruses have given rise to great interest on the part not only of the international scientific community but also of the general public because of their perceived potential as biological weapons. Much research has been performed on these viruses and major progress has been made in knowledge of their ecology, epidemiology and physiopathology and in development of vaccine candidates and therapeutic schemes. The purpose of this review is to present the main developments in these particular fields in the last decade.

  4. Biochemical and Functional Characterization of the Ebola Virus VP24 Protein: Implications for a Role in Virus Assembly and Budding

    PubMed Central

    Han, Ziying; Boshra, Hani; Sunyer, J. Oriol; Zwiers, Susan H.; Paragas, Jason; Harty, Ronald N.

    2003-01-01

    The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding. PMID:12525613

  5. Phylogenetic assessment of filoviruses: how many lineages of Marburg virus?

    PubMed Central

    Peterson, A Townsend; Holder, Mark T

    2012-01-01

    Filoviruses have to date been considered as consisting of one diverse genus (Ebola viruses) and one undifferentiated genus (Marburg virus). We reconsider this idea by means of detailed phylogenetic analyses of sequence data available for the Filoviridae: using coalescent simulations, we ascertain that two Marburg isolates (termed the “RAVN” strain) represent a quite-distinct lineage that should be considered in studies of biogeography and host associations, and may merit recognition at the level of species. In contrast, filovirus isolates recently obtained from bat tissues are not distinct from previously known strains, and should be considered as drawn from the same population. Implications for understanding the transmission geography and host associations of these viruses are discussed. PMID:22957185

  6. Infection and Activation of Monocytes by Marburg and Ebola Viruses

    PubMed Central

    Ströher, Ute; West, Elmar; Bugany, Harald; Klenk, Hans-Dieter; Schnittler, Hans-Joachim; Feldmann, Heinz

    2001-01-01

    In this study we investigated the effects of Marburg virus and Ebola virus (species Zaire and Reston) infections on freshly isolated suspended monocytes in comparison to adherent macrophages under culture conditions. Our data showed that monocytes are permissive for both filoviruses. As is the case in macrophages, infection resulted in the activation of monocytes which was largely independent of virus replication. The activation was triggered similarly by Marburg and Ebola viruses, species Zaire and Reston, as indicated by the release of the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor α, and IL-6 as well as the chemokines IL-8 and gro-α. Our data suggest that infected monocytes may play an important role in the spread of filoviruses and in the pathogenesis of filoviral hemorrhagic disease. PMID:11602743

  7. Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human infection.

    PubMed

    Amman, Brian R; Carroll, Serena A; Reed, Zachary D; Sealy, Tara K; Balinandi, Stephen; Swanepoel, Robert; Kemp, Alan; Erickson, Bobbie Rae; Comer, James A; Campbell, Shelley; Cannon, Deborah L; Khristova, Marina L; Atimnedi, Patrick; Paddock, Christopher D; Crockett, Rebekah J Kent; Flietstra, Timothy D; Warfield, Kelly L; Unfer, Robert; Katongole-Mbidde, Edward; Downing, Robert; Tappero, Jordan W; Zaki, Sherif R; Rollin, Pierre E; Ksiazek, Thomas G; Nichol, Stuart T; Towner, Jonathan S

    2012-01-01

    Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ~2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (~six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a

  8. Seasonal Pulses of Marburg Virus Circulation in Juvenile Rousettus aegyptiacus Bats Coincide with Periods of Increased Risk of Human Infection

    PubMed Central

    Amman, Brian R.; Carroll, Serena A.; Reed, Zachary D.; Sealy, Tara K.; Balinandi, Stephen; Swanepoel, Robert; Kemp, Alan; Erickson, Bobbie Rae; Comer, James A.; Campbell, Shelley; Cannon, Deborah L.; Khristova, Marina L.; Atimnedi, Patrick; Paddock, Christopher D.; Kent Crockett, Rebekah J.; Flietstra, Timothy D.; Warfield, Kelly L.; Unfer, Robert; Katongole-Mbidde, Edward; Downing, Robert; Tappero, Jordan W.; Zaki, Sherif R.; Rollin, Pierre E.; Ksiazek, Thomas G.; Nichol, Stuart T.; Towner, Jonathan S.

    2012-01-01

    Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ∼2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (∼six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide

  9. High-resolution Crystal Structure of Dimeric VP40 From Sudan ebolavirus.

    PubMed

    Clifton, Matthew C; Bruhn, Jessica F; Atkins, Kateri; Webb, Terry L; Baydo, Ruth O; Raymond, Amy; Lorimer, Donald D; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

    2015-10-01

    Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Rapid detection of EBOLA VP40 in microchip immunofiltration assay

    NASA Astrophysics Data System (ADS)

    Miethe, Peter; Gary, Dominik; Hlawatsch, Nadine; Gad, Anne-Marie

    2015-05-01

    In the spring of 2014, the Ebola virus (EBOV) strain Zaire caused a dramatic outbreak in several regions of West Africa. The RT-PCR and antigen capture diagnostic proved to be effective for detecting EBOV in blood and serum. In this paper, we present data of a rapid antigen capture test for the detection of VP40. The test was performed in a microfluidic chip for immunofiltration analysis. The chip integrates all necessary assay components. The analytical sensitivity of the rapid test was 8 ng/ml for recombinant VP40. In serum and whole blood samples spiked with virus culture material, the detection limit was 2.2 x 102 PFU/ml. The performance data of the rapid test (15 min) are comparable to that of the VP40 laboratory ELISA.

  11. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation

    SciTech Connect

    Elliott, L.H.; McCormick, J.B.; Johnson, K.M.

    1982-10-01

    Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with /sup 60/Co gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of /sup 60/Co radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose permore » rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. We found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents.« less

  12. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation

    SciTech Connect

    Elliott, L.H.; McCormick, J.B.; Johnson, K.M.

    1982-10-01

    Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with /sup 60/CO gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of /sup 60/CO radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose permore » rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. The authors found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents.« less

  13. Experimental respiratory Marburg virus haemorrhagic fever infection in the common marmoset (Callithrix jacchus)

    PubMed Central

    Smither, Sophie J; Nelson, Michelle; Eastaugh, Lin; Laws, Thomas R; Taylor, Christopher; Smith, Simon A; Salguero, Francisco J; Lever, Mark S

    2013-01-01

    Marburg virus causes a highly infectious and lethal haemorrhagic fever in primates and may be exploited as a potential biothreat pathogen. To combat the infection and threat of Marburg haemorrhagic fever, there is a need to develop and license appropriate medical countermeasures. To determine whether the common marmoset (Callithrix jacchus) would be an appropriate model to assess therapies against Marburg haemorrhagic fever, initial susceptibility, lethality and pathogenesis studies were performed. Low doses of virus, between 4 and 28 TCID50, were sufficient to cause a lethal, reproducible infection. Animals became febrile between days 5 and 6, maintaining a high fever before succumbing to disease between 8 and 11 days postchallenge. Typical signs of Marburg virus infection were observed including haemorrhaging and a transient rash. In pathogenesis studies, virus was isolated from the animals’ lungs from day 3 postchallenge and from the liver, spleen and blood from day 5 postchallenge. Early signs of histopathology were apparent in the kidney and liver from day 3. The most striking features were observed in animals exhibiting severe clinical signs, which included high viral titres in all organs, with the highest levels in the blood, increased levels in liver function enzymes and blood clotting times, decreased levels in platelets, multifocal moderate-to-severe hepatitis and perivascular oedema. PMID:23441639

  14. Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport.

    PubMed

    Takamatsu, Yuki; Kolesnikova, Larissa; Becker, Stephan

    2018-01-30

    The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different protein compositions. Only three of the five EBOV nucleocapsid proteins-nucleoprotein, VP35, and VP24-were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.

  15. Monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple Marburg viruses.

    PubMed

    Swenson, Dana L; Warfield, Kelly L; Larsen, Tom; Alves, D Anthony; Coberley, Sadie S; Bavari, Sina

    2008-05-01

    Virus-like particle (VLP)-based vaccines have the advantage of being morphologically and antigenically similar to the live virus from which they are derived. Expression of the glycoprotein and VP40 matrix protein from Lake Victoria marburgvirus (MARV) results in spontaneous production of VLPs in mammalian cells. Guinea pigs vaccinated with Marburg virus VLPs (mVLPs) or inactivated MARV (iMARV) develop homologous humoral and T-cell responses and are completely protected from a lethal homologous MARV challenge. To determine whether mVLPs based on the Musoke (aka Lake Victoria) isolate of MARV could broadly protect against diverse isolates of MARV, guinea pigs were vaccinated with mVLPs or iMARV-Musoke and challenged with MARV-Musoke, -Ravn or -Ci67. Prior to challenge, the mVLP- and iMARV-vaccinated guinea pigs had high levels of homologous MARV-Musoke and heterologous MARV-Ravn and -Ci67 antibodies. The Musoke-based mVLPs and iMARV vaccines provided complete protection in guinea pigs against viremia, viral replication and pathological changes in tissues, and lethal disease following challenge with MARV-Musoke, -Ravn or -Ci67. Guinea pigs vaccinated with RIBI adjuvant alone and infected with guinea pig-adapted MARV-Musoke, -Ravn or -Ci67 had histopathologic findings similar to those seen in the nonhuman primate model for MARV infection. Based on the strong protection observed in guinea pigs, we next vaccinated cynomolgus macaques with Musoke-based mVLPs and showed the VLP-vaccinated monkeys were broadly protected against three isolates of MARV (Musoke, Ravn and Ci67). Musoke mVLPs are effective at inducing broad heterologous immunity and protection against multiple MARV isolates.

  16. The VP35 protein of Ebola virus impairs dendritic cell maturation induced by virus and lipopolysaccharide.

    PubMed

    Jin, Huali; Yan, Zhipeng; Prabhakar, Bellur S; Feng, Zongdi; Ma, Yijie; Verpooten, Dustin; Ganesh, Balaji; He, Bin

    2010-02-01

    Ebola virus causes rapidly progressive haemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DCs), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T-cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse DCs. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86 and major histocompatibility complex class II. Further, it suppresses the induction of cytokines such as interleukin (IL)-6, IL-12, tumour necrosis factor alpha and alpha/beta interferon (IFN-alpha/beta). Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4(+) T cells. Addition of type I IFN to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolishes its activity, whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference in DC function with resultant deficiency in T-cell function, which may contribute to the profound virulence of Ebola virus infection.

  17. Non-active site mutations disturb the loop dynamics, dimerization, viral budding and egress of VP40 of the Ebola virus.

    PubMed

    Balmith, Marissa; Soliman, Mahmoud E S

    2017-02-28

    The first account of the dynamic features of the loop region of VP40 of the Ebola virus (EboV) using accelerated molecular dynamics (aMD) simulations is reported herein. Due to its major role in the Ebola life cycle, VP40 is considered a promising therapeutic target. The available experimental data on the N-terminal domain (NTD) loop indicates that mutations K127A, T129A and N130A demonstrate an unrecognized role for NTD-plasma membrane (PM) interaction for efficient VP40-PM localization, oligomerization, matrix assembly and egress. Despite experimental results, the molecular description of VP40 and the information it can provide still remain vague. Therefore, to gain further molecular insight into the effect of mutations on the loop region of VP40 and its effects on the overall protein conformation and VP40 dimerization, aMD simulations and post-dynamic analyses were employed for wildtype (WT) and mutant systems. The results showed significant variations in the presence of mutations as per RMSF, RMSD, R g , PCA and distance calculations in comparison to the WT. These results could provide researchers with insight with regards to the conformational aspects concerning VP40 and its close relation to the experimental data. We believe that the results presented in this study will ultimately provide a useful understanding of the structural landscape of the loop region of VP40, which would contribute towards the discovery of novel EboV inhibitors.

  18. Structure and dynamics of Ebola virus matrix protein VP40 by a coarse-grained Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Pandey, Ras; Farmer, Barry

    Ebola virus matrix protein VP40 (consisting of 326 residues) plays a critical role in viral assembly and its functions such as regulation of viral transcription, packaging, and budding of mature virions into the plasma membrane of infected cells. How does the protein VP40 go through structural evolution during the viral life cycle remains an open question? Using a coarse-grained Monte Carlo simulation we investigate the structural evolution of VP40 as a function of temperature with the input of a knowledge-based residue-residue interaction. A number local and global physical quantities (e.g. mobility profile, contact map, radius of gyration, structure factor) are analyzed with our large-scale simulations. Our preliminary data show that the structure of the protein evolves through different state with well-defined morphologies which can be identified and quantified via a detailed analysis of structure factor.

  19. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.

    PubMed

    Bharat, Tanmay A M; Noda, Takeshi; Riches, James D; Kraehling, Verena; Kolesnikova, Larissa; Becker, Stephan; Kawaoka, Yoshihiro; Briggs, John A G

    2012-03-13

    Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

  20. Experimental respiratory Marburg virus haemorrhagic fever infection in the common marmoset (Callithrix jacchus).

    PubMed

    Smither, Sophie J; Nelson, Michelle; Eastaugh, Lin; Laws, Thomas R; Taylor, Christopher; Smith, Simon A; Salguero, Francisco J; Lever, Mark S

    2013-04-01

    Marburg virus causes a highly infectious and lethal haemorrhagic fever in primates and may be exploited as a potential biothreat pathogen. To combat the infection and threat of Marburg haemorrhagic fever, there is a need to develop and license appropriate medical countermeasures. To determine whether the common marmoset (Callithrix jacchus) would be an appropriate model to assess therapies against Marburg haemorrhagic fever, initial susceptibility, lethality and pathogenesis studies were performed. Low doses of virus, between 4 and 28 TCID50 , were sufficient to cause a lethal, reproducible infection. Animals became febrile between days 5 and 6, maintaining a high fever before succumbing to disease between 8 and 11 days postchallenge. Typical signs of Marburg virus infection were observed including haemorrhaging and a transient rash. In pathogenesis studies, virus was isolated from the animals' lungs from day 3 postchallenge and from the liver, spleen and blood from day 5 postchallenge. Early signs of histopathology were apparent in the kidney and liver from day 3. The most striking features were observed in animals exhibiting severe clinical signs, which included high viral titres in all organs, with the highest levels in the blood, increased levels in liver function enzymes and blood clotting times, decreased levels in platelets, multifocal moderate-to-severe hepatitis and perivascular oedema. © 2013 Crown copyright. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

  1. Successful treatment of Marburg virus with orally administrated T-705 (Favipiravir) in a mouse model.

    PubMed

    Zhu, Wenjun; Zhang, Zirui; He, Shihua; Wong, Gary; Banadyga, Logan; Qiu, Xiangguo

    2018-03-01

    Filoviruses, such as Marburg and Ebola viruses, cause severe disease in humans with high case fatality rates and are therefore considered biological threat agents. To date, no licensed vaccine or therapeutic exists for their treatment. T-705 (favipiravir) is a pyrazinecarboxamide derivative that has shown broad antiviral activity against a number of viruses and is clinically licenced in Japan to treat influenza. Here we report the efficacy of T-705 against Marburg virus infection in vitro and in vivo. Notably, oral administration of T-705 beginning one or two days post-infection and continuing for eight days resulted in complete survival of mice that had been intraperitoneally infected with mouse-adapted Marburg virus (variant Angola). Moreover, lower doses of T-705 and higher doses administered later during infection (day 3 or 4 post-infection) showed partial efficacy, with at least half the infected mice surviving. Accordingly, we observed reductions in infectious virus particles and virus RNA levels following drug treatment that appeared to correlate with survival. Our findings suggest that T-705 may be an effective therapeutic against Marburg virus and might be especially promising for use in the event of an outbreak, where it could be orally administered quickly and safely even after exposure. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

  2. Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus

    PubMed Central

    Messaoudi, Ilhem; Amarasinghe, Gaya K.; Basler, Christopher F.

    2016-01-01

    Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people. The Ebola virus epidemic in West Africa, which was first recognized in early 2014, highlights the threat posed by these deadly viruses. Filovirus disease is characterized by uncontrolled virus replication and the activation of damaging host pathways. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon (IFN) response, which allows high levels of replication. Here we review the mechanisms deployed by filoviruses to block host innate immunity and discuss aspects of virus replication that promote disease. PMID:26439085

  3. Calculation of incubation period and serial interval from multiple outbreaks of Marburg virus disease.

    PubMed

    Pavlin, Boris I

    2014-12-13

    Marburg viruses have been responsible for a number of outbreaks throughout sub-Saharan Africa, as well as a number of laboratory infections. Despite many years of experience with the viruses, little is known about several important epidemiologic parameters relating to the development of Marburg virus disease. The analysis uses pooled data from all Marburg cases between 1967 and 2008 to develop estimates for the incubation period and the clinical onset serial interval (COSI). Data were obtained from original outbreak investigation forms (n=406) and from published data (n=45). Incubation periods were calculated for person-to-person exposure, for laboratory-acquired infections, and for presumed zoonotic exposures. Similar analysis was conducted for COSI, using only cases with unambiguous person-to-person transmission where both the primary and the secondary case patients had well-defined illness onsets. Seventy-six cases were retained for the incubation period analysis. Incubation periods ranged from a minimum of 2 days in the case of two laboratory workers to a maximum of at least 26 days for a person-to-person household transmission. Thirty-eight cases were retained for COSI analysis. The median COSI was 11 days, with an interquartile range of 8 to 15. This study extends the maximum known incubation period of Marburg virus disease to 26 days. The analysis was severely hampered by a lack of completeness in epidemiologic data. It is necessary to prioritize obtaining more accurate epidemiologic data in future outbreaks; greater use of COSI may facilitate an improved understanding of outbreak dynamics in Marburg and other diseases.

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

    PubMed

    Fusco, Marnie L; Hashiguchi, Takao; Cassan, Robyn; Biggins, Julia E; Murin, Charles D; Warfield, Kelly L; Li, Sheng; Holtsberg, Frederick W; Shulenin, Sergey; Vu, Hong; Olinger, Gene G; Kim, Do H; Whaley, Kevin J; Zeitlin, Larry; Ward, Andrew B; Nykiforuk, Cory; Aman, M Javad; Berry, Jody D; Berry, Jody; Saphire, Erica Ollmann

    2015-06-01

    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.

  5. Vaccine to Confer to Nonhuman Primates Complete Protection Against Multistrain Ebola and Marburg Virus Infections

    DTIC Science & Technology

    2008-01-01

    current filovirus threats in the event of natural hemorrhagic fever outbreak or biological attack. Ebola virus (EBOV) and Marburg virus (MARV) are mem...lethal, causing severe hemorrhagic fever disease in humans and apes with high mortality rates (up to 90%). The recent description of massive gorilla...threats in the event of natural hemorrhagic fever outbreak or biological attack. 15. SUBJECT TERMS filovirus, Ebola, Marburg, adenovirus-based vaccine

  6. Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus.

    PubMed

    Messaoudi, Ilhem; Amarasinghe, Gaya K; Basler, Christopher F

    2015-11-01

    Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirus pathogenesis.

  7. Prospects for immunisation against Marburg and Ebola viruses.

    PubMed

    Geisbert, Thomas W; Bausch, Daniel G; Feldmann, Heinz

    2010-11-01

    For more than 30 years the filoviruses, Marburg virus and Ebola virus, have been associated with periodic outbreaks of hemorrhagic fever that produce severe and often fatal disease. The filoviruses are endemic primarily in resource-poor regions in Central Africa and are also potential agents of bioterrorism. Although no vaccines or antiviral drugs for Marburg or Ebola are currently available, remarkable progress has been made over the last decade in developing candidate preventive vaccines against filoviruses in nonhuman primate models. Due to the generally remote locations of filovirus outbreaks, a single-injection vaccine is desirable. Among the prospective vaccines that have shown efficacy in nonhuman primate models of filoviral hemorrhagic fever, two candidates, one based on a replication-defective adenovirus serotype 5 and the other on a recombinant VSV (rVSV), were shown to provide complete protection to nonhuman primates when administered as a single injection. The rVSV-based vaccine has also shown utility when administered for postexposure prophylaxis against filovirus infections. A VSV-based Ebola vaccine was recently used to manage a potential laboratory exposure. 2010 John Wiley & Sons, Ltd.

  8. Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody.

    PubMed

    Mire, Chad E; Geisbert, Joan B; Borisevich, Viktoriya; Fenton, Karla A; Agans, Krystle N; Flyak, Andrew I; Deer, Daniel J; Steinkellner, Herta; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Hiatt, Andrew; Kim, Do H; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Crowe, James E; Zeitlin, Larry; Geisbert, Thomas W

    2017-04-05

    As observed during the 2013-2016 Ebola virus disease epidemic, containment of filovirus outbreaks is challenging and made more difficult by the lack of approved vaccine or therapeutic options. Marburg and Ravn viruses are highly virulent and cause severe and frequently lethal disease in humans. Monoclonal antibodies (mAbs) are a platform technology in wide use for autoimmune and oncology indications. Previously, we described human mAbs that can protect mice from lethal challenge with Marburg virus. We demonstrate that one of these mAbs, MR191-N, can confer a survival benefit of up to 100% to Marburg or Ravn virus-infected rhesus macaques when treatment is initiated up to 5 days post-inoculation. These findings extend the small but growing body of evidence that mAbs can impart therapeutic benefit during advanced stages of disease with highly virulent viruses and could be useful in epidemic settings. Copyright © 2017, American Association for the Advancement of Science.

  9. Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus

    SciTech Connect

    Messaoudi, Ilhem; Amarasinghe, Gaya K.; Basler, Christopher F.

    Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirusmore » pathogenesis.« less

  10. Oral shedding of Marburg virus in experimentally infected Egyptian fruit bats (Rousettus aegyptiacus).

    PubMed

    Amman, Brian R; Jones, Megan E B; Sealy, Tara K; Uebelhoer, Luke S; Schuh, Amy J; Bird, Brian H; Coleman-McCray, JoAnn D; Martin, Brock E; Nichol, Stuart T; Towner, Jonathan S

    2015-01-01

    Marburg virus (Marburg marburgvirus; MARV) causes sporadic outbreaks of Marburg hemorrhagic fever (MHF) in Africa. The Egyptian fruit bat (Rousettus aegyptiacus) has been identified as a natural reservoir based most-recently on the repeated isolation of MARV directly from bats caught at two locations in southwestern Uganda where miners and tourists separately contracted MHF from 2007-08. Despite learning much about the ecology of MARV through extensive field investigations, there remained unanswered questions such as determining the primary routes of virus shedding and the severity of disease, if any, caused by MARV in infected bats. To answer these questions and others, we experimentally infected captive-bred R. aegyptiacus with MARV under high (biosafety level 4) containment. These experiments have shown infection profiles consistent with R. aegyptiacus being a bona fide natural reservoir host for MARV and demonstrated routes of viral shedding capable of infecting humans and other animals.

  11. Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

    PubMed Central

    Ren, Shoufeng; Wei, Qimei; Cai, Liya; Yang, Xuejing; Xing, Cuicui; Tan, Feng; Leavenworth, Jianmei W.; Liang, Shaohui; Liu, Wenquan

    2018-01-01

    Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention. PMID:29375526

  12. Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus.

    PubMed

    Qiu, Xiangguo; Wong, Gary; Audet, Jonathan; Cutts, Todd; Niu, Yulian; Booth, Stephanie; Kobinger, Gary P

    2014-11-01

    Infections with Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in humans and nonhuman primates (NHPs) with fatality rates up to 90%. A number of experimental vaccine and treatment platforms have previously been shown to be protective against EBOV infection. However, the rate of development for prophylactics and therapeutics against MARV has been lower in comparison, possibly because a small-animal model is not widely available. Here we report the development of a mouse model for studying the pathogenesis of MARV Angola (MARV/Ang), the most virulent strain of MARV. Infection with the wild-type virus does not cause disease in mice, but the adapted virus (MARV/Ang-MA) recovered from liver homogenates after 24 serial passages in severe combined immunodeficient (SCID) mice caused severe disease when administered intranasally (i.n.) or intraperitoneally (i.p.). The median lethal dose (LD50) was determined to be 0.015 50% TCID50 (tissue culture infective dose) of MARV/Ang-MA in SCID mice, and i.p. infection at a dose of 1,000× LD50 resulted in death between 6 and 8 days postinfection in SCID mice. Similar results were obtained with immunocompetent BALB/c and C57BL/6 mice challenged i.p. with 2,000× LD50 of MARV/Ang-MA. Virological and pathological analyses of MARV/Ang-MA-infected BALB/c mice revealed that the associated pathology was reminiscent of observations made in NHPs with MARV/Ang. MARV/Ang-MA-infected mice showed most of the clinical hallmarks observed with Marburg hemorrhagic fever, including lymphopenia, thrombocytopenia, marked liver damage, and uncontrolled viremia. Virus titers reached 10(8) TCID50/ml in the blood and between 10(6) and 10(10) TCID50/g tissue in the intestines, kidney, lungs, brain, spleen, and liver. This model provides an important tool to screen candidate vaccines and therapeutics against MARV infections. The Angola strain of Marburg virus (MARV/Ang) was responsible for the largest outbreak ever documented for

  13. Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

    PubMed

    Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

    2005-09-01

    Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

  14. Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

    PubMed

    Marín-López, Alejandro; Ortego, Javier

    2016-01-01

    Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

  15. Natural History of Aerosol Exposure with Marburg Virus in Rhesus Macaques

    PubMed Central

    Ewers, Evan C.; Pratt, William D.; Twenhafel, Nancy A.; Shamblin, Joshua; Donnelly, Ginger; Esham, Heather; Wlazlowski, Carly; Johnson, Joshua C.; Botto, Miriam; Hensley, Lisa E.; Goff, Arthur J.

    2016-01-01

    Marburg virus causes severe and often lethal viral disease in humans, and there are currently no Food and Drug Administration (FDA) approved medical countermeasures. The sporadic occurrence of Marburg outbreaks does not allow for evaluation of countermeasures in humans, so therapeutic and vaccine candidates can only be approved through the FDA animal rule—a mechanism requiring well-characterized animal models in which efficacy would be evaluated. Here, we describe a natural history study where rhesus macaques were surgically implanted with telemetry devices and central venous catheters prior to aerosol exposure with Marburg-Angola virus, enabling continuous physiologic monitoring and blood sampling without anesthesia. After a three to four day incubation period, all animals developed fever, viremia, and lymphopenia before developing tachycardia, tachypnea, elevated liver enzymes, decreased liver function, azotemia, elevated D-dimer levels and elevated pro-inflammatory cytokines suggesting a systemic inflammatory response with organ failure. The final, terminal period began with the onset of sustained hypotension, dehydration progressed with signs of major organ hypoperfusion (hyperlactatemia, acute kidney injury, hypothermia), and ended with euthanasia or death. The most significant pathologic findings were marked infection of the respiratory lymphoid tissue with destruction of the tracheobronchial and mediastinal lymph nodes, and severe diffuse infection in the liver, and splenitis. PMID:27043611

  16. Natural History of Aerosol Exposure with Marburg Virus in Rhesus Macaques.

    PubMed

    Ewers, Evan C; Pratt, William D; Twenhafel, Nancy A; Shamblin, Joshua; Donnelly, Ginger; Esham, Heather; Wlazlowski, Carly; Johnson, Joshua C; Botto, Miriam; Hensley, Lisa E; Goff, Arthur J

    2016-03-30

    Marburg virus causes severe and often lethal viral disease in humans, and there are currently no Food and Drug Administration (FDA) approved medical countermeasures. The sporadic occurrence of Marburg outbreaks does not allow for evaluation of countermeasures in humans, so therapeutic and vaccine candidates can only be approved through the FDA animal rule-a mechanism requiring well-characterized animal models in which efficacy would be evaluated. Here, we describe a natural history study where rhesus macaques were surgically implanted with telemetry devices and central venous catheters prior to aerosol exposure with Marburg-Angola virus, enabling continuous physiologic monitoring and blood sampling without anesthesia. After a three to four day incubation period, all animals developed fever, viremia, and lymphopenia before developing tachycardia, tachypnea, elevated liver enzymes, decreased liver function, azotemia, elevated D-dimer levels and elevated pro-inflammatory cytokines suggesting a systemic inflammatory response with organ failure. The final, terminal period began with the onset of sustained hypotension, dehydration progressed with signs of major organ hypoperfusion (hyperlactatemia, acute kidney injury, hypothermia), and ended with euthanasia or death. The most significant pathologic findings were marked infection of the respiratory lymphoid tissue with destruction of the tracheobronchial and mediastinal lymph nodes, and severe diffuse infection in the liver, and splenitis.

  17. Ebola and Marburg virus vaccines.

    PubMed

    Reynolds, Pierce; Marzi, Andrea

    2017-08-01

    The filoviruses, Ebola virus (EBOV), and Marburg virus (MARV), are among the most pathogenic viruses known to man and the causative agents of viral hemorrhagic fever outbreaks in Africa with case fatality rates of up to 90%. Nearly 30,000 infections were observed in the latest EBOV epidemic in West Africa; previous outbreaks were much smaller, typically only affecting less than a few hundred people. Compared to other diseases such as AIDS or Malaria with millions of cases annually, filovirus hemorrhagic fever (FHF) is one of the neglected infectious diseases. There are no licensed vaccines or therapeutics available to treat EBOV and MARV infections; therefore, these pathogens can only be handled in maximum containment laboratories and are classified as select agents. Under these limitations, a very few laboratories worldwide conducted basic research and countermeasure development for EBOV and MARV since their respective discoveries in 1967 (MARV) and 1976 (EBOV). In this review, we discuss several vaccine platforms against EBOV and MARV, which have been assessed for their protective efficacy in animal models of FHF. The focus is on the most promising approaches, which were accelerated in clinical development (phase I-III trials) during the EBOV epidemic in West Africa.

  18. Generation and characterization of protective antibodies to Marburg virus.

    PubMed

    Froude, Jeffrey W; Pelat, Thibaut; Miethe, Sebastian; Zak, Samantha E; Wec, Anna Z; Chandran, Kartik; Brannan, Jennifer Mary; Bakken, Russell R; Hust, Michael; Thullier, Philippe; Dye, John M

    Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP 1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct V H and V L sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 µg of scFv-Fc on days -1, 1 and 3 demonstrated protective efficacies ranging from 75-100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.

  19. Full protection against African horsesickness (AHS) in horses induced by baculovirus-derived AHS virus serotype 4 VP2, VP5 and VP7.

    PubMed

    Martínez-Torrecuadrada, J L; Díaz-Laviada, M; Roy, P; Sánchez, C; Vela, C; Sánchez-Vizcaíno, J M; Casal, J I

    1996-06-01

    African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors.

  20. Evaluation of the Protective Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against Marburg Hemorrhagic Fever in Nonhuman Primate Models

    DTIC Science & Technology

    2007-01-19

    fever in Nonhuman Primate Models" Date d?JO )oi Date )&*7 Date Dissertation and Abstract Approved: Robert Friedm ,M.D. Department of Pathology Committee...thesis manuscript entitled: "Evaluation of the Protective Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against Marburg Hemorrhagic fever ...stomatitis virus vectors against Marburg hemorrhagic fever in nonhuman primate models By Kathleen Daddario-DiCaprio Dissertation

  1. VP3 is crucial for the stability of Nora virus virions.

    PubMed

    Sadanandan, Sajna Anand; Ekström, Jens-Ola; Jonna, Venkateswara Rao; Hofer, Anders; Hultmark, Dan

    2016-09-02

    Nora virus is an enteric virus that causes persistent, non-pathological infection in Drosophila melanogaster. It replicates in the fly gut and is transmitted via the fecal-oral route. Nora virus has a single-stranded positive-sense RNA genome, which is translated in four open reading frames. Reading frame three encodes the VP3 protein, the structure and function of which we have investigated in this work. We have shown that VP3 is a trimer that has an α-helical secondary structure, with a functionally important coiled-coil domain. In order to identify the role of VP3 in the Nora virus life cycle, we constructed VP3-mutants using the cDNA clone of the virus. Our results show that VP3 does not have a role in the actual assembly of the virus particles, but virions that lack VP3 or harbor VP3 with a disrupted coiled coil domain are incapable of transmission via the fecal-oral route. Removing the region downstream of the putative coiled coil appears to have an effect on the fitness of the virus but does not hamper its replication or transmission. We also found that the VP3 protein and particularly the coiled coil domain are crucial for the stability of Nora virus virions when exposed to heat or proteases. Hence, we propose that VP3 is imperative to Nora virus virions as it confers stability to the viral capsid. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Post-exposure treatments for Ebola and Marburg virus infections.

    PubMed

    Cross, Robert W; Mire, Chad E; Feldmann, Heinz; Geisbert, Thomas W

    2018-06-01

    The filoviruses - Ebola virus and Marburg virus - cause lethal haemorrhagic fever in humans and non-human primates (NHPs). Filoviruses present a global health threat both as naturally acquired diseases and as potential agents of bioterrorism. In the recent 2013-2016 outbreak of Ebola virus, the most promising therapies for post-exposure use with demonstrated efficacy in the gold-standard NHP models of filovirus disease were unable to show statistically significant protection in patients infected with Ebola virus. This Review briefly discusses these failures and what has been learned from these experiences, and summarizes the current status of post-exposure medical countermeasures in development, including antibodies, small interfering RNA and small molecules. We outline how our current knowledge could be applied to the identification of novel interventions and ways to use interventions more effectively.

  3. Structural Basis for Marburg Virus Neutralization by a Cross-Reactive Human Antibody

    DOE PAGES

    Hashiguchi, Takao; Fusco, Marnie L.; Bornholdt, Zachary A.; ...

    2015-02-26

    The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of theirmore » NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. We find that these structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.« less

  4. Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

    PubMed

    Hashiguchi, Takao; Fusco, Marnie L; Bornholdt, Zachary A; Lee, Jeffrey E; Flyak, Andrew I; Matsuoka, Rei; Kohda, Daisuke; Yanagi, Yusuke; Hammel, Michal; Crowe, James E; Saphire, Erica Ollmann

    2015-02-26

    The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle.

    PubMed

    Biedenkopf, Nadine; Lier, Clemens; Becker, Stephan

    2016-05-15

    Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that

  6. Sequencing ebola and marburg viruses genomes using microarrays.

    PubMed

    Hardick, Justin; Woelfel, Roman; Gardner, Warren; Ibrahim, Sofi

    2016-08-01

    Periodic outbreaks of Ebola and Marburg hemorrhagic fevers have occurred in Africa over the past four decades with case fatality rates reaching as high as 90%. The latest Ebola outbreak in West Africa in 2014 raised concerns that these infections can spread across continents and pose serious health risks. Early and accurate identification of the causative agents is necessary to contain outbreaks. In this report, we describe sequencing-by-hybridization (SBH) technique using high density microarrays to identify Ebola and Marburg viruses. The microarrays were designed to interrogate the sequences of entire viral genomes, and were evaluated with three species of Ebolavirus (Reston, Sudan, and Zaire), and three strains of Marburgvirus (Angola, Musoke, and Ravn). The results showed that the consensus sequences generated with four or more hybridizations had 92.1-98.9% accuracy over 95-99% of the genomes. Additionally, with SBH microarrays it was possible to distinguish between different strains of the Lake Victoria Marburgvirus. J. Med. Virol. 88:1303-1308, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Interaction of monomeric Ebola VP40 protein with a plasma membrane: A coarse-grained molecular dynamics (CGMD) simulation study.

    PubMed

    Mohamad Yusoff, Mohamad Ariff; Abdul Hamid, Azzmer Azzar; Mohammad Bunori, Noraslinda; Abd Halim, Khairul Bariyyah

    2018-06-01

    Ebola virus is a lipid-enveloped filamentous virus that affects human and non-human primates and consists of several types of protein: nucleoprotein, VP30, VP35, L protein, VP40, VP24, and transmembrane glycoprotein. Among the Ebola virus proteins, its matrix protein VP40 is abundantly expressed during infection and plays a number of critical roles in oligomerization, budding and egress from the host cell. VP40 exists predominantly as a monomer at the inner leaflet of the plasma membrane, and has been suggested to interact with negatively charged lipids such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylserine (PS) via its cationic patch. The hydrophobic loop at the C-terminal domain has also been shown to be important in the interaction between the VP40 and the membrane. However, details of the molecular mechanisms underpinning their interactions are not fully understood. This study aimed at investigating the effects of mutation in the cationic patch and hydrophobic loop on the interaction between the VP40 monomer and the plasma membrane using coarse-grained molecular dynamics simulation (CGMD). Our simulations revealed that the interaction between VP40 and the plasma membrane is mediated by the cationic patch residues. This led to the clustering of PIP 2 around the protein in the inner leaflet as a result of interactions between some cationic residues including R52, K127, K221, K224, K225, K256, K270, K274, K275 and K279 and PIP 2 lipids via electrostatic interactions. Mutation of the cationic patch or hydrophobic loop amino acids caused the protein to bind at the inner leaflet of the plasma membrane in a different orientation, where no significant clustering of PIP 2 was observed around the mutated protein. This study provides basic understanding of the interaction of the VP40 monomer and its mutants with the plasma membrane. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. [Marburg and Ebola hemorrhagic fevers--pathogens, epidemiology and therapy].

    PubMed

    Stock, Ingo

    2014-09-01

    Marburg and Ebola hemorrhagic fevers are severe, systemic viral diseases affecting humans and non-human primates. They are characterized by multiple symptoms such as hemorrhages, fever, headache, muscle and abdominal pain, chills, sore throat, nausea, vomiting and diarrhea. Elevated liver-associated enzyme levels and coagulopathy are also associated with these diseases. Marburg and Ebola hemorrhagic fevers are caused by (Lake victoria) Marburg virus and different species of Ebola viruses, respectively. They are enveloped, single-stranded RNA viruses and belong to the family of filoviridae. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, ranging from 25 to 90% or more. Outbreaks of Marburg and Ebola hemorrhagic fever occur in certain regions of equatorial Africa at irregular intervals. Since 2000, the number of outbreaks has increased. In 2014, the biggest outbreak of a filovirus-induced hemorrhagic fever that has been documented so far occurred from March to July 2014 in Guinea, Sierra Leone, Liberia and Nigeria. The outbreak was caused by a new variant of Zaire Ebola-Virus, affected more than 2600 people (stated 20 August) and was associated with case-fatality rates of up to 67% (Guinea). Treatment of Marburg and Ebola hemorrhagic fevers is symptomatic and supportive, licensed antiviral agents are currently not available. Recently, BCX4430, a promising synthetic adenosine analogue with high in vitro and in vivo activity against filoviruses and other RNA viruses, has been described. BCX4430 inhibits viral RNA polymerase activity and protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. Nucleic acid-based products, recombinant vaccines and antibodies appear to be less suitable for the treatment of Marburg and Ebola hemorrhagic fevers.

  9. A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

    PubMed

    Watt, Ari; Moukambi, Felicien; Banadyga, Logan; Groseth, Allison; Callison, Julie; Herwig, Astrid; Ebihara, Hideki; Feldmann, Heinz; Hoenen, Thomas

    2014-09-01

    Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study

  10. Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro

    PubMed Central

    van de Water, Sandra G. P.; Potgieter, Christiaan A.; van Rijn, Piet A.

    2016-01-01

    ABSTRACT The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro. In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro. Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has

  11. Ebola virus VP35 blocks stress granule assembly.

    PubMed

    Le Sage, Valerie; Cinti, Alessandro; McCarthy, Stephen; Amorim, Raquel; Rao, Shringar; Daino, Gian Luca; Tramontano, Enzo; Branch, Donald R; Mouland, Andrew J

    2017-02-01

    Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Multidistrict Outbreak of Marburg Virus Disease-Uganda, 2012.

    PubMed

    Knust, Barbara; Schafer, Ilana J; Wamala, Joseph; Nyakarahuka, Luke; Okot, Charles; Shoemaker, Trevor; Dodd, Kimberly; Gibbons, Aridth; Balinandi, Stephen; Tumusiime, Alex; Campbell, Shelley; Newman, Edmund; Lasry, Estrella; DeClerck, Hilde; Boum, Yap; Makumbi, Issa; Bosa, Henry Kyobe; Mbonye, Anthony; Aceng, Jane Ruth; Nichol, Stuart T; Ströher, Ute; Rollin, Pierre E

    2015-10-01

    In October 2012, a cluster of illnesses and deaths was reported in Uganda and was confirmed to be an outbreak of Marburg virus disease (MVD). Patients meeting the case criteria were interviewed using a standard investigation form, and blood specimens were tested for evidence of acute or recent Marburg virus infection by reverse transcription-polymerase chain reaction (RT-PCR) and antibody enzyme-linked immunosorbent assay. The total count of confirmed and probable MVD cases was 26, of which 15 (58%) were fatal. Four of 15 laboratory-confirmed cases (27%) were fatal. Case patients were located in 4 different districts in Uganda, although all chains of transmission originated in Ibanda District, and the earliest case detected had an onset in July 2012. No zoonotic exposures were identified. Symptoms significantly associated with being a MVD case included hiccups, anorexia, fatigue, vomiting, sore throat, and difficulty swallowing. Contact with a case patient and attending a funeral were also significantly associated with being a case. Average RT-PCR cycle threshold values for fatal cases during the acute phase of illness were significantly lower than those for nonfatal cases. Following the institution of contact tracing, active case surveillance, care of patients with isolation precautions, community mobilization, and rapid diagnostic testing, the outbreak was successfully contained 14 days after its initial detection. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  13. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Lassa and Marburg viruses elicit distinct host transcriptional responses early after infection.

    PubMed

    Caballero, Ignacio S; Yen, Judy Y; Hensley, Lisa E; Honko, Anna N; Goff, Arthur J; Connor, John H

    2014-11-06

    Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosis is difficult because patients infected with either pathogen present similar nonspecific symptoms early after infection. Current diagnostic tests are based on detecting viral proteins or nucleic acids in the blood, but these cannot be found during the early stages of disease, before the virus starts replicating in the blood. Using the transcriptional response of the host during infection can lead to earlier diagnoses compared to those of traditional methods. In this study, we use RNA sequencing to obtain a high-resolution view of the in vivo transcriptional dynamics of peripheral blood mononuclear cells (PBMCs) throughout both types of infection. We report a subset of host mRNAs, including heat-shock proteins like HSPA1B, immunoglobulins like IGJ, and cell adhesion molecules like SIGLEC1, whose differences in expression are strong enough to distinguish Lassa infection from Marburg infection in non-human primates. We have validated these infection-specific expression differences by using microarrays on a larger set of samples, and by quantifying the expression of individual genes using RT-PCR. These results suggest that host transcriptional signatures are correlated with specific viral infections, and that they can be used to identify highly pathogenic viruses during the early stages of disease, before standard detection methods become effective.

  15. Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with Ebola and Marburg viruses.

    PubMed

    Geisbert, Thomas W; Daddario-Dicaprio, Kathleen M; Geisbert, Joan B; Reed, Douglas S; Feldmann, Friederike; Grolla, Allen; Ströher, Ute; Fritz, Elizabeth A; Hensley, Lisa E; Jones, Steven M; Feldmann, Heinz

    2008-12-09

    Considerable progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against Ebola and Marburg viruses. A vaccine based on recombinant vesicular stomatitis virus (VSV) seems to be particularly robust as it can also confer protection when administered as a postexposure treatment. While filoviruses are not thought to be transmitted by aerosol in nature the inhalation route is among the most likely portals of entry in the setting of a bioterrorist event. At present, all candidate filoviral vaccines have been evaluated against parenteral challenges but none have been tested against an aerosol exposure. Here, we evaluated our recombinant VSV-based Zaire ebolavirus (ZEBOV) and Marburg virus (MARV) vaccines against aerosol challenge in cynomolgus macaques. All monkeys vaccinated with a VSV vector expressing the glycoprotein of ZEBOV were completely protected against an aerosol exposure of ZEBOV. Likewise, all monkeys vaccinated with a VSV vector expressing the glycoprotein of MARV were completely protected against an aerosol exposure of MARV. All control animals challenged by the aerosol route with either ZEBOV or MARV succumbed. Interestingly, disease in control animals appeared to progress slower than previously seen in macaques exposed to comparable doses by intramuscular injection.

  16. [Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

    PubMed

    Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

    2009-09-01

    To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

  17. How severe and prevalent are Ebola and Marburg viruses? A systematic review and meta-analysis of the case fatality rates and seroprevalence.

    PubMed

    Nyakarahuka, Luke; Kankya, Clovice; Krontveit, Randi; Mayer, Benjamin; Mwiine, Frank N; Lutwama, Julius; Skjerve, Eystein

    2016-11-25

    Ebola and Marburg virus diseases are said to occur at a low prevalence, but are very severe diseases with high lethalities. The fatality rates reported in different outbreaks ranged from 24-100%. In addition, sero-surveys conducted have shown different seropositivity for both Ebola and Marburg viruses. We aimed to use a meta-analysis approach to estimate the case fatality and seroprevalence rates of these filoviruses, providing vital information for epidemic response and preparedness in countries affected by these diseases. Published literature was retrieved through a search of databases. Articles were included if they reported number of deaths, cases, and seropositivity. We further cross-referenced with ministries of health, WHO and CDC databases. The effect size was proportion represented by case fatality rate (CFR) and seroprevalence. Analysis was done using the metaprop command in STATA. The weighted average CFR of Ebola virus disease was estimated to be 65.0% [95% CI (54.0-76.0%), I 2  = 97.98%] whereas that of Marburg virus disease was 53.8% (26.5-80.0%, I 2  = 88.6%). The overall seroprevalence of Ebola virus was 8.0% (5.0%-11.0%, I 2  = 98.7%), whereas that for Marburg virus was 1.2% (0.5-2.0%, I 2  = 94.8%). The most severe species of ebolavirus was Zaire ebolavirus while Bundibugyo Ebolavirus was the least severe. The pooled CFR and seroprevalence for Ebola and Marburg viruses were found to be lower than usually reported, with species differences despite high heterogeneity between studies. Countries with an improved health surveillance and epidemic response have lower CFR, thereby indicating need for improving early detection and epidemic response in filovirus outbreaks.

  18. Ebola Virus Glycoprotein Promotes Enhanced Viral Egress by Preventing Ebola VP40 From Associating With the Host Restriction Factor BST2/Tetherin.

    PubMed

    Gustin, Jean K; Bai, Ying; Moses, Ashlee V; Douglas, Janet L

    2015-10-01

    BST2/tetherin is an innate immune molecule with the unique ability to restrict the egress of human immunodeficiency virus (HIV) and other enveloped viruses, including Ebola virus (EBOV). Coincident with this discovery was the finding that the HIV Vpu protein down-regulates BST2 from the cell surface, thereby promoting viral release. Evidence suggests that the EBOV envelope glycoprotein (GP) also counteracts BST2, although the mechanism is unclear. We find that total levels of BST2 remain unchanged in the presence of GP, whereas surface BST2 is significantly reduced. GP is known to sterically mask surface receptors via its mucin domain. Our evaluation of mutant GP molecules indicate that masking of BST2 by GP is probably responsible for the apparent surface BST2 down-regulation; however, this masking does not explain the observed virus-like particle egress enhancement. We discovered that VP40 coimmunoprecipitates and colocalizes with BST2 in the absence but not in the presence of GP. These results suggest that GP may overcome the BST2 restriction by blocking an interaction between VP40 and BST2. Furthermore, we have observed that GP may enhance BST2 incorporation into virus-like particles. Understanding this novel EBOV immune evasion strategy will provide valuable insights into the pathogenicity of this deadly pathogen. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. How Ebola and Marburg viruses battle the immune system.

    PubMed

    Mohamadzadeh, Mansour; Chen, Lieping; Schmaljohn, Alan L

    2007-07-01

    The filoviruses Ebola and Marburg have emerged in the past decade from relative obscurity to serve now as archetypes for some of the more intriguing and daunting challenges posed by such agents. Public imagination is captured by deadly outbreaks of these viruses and reinforced by the specter of bioterrorism. As research on these agents has accelerated, it has been found increasingly that filoviruses use a combination of familiar and apparently new ways to baffle and battle the immune system. Filoviruses have provided thereby a new lens through which to examine the immune system itself.

  20. RNA binding specificity of Ebola virus transcription factor VP30.

    PubMed

    Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

    2016-09-01

    The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

  1. Ebola and marburg hemorrhagic fever.

    PubMed

    Hartman, Amy L; Towner, Jonathan S; Nichol, Stuart T

    2010-03-01

    Ebola and Marburg viruses cause a severe viral hemorrhagic fever disease mainly in Sub-Saharan Africa. Although outbreaks are sporadic, there is the potential for filoviruses to spread to other continents unintentionally because of air travel or intentionally because of bioterrorism. This article discusses the natural history, epidemiology, and clinical presentation of patients infected with Ebola and Marburg viruses. Clinicians in the United States should be aware of the symptoms of these viral infections in humans and know the appropriate procedures for contacting local, state, and national reference laboratories in the event of a suspected case of filoviral hemorrhagic fever. 2010 Elsevier Inc. All rights reserved.

  2. How Ebola and Marburg Viruses Battle the Immune System

    DTIC Science & Technology

    2007-07-01

    macrophages, neutrophils) Asialoglycoprotein receptor ( hepatocytes ) TLR Other? NP VP35 VP40 GP VP30 VP24 L 3′ 5′ Cell-surface GP Filovirus Matrix... hepatocytes are particularly susceptible, elevated liver enzymes are among the first telling signs of disease and liver damage seems to account for much...of monocytic origin (such as immature DCs) also promotes filoviral entry10. Another C-type lectin, the asialoglycoprotein *US Army Medical Research

  3. Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

    PubMed

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

    2015-10-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Immunogenicity of Newcastle Disease Virus Vectors Expressing Norwalk Virus Capsid Protein in the Presence or Absence of VP2 Protein

    PubMed Central

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y.; Samal, Siba K.

    2015-01-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirs-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. PMID:26099695

  5. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

  6. Ebola Virus and Marburg Virus in Human Milk Are Inactivated by Holder Pasteurization.

    PubMed

    Hamilton Spence, Erin; Huff, Monica; Shattuck, Karen; Vickers, Amy; Yun, Nadezda; Paessler, Slobodan

    2017-05-01

    Potential donors of human milk are screened for Ebola virus (EBOV) using standard questions, but testing for EBOV and Marburg virus (MARV) is not part of routine serological testing performed by milk banks. Research aim: This study tested the hypothesis that EBOV would be inactivated in donor human milk (DHM) by standard pasteurization techniques (Holder) used in all North American nonprofit milk banks. Milk samples were obtained from a nonprofit milk bank. They were inoculated with EBOV (Zaire strain) and MARV (Angola strain) and processed by standard Holder pasteurization technique. Plaque assays for EBOV and MARV were performed to detect the presence of virus after pasteurization. Neither EBOV nor MARV was detectable by viral plaque assay in DHM or culture media samples, which were pasteurized by the Holder process. EBOV and MARV are safely inactivated in human milk by standard Holder pasteurization technique. Screening for EBOV or MARV beyond questionnaire and self-deferral is not needed to ensure safety of DHM for high-risk infants.

  7. Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells.

    PubMed

    Dolnik, Olga; Kolesnikova, Larissa; Welsch, Sonja; Strecker, Thomas; Schudt, Gordian; Becker, Stephan

    2014-10-01

    Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV(PSAPmut)). rMARV(PSAPmut) was attenuated by up to one log compared with recombinant wild-type MARV (rMARV(wt)), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV(PSAPmut)-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV(wt)-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV(wt)-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the

  8. Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors.

    PubMed

    Anantpadma, Manu; Kouznetsova, Jennifer; Wang, Hang; Huang, Ruili; Kolokoltsov, Andrey; Guha, Rajarshi; Lindstrom, Aaron R; Shtanko, Olena; Simeonov, Anton; Maloney, David J; Maury, Wendy; LaCount, Douglas J; Jadhav, Ajit; Davey, Robert A

    2016-08-01

    Filoviruses are highly infectious, and no FDA-approved drug therapy for filovirus infection is available. Most work to find a treatment has involved only a few strains of Ebola virus and testing of relatively small drug libraries or compounds that have shown efficacy against other virus types. Here we report the findings of a high-throughput screening of 319,855 small molecules from the Molecular Libraries Small Molecule Repository library for their activities against Marburg virus and Ebola virus. Nine of the most potent, novel compounds that blocked infection by both viruses were analyzed in detail for their mechanisms of action. The compounds inhibited known key steps in the Ebola virus infection mechanism by blocking either cell surface attachment, macropinocytosis-mediated uptake, or endosomal trafficking. To date, very few specific inhibitors of macropinocytosis have been reported. The 2 novel macropinocytosis inhibitors are more potent inhibitors of Ebola virus infection and less toxic than ethylisopropylamiloride, one commonly accepted macropinocytosis inhibitor. Each compound blocked infection of primary human macrophages, indicating their potential to be developed as new antifiloviral therapies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Expression of the VP40 antigen from the Zaire ebolavirus in tobacco plants.

    PubMed

    Monreal-Escalante, Elizabeth; Ramos-Vega, Abel A; Salazar-González, Jorge A; Bañuelos-Hernández, Bernardo; Angulo, Carlos; Rosales-Mendoza, Sergio

    2017-07-01

    The plant cell is able to produce the VP40 antigen from the Zaire ebolavirus , retaining the antigenicity and the ability to induce immune responses in BALB/c mice. The recent Ebola outbreak evidenced the need for having vaccines approved for human use. Herein we report the expression of the VP40 antigen from the Ebola virus as an initial effort in the development of a plant-made vaccine that could offer the advantages of being cheap and scalable, which is proposed to overcome the rapid need for having vaccines to deal with future outbreaks. Tobacco plants were transformed by stable DNA integration into the nuclear genome using the CaMV35S promoter and a signal peptide to access the endoplasmic reticulum, reaching accumulation levels up to 2.6 µg g -1 FW leaf tissues. The antigenicity of the plant-made VP40 antigen was evidenced by Western blot and an initial immunogenicity assessment in test animals that revealed the induction of immune responses in BALB/c mice following three weekly oral or subcutaneous immunizations at very low doses (125 and 25 ng, respectively) without accessory adjuvants. Therefore, this plant-based vaccination prototype is proposed as an attractive platform for the production of vaccines in the fight against Ebola virus disease outbreaks.

  10. Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells.

    PubMed

    Hölzer, Martin; Krähling, Verena; Amman, Fabian; Barth, Emanuel; Bernhart, Stephan H; Carmelo, Victor A O; Collatz, Maximilian; Doose, Gero; Eggenhofer, Florian; Ewald, Jan; Fallmann, Jörg; Feldhahn, Lasse M; Fricke, Markus; Gebauer, Juliane; Gruber, Andreas J; Hufsky, Franziska; Indrischek, Henrike; Kanton, Sabina; Linde, Jörg; Mostajo, Nelly; Ochsenreiter, Roman; Riege, Konstantin; Rivarola-Duarte, Lorena; Sahyoun, Abdullah H; Saunders, Sita J; Seemann, Stefan E; Tanzer, Andrea; Vogel, Bertram; Wehner, Stefanie; Wolfinger, Michael T; Backofen, Rolf; Gorodkin, Jan; Grosse, Ivo; Hofacker, Ivo; Hoffmann, Steve; Kaleta, Christoph; Stadler, Peter F; Becker, Stephan; Marz, Manja

    2016-10-07

    The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections.

  11. Localization of the herpes simplex virus type 1 major capsid protein VP5 to the cell nucleus requires the abundant scaffolding protein VP22a.

    PubMed

    Nicholson, P; Addison, C; Cross, A M; Kennard, J; Preston, V G; Rixon, F J

    1994-05-01

    The intracellular distributions of three herpes simplex virus type 1 (HSV-1) capsid proteins, VP23, VP5 and VP22a, were examined using vaccinia virus and plasmid expression systems. During infection of cells with HSV-1 wild-type virus, all three proteins were predominantly located in the nucleus, which is the site of capsid assembly. However, when expressed in the absence of any other HSV-1 proteins, although VP22a was found exclusively in the nucleus as expected, VP5 and VP23 were distributed throughout the cell. Thus nuclear localization is not an intrinsic property of these proteins but must be mediated by one or more HSV-1-induced proteins. Co-expression experiments demonstrated that VP5 was efficiently transported to the nucleus in the presence of VP22a, but the distribution of VP23 was unaffected by the presence of either or both of the other two proteins.

  12. The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

    PubMed

    Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

    2017-09-15

    Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35

  13. The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

    PubMed Central

    Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E.; Miorin, Lisa; Johnson, Jeffrey R.; Krogan, Nevan J.; Basler, Christopher F.; Freiberg, Alexander N.

    2017-01-01

    ABSTRACT Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP

  14. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

    PubMed

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-06-16

    Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  15. Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus

    PubMed Central

    Graff, Judith; Richards, Oliver C.; Swiderek, Kristine M.; Davis, Michael T.; Rusnak, Felicia; Harmon, Shirley A.; Jia, Xi-Yu; Summers, Donald F.; Ehrenfeld, Ellie

    1999-01-01

    Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1. PMID:10364353

  16. Recombinant infectious hematopoietic necrosis virus expressing infectious pancreatic necrosis virus VP2 protein induces immunity against both pathogens.

    PubMed

    Guo, Mengting; Shi, Wen; Wang, Yanxue; Wang, Yuting; Chen, Yaping; Li, Dechuan; Ren, Xuanyu; Hua, Xiaojing; Tang, Lijie; Li, Yijing; Liu, Min

    2018-04-21

    Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Although vaccines against IHNV and IPNV have been commercialized in many countries, the prevalence of IHNV and IPNV is still widespread in modern aquaculture. In the present study, two IHNV recombinant viruses displaying IPNV VP2 protein (rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE) were generated using the RNA polymerase Ⅱ system to explore the immunogenicity of IHNV and IPNV. The recombinant IHNV viruses were stable, which was confirmed by sequencing, indirect immunofluorescence assay, western blotting, transmission electron microscopy and viral growth curve assay. IHNV and IPNV challenge showed that the recombinant viruses had high protection rates against IHNV and IPNV with approximately 65% relative percent survival rates. Rainbow trout (mean weight 20 g) vaccinated with these two recombinant viruses showed a high level of antibodies against IHNV and IPNV infection. Taken together, our findings demonstrate that rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE might be promising vaccine candidates against IHNV and IPNV. Copyright © 2018. Published by Elsevier Ltd.

  17. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    PubMed Central

    2011-01-01

    Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

  18. Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens

    PubMed Central

    Zanetti, Flavia Adriana; Grand, María Daniela Conte; Mitarotonda, Romina Cristina; Taboga, Oscar Alberto; Calamante, Gabriela

    2014-01-01

    Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens. PMID:24948937

  19. Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.

    PubMed

    Martínez-Torrecuadrada, J L; Langeveld, J P; Venteo, A; Sanz, A; Dalsgaard, K; Hamilton, W D; Meloen, R H; Casal, J I

    1999-05-10

    African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes. Copyright 1999 Academic Press.

  20. VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

    PubMed

    Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

    1997-07-01

    The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

  1. [Immune Response of Recombinant Pseudorabies Virus rPRV-VP2 Expressing VP2 Gene of Porcine Parvovirus in Mice].

    PubMed

    Fu, Pengfei; Pan, Xinlong; Han, Qiao; Yang, Xingwu; Zhu, Qianlei; Guo, Xiaoqing; Zhang, Yu; Chen, Hongying

    2016-03-01

    In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.

  2. Lessons learned during active epidemiological surveillance of Ebola and Marburg viral hemorrhagic fever epidemics in Africa.

    PubMed

    Allaranga, Yokouide; Kone, Mamadou Lamine; Formenty, Pierre; Libama, Francois; Boumandouki, Paul; Woodfill, Celia J I; Sow, Idrissa; Duale, Sambe; Alemu, Wondimagegnehu; Yada, Adamou

    2010-03-01

    To review epidemiological surveillance approaches used during Ebola and Marburg hemorrhagic fever epidemics in Africa in the past fifteen years. Overall, 26 hemorrhagic epidemic outbreaks have been registered in 12 countries; 18 caused by the Ebola virus and eight by the Marburg virus. About 2551 cases have been reported, among which 268 were health workers (9,3%). Based on articles and epidemic management reports, this review analyses surveillance approaches, route of introduction of the virus into the population (urban and rural), the collaboration between the human health sector and the wildlife sector and factors that have affected epidemic management. Several factors affecting the epidemiological surveillance during Ebola and Marburg viruses hemorrhagic epidemics have been observed. During epidemics in rural settings, outbreak investigations have shown multiple introductions of the virus into the human population through wildlife. In contrast, during epidemics in urban settings a single introduction of the virus in the community was responsible for the epidemic. Active surveillance is key to containing outbreaks of Ebola and Marburg viruses Collaboration with those in charge of the conservation of wildlife is essential for the early detection of viral hemorrhagic fever epidemics. Hemorrhagic fever epidemics caused by Ebola and Marburg viruses are occurring more and more frequently in Sub-Saharan Africa and only an adapted epidemiological surveillance system will allow for early detection and effective response.

  3. The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host.

    PubMed

    Calvo-Pinilla, Eva; Gubbins, Simon; Mertens, Peter; Ortego, Javier; Castillo-Olivares, Javier

    2018-06-01

    African horse sickness (AHS) is a lethal equine disease transmitted by Culicoides biting midges and caused by African horse sickness virus (AHSV). AHS is endemic to sub-Saharan Africa, but devastating outbreaks have been recorded periodically outside this region. The perceived risk of an AHS outbreak occurring in Europe has increased following the frequent epidemics caused in ruminants by bluetongue virus, closely related to AHSV. Attenuated vaccines for AHS are considered unsuitable for use in non-endemic countries due bio-safety concerns. Further, attenuated and inactivated vaccines are not compatible with DIVA (differentiate infected from vaccinated animals) strategies. All these factors stimulated the development of novel AHS vaccines that are safer, more efficacious and DIVA compatible. We showed previously that recombinant modified Vaccinia Ankara virus (MVA) vaccines encoding the outer capsid protein of AHSV (AHSV-VP2) induced virus neutralising antibodies (VNAb) and protection against AHSV in a mouse model and also in the horse. Passive immunisation studies demonstrated that immunity induced by MVA-VP2 was associated with pre-challenge VNAb titres in the vaccinates. Analyses of the inoculum of these MVA-VP2 experimental vaccines showed that they contained pre-formed AHSV-VP2. We continued studying the influence of pre-formed AHSV-VP2, present in the inoculum of MVA-VP2 vaccines, in the immunogenicity of MVA-VP2 vaccines. Thus, we compared correlates of immunity in challenged mice that were previously vaccinated with: a) MVA-VP2 (live); b) MVA-VP2 (live and sucrose gradient purified); c) MVA-VP2 (UV light inactivated); d) MVA-VP2 (UV light inactivated and diluted); e) MVA-VP2 (heat inactivated); f) MVA-VP2 (UV inactivated) + MVA-VP2 (purified); g) MVA-VP2 (heat inactivated) + MVA-VP2 (purified); and h) wild type-MVA (no insert). The results of these experiments showed that protection was maximal using MVA-VP2 (live) vaccine and that the protection

  4. Crystal Structures of Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus Shed Light on Their Evolutionary Relationship

    SciTech Connect

    Tang,X.; Wu, J.; Sivaraman, J.

    2007-01-01

    White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 {angstrom}, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelopemore » proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt {beta}-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core {beta}-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.« less

  5. Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

    PubMed

    Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

    2015-10-01

    Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious

  6. Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists

    PubMed Central

    Cheng, Han; Lear-Rooney, Calli M.; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W.; Olinger, Gene G.

    2015-01-01

    ABSTRACT Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of

  7. Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

    SciTech Connect

    Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.

    2010-10-11

    Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that lossmore » of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.« less

  8. Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

    PubMed

    Daaboul, George G; Lopez, Carlos A; Chinnala, Jyothsna; Goldberg, Bennett B; Connor, John H; Ünlü, M Selim

    2014-06-24

    Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

  9. One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses.

    PubMed

    Park, Sun-Whan; Lee, Ye-Ji; Lee, Won-Ja; Jee, Youngmee; Choi, WooYoung

    2016-06-01

    Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection. We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-transcribed RNA. We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses. The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers.

  10. A free VP3 C-terminus is essential for the replication of infectious bursal disease virus.

    PubMed

    Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

    2017-03-15

    Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Crystal Structures of Yellowtail Ascites Virus VP4 Protease

    PubMed Central

    Chung, Ivy Yeuk Wah; Paetzel, Mark

    2013-01-01

    Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala716) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser633 as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water. PMID:23511637

  12. Durability of a vesicular stomatitis virus-based marburg virus vaccine in nonhuman primates.

    PubMed

    Mire, Chad E; Geisbert, Joan B; Agans, Krystle N; Satterfield, Benjamin A; Versteeg, Krista M; Fritz, Elizabeth A; Feldmann, Heinz; Hensley, Lisa E; Geisbert, Thomas W

    2014-01-01

    The filoviruses, Marburg virus (MARV) and Ebola virus, causes severe hemorrhagic fever with high mortality in humans and nonhuman primates. A promising filovirus vaccine under development is based on a recombinant vesicular stomatitis virus (rVSV) that expresses individual filovirus glycoproteins (GPs) in place of the VSV glycoprotein (G). These vaccines have shown 100% efficacy against filovirus infection in nonhuman primates when challenge occurs 28-35 days after a single injection immunization. Here, we examined the ability of a rVSV MARV-GP vaccine to provide protection when challenge occurs more than a year after vaccination. Cynomolgus macaques were immunized with rVSV-MARV-GP and challenged with MARV approximately 14 months after vaccination. Immunization resulted in the vaccine cohort of six animals having anti-MARV GP IgG throughout the pre-challenge period. Following MARV challenge none of the vaccinated animals showed any signs of clinical disease or viremia and all were completely protected from MARV infection. Two unvaccinated control animals exhibited signs consistent with MARV infection and both succumbed. Importantly, these data are the first to show 100% protective efficacy against any high dose filovirus challenge beyond 8 weeks after final vaccination. These findings demonstrate the durability of VSV-based filovirus vaccines.

  13. A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus.

    PubMed

    Cheng, Han; Koning, Katie; O'Hearn, Aileen; Wang, Minxiu; Rumschlag-Booms, Emily; Varhegyi, Elizabeth; Rong, Lijun

    2015-11-24

    Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study. This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.

  14. Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus.

    PubMed

    He, Lei; Hu, Xiaolong; Zhu, Min; Liang, Zi; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2017-09-05

    The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1-S10). The segment 7 (S7) encodes 50kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. VP24 Is a Chitin-Binding Protein Involved in White Spot Syndrome Virus Infection

    PubMed Central

    Li, Zaipeng; Han, Yali; Xu, Limei

    2015-01-01

    ABSTRACT Oral ingestion is the major route of infection for the white spot syndrome virus (WSSV). However, the mechanism by which virus particles in the digestive tract invade host cells is unknown. In the present study, we demonstrate that WSSV virions can bind to chitin through one of the major envelope proteins (VP24). Mutagenesis analysis indicated that amino acids (aa) 186 to 200 in the C terminus of VP24 were required for chitin binding. Moreover, the P-VP24186–200 peptide derived from the VP24 chitin binding region significantly inhibited the VP24-chitin interaction and the WSSV-chitin interaction, implying that VP24 participates in WSSV binding to chitin. Oral inoculation experiments showed that P-VP24186–200 treatment reduced the number of virus particles remaining in the digestive tract during the early stage of infection and greatly hindered WSSV proliferation in shrimp. These data indicate that binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection and provide new ideas for preventing WSSV infection in shrimp farms. IMPORTANCE In this study, we show that WSSV can bind to chitin through the envelope protein VP24. The chitin-binding domain of VP24 maps to amino acids 186 to 200 in the C terminus. Binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection. These findings not only extend our knowledge of WSSV infection but also provide new insights into strategies to prevent WSSV infection in shrimp farms. PMID:26512091

  16. Virtual screening of the inhibitors targeting at the viral protein 40 of Ebola virus.

    PubMed

    Karthick, V; Nagasundaram, N; Doss, C George Priya; Chakraborty, Chiranjib; Siva, R; Lu, Aiping; Zhang, Ge; Zhu, Hailong

    2016-02-17

    The Ebola virus is highly pathogenic and destructive to humans and other primates. The Ebola virus encodes viral protein 40 (VP40), which is highly expressed and regulates the assembly and release of viral particles in the host cell. Because VP40 plays a prominent role in the life cycle of the Ebola virus, it is considered as a key target for antiviral treatment. However, there is currently no FDA-approved drug for treating Ebola virus infection, resulting in an urgent need to develop effective antiviral inhibitors that display good safety profiles in a short duration. This study aimed to screen the effective lead candidate against Ebola infection. First, the lead molecules were filtered based on the docking score. Second, Lipinski rule of five and the other drug likeliness properties are predicted to assess the safety profile of the lead candidates. Finally, molecular dynamics simulations was performed to validate the lead compound. Our results revealed that emodin-8-beta-D-glucoside from the Traditional Chinese Medicine Database (TCMD) represents an active lead candidate that targets the Ebola virus by inhibiting the activity of VP40, and displays good pharmacokinetic properties. This report will considerably assist in the development of the competitive and robust antiviral agents against Ebola infection.

  17. Physicochemical inactivation of Lassa, Ebola, and Marburg viruses and effect on clinical laboratory analyses

    SciTech Connect

    Mitchell, S.W.; McCormick, J.B.

    1984-09-01

    Clinical specimens from patients infected with Lassa, Ebola, or Marburg virus may present a serious biohazard to laboratory workers. The authors have examined the effects of heat, alteration of pH, and gamma radiation on these viruses in human blood and on the electrolytes, enzymes, and coagulation factors measured in laboratory tests that are important in the care of an infected patient. Heating serum at 60 degrees C for 1 h reduced high titers of these viruses to noninfectious levels without altering the serum levels of glucose, blood urea nitrogen, and electrolytes. Dilution of blood in 3% acetic acid, diluent formore » a leukocyte count, inactivated all of these viruses. All of the methods tested for viral inactivation markedly altered certain serum proteins, making these methods unsuitable for samples that are to be tested for certain enzyme levels and coagulation factors.« less

  18. Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of ebola virus.

    PubMed

    Hartman, Amy L; Bird, Brian H; Towner, Jonathan S; Antoniadou, Zoi-Anna; Zaki, Sherif R; Nichol, Stuart T

    2008-03-01

    Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

  19. Prevalence and stability of human serum antibodies to simian virus 40 VP1 virus-like particles.

    PubMed

    Lundstig, Annika; Eliasson, Linda; Lehtinen, Matti; Sasnauskas, Kestutis; Koskela, Pentti; Dillner, Joakim

    2005-06-01

    Possible human infection with simian virus 40 (SV40) has been of great concern ever since SV40 was discovered in polio vaccines. Human populations are SV40-seropositive, but because of serological cross-reactivity between SV40 and the human polyomaviruses BK virus (BKV) and JC virus (JCV), it is debatable whether these antibodies are specific. An SV40-specific serological assay was established, based on purified virus-like particles (VLPs), where the SV40 VLPs were blocked with hyperimmune sera to BKV and JCV. Competition with SV40 hyperimmune sera was used as a confirmatory test. Among 288 Swedish children of between 1 and 13 years of age, 7.6 % had SV40-specific antibodies. SV40 seroprevalence reached a peak of 14 % at 7-9 years of age. Among 100 control patients with benign tumours, 9 % were SV40-seropositive. However, SV40 DNA was not detectable in corresponding buffy-coat samples. In serial samples taken up to 5 years apart from 141 Finnish women participating in the population-based serological screening for congenital infections, only two of 141 women were SV40-seropositive in both samples. Six women seroconverted and eight women had a loss of antibodies over time. None of the SV40-seropositive samples contained detectable SV40 DNA. In conclusion, there is a low prevalence of SV40-specific antibodies in the Nordic population. The SV40 antibodies appear to have a low stability over time and their origin is not clear.

  20. The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

    PubMed

    Feldmann, H; Volchkov, V E; Volchkova, V A; Klenk, H D

    1999-01-01

    Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

  1. VP08R from Infectious Spleen and Kidney Necrosis Virus Is a Novel Component of the Virus-Mock Basement Membrane

    PubMed Central

    Xu, Xiaopeng; Yan, Muting; Wang, Rui; Lin, Ting; Tang, Junliang; Li, Chaozheng; Weng, Shaoping

    2014-01-01

    ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement membrane (BM)-like structure, termed virus-mock basement membrane (VMBM), on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R is located on the plasma membrane of infected cells and shows an expression profile similar to that of VP23R. Coimmunoprecipitation showed that VP08R interacts with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in the maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions. IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV; genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein, VP23R, binds and recruits the host

  2. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

    PubMed

    Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J

    2017-03-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

  3. Genetic Diversity of Hepatitis A Virus in China: VP3-VP1-2A Genes and Evidence of Quasispecies Distribution in the Isolates

    PubMed Central

    Cao, Jingyuan; Zhou, Wenting; Yi, Yao; Jia, Zhiyuan; Bi, Shengli

    2013-01-01

    Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22x10-4 -2.33x10-3 substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed. PMID:24069343

  4. Temporal Characterization of Marburg Virus Angola Infection following Aerosol Challenge in Rhesus Macaques.

    PubMed

    Lin, Kenny L; Twenhafel, Nancy A; Connor, John H; Cashman, Kathleen A; Shamblin, Joshua D; Donnelly, Ginger C; Esham, Heather L; Wlazlowski, Carly B; Johnson, Joshua C; Honko, Anna N; Botto, Miriam A; Yen, Judy; Hensley, Lisa E; Goff, Arthur J

    2015-10-01

    Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, 15 rhesus macaques were sequentially sacrificed following aerosol exposure to the MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 postexposure (p.e.). The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 p.e. High (>10(7) PFU/ml) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 p.e. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as interleukin 10 (IL-10) and IL-5 and late-stage increases in Th1 cytokines such as IL-2, IL-15, and granulocyte-macrophage colony-stimulating factor (GM-CSF). This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model. In this study, we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following aerosol exposure. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

    SciTech Connect

    Luthra, Priya; Jordan, David S.; Leung, Daisy W.

    2015-03-04

    Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

  6. Considerations in the Use of Nonhuman Primate Models of Ebola Virus and Marburg Virus Infection.

    PubMed

    Geisbert, Thomas W; Strong, James E; Feldmann, Heinz

    2015-10-01

    The filoviruses, Ebola virus and Marburg virus, are zoonotic pathogens that cause severe hemorrhagic fever in humans and nonhuman primates (NHPs), with case-fatality rates ranging from 23% to 90%. The current outbreak of Ebola virus infection in West Africa, with >26 000 cases, demonstrates the long-underestimated public health danger that filoviruses pose as natural human pathogens. Currently, there are no vaccines or treatments licensed for human use. Licensure of any medical countermeasure may require demonstration of efficacy in the gold standard cynomolgus or rhesus macaque models of filovirus infection. Substantial progress has been made over the last decade in characterizing the filovirus NHP models. However, there is considerable debate over a variety of experimental conditions, including differences among filovirus isolates used, routes and doses of exposure, and euthanasia criteria, all of which may contribute to variability of results among different laboratories. As an example of the importance of understanding these differences, recent data with Ebola virus shows that an addition of a single uridine residue in the glycoprotein gene at the editing site attenuates the virus. Here, we draw on decades of experience working with filovirus-infected NHPs to provide a perspective on the importance of various experimental conditions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  7. A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system.

    PubMed

    Zhang, Li; Li, Qianqian; Liu, Qiang; Huang, Weijin; Nie, Jianhui; Wang, Youchun

    2017-08-03

    Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

  8. Novel activities by ebolavirus and marburgvirus interferon antagonists revealed using a standardized in vitro reporter system.

    PubMed

    Guito, Jonathan C; Albariño, César G; Chakrabarti, Ayan K; Towner, Jonathan S

    2017-01-15

    Filoviruses are highly lethal in humans and nonhuman primates, likely due to potent antagonism of host interferon (IFN) responses early in infection. Filoviral protein VP35 is implicated as the major IFN induction antagonist, while Ebola virus (EBOV) VP24 or Marburg virus (MARV) VP40 are known to block downstream IFN signaling. Despite progress elucidating EBOV and MARV antagonist function, those for most other filoviruses, including Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV), Bundibugyo (BDBV) and Ravn (RAVV) viruses, remain largely neglected. Thus, using standardized vectors and reporter assays, we characterized activities by each IFN antagonist from all known ebolavirus and marburgvirus species side-by-side. We uncover noncanonical suppression of IFN induction by ebolavirus VP24, differing potencies by MARV and RAVV proteins, and intriguingly, weaker antagonism by VP24 of RESTV. These underlying molecular explanations for differential virulence in humans could guide future investigations of more-neglected filoviruses as well as treatment and vaccine studies. Published by Elsevier Inc.

  9. Geographic potential of disease caused by Ebola and Marburg viruses in Africa.

    PubMed

    Peterson, A Townsend; Samy, Abdallah M

    2016-10-01

    Filoviruses represent a significant public health threat worldwide. West Africa recently experienced the largest-scale and most complex filovirus outbreak yet known, which underlines the need for a predictive understanding of the geographic distribution and potential for transmission to humans of these viruses. Here, we used ecological niche modeling techniques to understand the relationship between known filovirus occurrences and environmental characteristics. Our study derived a picture of the potential transmission geography of Ebola virus species and Marburg, paired with views of the spatial uncertainty associated with model-to-model variation in our predictions. We found that filovirus species have diverged ecologically, but only three species are sufficiently well known that models could be developed with significant predictive power. We quantified uncertainty in predictions, assessed potential for outbreaks outside of known transmission areas, and highlighted the Ethiopian Highlands and scattered areas across East Africa as additional potentially unrecognized transmission areas. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2▿†

    PubMed Central

    Bolstad, M.; Abaitua, F.; Crump, C. M.; O'Hare, P.

    2011-01-01

    The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances. PMID:21715485

  11. Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo.

    PubMed

    Moyen, Nanikaly; Thirion, Laurence; Emmerich, Petra; Dzia-Lepfoundzou, Amelia; Richet, Hervé; Boehmann, Yannik; Dimi, Yannick; Gallian, Pierre; Gould, Ernest A; Günther, Stephan; de Lamballerie, Xavier

    2015-01-01

    Ebola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV. In 2011, we conducted a MARV and EBOV seroprevalence study amongst 809 blood donors recruited in rural (75; 9.3%) and urban (734; 90.7%) areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds. This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors.

  12. Egyptian rousette bats maintain long-term protective immunity against Marburg virus infection despite diminished antibody levels.

    PubMed

    Schuh, Amy J; Amman, Brian R; Sealy, Tara K; Spengler, Jessica R; Nichol, Stuart T; Towner, Jonathan S

    2017-08-18

    Although bats are natural reservoir hosts for numerous zoonotic viruses, little is known about the long-term dynamics of the host immune response following infection and how these viruses are maintained in nature. The Egyptian rousette bat (ERB) is a known reservoir host for Marburg virus (MARV). Following infection of ERBs with MARV, virus-specific IgG antibodies are induced but rapidly wane and by 3 months post-infection the bats are seronegative. To determine whether reinfection of ERBs plays a role in MARV maintenance, we challenge groups of ERBs that were "naturally" or experimentally infected with MARV 17-24 months prior. No bats in either group exhibit evidence of MARV replication or shedding and all bats develop virus-specific secondary immune responses. This study demonstrates that infection of ERBs with MARV induces long-term protective immunity against reinfection and indicates that other factors, such as host population dynamics, drive MARV maintenance in nature.

  13. Animal models for Ebola and Marburg virus infections

    PubMed Central

    Nakayama, Eri; Saijo, Masayuki

    2013-01-01

    Ebola and Marburg hemorrhagic fevers (EHF and MHF) are caused by the Filoviridae family, Ebolavirus and Marburgvirus (ebolavirus and marburgvirus), respectively. These severe diseases have high mortality rates in humans. Although EHF and MHF are endemic to sub-Saharan Africa. A novel filovirus, Lloviu virus, which is genetically distinct from ebolavirus and marburgvirus, was recently discovered in Spain where filoviral hemorrhagic fever had never been reported. The virulence of this virus has not been determined. Ebolavirus and marburgvirus are classified as biosafety level-4 (BSL-4) pathogens and Category A agents, for which the US government requires preparedness in case of bioterrorism. Therefore, preventive measures against these viral hemorrhagic fevers should be prepared, not only in disease-endemic regions, but also in disease-free countries. Diagnostics, vaccines, and therapeutics need to be developed, and therefore the establishment of animal models for EHF and MHF is invaluable. Several animal models have been developed for EHF and MHF using non-human primates (NHPs) and rodents, which are crucial to understand pathophysiology and to develop diagnostics, vaccines, and therapeutics. Rhesus and cynomolgus macaques are representative models of filovirus infection as they exhibit remarkably similar symptoms to those observed in humans. However, the NHP models have practical and ethical problems that limit their experimental use. Furthermore, there are no inbred and genetically manipulated strains of NHP. Rodent models such as mouse, guinea pig, and hamster, have also been developed. However, these rodent models require adaptation of the virus to produce lethal disease and do not mirror all symptoms of human filovirus infection. This review article provides an outline of the clinical features of EHF and MHF in animals, including humans, and discusses how the animal models have been developed to study pathophysiology, vaccines, and therapeutics. PMID:24046765

  14. Animal models for Ebola and Marburg virus infections.

    PubMed

    Nakayama, Eri; Saijo, Masayuki

    2013-09-05

    Ebola and Marburg hemorrhagic fevers (EHF and MHF) are caused by the Filoviridae family, Ebolavirus and Marburgvirus (ebolavirus and marburgvirus), respectively. These severe diseases have high mortality rates in humans. Although EHF and MHF are endemic to sub-Saharan Africa. A novel filovirus, Lloviu virus, which is genetically distinct from ebolavirus and marburgvirus, was recently discovered in Spain where filoviral hemorrhagic fever had never been reported. The virulence of this virus has not been determined. Ebolavirus and marburgvirus are classified as biosafety level-4 (BSL-4) pathogens and Category A agents, for which the US government requires preparedness in case of bioterrorism. Therefore, preventive measures against these viral hemorrhagic fevers should be prepared, not only in disease-endemic regions, but also in disease-free countries. Diagnostics, vaccines, and therapeutics need to be developed, and therefore the establishment of animal models for EHF and MHF is invaluable. Several animal models have been developed for EHF and MHF using non-human primates (NHPs) and rodents, which are crucial to understand pathophysiology and to develop diagnostics, vaccines, and therapeutics. Rhesus and cynomolgus macaques are representative models of filovirus infection as they exhibit remarkably similar symptoms to those observed in humans. However, the NHP models have practical and ethical problems that limit their experimental use. Furthermore, there are no inbred and genetically manipulated strains of NHP. Rodent models such as mouse, guinea pig, and hamster, have also been developed. However, these rodent models require adaptation of the virus to produce lethal disease and do not mirror all symptoms of human filovirus infection. This review article provides an outline of the clinical features of EHF and MHF in animals, including humans, and discusses how the animal models have been developed to study pathophysiology, vaccines, and therapeutics.

  15. Integrated Computational Approach for Virtual Hit Identification against Ebola Viral Proteins VP35 and VP40.

    PubMed

    Mirza, Muhammad Usman; Ikram, Nazia

    2016-10-26

    The Ebola virus (EBOV) has been recognised for nearly 40 years, with the most recent EBOV outbreak being in West Africa, where it created a humanitarian crisis. Mortalities reported up to 30 March 2016 totalled 11,307. However, up until now, EBOV drugs have been far from achieving regulatory (FDA) approval. It is therefore essential to identify parent compounds that have the potential to be developed into effective drugs. Studies on Ebola viral proteins have shown that some can elicit an immunological response in mice, and these are now considered essential components of a vaccine designed to protect against Ebola haemorrhagic fever. The current study focuses on chemoinformatic approaches to identify virtual hits against Ebola viral proteins (VP35 and VP40), including protein binding site prediction, drug-likeness, pharmacokinetic and pharmacodynamic properties, metabolic site prediction, and molecular docking. Retrospective validation was performed using a database of non-active compounds, and early enrichment of EBOV actives at different false positive rates was calculated. Homology modelling and subsequent superimposition of binding site residues on other strains of EBOV were carried out to check residual conformations, and hence to confirm the efficacy of potential compounds. As a mechanism for artefactual inhibition of proteins through non-specific compounds, virtual hits were assessed for their aggregator potential compared with previously reported aggregators. These systematic studies have indicated that a few compounds may be effective inhibitors of EBOV replication and therefore might have the potential to be developed as anti-EBOV drugs after subsequent testing and validation in experiments in vivo.

  16. Immunogenicity of virus-like particles containing modified goose parvovirus VP2 protein.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Zhu, Yingqi; Wang, Binbin; Meng, Chunchun; Liu, Guangqing

    2012-10-01

    The major capsid protein VP2 of goose parvovirus (GPV) expressed using a baculovirus expression system (BES) assembles into virus-like particles (VLPs). To optimize VP2 gene expression in Sf9 cells, we converted wild-type VP2 (VP2) codons into codons that are more common in insect genes. This change greatly increased VP2 protein production in Sf9 cells. The protein generated from the codon-optimized VP2 (optVP2) was detected by immunoblotting and an indirect immunofluorescence assay (IFA). Transmission electron microscopy analysis revealed the formation of VLPs. These findings indicate that optVP2 yielded stable and high-quality VLPs. Immunogenicity assays revealed that the VLPs are highly immunogenic, elicit a high level of neutralizing antibodies and provide protection against lethal challenge. The antibody levels appeared to be directly related to the number of GP-Ag-positive hepatocytes. The variation trends for GP-Ag-positive hepatocytes were similar in the vaccine groups. In comparison with the control group, the optVP2 VLPs groups exhibited obviously better responses. These data indicate that the VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Thus, GPV optVP2 appears to be a good candidate for the vaccination of goslings. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Secondary metabolites extracted from marine sponge associated Comamonas testosteroni and Citrobacter freundii as potential antimicrobials against MDR pathogens and hypothetical leads for VP40 matrix protein of Ebola virus: an in vitro and in silico investigation.

    PubMed

    Skariyachan, Sinosh; Acharya, Archana B; Subramaniyan, Saumya; Babu, Sumangala; Kulkarni, Shruthi; Narayanappa, Rajeswari

    2016-09-01

    The current study explores therapeutic potential of metabolites extracted from marine sponge (Cliona sp.)-associated bacteria against MDR pathogens and predicts the binding prospective of probable lead molecules against VP40 target of Ebola virus. The metabolite-producing bacteria were characterized by agar overlay assay and as per the protocols in Bergey's manual of determinative bacteriology. The antibacterial activities of extracted metabolites were tested against clinical pathogens by well-diffusion assay. The selected metabolite producers were characterized by 16S rDNA sequencing. Chemical screening and Fourier Transform Infrared (FTIR) analysis for selected compounds were performed. The probable lead molecules present in the metabolites were hypothesized based on proximate analysis, FTIR data, and literature survey. The drug-like properties and binding potential of lead molecules against VP40 target of Ebola virus were hypothesized by computational virtual screening and molecular docking. The current study demonstrated that clear zones around bacterial colonies in agar overlay assay. Antibiotic sensitivity profiling demonstrated that the clinical isolates were multi-drug resistant, however; most of them showed sensitivity to secondary metabolites (MIC-15 μl/well). The proximate and FTIR analysis suggested that probable metabolites belonged to alkaloids with O-H, C-H, C=O, and N-H groups. 16S rDNA characterization of selected metabolite producers demonstrated that 96% and 99% sequence identity to Comamonas testosteroni and Citrobacter freundii, respectively. The docking studies suggested that molecules such as Gymnastatin, Sorbicillactone, Marizomib, and Daryamide can designed as probable lead candidates against VP40 target of Ebola virus.

  18. Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

    PubMed Central

    Martin, Annette; Bénichou, Danièle; Chao, Shih-Fong; Cohen, Lisette M.; Lemon, Stanley M.

    1999-01-01

    Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase. PMID:10400711

  19. Fold Prediction of VP24 Protein of Ebola and Marburg Viruses using de novo Fragment Assembly

    DTIC Science & Technology

    2009-05-15

    Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D., Karplus, M., 1998. All-atom empirical potential for molecular modeling and dynamics studies of...aqueous forms that may be related to its role as a matrix protein. Specifically, the molecular weight of VP24 oligomers was determined using differ...dielectric electrostatic function. Next, the PARAM22 plus generalized Born molecular volume solvation (GBMV2) (Lee et al., 2003) energy (including a 15

  20. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    NASA Technical Reports Server (NTRS)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  1. Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo

    PubMed Central

    Moyen, Nanikaly; Thirion, Laurence; Emmerich, Petra; Dzia-Lepfoundzou, Amelia; Richet, Hervé; Boehmann, Yannik; Dimi, Yannick; Gallian, Pierre; Gould, Ernest A.; Günther, Stephan; de Lamballerie, Xavier

    2015-01-01

    Background Ebola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV. Methodology and Findings In 2011, we conducted a MARV and EBOV seroprevalence study amongst 809 blood donors recruited in rural (75; 9.3%) and urban (734; 90.7%) areas of the Republic of Congo. Serum titres of IgG antibodies to MARV and EBOV were assessed by indirect double-immunofluorescence microscopy. MARV seroprevalence was 0.5% (4 in 809) without any identified risk factors. Prevalence of IgG to EBOV was 2.5%, peaking at 4% in rural areas and in Pointe Noire. Independent risk factors identified by multivariate analysis were contact with bats and exposure to birds. Conclusions/Significance This MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors. PMID:26047124

  2. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses.

    PubMed

    Martines, Roosecelis Brasil; Ng, Dianna L; Greer, Patricia W; Rollin, Pierre E; Zaki, Sherif R

    2015-01-01

    Ebola viruses and Marburg viruses include some of the most virulent and fatal pathogens known to humans. These viruses cause severe haemorrhagic fevers, with case fatality rates in the range 25-90%. The diagnosis of filovirus using formalin-fixed tissues from fatal cases poses a significant challenge. The most characteristic histopathological findings are seen in the liver; however, the findings overlap with many other viral and non-viral haemorrhagic diseases. The need to distinguish filovirus infections from other haemorrhagic fevers, particularly in areas with multiple endemic viral haemorrhagic agents, is of paramount importance. In this review we discuss the current state of knowledge of filovirus infections and their pathogenesis, including histopathological findings, epidemiology, modes of transmission and filovirus entry and spread within host organisms. The pathogenesis of filovirus infections is complex and involves activation of the mononuclear phagocytic system, with release of pro-inflammatory cytokines, chemokines and growth factors, endothelial dysfunction, alterations of the innate and adaptive immune systems, direct organ and endothelial damage from unrestricted viral replication late in infection, and coagulopathy. Although our understanding of the pathogenesis of filovirus infections has rapidly increased in the past few years, many questions remain unanswered. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  3. Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles.

    PubMed

    Hu, Lei; Trefethen, Jared M; Zeng, Yuhong; Yee, Luisa; Ohtake, Satoshi; Lechuga-Ballesteros, David; Warfield, Kelly L; Aman, M Javad; Shulenin, Sergey; Unfer, Robert; Enterlein, Sven G; Truong-Le, Vu; Volkin, David B; Joshi, Sangeeta B; Middaugh, C Russell

    2011-12-01

    The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs. Copyright © 2011 Wiley-Liss, Inc.

  4. A Comparison of the Pathogenesis of Marburg Virus Disease in Humans and Nonhuman Primates and Evaluation of the Suitability of These Animal Models for Predicting Clinical Efficacy under the 'Animal Rule'.

    PubMed

    Glaze, Elizabeth R; Roy, Michael J; Dalrymple, Lonnie W; Lanning, Lynda L

    2015-06-01

    Marburg virus outbreaks are sporadic, infrequent, brief, and relatively small in terms of numbers of subjects affected. In addition, outbreaks most likely will occur in remote regions where clinical trials are not feasible; therefore, definitive, well-controlled human efficacy studies to test the effectiveness of a drug or biologic product are not feasible. Healthy human volunteers cannot ethically be deliberately exposed to a lethal agent such as Marburg virus in order to test the efficacy of a therapy or preventive prior to licensure. When human efficacy studies are neither ethical nor feasible, the US Food and Drug Administration may grant marketing approval of a drug or biologic product under the 'Animal Rule,' through which demonstration of the efficacy of a product can be 'based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is reasonably likely to produce clinical benefit in humans.' This process requires that the pathogenic determinants of the disease in the animal model are similar to those that have been identified in humans. After reviewing primarily English-language, peer-reviewed journal articles, we here summarize the clinical manifestations of Marburg virus disease and the results of studies in NHP showing the characteristics and progression of the disease. We also include a detailed comparison of the characteristics of the human disease relative to those for NHP. This review reveals that the disease characteristics of Marburg virus disease are generally similar for humans and 3 NHP species: cynomolgus macaques (Macaca fascicularis), rhesus macaques (Macaca mulatta), and African green monkeys (Chlorocebus aethiops).

  5. Marburg virus survivor immune responses are Th1 skewed with limited neutralizing antibody responses.

    PubMed

    Stonier, Spencer W; Herbert, Andrew S; Kuehne, Ana I; Sobarzo, Ariel; Habibulin, Polina; Dahan, Chen V Abramovitch; James, Rebekah M; Egesa, Moses; Cose, Stephen; Lutwama, Julius Julian; Lobel, Leslie; Dye, John M

    2017-09-04

    Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4 + T cell responses but limited CD8 + T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity. © 2017 Stonier et al.

  6. Recombinant VP1 protein of duck hepatitis virus 1 expressed in Pichia pastoris and its immunogenicity in ducks.

    PubMed

    Wang, C; Li, X K; Wu, T C; Wang, Y; Zhang, C J; Cheng, X C; Chen, P Y

    2014-01-01

    The VP1 gene of duck hepatitis virus type 1 (DHV-1) strain VJ09 was amplified by reverse transcription PCR from the liver of a duckling with clinical symptoms of viral hepatitis. The resulting VP1 cDNA was 720 bp in length and encoded a 240-amino-acid protein. In VP1 gene-based phylogenetic analysis, the VJ09 strain grouped with DHV-1 genotype C. The VP1 gene was inserted into the expression vector pPICZαA and expressed in Pichia pastoris. The expressed VP1 protein was purified and identified by western blot analysis. To evaluate the recombinant VP1's immunogenic potential in ducklings, the antibodies raised in the immunized ducklings were titrated by ELISA, and lymphocyte proliferation and virus neutralization assays were performed. The results show that the recombinant VP1 protein induced a significant immune response in ducklings and this could be a candidate for the development of a subunit vaccine against DHV-1 genotype C.

  7. Ebola virus disease and Marburg disease in pregnancy: a review and management considerations for filovirus infection.

    PubMed

    Bebell, Lisa M; Riley, Laura E

    2015-06-01

    The largest-ever recorded outbreak of viral hemorrhagic fever is ongoing. As a result of the epidemic and rural nature of outbreaks, little is published about the Filovirus infections Ebola virus disease and Marburg disease in pregnancy. This review of viral hemorrhagic fever focusing on Marburg and Ebola uses knowledge of disease in nonpregnant individuals and pregnancy-specific data to inform management for pregnant women. Filovirus infection presentation is similar between pregnant and nonpregnant patients, although infections may be more severe in pregnancy. Although labeled as hemorrhagic fevers, Marburg and Ebola do not commonly cause gross bleeding and should be conceptualized as diseases of high gastrointestinal losses. Early, aggressive supportive care is the mainstay of Filovirus infection management with massive fluid resuscitation as the key management principle. Patients often require 5-10 L or more per day of intravenous or oral fluid to maintain circulating blood volume in the setting of ongoing gastrointestinal loss. Fluid shifts warrant aggressive monitoring and correction of potassium levels and acid-base disturbances to prevent life-threatening arrhythmias and metabolic complications. Regardless of maternal survival, fetal loss rates are nearly 100% in Filovirus infection, likely resulting from unchecked transplacental and hematogenous viral spread. High fetal loss rates support the placenta as a difficult-to-eradicate Filovirus infection reservoir. In conclusion, the management of Filovirus infection in pregnancy should focus on stabilizing the mother with intensive monitoring and aggressive fluid and electrolyte repletion as well as maintaining strict infection control to minimize transmission to others.

  8. Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3

    PubMed Central

    Martínez-Torrecuadrada, Jorge L.; Lázaro, Beatriz; Rodriguez, José F.; Casal, J. Ignacio

    2000-01-01

    The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

  9. Antigenic properties and diagnostic potential of baculovirus-expressed infectious bursal disease virus proteins VPX and VP3.

    PubMed

    Martínez-Torrecuadrada, J L; Lázaro, B; Rodriguez, J F; Casal, J I

    2000-07-01

    The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.

  10. High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses.

    PubMed

    Uebelhoer, Luke S; Albariño, César G; McMullan, Laura K; Chakrabarti, Ayan K; Vincent, Joel P; Nichol, Stuart T; Towner, Jonathan S

    2014-06-01

    Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle. Published by Elsevier B.V.

  11. Vaccination with recombinant Modified Vaccinia Ankara (MVA) viruses expressing single African horse sickness virus VP2 antigens induced cross-reactive virus neutralising antibodies (VNAb) in horses when administered in combination.

    PubMed

    Manning, Nicola Mary; Bachanek-Bankowska, Katarzyna; Mertens, Peter Paul Clement; Castillo-Olivares, Javier

    2017-10-20

    African horse sickness is a lethal viral disease of equids transmitted by biting midges of the Genus Culicoides. The disease is endemic to sub-Saharan Africa but outbreaks of high mortality and economic impact have occurred in the past in non-endemic regions of Africa, Asia and Southern Europe. Vaccination is critical for the control of this disease but only live attenuated vaccines are currently available. However, there are bio-safety concerns over the use of this type of vaccines, especially in non-endemic countries, and live attenuated vaccines do not have DIVA (Differentiation of Infected from Vaccinated Animals) capacity. In addition, large scale manufacturing of live attenuated vaccines of AHSV represents a significant environmental and health risk and level 3 bio-safety containment facilities are required for their production. A variety of different technologies have been investigated over the years to develop alternative AHSV vaccines, including the use of viral vaccine vectors such Modified Vaccinia Ankara virus (MVA). In previous studies we demonstrated that recombinant MVA expressing outer capsid protein AHSV-VP2 induced virus neutralising antibodies and protection against virulent challenge both in a mouse model and in the horse. However, AHSV-VP2 is antigenically variable and determines the existence of 9 different AHSV serotypes. Immunity against AHSV is serotype-specific and there is limited cross-reactivity between certain AHSV serotypes: 1 and 2, 3 and 7, 5 and 8, 6 and 9. In Africa, multiple serotypes circulate simultaneously and a polyvalent attenuated vaccine comprising different AHSV serotypes is used. We investigated the potential of a polyvalent AHSV vaccination strategy based on combinations of MVA-VP2 viruses each expressing a single VP2 antigen from a specific serotype. We showed that administration of 2 different recombinant MVA viruses, each expressing a single VP2 protein from AHSV serotype 4 or 9, denoted respectively as MVA-VP2

  12. Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity.

    PubMed Central

    Hoshino, Y; Sereno, M M; Midthun, K; Flores, J; Kapikian, A Z; Chanock, R M

    1985-01-01

    Antiserum prepared against the M37 strain of rotavirus, recovered from an asymptomatic newborn infant in Venezuela, neutralized two prototype human rotaviruses that define two separate serotypes: serotype 1 (Wa) and serotype 4 (ST3). Thus, the M37 strain is a naturally occurring intertypic rotavirus. Analysis of reassortant viruses produced during coinfection in vitro indicated that the observed dual serotype specificity of M37 resulted from sharing a related outer capsid protein, VP3, with the ST3 virus and another related outer capsid protein, VP7, with the Wa virus. Analysis of single (VP3)-gene-substitution reassortants indicated that VP3 was as potent an immunogen as VP7. In addition, direct evidence was obtained that the serotype specificity of neutralizing antibody elicited by VP3 can differ from the serotype specificity of neutralizing antibody elicited by VP7, indicating the need for a dual system of rotavirus classification in which the neutralization specificity of both VP3 and VP7 outer capsid proteins are identified. Images PMID:3001716

  13. VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

    SciTech Connect

    Schwarz, Toni M.; Edwards, Megan R.; Diederichs, Audrey

    ABSTRACT Zaire ebolavirus(EBOV),Bundibugyo ebolavirus(BDBV), andReston ebolavirus(RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA bindingmore » affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCEThe interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of theEbolavirusgenus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA

  14. VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

    PubMed

    Schwarz, Toni M; Edwards, Megan R; Diederichs, Audrey; Alinger, Joshua B; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2017-02-15

    Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition

  15. Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus.

    PubMed

    Bixler, Sandra L; Bocan, Thomas M; Wells, Jay; Wetzel, Kelly S; Van Tongeren, Sean A; Dong, Lian; Garza, Nicole L; Donnelly, Ginger; Cazares, Lisa H; Nuss, Jonathan; Soloveva, Veronica; Koistinen, Keith A; Welch, Lisa; Epstein, Carol; Liang, Li-Fang; Giesing, Dennis; Lenk, Robert; Bavari, Sina; Warren, Travis K

    2018-03-01

    Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV). While no survival benefit was observed in two studies employing once- or twice-daily oral dosing of favipiravir during EBOV infection of NHPs, an antiviral effect was observed in terms of extended time-to-death and reduced levels of viral RNA. However, oral dosing in biosafety level-4 (BSL-4) presents logistical and technical challenges, and repeated anesthesia events may potentially worsen survival outcome in animals. For the third study of treatment of MARV infection, we therefore made use of catheters, jackets, and tethers for intravenous (IV) dosing and blood collection, which minimized the requirement for repeated anesthesia events. When MARV infection was treated with IV favipiravir, five of six animals (83%) survived infection, while all untreated NHPs succumbed. An accompanying report presents the results of favipiravir treatment of EBOV infection in mice. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  16. The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold.

    PubMed

    Zhang, Adrianna P P; Bornholdt, Zachary A; Liu, Tong; Abelson, Dafna M; Lee, David E; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

    2012-02-01

    Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

  17. Mutual antagonism between the Ebola virus VP35 protein and the RIG-I activator PACT determines infection outcome.

    PubMed

    Luthra, Priya; Ramanan, Parameshwaran; Mire, Chad E; Weisend, Carla; Tsuda, Yoshimi; Yen, Benjamin; Liu, Gai; Leung, Daisy W; Geisbert, Thomas W; Ebihara, Hideki; Amarasinghe, Gaya K; Basler, Christopher F

    2013-07-17

    The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

    PubMed

    Wu, Xiaoying; Li, Xiaojun; Zhang, Qingshan; Wulin, Shaozhou; Bai, Xiaofei; Zhang, Tingting; Wang, Yue; Liu, Ming; Zhang, Yun

    2015-01-01

    The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

  19. Lack of Marburg Virus Transmission From Experimentally Infected to Susceptible In-Contact Egyptian Fruit Bats.

    PubMed

    Paweska, Janusz T; Jansen van Vuren, Petrus; Fenton, Karla A; Graves, Kerry; Grobbelaar, Antoinette A; Moolla, Naazneen; Leman, Patricia; Weyer, Jacqueline; Storm, Nadia; McCulloch, Stewart D; Scott, Terence P; Markotter, Wanda; Odendaal, Lieza; Clift, Sarah J; Geisbert, Thomas W; Hale, Martin J; Kemp, Alan

    2015-10-01

    Egyptian fruit bats (Rousettus aegyptiacus) were inoculated subcutaneously (n = 22) with Marburg virus (MARV). No deaths, overt signs of morbidity, or gross lesions was identified, but microscopic pathological changes were seen in the liver of infected bats. The virus was detected in 15 different tissues and plasma but only sporadically in mucosal swab samples, urine, and fecal samples. Neither seroconversion nor viremia could be demonstrated in any of the in-contact susceptible bats (n = 14) up to 42 days after exposure to infected bats. In bats rechallenged (n = 4) on day 48 after infection, there was no viremia, and the virus could not be isolated from any of the tissues tested. This study confirmed that infection profiles are consistent with MARV replication in a reservoir host but failed to demonstrate MARV transmission through direct physical contact or indirectly via air. Bats develop strong protective immunity after infection with MARV. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

    PubMed

    Kruse, Thomas; Biedenkopf, Nadine; Hertz, Emil Peter Thrane; Dietzel, Erik; Stalmann, Gertrud; López-Méndez, Blanca; Davey, Norman E; Nilsson, Jakob; Becker, Stephan

    2018-01-04

    Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. A Comparison of the Pathogenesis of Marburg Virus Disease in Humans and Nonhuman Primates and Evaluation of the Suitability of These Animal Models for Predicting Clinical Efficacy under the ‘Animal Rule’

    PubMed Central

    Glaze, Elizabeth R; Roy, Michael J; Dalrymple, Lonnie W; Lanning, Lynda L

    2015-01-01

    Marburg virus outbreaks are sporadic, infrequent, brief, and relatively small in terms of numbers of subjects affected. In addition, outbreaks most likely will occur in remote regions where clinical trials are not feasible; therefore, definitive, well-controlled human efficacy studies to test the effectiveness of a drug or biologic product are not feasible. Healthy human volunteers cannot ethically be deliberately exposed to a lethal agent such as Marburg virus in order to test the efficacy of a therapy or preventive prior to licensure. When human efficacy studies are neither ethical nor feasible, the US Food and Drug Administration may grant marketing approval of a drug or biologic product under the ‘Animal Rule,’ through which demonstration of the efficacy of a product can be ‘based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is reasonably likely to produce clinical benefit in humans.’ This process requires that the pathogenic determinants of the disease in the animal model are similar to those that have been identified in humans. After reviewing primarily English-language, peer-reviewed journal articles, we here summarize the clinical manifestations of Marburg virus disease and the results of studies in NHP showing the characteristics and progression of the disease. We also include a detailed comparison of the characteristics of the human disease relative to those for NHP. This review reveals that the disease characteristics of Marburg virus disease are generally similar for humans and 3 NHP species: cynomolgus macaques (Macaca fascicularis), rhesus macaques (Macaca mulatta), and African green monkeys (Chlorocebus aethiops). PMID:26141449

  2. Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging.

    PubMed

    Banadyga, Logan; Hoenen, Thomas; Ambroggio, Xavier; Dunham, Eric; Groseth, Allison; Ebihara, Hideki

    2017-08-09

    Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.

  3. Elucidation of the Ebola virus VP24 cellular interactome and disruption of virus biology through targeted inhibition of host-cell protein function.

    PubMed

    García-Dorival, Isabel; Wu, Weining; Dowall, Stuart; Armstrong, Stuart; Touzelet, Olivier; Wastling, Jonathan; Barr, John N; Matthews, David; Carroll, Miles; Hewson, Roger; Hiscox, Julian A

    2014-11-07

    Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

  4. Ebola and Marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for Africa.

    PubMed

    Leroy, E M; Gonzalez, J-P; Baize, S

    2011-07-01

    Ebola and Marburg viruses are the only members of the Filoviridae family (order Mononegavirales), a group of viruses characterized by a linear, non-segmented, single-strand negative RNA genome. They are among the most virulent pathogens for humans and great apes, causing acute haemorrhagic fever and death within a matter of days. Since their discovery 50 years ago, filoviruses have caused only a few outbreaks, with 2317 clinical cases and 1671 confirmed deaths, which is negligible compared with the devastation caused by malnutrition and other infectious diseases prevalent in Africa (malaria, cholera, AIDS, dengue, tuberculosis …). Yet considerable human and financial resourses have been devoted to research on these viruses during the past two decades, partly because of their potential use as bioweapons. As a result, our understanding of the ecology, host interactions, and control of these viruses has improved considerably. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

  5. Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus.

    PubMed

    Wang, Nian; Zhang, Lizhou; Chen, Yuming; Lu, Zhen; Gao, Li; Wang, Yongqiang; Gao, Yulong; Gao, Honglei; Cui, Hongyu; Li, Kai; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2015-01-01

    Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.

  6. Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells.

    PubMed

    Ilinykh, Philipp A; Lubaki, Ndongala M; Widen, Steven G; Renn, Lynnsey A; Theisen, Terence C; Rabin, Ronald L; Wood, Thomas G; Bukreyev, Alexander

    2015-08-01

    Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC. This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal

  7. Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA.

    PubMed

    Thi, Emily P; Mire, Chad E; Ursic-Bedoya, Raul; Geisbert, Joan B; Lee, Amy C H; Agans, Krystle N; Robbins, Marjorie; Deer, Daniel J; Fenton, Karla A; MacLachlan, Ian; Geisbert, Thomas W

    2014-08-20

    Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)-targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti-MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease. Copyright © 2014, American Association for the Advancement of Science.

  8. Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

    PubMed

    Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

    2014-12-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  9. A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge.

    PubMed

    Grant-Klein, Rebecca J; Van Deusen, Nicole M; Badger, Catherine V; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

    2012-11-01

    We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.

  10. Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

    PubMed

    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

    2013-11-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

  11. Definition of neutralizing sites on African horse sickness virus serotype 4 VP2 at the level of peptides.

    PubMed

    Martínez-Torrecuadrada, J L; Langeveld, J P; Meloen, R H; Casal, J I

    2001-10-01

    The antigenic structure of African horse sickness virus (AHSV) serotype 4 capsid protein VP2 has been determined at the peptide level by PEPSCAN analysis in combination with a large collection of polyclonal antisera and monoclonal antibodies. VP2, the determinant for the virus serotype and an important target in virus neutralization, was found to contain 15 antigenic sites. A major antigenic region containing 12 of the 15 sites was identified in the region between residues 223 and 400. A second domain between residues 568 and 681 contained the three remaining sites. These sites were used for the synthesis of peptides, which were later tested in rabbits. Of the 15 synthetic peptides, three were able to induce neutralizing antibodies for AHSV-4, defining two neutralizing epitopes, 'a' and 'b', between residues 321 and 339, and 377 and 400, respectively. A combination of peptides representing both sites induced a more effective neutralizing response. Still, the relatively low neutralization titres make the possibility of producing a synthetic vaccine for AHSV unlikely. The complex protein-protein interaction of the outer shell of the viral capsid would probably require the presence of either synthetic peptides in the correct conformation or peptide segments from the different proteins VP2, VP5 and VP7.

  12. Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

    PubMed

    Liu, Nina; Tao, Yisong; Brenowitz, Michael D; Girvin, Mark E; Lai, Jonathan R

    2015-10-01

    Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges.

    PubMed

    Wang, Danher; Hevey, Michael; Juompan, Laure Y; Trubey, Charles M; Raja, Nicholas U; Deitz, Stephen B; Woraratanadharm, Jan; Luo, Min; Yu, Hong; Swain, Benjamin M; Moore, Kevin M; Dong, John Y

    2006-09-30

    The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guinea pigs. Significantly, guinea pigs vaccinated with at least 5 x 10(7) pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV.

  14. Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges

    SciTech Connect

    Wang Danher; Hevey, Michael; Juompan, Laure Y.

    2006-09-30

    The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guineamore » pigs. Significantly, guinea pigs vaccinated with at least 5 x 10{sup 7} pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV.« less

  15. Comparison of the Pathogenesis of the Angola and Ravn Strains of Marburg Virus in the Outbred Guinea Pig Model.

    PubMed

    Cross, Robert W; Fenton, Karla A; Geisbert, Joan B; Ebihara, Hideki; Mire, Chad E; Geisbert, Thomas W

    2015-10-01

    Phylogenetic comparisons of known Marburg virus (MARV) strains reveal 2 distinct genetic lineages: Ravn and the Lake Victoria Marburg complex (eg, Musoke, Popp, and Angola strains). Nucleotide variances of >20% between Ravn and other MARV genomes suggest that differing virulence between lineages may accompany this genetic divergence. To date, there exists limited systematic experimental evidence of pathogenic differences between MARV strains. Uniformly lethal outbred guinea pig models of MARV-Angola (MARV-Ang) and MARV-Ravn (MARV-Rav) were developed by serial adaptation. Changes in genomic sequence, weight, temperature, histopathologic findings, immunohistochemical findings, hematologic profiles, circulating biochemical enzyme levels, coagulation parameters, viremia levels, cytokine levels, eicanosoid levels, and nitric oxide production were compared between strains. MARV-Rav infection resulted in delayed increases in circulating inflammatory and prothrombotic elements, notably lower viremia levels, less severe histologic alterations, and a delay in mean time to death, compared with MARV-Ang infection. Both strains produced more marked coagulation abnormalities than previously seen in MARV-infected mice or inbred guinea pigs. Although both strains exhibit great similarity to pathogenic markers of human and nonhuman primate MARV infection, these data highlight several key differences in pathogenicity that may serve to guide the choice of strain and model used for development of vaccines or therapeutics for Marburg hemorrhagic fever. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  16. Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability

    PubMed Central

    Ma, Xiuli; Sheng, Zizhang; Huang, Bing; Qi, Lihong; Li, Yufeng; Yu, Kexiang; Liu, Cunxia; Qin, Zhuoming; Wang, Dan; Song, Minxun; Li, Feng

    2015-01-01

    The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10-4 per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses. PMID:26173145

  17. Lectin Affinity Plasmapheresis for Middle East Respiratory Syndrome-Coronavirus and Marburg Virus Glycoprotein Elimination.

    PubMed

    Koch, Benjamin; Schult-Dietrich, Patricia; Büttner, Stefan; Dilmaghani, Bijan; Lohmann, Dario; Baer, Patrick C; Dietrich, Ursula; Geiger, Helmut

    2018-04-26

    Middle East respiratory syndrome coronavirus (MERS-CoV) and Marburg virus (MARV) are among the World Health Organization's top 8 emerging pathogens. Both zoonoses share nonspecific early symptoms, a high lethality rate, and a reduced number of specific treatment options. Therefore, we evaluated extracorporeal virus and glycoprotein (GP) elimination by lectin affinity plasmapheresis (LAP). For both MERS-CoV (pseudovirus) as well as MARV (GPs), 4 LAP devices (Mini Hemopurifiers, Aethlon Medical, San Diego, CA, USA) and 4 negative controls were tested. Samples were collected every 30 min and analyzed for reduction in virus infectivity by a flow cytometry-based infectivity assay (MERS-CoV) and in soluble GP content (MARV) by an immunoassay. The experiments show a time-dependent clearance of MERS-CoV of up to 80% within 3 h (pseudovirus). Up to 70% of MARV-soluble GPs were eliminated at the same time. Substantial saturation of the binding resins was detected within the first treatment hour. MERS-CoV (pseudovirus) and MARV soluble GPs are eliminated by LAP in vitro. Considering the high lethality and missing established treatment options, LAP should be evaluated in vivo. Especially early initiation, continuous therapy, and timed cartridge exchanges could be of importance. The Author(s). Published by S. Karger AG, Basel.

  18. Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges.

    PubMed

    Grant-Klein, Rebecca J; Altamura, Louis A; Badger, Catherine V; Bounds, Callie E; Van Deusen, Nicole M; Kwilas, Steven A; Vu, Hong A; Warfield, Kelly L; Hooper, Jay W; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

    2015-01-01

    Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

  19. Homologous and heterologous protection of nonhuman primates by Ebola and Sudan virus-like particles.

    PubMed

    Warfield, Kelly L; Dye, John M; Wells, Jay B; Unfer, Robert C; Holtsberg, Frederick W; Shulenin, Sergey; Vu, Hong; Swenson, Dana L; Bavari, Sina; Aman, M Javad

    2015-01-01

    Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

  20. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge.

    PubMed

    Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

    2014-06-17

    African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR -/-) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Safety and immunogenicity of Ebola virus and Marburg virus glycoprotein DNA vaccines assessed separately and concomitantly in healthy Ugandan adults: a phase 1b, randomised, double-blind, placebo-controlled clinical trial.

    PubMed

    Kibuuka, Hannah; Berkowitz, Nina M; Millard, Monica; Enama, Mary E; Tindikahwa, Allan; Sekiziyivu, Arthur B; Costner, Pamela; Sitar, Sandra; Glover, Deline; Hu, Zonghui; Joshi, Gyan; Stanley, Daphne; Kunchai, Meghan; Eller, Leigh Anne; Bailer, Robert T; Koup, Richard A; Nabel, Gary J; Mascola, John R; Sullivan, Nancy J; Graham, Barney S; Roederer, Mario; Michael, Nelson L; Robb, Merlin L; Ledgerwood, Julie E

    2015-04-18

    Ebola virus and Marburg virus cause serious disease outbreaks with high case fatality rates. We aimed to assess the safety and immunogenicity of two investigational DNA vaccines, one (EBO vaccine) encoding Ebola virus Zaire and Sudan glycoproteins and one (MAR) encoding Marburg virus glycoprotein. RV 247 was a phase 1b, double-blinded, randomised, placebo-controlled clinical trial in Kampala, Uganda to examine the safety and immunogenicity of the EBO and MAR vaccines given individually and concomitantly. Healthy adult volunteers aged 18-50 years were randomly assigned (5:1) to receive three injections of vaccine or placebo at weeks 0, 4, and 8, with vaccine allocations divided equally between three active vaccine groups: EBO vaccine only, MAR vaccine only, and both vaccines. The primary study objective was to investigate the safety and tolerability of the vaccines, as assessed by local and systemic reactogenicity and adverse events. We also assessed immunogenicity on the basis of antibody responses (ELISA) and T-cell responses (ELISpot and intracellular cytokine staining assays) 4 weeks after the third injection. Participants and investigators were masked to group assignment. Analysis was based on the intention-to-treat principle. This trial is registered at ClinicalTrials.gov, number NCT00997607. 108 participants were enrolled into the study between Nov 2, 2009, and April 15, 2010. All 108 participants received at least one study injection (including 100 who completed the injection schedule) and were included in safety and tolerability analyses; 107 for whom data were available were included in the immunogenicity analyses. Study injections were well tolerated, with no significant differences in local or systemic reactions between groups. The vaccines elicited antibody and T-cell responses specific to the glycoproteins received and we detected no differences between the separate and concomitant use of the two vaccines. 17 of 30 (57%, 95% CI 37-75) participants in

  2. Investigating Ebola virus pathogenicity using molecular dynamics.

    PubMed

    Pappalardo, Morena; Collu, Francesca; Macpherson, James; Michaelis, Martin; Fraternali, Franca; Wass, Mark N

    2017-08-11

    Ebolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5. As a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling. Our results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.

  3. Protection against lethal Marburg virus infection mediated by lipid encapsulated small interfering RNA.

    PubMed

    Ursic-Bedoya, Raul; Mire, Chad E; Robbins, Marjorie; Geisbert, Joan B; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W

    2014-02-15

    Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Treatment resulted in 60%-100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection.

  4. Protection Against Lethal Marburg Virus Infection Mediated by Lipid Encapsulated Small Interfering RNA

    PubMed Central

    Ursic-Bedoya, Raul; Mire, Chad E.; Robbins, Marjorie; Geisbert, Joan B.; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W.

    2014-01-01

    Background. Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. Methods. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Results. Treatment resulted in 60%–100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. Conclusions. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection. PMID:23990568

  5. A conserved carboxy-terminal domain in the major tegument structural protein VP22 facilitates virion packaging of a chimeric protein during productive herpes simplex virus 1 infection

    SciTech Connect

    Schlegel, Elisabeth F.M.; Blaho, John A., E-mail: john.blaho@mssm.ed

    2009-05-10

    Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least amore » portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.« less

  6. Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches

    PubMed Central

    Fu, Xu; Wang, Zhihua; Li, Lixin; Dong, Shishang; Li, Zhucui; Jiang, Zhenzuo; Wang, Yuefei; Shui, Wenqing

    2016-01-01

    The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development. PMID:27403722

  7. Recombinant VP1 protein as a potential marker for the diagnosis of acute hepatitis A virus infection.

    PubMed

    da Silva Junior, Haroldo Cid; da Silva, Edimilson Domingos; Lewis-Ximenez de Souza Rodrigues, Lia Laura; Medeiros, Marco Alberto

    2017-07-01

    Since hepatitis A virus (HAV) production is time-consuming and expensive, the use of recombinant proteins may represent an alternative source of antigens for diagnostic purposes. The present study aimed to express, purify and evaluate the potential of recombinant VP1 protein (rVP1) as a marker for the diagnosis of acute HAV infection. The rVP1 was expressed and purified successfully from Escherichia coli. The purified rVP1 was used to establish an in-house enzyme-linked immunosorbent assay (ELISA-rVP1) for detection of IgM antibodies in sera from HAV-positive patients. For a cut-off point of 0.351, the sensitivity and specificity of ELISA-rVP1 were 100.0% and 95.0%, respectively. These results indicate that rVP1 may be a useful antigen for detection of IgM antibodies against HAV. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Molecular characterization of the VP1 gene of a Mexican isolate of infectious pancreatic necrosis virus.

    PubMed

    Barrera-Mejía, Magda; Simón-Martínez, José; Ulloa-Arvizu, Raúl; Salgado-Miranda, Celene; Soriano-Vargas, Edgardo

    2010-07-01

    The presence of infectious pancreatic necrosis virus (IPNV) in salmonids predominantly produces a high mortality rate in first-feeding fry. Genomic analysis of the vp2 gene sequence is most commonly used to determine the genetic diversity of IPNV isolates. Recently, information obtained from the vp1 gene allowed for efficient analysis of the genetic diversity of IPNV. In this study, the vp1 gene from a Mexican IPNV isolate was characterized and compared with IPNV isolates from Europe, North America, and Asia. The results indicate that the Mexican isolate is most closely related genetically to the 2310 strain from Spain.

  9. Marburg Virus Glycoprotein GP2: pH-Dependent Stability of the Ectodomain α-Helical Bundle†

    PubMed Central

    Harrison, Joseph S.; Koellhoffer, Jayne F.; Chandran, Kartik; Lai, Jonathan R.

    2012-01-01

    Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV is required for fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔGunf H2O, of 33.4 ± 2.5 kcal/mol and melting temperature, Tm, of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH with higher stability under lower pH conditions (Tm values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. Based on these results, we hypothesize that pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism to control conformational preferences such that the six-helix bundle ‘post-fusion’ state is preferred under conditions of appropriately matured endosomes. PMID:22369502

  10. Immune Response to Marburg Virus Angola Infection in Nonhuman Primates.

    PubMed

    Fernando, Lisa; Qiu, Xiangguo; Melito, P Leno; Williams, Kinola J N; Feldmann, Friederike; Feldmann, Heinz; Jones, Steven M; Alimonti, Judie B

    2015-10-01

    The 2005 outbreak of Marburg virus (MARV) infection in Angola was the most lethal MARV infection outbreak in history, with a case-fatality rate (90%) similar to that for Zaire ebolavirus (EBOV) infection. However, very little is known about the pathogenicity of MARV Angola, as few studies have been conducted to date. Therefore, the immune response was examined in MARV Angola-infected nonhuman primates. Cynomolgus macaques were infected with MARV Angola and monitored for survival. The effect of MARV Angola on the immune system was examined by immunophenotyping whole-blood and by analyzing cytokine and chemokine levels in plasma and spleen specimens, using flow cytometry. The prominent clinical findings were rapid onset of disease and death (mean time after infection, 6.7 days), fever, depression, anorexia, petechial rash, and lymphopenia. Specifically, T, B, and natural killer cells were severely depleted in the blood by day 6. The typical cytokine storm was present, with levels of interferon γ, tumor necrosis factor, interleukin 6, and CCL2 rising in the blood early during infection. MARV Angola displayed the same virulence and disease pathology as EBOV. MARV Angola appears to cause a more rapid onset and severe outcome of infection than other MARV strains. © Crown copyright 2015.

  11. Single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus.

    PubMed

    Geisbert, Thomas W; Geisbert, Joan B; Leung, Anders; Daddario-DiCaprio, Kathleen M; Hensley, Lisa E; Grolla, Allen; Feldmann, Heinz

    2009-07-01

    The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (CIEBOV), and Marburgvirus (MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVDeltaG/SEBOVGP, VSVDeltaG/ZEBOVGP, and VSVDeltaG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVDeltaG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic

  12. Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice.

    PubMed

    Lee, John S; Groebner, Jennifer L; Hadjipanayis, Angela G; Negley, Diane L; Schmaljohn, Alan L; Welkos, Susan L; Smith, Leonard A; Smith, Jonathan F

    2006-11-17

    The development of multiagent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. We report here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease, anthrax; a viral disease, Marburg fever; and against a toxin-mediated disease, botulism. The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)). CBA/J mice inoculated with a mixture of VRP expressing BoNT H(C) serotype C (BoNT/C H(C)) and MAT-PA were 80% protected from a B. anthracis (Sterne strain) challenge and then 100% protected from a sequential BoNT/C challenge. Swiss mice inoculated with individual VRP or with mixtures of VRP vaccines expressing BoNT H(C) serotype A (BoNT/A H(C)), MAT-PA, and MBGV-GP produced antibody responses specific to the corresponding replicon-expressed protein. Combination of the different VRP vaccines did not diminish the antibody responses measured for Swiss mice inoculated with formulations of two or three VRP vaccines as compared to mice that received only one VRP vaccine. Swiss mice inoculated with VRP expressing BoNT/A H(C) alone or in combination with VRP expressing MAT-PA and MBGV GP, were completely protected from a BoNT/A challenge. These studies demonstrate the utility of combining individual VRP vaccines into multiagent formulations for eliciting protective immune responses to various types of diseases.

  13. C Terminus of Infectious Bursal Disease Virus Major Capsid Protein VP2 Is Involved in Definition of the T Number for Capsid Assembly

    PubMed Central

    Castón, José R.; Martínez-Torrecuadrada, Jorge L.; Maraver, Antonio; Lombardo, Eleuterio; Rodríguez, José F.; Casal, J. Ignacio; Carrascosa, José L.

    2001-01-01

    Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis. PMID:11602723

  14. Ebola Virus Disease Candidate Vaccines Under Evaluation in Clinical Trials

    DTIC Science & Technology

    2016-06-02

    studies in HPIV-3-immune guinea pigs with EBOV GP1,2-expressing HPIV-3 have suggested that while pre-existing immunity to the vector suppressed... guinea pigs and nonhuman primates against infection with multiple Marburg viruses. Expert Rev Vaccines, 7(4), 417-429 (2008). 98. Warfield KL...Swenson DL, Negley DL et al. Marburg virus-like particles protect guinea pigs from lethal Marburg virus infection. Vaccine, 22(25-26), 3495-3502 (2004

  15. Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3

    PubMed Central

    Iverson, Eric A.; Goodman, David A.; Gorchels, Madeline E.

    2017-01-01

    ABSTRACT Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3, allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure

  16. Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

    PubMed

    Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

    2017-05-15

    Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of

  17. Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells.

    PubMed

    Zhu, Yali; Cherukuri, Nil Celebi; Jackel, Jamie N; Wu, Zetang; Crary, Monica; Buckley, Kenneth J; Bisaro, David M; Parris, Deborah S

    2012-03-01

    Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

  18. Molecular docking and simulation studies of 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 against VP26 and VP28 proteins of white spot syndrome virus.

    PubMed

    Sudharsana, S; Rajashekar Reddy, C B; Dinesh, S; Rajasekhara Reddy, S; Mohanapriya, A; Itami, T; Sudhakaran, R

    2016-10-01

    White spot syndrome virus (WSSV), an aquatic virus infecting shrimps and other crustaceans, is widely distributed in Asian subcontinents including India. The infection has led to a serious economic loss in shrimp farming. The WSSV genome is approximately 300 kb and codes for several proteins mediating the infection. The envelope proteins VP26 and VP28 play a major role in infection process and also in the interaction with the host cells. A comprehensive study on the viral proteins leading to the development of safe and potent antiviral therapeutic is of adverse need. The novel synthesized compound 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 is proved to have potent antiviral activity against WSSV. The compound antiviral activity is validated in freshwater crabs (Paratelphusa hydrodomous). An in silico molecular docking and simulation analysis of the envelope proteins VP26 and VP28 with the ligand 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 are carried out. The docking analysis reveals that the polar amino acids in the pore region of the envelope proteins were involved in the ligand binding. The influence of the ligand binding on the proteins is validated by the molecular dynamics and simulation study. These in silico approaches together demonstrate the ligand's efficiency in preventing the trimers from exhibiting their physiological function. © 2016 John Wiley & Sons Ltd.

  19. Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

    PubMed Central

    Warfield, Kelly L.; Dye, John M.; Wells, Jay B.; Unfer, Robert C.; Holtsberg, Frederick W.; Shulenin, Sergey; Vu, Hong; Swenson, Dana L.; Bavari, Sina; Aman, M. Javad

    2015-01-01

    Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components. PMID:25793502

  20. Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses.

    PubMed

    Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fuller, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

    2006-07-01

    Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.

  1. A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease.

    PubMed

    Alfson, Kendra J; Avena, Laura E; Delgado, Jenny; Beadles, Michael W; Patterson, Jean L; Carrion, Ricardo; Griffiths, Anthony

    2018-01-01

    Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA's Animal Efficacy Rule, which requires well-characterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low- and high-passage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo . These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures. IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV

  2. A single phosphorodiamidate morpholino oligomer targeting VP24 protects rhesus monkeys against lethal Ebola virus infection.

    PubMed

    Warren, Travis K; Whitehouse, Chris A; Wells, Jay; Welch, Lisa; Heald, Alison E; Charleston, Jay S; Sazani, Pete; Reid, St Patrick; Iversen, Patrick L; Bavari, Sina

    2015-02-10

    Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMOplus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMOplus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures. Several West African countries are currently being ravaged by an outbreak of Ebola virus (EBOV) that has become a major epidemic affecting not only these African countries but also Europe and the United States. A better understanding of the mechanism of virulence of EBOV is important for the development of effective treatments, as no licensed treatments or vaccines for EBOV disease are currently available. This study of phosphorodiamidate morpholino oligomers (PMOs) targeting the mRNAs of two different EBOV proteins, alone and in combination, demonstrated that targeting a single protein was effective at conferring a significant survival benefit in an EBOV lethal primate model. Future development of PMOs with efficacy against EBOV will be simplified if only one PMO is required instead of a combination, particularly in terms of regulatory approval. Copyright © 2015 Warren et al.

  3. Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

    PubMed Central

    Tornow, J; Polvino-Bodnar, M; Santangelo, G; Cole, C N

    1985-01-01

    The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain. Images PMID:2982029

  4. Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings.

    PubMed

    Wang, Jianzhong; Cong, Yanlong; Yin, Renfu; Feng, Na; Yang, Songtao; Xia, Xianzhu; Xiao, Yueqiang; Wang, Wenxiu; Liu, Xiufan; Hu, Shunlin; Ding, Chan; Yu, Shengqing; Wang, Chunfeng; Ding, Zhuang

    2015-05-04

    Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Complete protection of cats against feline panleukopenia virus challenge by a recombinant canine adenovirus type 2 expressing VP2 from FPV.

    PubMed

    Yang, Songtao; Xia, Xianzhu; Qiao, Jun; Liu, Quan; Chang, Shuang; Xie, Zhijing; Ju, Huiyan; Zou, Xiaohuan; Gao, Yuwei

    2008-03-10

    Feline panleukopenia virus (FPV) is an important infectious pathogen of all members of the family Felidae. Here, we describe construction of a replication-competent recombinant canine adenovirus type 2 (CAV-2) expressing the VP2 protein of FPV (CAV-2-VP2) by transfection of MDCK cells with recombinant CAV-2 genome carrying a VP2 expression cassette. Ten 3-month-old cats were vaccinated with the recombinant virus with two boosters at 15-day intervals. All cats developed neutralizing antibodies of titers 1:16-1:32 by day 15 post-primary vaccination, increasing to 1:64-1:128 by day 45. Examination for clinical signs and viral presence, and total white blood cell counts in peripheral blood following FPV challenge, showed that all were completely protected. This recombinant virus appears to provide an effective alternative to attenuated and inactivated vaccines in immunizing cats against feline panleukopenia.

  6. [Recombinant Vp2 protein of infectious bursal disease virus AH1 strain expressed in insect cells: a vaccine candidate].

    PubMed

    Ouyang, Wei; Wang, Yongshan; Zhou, Yu; Zhang, Haibin; Tang, Yude

    2010-05-01

    Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus (IBDV). Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA), which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) and "inclusion body-like"structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2 x 10(3) and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8 x 10(2) and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control (P < 0.05). The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate

  7. Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

    SciTech Connect

    Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.

    2009-09-01

    White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed thatmore » rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.« less

  8. Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

    NASA Technical Reports Server (NTRS)

    Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

  9. Structural insights into the multifunctional protein VP3 of birnaviruses.

    PubMed

    Casañas, Arnau; Navarro, Aitor; Ferrer-Orta, Cristina; González, Dolores; Rodríguez, José F; Verdaguer, Núria

    2008-01-01

    Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most harmful poultry diseases. The IBDV genome encodes five mature proteins; of these, the multifunctional protein VP3 plays an essential role in virus morphogenesis. This protein, which interacts with the structural protein VP2, with the double-stranded RNA genome, and with the virus-encoded, RNA-dependent RNA polymerase, VP1, is involved not only in the formation of the viral capsid, but also in the recruitment of VP1 into the capsid and in the encapsidation of the viral genome. Here, we report the X-ray structure of the central region of VP3, residues 92-220, consisting of two alpha-helical domains connected by a long and flexible hinge that are organized as a dimer. Unexpectedly, the overall fold of the second VP3 domain shows significant structural similarities with different transcription regulation factors.

  10. Identification of a novel NLS of herpes simplex virus type 1 (HSV-1) VP19C and its nuclear localization is required for efficient production of HSV-1.

    PubMed

    Li, You; Zhao, Lei; Wang, Shuai; Xing, Junji; Zheng, Chunfu

    2012-09-01

    Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of two copies of VP23 and one copy of VP19C. Here, we identified a non-classical NLS of VP19C between aa 50 and 61, and the nuclear import of VP19C was mediated by RanGTP and importin β1-, but not importin α5-, dependent pathway. Additionally, recombinant virus harbouring this NLS mutation (NLSm) replicates less efficiently as wild-type. These data strongly suggested that the nuclear import of VP19C is required for efficient HSV-1 production.

  11. The study of the intercellular trafficking of the fusion proteins of herpes simplex virus protein VP22.

    PubMed

    Xue, Xiaodong; Huang, Jianhua; Wang, Huishan

    2014-01-01

    Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification. Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot. VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components. The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP22-hBcl-xL and hBcl-xL-VP22 failed to

  12. The Study of the Intercellular Trafficking of the Fusion Proteins of Herpes Simplex Virus Protein VP22

    PubMed Central

    Xue, Xiaodong; Huang, Jianhua; Wang, Huishan

    2014-01-01

    Background Genetic modifications can improve the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in myocardial infarction. However, so far, the efficiency of MSC modification is very low. Seeking for a more efficient way of MSC modification, we investigated the possibility of employing the intercellular trafficking capacity of the herpes simplex virus type-1 tegument protein VP22 on the enhancement of MSC modification. Methods Plasmids pVP22-myc, pVP22-EGFP, pEGFP-VP22, pVP22-hBcl-xL and phBcl-xL-VP22 were constructed for the expressions of the myc-tagged VP22 and the fusion proteins VP22-EGFP, EGFP-VP22, VP22-hBcl-xL and hBcl-xL-VP22. MSCs were isolated from rat bone marrow and the surface markers were identified by Flowcytometry. COS-1 cells were transfected with the above plasmids and co-cultured with untransfected MSCs, the intercellular transportations of the constructed proteins were studied by immunofluorescence. The solubility of VP22-hBcl-xL and hBcl-xL-VP22 was analyzed by Western blot. Results VP22-myc could be expressed in and spread between COS-1 cells, which indicates the validity of our VP22 expression construct. Flowcytometry analysis revealed that the isolated MSCs were CD29, CD44, and CD90 positive and were negative for the hematopoietic markers, CD34 and CD45. The co-culturing and immunofluorescence assay showed that VP22-myc, VP22-EGFP and EGFP-VP22 could traffic between COS-1 cells and MSCs, while the evidence of intercellular transportation of VP22-hBcl-xL and hBcl-xL-VP22 was not detected. Western blot analysis showed that VP22-hBcl-xL and hBcl-xL-VP22 were both insoluble in the cell lysate suggesting interactions of the fusion proteins with other cellular components. Conclusions The intercellular trafficking of VP22-myc, VP22-EGFP and EGFP-VP22 between COS-1 cells and MSCs presents an intriguing prospect in the therapeutic application of VP22 as a delivery vehicle which enhances genetic modifications of MSCs. However, VP

  13. Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

    PubMed

    Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

    2002-12-01

    Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

  14. Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

    SciTech Connect

    Geiss, Brian J.; Cano, Gina L.; Tavis, John E.

    2004-12-05

    Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22.more » Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.« less

  15. A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

    PubMed Central

    Alfson, Kendra J.; Avena, Laura E.; Delgado, Jenny; Beadles, Michael W.; Patterson, Jean L.; Carrion, Ricardo

    2018-01-01

    ABSTRACT Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA’s Animal Efficacy Rule, which requires well-characterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low- and high-passage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo. These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures. IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages

  16. A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

    PubMed

    Qi, Xiaole; Gao, Xiang; Lu, Zhen; Zhang, Lizhou; Wang, Yongqiang; Gao, Li; Gao, Yulong; Li, Kai; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Wang, Xiaomei

    2016-07-01

    To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

  17. Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications

    PubMed Central

    2013-01-01

    Background Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Results Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other

  18. Insights into the homo-oligomerization properties of N-terminal coiled-coil domain of Ebola virus VP35 protein.

    PubMed

    Ramaswamy, Venkata Krishnan; Di Palma, Francesco; Vargiu, Attilio V; Corona, Angela; Piano, Dario; Ruggerone, Paolo; Zinzula, Luca; Tramontano, Enzo

    2018-03-02

    The multifunctional Ebola virus (EBOV) VP35 protein is a key determinant of virulence. VP35 is essential for EBOV replication, is a component of the viral RNA polymerase and participates in nucleocapsid formation. Furthermore, VP35 contributes to EBOV evasion of the host innate immune response by suppressing RNA silencing and blocking RIG-I like receptors' pathways that lead to type I interferon (IFN) production. VP35 homo-oligomerization has been reported to be critical for its replicative function and to increase its IFN-antagonism properties. Moreover, homo-oligomerization is mediated by a predicted coiled-coil (CC) domain located within its N-terminal region. Here we report the homo-oligomerization profile of full-length recombinant EBOV VP35 (rVP35) assessed by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Based on our biochemical results and in agreement with previous experimental observations, we have built an in silico 3D model of the so-far structurally unsolved EBOV VP35 CC domain and performed self-assembly homo-oligomerization simulations by means of molecular dynamics. Our model advances the understanding of how VP35 may associate in different homo-oligomeric species, a crucial process for EBOV replication and pathogenicity. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Development and characterization of a guinea pig model for Marburg virus.

    PubMed

    Wong, Gary; Cao, Wen-Guang; He, Shi-Hua; Zhang, Zi-Rui; Zhu, Wen-Jun; Moffat, Estella; Ebihara, Hideki; Embury-Hyatt, Carissa; Qiu, Xiang-Guo

    2018-01-18

    The Angolan strain of Marburg virus (MARV/Ang) can cause lethal disease in humans with a case fatality rate of up to 90%, but infection of immunocompetent rodents do not result in any observable symptoms. Our previous work includes the development and characterization of a MARV/Ang variant that can cause lethal disease in mice (MARV/Ang-MA), with the aim of using this tool to screen for promising prophylactic and therapeutic candidates. An intermediate animal model is needed to confirm any findings from mice studies before testing in the gold-standard non-human primate (NHP) model. In this study, we serially passaged the clinical isolate of MARV/Ang in the livers and spleens of guinea pigs until a variant emerged that causes 100% lethality in guinea pigs (MARV/Ang-GA). Animals infected with MARV/Ang-GA showed signs of filovirus infection including lymphocytopenia, thrombocytopenia, and high viremia leading to spread to major organs, including the liver, spleen, lungs, and kidneys. The MARV/Ang-GA guinea pigs died between 7-9 days after infection, and the LD 50 was calculated to be 1.1×10 -1 TCID 50 (median tissue culture infective dose). Mutations in MARV/Ang-GA were identified and compared to sequences of known rodent-adapted MARV/Ang variants, which may benefit future studies characterizing important host adaptation sites in the MARV/Ang viral genome.

  20. Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

    PubMed

    Zhang, Ruihua; Zhou, Guomei; Xin, Yinghao; Chen, Junhao; Lin, Shaoli; Tian, Ye; Xie, Zhijing; Jiang, Shijin

    2015-11-18

    Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Application of bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2.

    PubMed

    Feenstra, Femke; Pap, Janny S; van Rijn, Piet A

    2015-02-04

    Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks can be controlled by vaccination, however, currently available vaccines have several drawbacks. Further, there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based on live-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infectious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotype determining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectable viremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1 for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects on virus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimeric VP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1, expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutralizing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchange with the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA vaccine with 1/16 chimeric VP2 containing amino acid region 249-398 of serotype 16 raised antibodies in sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for both parental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccine platform for several serotypes and further extend the application for serotypes that are unsuccessful in single VP2 exchange. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Membrane insertion of fusion peptides from Ebola and Marburg viruses studied by replica-exchange molecular dynamics simulations.

    PubMed

    Olson, Mark A; Lee, Michael S; Yeh, In-Chul

    2017-06-15

    This work presents replica-exchange molecular dynamics simulations of inserting a 16-residue Ebola virus fusion peptide into a membrane bilayer. A computational approach is applied for modeling the peptide at the explicit all-atom level and the membrane-aqueous bilayer by a generalized Born continuum model with a smoothed switching function (GBSW). We provide an assessment of the model calculations in terms of three metrics: (1) the ability to reproduce the NMR structure of the peptide determined in the presence of SDS micelles and comparable structural data on other fusion peptides; (2) determination of the effects of the mutation Trp-8 to Ala and sequence discrimination of the homologous Marburg virus; and (3) calculation of potentials of mean force for estimating the partitioning free energy and their comparison to predictions from the Wimley-White interfacial hydrophobicity scale. We found the GBSW implicit membrane model to produce results of limited accuracy in conformational properties of the peptide when compared to the NMR structure, yet the model resolution is sufficient to determine the effect of sequence differentiation on peptide-membrane integration. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Antibody Responses to Marburg Virus in Egyptian Rousette Bats and Their Role in Protection against Infection.

    PubMed

    Storm, Nadia; Jansen Van Vuren, Petrus; Markotter, Wanda; Paweska, Janusz T

    2018-02-10

    Egyptian rousette bats (ERBs) are reservoir hosts for the Marburg virus (MARV). The immune dynamics and responses to MARV infection in ERBs are poorly understood, and limited information exists on the role of antibodies in protection of ERBs against MARV infection. Here, we determine the duration of maternal immunity to MARV in juvenile ERBs, and evaluate the duration of the antibody response to MARV in bats naturally or experimentally infected with the virus. We further explore whether antibodies in previously naturally exposed bats is fully protective against experimental reinfection with MARV. Maternal immunity was lost in juvenile ERBs by 5 months of age. Antibodies to MARV remained detectable in 67% of experimentally infected bats approximately 4 months post inoculation (p.i.), while antibodies to MARV remained present in 84% of naturally exposed bats at least 11 months after capture. Reinfection of seropositive ERBs with MARV produced an anamnestic response from day 5 p.i. Although PCR-defined viremia was present in 73.3% of reinfected ERBs, replicating virus was recovered from the serum of only one bat on day 3 p.i. The negative PCR results in the salivary glands, intestines, bladders and reproductive tracts of reinfected bats, and the apparent absence of MARV in the majority of swabs collected from these bats suggest that reinfection may only play a minor role in the transmission and maintenance of MARV amongst ERBs in nature.

  4. Marine derived compounds as binders of the White spot syndrome virus VP28 envelope protein: In silico insights from molecular dynamics and binding free energy calculations.

    PubMed

    Sivakumar, K C; Sajeevan, T P; Bright Singh, I S

    2016-10-01

    White spot syndrome virus (WSSV) remains as one of the most dreadful pathogen of the shrimp aquaculture industry owing to its high virulence. The cumulative mortality reaches up to 100% within in 2-10days in a shrimp farm. Currently, no chemotherapeutics are available to control WSSV. The viral envelope protein, VP28, located on the surface of the virus particle acts as a vital virulence factor in the initial phases of inherent WSSV infection in shrimp. Hence, inhibition of envelope protein VP28 could be a novel way to deal with infection by inhibiting its interaction in the endocytic pathway. In this direction, a timely attempt was made to recognize a potential drug candidate of marine origin against WSSV using VP28 as a target by employing in silico docking and molecular dynamic simulations. A virtual library of 388 marine bioactive compounds was extracted from reports published in Marine Drugs. The top ranking compounds from docking studies were chosen from the flexible docking based on the binding affinities (ΔGb). In addition, the MD simulation and binding free energy analysis were implemented to validate and capture intermolecular interactions. The results suggested that the two compounds obtained a negative binding free energy with -40.453kJ/mol and -31.031kJ/mol for compounds with IDs 30797199 and 144162 respectively. The RMSD curve indicated that 30797199 moves into the hydrophobic core, while the position of 144162 atoms changes abruptly during simulation and is mostly stabilized by water bridges. The shift in RMSD values of VP28 corresponding to ligand RMSD gives an insight into the ligand induced conformational changes in the protein. This study is first of its kind to elucidate the explicit binding of chemical inhibitor to WSSV major structural protein VP28. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Protection against Foot-and-Mouth Disease Virus in Guinea Pigs via Oral Administration of Recombinant Lactobacillus plantarum Expressing VP1

    PubMed Central

    Wang, Miao; Pan, Li; Zhou, Peng; Lv, Jianliang; Zhang, Zhongwang; Wang, Yonglu; Zhang, Yongguang

    2015-01-01

    Mucosal vaccination is an effective strategy for generating antigen-specific immune responses against mucosal infections of foot-and-mouth disease virus (FMDV). In this study, Lactobacillus plantarum strains NC8 and WCFS1 were used as oral delivery vehicles containing a pSIP411-VP1 recombinant plasmid to initiate mucosal and systemic immune responses in guinea pigs. Guinea pigs were orally vaccinated (three doses) with NC8-pSIP411, NC8-pSIP411-VP1, WCFS1-pSIP411, WCFS1-pSIP411-VP1 or milk. Animals immunized with NC8-pSIP411-VP1 and WCFS1-pSIP411-VP1 developed high levels of antigen-specific serum IgG, IgA, IgM, mucosal secretory IgA (sIgA) and neutralizing antibodies, and revealed stronger cell-mediated immune responses and enhanced protection against FMDV challenge compared with control groups. The recombinant pSIP411-VP1 effectively improved immunoprotection against FMDV in guinea pigs. PMID:26629822

  6. Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations

    PubMed Central

    Kuhn, Jens H.; Becker, Stephan; Ebihara, Hideki; Geisbert, Thomas W.; Johnson, Karl M.; Kawaoka, Yoshihiro; Lipkin, W. Ian; Negredo, Ana I.; Netesov, Sergey V.; Nichol, Stuart T.; Palacios, Gustavo; Peters, Clarence J.; Tenorio, Antonio; Volchkov, Viktor E.; Jahrling, Peter B.

    2011-01-01

    The taxonomy of the family Filoviridae (marburgviruses and ebolaviruses) has changed several times since the discovery of its members, resulting in a plethora of species and virus names and abbreviations. The current taxonomy has only been partially accepted by most laboratory virologists. Confusion likely arose for several reasons: species names that consist of several words or which (should) contain diacritical marks, the current orthographic identity of species and virus names, and the similar pronunciation of several virus abbreviations in the absence of guidance for the correct use of vernacular names. To rectify this problem, we suggest (1) to retain the current species names Reston ebolavirus, Sudan ebolavirus, and Zaire ebolavirus, but to replace the name Cote d'Ivoire ebolavirus [sic] with Taï Forest ebolavirus and Lake Victoria marburgvirus with Marburg marburgvirus; (2) to revert the virus names of the type marburgviruses and ebolaviruses to those used for decades in the field (Marburg virus instead of Lake Victoria marburgvirus and Ebola virus instead of Zaire ebolavirus); (3) to introduce names for the remaining viruses reminiscent of jargon used by laboratory virologists but nevertheless different from species names (Reston virus, Sudan virus, Taï Forest virus), and (4) to introduce distinct abbreviations for the individual viruses (RESTV for Reston virus, SUDV for Sudan virus, and TAFV for Taï Forest virus), while retaining that for Marburg virus (MARV) and reintroducing that used over decades for Ebola virus (EBOV). Paying tribute to developments in the field, we propose (a) to create a new ebolavirus species (Bundibugyo ebolavirus) for one member virus (Bundibugyo virus, BDBV); (b) to assign a second virus to the species Marburg marburgvirus (Ravn virus, RAVV) for better reflection of now available high-resolution phylogeny; and (c) to create a new tentative genus (Cuevavirus) with one tentative species (Lloviu cuevavirus) for the recently

  7. Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations.

    PubMed

    Kuhn, Jens H; Becker, Stephan; Ebihara, Hideki; Geisbert, Thomas W; Johnson, Karl M; Kawaoka, Yoshihiro; Lipkin, W Ian; Negredo, Ana I; Netesov, Sergey V; Nichol, Stuart T; Palacios, Gustavo; Peters, Clarence J; Tenorio, Antonio; Volchkov, Viktor E; Jahrling, Peter B

    2010-12-01

    The taxonomy of the family Filoviridae (marburgviruses and ebolaviruses) has changed several times since the discovery of its members, resulting in a plethora of species and virus names and abbreviations. The current taxonomy has only been partially accepted by most laboratory virologists. Confusion likely arose for several reasons: species names that consist of several words or which (should) contain diacritical marks, the current orthographic identity of species and virus names, and the similar pronunciation of several virus abbreviations in the absence of guidance for the correct use of vernacular names. To rectify this problem, we suggest (1) to retain the current species names Reston ebolavirus, Sudan ebolavirus, and Zaire ebolavirus, but to replace the name Cote d'Ivoire ebolavirus [sic] with Taï Forest ebolavirus and Lake Victoria marburgvirus with Marburg marburgvirus; (2) to revert the virus names of the type marburgviruses and ebolaviruses to those used for decades in the field (Marburg virus instead of Lake Victoria marburgvirus and Ebola virus instead of Zaire ebolavirus); (3) to introduce names for the remaining viruses reminiscent of jargon used by laboratory virologists but nevertheless different from species names (Reston virus, Sudan virus, Taï Forest virus), and (4) to introduce distinct abbreviations for the individual viruses (RESTV for Reston virus, SUDV for Sudan virus, and TAFV for Taï Forest virus), while retaining that for Marburg virus (MARV) and reintroducing that used over decades for Ebola virus (EBOV). Paying tribute to developments in the field, we propose (a) to create a new ebolavirus species (Bundibugyo ebolavirus) for one member virus (Bundibugyo virus, BDBV); (b) to assign a second virus to the species Marburg marburgvirus (Ravn virus, RAVV) for better reflection of now available high-resolution phylogeny; and (c) to create a new tentative genus (Cuevavirus) with one tentative species (Lloviu cuevavirus) for the recently

  8. Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

    PubMed Central

    Adu-Gyamfi, Emmanuel; Johnson, Kristen A.; Fraser, Mark E.; Scott, Jordan L.; Soni, Smita P.; Jones, Keaton R.; Digman, Michelle A.; Gratton, Enrico; Tessier, Charles R.

    2015-01-01

    ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. PMID

  9. Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

    PubMed

    Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

    2015-09-01

    Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

  10. Ebola Virus and Marburg Virus

    MedlinePlus

    ... chimps and fruit bats in Africa. Transmission from animals to humans Experts suspect that both viruses are transmitted to humans through an infected animal's bodily fluids. Examples include: Blood. Butchering or eating ...

  11. Evolutionary Analysis of Structural Protein Gene VP1 of Foot-and-Mouth Disease Virus Serotype Asia 1

    PubMed Central

    Zhang, Qingxun; Liu, Xinsheng; Fang, Yuzhen; Pan, Li; Lv, Jianliang; Zhang, Zhongwang; Zhou, Peng; Ding, Yaozhong; Chen, Haotai; Shao, Junjun; Zhao, Furong; Lin, Tong; Chang, Huiyun; Zhang, Jie; Wang, Yonglu; Zhang, Yongguang

    2015-01-01

    Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I–VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10−3 substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown. PMID:25793223

  12. Ebola Virus Neutralizing Antibodies Detectable in Survivors of theYambuku, Zaire Outbreak 40 Years after Infection.

    PubMed

    Rimoin, Anne W; Lu, Kai; Bramble, Matthew S; Steffen, Imke; Doshi, Reena H; Hoff, Nicole A; Mukadi, Patrick; Nicholson, Bradly P; Alfonso, Vivian H; Olinger, Gerrard; Sinai, Cyrus; Yamamoto, Lauren K; Ramirez, Christina M; Okitolonda Wemakoy, Emile; Kebela Illunga, Benoit; Pettitt, James; Logue, James; Bennett, Richard S; Jahrling, Peter; Heymann, David L; Piot, Peter; Muyembe-Tamfum, Jean Jacques; Hensley, Lisa E; Simmons, Graham

    2018-01-04

    The first reported outbreak of Ebola virus disease occurred in 1976 in Yambuku, Democratic Republic of Congo. Antibody responses in survivors 11 years after infection have been documented. However, this report is the first characterization of anti-Ebola virus antibody persistence and neutralization capacity 40 years after infection. Using ELISAs we measured survivor's immunological response to Ebola virus Zaire (EBOV) glycoprotein and nucleoprotein, and assessed VP40 reactivity. Neutralization of EBOV was measured using a pseudovirus approach and plaque reduction neutralization test with live EBOV. Some survivors from the original EBOV outbreak still harbor antibodies against all 3 measures. Interestingly, a subset of these survivors' serum antibodies could still neutralize live virus 40 years postinitial infection. These data provide the longest documentation of both anti-Ebola serological response and neutralization capacity within any survivor cohort, extending the known duration of response from 11 years postinfection to at least 40 years after symptomatic infection. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  13. [Protective immune response of guinea pigs against challenge with foot and mouth disease virus by immunization with foliar extracts from transgenic tomato plants expressing the FMDV structural protein VP1].

    PubMed

    Pan, Li; Zhang, Yong-Guang; Wang, Yong-Lu; Wang, Bao-Qin; Xie, Qing-Ge

    2006-10-01

    The plant constitutive expression vector pBin438/VP1 for VP1 gene of foot-and-mouth disease virus strain O/ China/99 was constructed. Mediated with Agrobacterium tumefaciens GV3101 harboring pBin438/VP1, VP1 gene was transferred into cotyledons of tomato. After selected by Kanamysin, sixty resistant lines were obtained. The integration and transcription of the VP1 gene in transformed plants was detected by PCR and RT-PCR. After being detected by sandwich-ELISA assays, about 40% transformed plants confirmed to express the recombinant protein. The leave extracts of two positive lines were respectively emulsified in Freund's adjuvant and guinea pigs were intramuscular inoculation at days 0, 15 and 30d. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID50 FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants. Experimental results showed that the plant expression vector was successfully constructed. PCR and RT-PCR analyses confirmed VP1 gene was transformed into tomato plants and got expression at the transcription levels. The expressed VP1 protein of FMDV, which was identified by ELISA and Western blot, can be specifically recognized by polyclonal antibodies against FMDV. Indirect-ELISA antibody titers reached 1:64 twenty-one days after the third inoculation. In the challenge test, the protection against FMDV challenge in two groups was 80% and 40% respectively. The immunization test in guinea pigs indicated that the expression product of transgenic tomato plants had immunogenicity and could effectively induce the specific antibodies against FMDV.

  14. T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity

    PubMed Central

    Franssila, R; Auramo, J; Modrow, S; Möbs, M; Oker-Blom, C; Käpylä, P; Söderlund-Venermo, M; Hedman, K

    2005-01-01

    Human parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-γ) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactivity with VP1u was determined specifically with a prokaryotically expressed VP1u antigen. In general, B19-specific IFN-γ responses were stronger than IL-10 responses in both recent and remote infection; however, IL-10 responses were readily detectable among both groups, with the exception of patients with relapsed or persisting symptoms who showed strikingly low IL-10 responses. Whereas VP1u-specific IFN-γ responses were very strong among the recently infected subjects, the VP1u-specific IFN-γ and IL-10 responses were virtually absent among the remotely infected subjects. The disappearance of VP1u-specific IFN-γ expression is surprising, as B-cell immunity against VP1u is well maintained. PMID:16178856

  15. Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

    PubMed

    Biswal, Jitendra K; Das, Biswajit; Sharma, Gaurav K; Khulape, Sagar A; Pattnaik, Bramhadev

    2016-04-01

    Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142 → R or H142 → F or H142 → A substitutions resulted in non-infectious FMDV, H142 → D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

  16. Case definition for Ebola and Marburg haemorrhagic fevers: a complex challenge for epidemiologists and clinicians.

    PubMed

    Pittalis, Silvia; Fusco, Francesco Maria; Lanini, Simone; Nisii, Carla; Puro, Vincenzo; Lauria, Francesco Nicola; Ippolito, Giuseppe

    2009-10-01

    Viral haemorrhagic fevers (VHFs) represent a challenge for public health because of their epidemic potential, and their possible use as bioterrorism agents poses particular concern. In 1999 the World Health Organization (WHO) proposed a case definition for VHFs, subsequently adopted by other international institutions with the aim of early detection of initial cases/outbreaks in western countries. We applied this case definition to reports of Ebola and Marburg virus infections to estimate its sensitivity to detect cases of the disease. We analyzed clinical descriptions of 795 reported cases of Ebola haemorrhagic fever: only 58.5% of patients met the proposed case definition. A similar figure was obtained reviewing 169 cases of Marburg diseases, of which only 64.5% were in accordance with the case definition. In conclusion, the WHO case definition for hemorrhagic fevers is too specific and has poor sensitivity both for case finding during Ebola or Marburg outbreaks, and for early detection of suspected cases in western countries. It can lead to a hazardous number of false negatives and its use should be discouraged for early detection of cases.

  17. Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques.

    PubMed

    Warren, Travis K; Whitehouse, Chris A; Wells, Jay; Welch, Lisa; Charleston, Jay S; Heald, Alison; Nichols, Donald K; Mattix, Marc E; Palacios, Gustavo; Kugleman, Jeffrey R; Iversen, Patrick L; Bavari, Sina

    2016-02-01

    Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83-100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI.

  18. De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia.

    PubMed

    Sawtell, Nancy M; Thompson, Richard L

    2016-09-01

    The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.

  19. De Novo Herpes Simplex Virus VP16 Expression Gates a Dynamic Programmatic Transition and Sets the Latent/Lytic Balance during Acute Infection in Trigeminal Ganglia

    PubMed Central

    Sawtell, Nancy M.; Thompson, Richard L.

    2016-01-01

    The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system. PMID:27607440

  20. Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

    PubMed

    Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

    2016-08-28

    The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

  1. Conformational plasticity of the Ebola virus matrix protein.

    PubMed

    Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

    2014-11-01

    Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. © 2014 The Protein Society.

  2. Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus.

    PubMed

    Martínez-Torrecuadrada, J L; Casal, J I

    1995-07-10

    Overlapping fragments of the outermost capsid protein VP2 of African horse sickness virus serotype 4 (AHSV-4) have been expressed in Escherichia coli. Horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for AHSV were used to screen these fragments for antigenic regions. The screening revealed that the major antigenic domain of the AHSV-4 VP2 is localized in a central region (amino acids 200 to 413) and that both the N-terminal region (aa 1-159) and the half C-terminal region (aa 414-1060) are not immunogenic. All the fragments containing a region between amino acids 253 and 413 (fragment H) were able to elicit consistently high titers of neutralizing antibodies. The ability of several subfragments of this region to evoke neutralizing antibodies indicates the presence of several sites inside this domain. However, neutralizing antibodies in sera of horse infected or vaccinated with attenuated viruses were not absorbed by fragment H, indicating that this domain is not immunodominant in AHSV. This information might be useful in designing a subunit vaccine against AHSV infection.

  3. Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

    PubMed Central

    Graham, Kate L.; Halasz, Peter; Tan, Yan; Hewish, Marilyn J.; Takada, Yoshikazu; Mackow, Erich R.; Robinson, Martyn K.; Coulson, Barbara S.

    2003-01-01

    Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection. PMID:12941907

  4. Expression and immunogenic analysis of recombinant polypeptides derived from capsid protein VP1 for developing subunit vaccine material against hepatitis A virus.

    PubMed

    Jang, Kyoung Ok; Park, Jong-Hwa; Lee, Hyun Ho; Chung, Dae Kyun; Kim, Wonyong; Chung, In Sik

    2014-08-01

    Three recombinant polypeptides, VP1-His, VP1-3N-His, and 3D2-His, were produced by Escherichia coli expression system. Recombinant VP1-His, VP1-3N-His, and 3D2-His were expressed as bands with molecular weights of 32, 38, and 30 kDa, respectively. These were purified by affinity chromatography using Ni-NTA Fast-flow resin and/or ion-exchange chromatography using DEAE-Sepharose Fast-flow resin. Intraperitoneal immunizations of recombinant polypeptides successfully elicited the productions of VP1-His, VP1-3N-His, and 3D2-His specific IgG antibodies (IgG subclass distribution of IgG1>IgG2a>IgG2b>IgG3) in sera and induced the secretions of cytokines IFN-γ and IL-6 in spleen cells. Sera from recombinant VP1-His-, VP1-3N-His-, and 3D2-His-immunized mice neutralized the propagation of HAV. The highest neutralizing activity was shown in sera from recombinant VP1-3N-His-immunized mice. These results suggest that recombinant VP1-3N-His can be a useful source for developing hepatitis A virus (HAV) subunit vaccine candidates. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.) fruit by expression of capsid proteins VP2 and VP6 and immunological studies.

    PubMed

    Saldaña, Sergio; Esquivel Guadarrama, Fernando; Olivera Flores, Teresa De Jesús; Arias, Nancy; López, Susana; Arias, Carlos; Ruiz-Medrano, Roberto; Mason, Hugh; Mor, Tsafrir; Richter, Liz; Arntzen, Charles J; Gómez Lim, Miguel A

    2006-01-01

    A number of different antigens have been successfully expressed in transgenic plants, and some are currently being evaluated as orally delivered vaccines. Here we report the successful expression of rotavirus capsid proteins VP2 and VP6 in fruits of transgenic tomato plants. By western blot analysis, using specific antibodies, we determined that the VP2 and VP6 produced in plants have molecular weights similar to those found in native rotavirus. The plant-synthesized VP6 protein retained the capacity to form trimers. We were able to recover rotavirus virus-like particles from tomato fruit (i.e., tomatoes) by centrifugation on a sucrose cushion and to visualize them by electron microscopy. This result indicated that VP2/VP6 can self-assemble into virus-like particles (VLPs) in plant cells, even though only a small proportion of VP2/VP6 assembled into VLPs. To investigate immunogenicity, adult mice were immunized intraperitoneally (i.p.) three times with a protein extract from a transgenic tomatoes in adjuvant. We found that the transgenic tomato extract induced detectable levels of anti-rotavirus antibodies in serum; however, we did not determine the contribution of either the free rotavirus proteins or the VLPs to the induction of the antibody response. These results suggest the potential of plant-based rotavirus VLPs for the development of a vaccine against rotavirus infection.

  6. Ecological Niche Modeling for Filoviruses: A Risk Map for Ebola and Marburg Virus Disease Outbreaks in Uganda.

    PubMed

    Nyakarahuka, Luke; Ayebare, Samuel; Mosomtai, Gladys; Kankya, Clovice; Lutwama, Julius; Mwiine, Frank Norbert; Skjerve, Eystein

    2017-09-05

    Uganda has reported eight outbreaks caused by filoviruses between 2000 to 2016, more than any other country in the world. We used species distribution modeling to predict where filovirus outbreaks are likely to occur in Uganda to help in epidemic preparedness and surveillance. The MaxEnt software, a machine learning modeling approach that uses presence-only data was used to establish filovirus - environmental relationships. Presence-only data for filovirus outbreaks were collected from the field and online sources. Environmental covariates from Africlim that have been downscaled to a nominal resolution of 1km x 1km were used. The final model gave the relative probability of the presence of filoviruses in the study area obtained from an average of 100 bootstrap runs. Model evaluation was carried out using Receiver Operating Characteristic (ROC) plots. Maps were created using ArcGIS 10.3 mapping software. We showed that bats as potential reservoirs of filoviruses are distributed all over Uganda. Potential outbreak areas for Ebola and Marburg virus disease were predicted in West, Southwest and Central parts of Uganda, which corresponds to bat distribution and previous filovirus outbreaks areas. Additionally, the models predicted the Eastern Uganda region and other areas that have not reported outbreaks before to be potential outbreak hotspots. Rainfall variables were the most important in influencing model prediction compared to temperature variables. Despite the limitations in the prediction model due to lack of adequate sample records for outbreaks, especially for the Marburg cases, the models provided risk maps to the Uganda surveillance system on filovirus outbreaks. The risk maps will aid in identifying areas to focus the filovirus surveillance for early detection and responses hence curtailing a pandemic. The results from this study also confirm previous findings that suggest that filoviruses are mainly limited by the amount of rainfall received in an area.

  7. Ecological Niche Modeling for Filoviruses: A Risk Map for Ebola and Marburg Virus Disease Outbreaks in Uganda

    PubMed Central

    Nyakarahuka, Luke; Ayebare, Samuel; Mosomtai, Gladys; Kankya, Clovice; Lutwama, Julius; Mwiine, Frank Norbert; Skjerve, Eystein

    2017-01-01

    Introduction: Uganda has reported eight outbreaks caused by filoviruses between 2000 to 2016, more than any other country in the world. We used species distribution modeling to predict where filovirus outbreaks are likely to occur in Uganda to help in epidemic preparedness and surveillance. Methods: The MaxEnt software, a machine learning modeling approach that uses presence-only data was used to establish filovirus – environmental relationships. Presence-only data for filovirus outbreaks were collected from the field and online sources. Environmental covariates from Africlim that have been downscaled to a nominal resolution of 1km x 1km were used. The final model gave the relative probability of the presence of filoviruses in the study area obtained from an average of 100 bootstrap runs. Model evaluation was carried out using Receiver Operating Characteristic (ROC) plots. Maps were created using ArcGIS 10.3 mapping software. Results: We showed that bats as potential reservoirs of filoviruses are distributed all over Uganda. Potential outbreak areas for Ebola and Marburg virus disease were predicted in West, Southwest and Central parts of Uganda, which corresponds to bat distribution and previous filovirus outbreaks areas. Additionally, the models predicted the Eastern Uganda region and other areas that have not reported outbreaks before to be potential outbreak hotspots. Rainfall variables were the most important in influencing model prediction compared to temperature variables. Conclusions: Despite the limitations in the prediction model due to lack of adequate sample records for outbreaks, especially for the Marburg cases, the models provided risk maps to the Uganda surveillance system on filovirus outbreaks. The risk maps will aid in identifying areas to focus the filovirus surveillance for early detection and responses hence curtailing a pandemic. The results from this study also confirm previous findings that suggest that filoviruses are mainly limited by

  8. [Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication].

    PubMed

    Huang, Yu; Zhu, Yumin; Dong, Shijuan; Yu, Ruisong; Zhang, Yuanshu; Li, Zhen

    2015-01-01

    New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

  9. High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli.

    PubMed

    Jung, Joon-Goo; Lee, Yong Jae; Velmurugan, Natarajan; Ko, Young-Joon; Lee, Hyang-Sim; Jeong, Ki Jun

    2013-07-01

    For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.

  10. Accelerating Biomedical Research in Designing Diagnostic Assays, Drugs, and Vaccines

    DTIC Science & Technology

    2010-10-01

    biodefense. For example, USAMRIID researchers are using Dovis to initiate drug discovery efforts against the ricin A-chain toxin and the Ebola virus...in host cell invasion and bacterial toxin production). Traditional experimental methods to determine the functions of proteins encoded in genomic...readily modeled. A second study involved determining the pro- tein structure of VP24, the smallest protein in the Ebola and Marburg virus genomes.9

  11. Enhancement of VP6 immunogenicity and protective efficacy against rotavirus by VP2 in a genetic immunization.

    PubMed

    Lopez-Guerrero, D V; Arias, N; Gutierrez-Xicotencatl, L; Chihu-Amparan, L; González, A; Pedroza-Saavedra, A; Rosas-Salgado, G; Villegas-Garcia, J C; Badillo-Godinez, O; Fernandez, G; Lopez, S; Esquivel-Guadarrama, F

    2018-05-24

    VP2/VP6 virus like particles (VLPs) are very effective in inducing protection against the rotavirus infection in animal models. Individually, VP6 can also induce protection. However, there is no information about the immunogenicity of VP2. The aim of this work was to evaluate the efficacy of DNA vaccines codifying for VP2 or VP6, alone or combined, to induce protection against the rotavirus infection. Murine rotavirus VP2 and VP6 genes were cloned into the pcDNA3 vector. Adult BALB/c mice were inoculated three times by intramuscular (i.m.) injections with 100 or 200µg of pcDNA3-VP2 or pcDNA3-VP6 alone or co-administered with 100µg of pcDNA3-VP2/100µg of pcDNA3-VP6. Two weeks after the last inoculation, mice were challenged with the wild type murine rotavirus strain epizootic diarrhea of infant mice (EDIM wt ). We found that both plasmids, pcDNA3-VP2 and pcDNA3-VP6, were able to induce rotavirus-specific serum antibodies, but not intestinal rotavirus-specific IgA; only 200µg of pcDNA3-VP6 induced 35% protection against the infection. A similar level of protection was found when mice were co-administered with 100µg of pcDNA3-VP2/100µg of pcDNA3-VP6 (1:1 ratio). However, the best protection (up to 58%) occurred when mice were inoculated with 10µg of pcDNA3-VP2/100µg of pcDNA3-VP6 (1:10 ratio). These results indicate that the DNA plasmid expressing VP6 is a better vaccine candidate that the one expressing VP2. However, when co-expressed, VP2 potentiates the immunogenicity and protective efficacy of VP6. Copyright © 2017. Published by Elsevier Ltd.

  12. Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus.

    PubMed

    Chen, Xiaowei; Song, Cailing; Liu, Yun; Qu, Liandong; Liu, Dafei; Zhang, Yun; Liu, Ming

    2016-02-01

    For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. VP1 amino acid residue 145 of enterovirus 71 is a key residue for its receptor attachment and resistance to neutralizing antibody during cynomolgus monkey infection.

    PubMed

    Fujii, Ken; Sudaka, Yui; Takashino, Ayako; Kobayashi, Kyousuke; Kataoka, Chikako; Suzuki, Tadaki; Iwata-Yoshikawa, Naoko; Kotani, Osamu; Ami, Yasushi; Shimizu, Hiroyuki; Nagata, Noriyo; Mizuta, Katsumi; Matsuzaki, Yoko; Koike, Satoshi

    2018-05-30

    Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G to E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence. IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some reports, VP1-145G/Q viruses were more frequently isolated from severely affected than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper, we showed that VP1-145E viruses are more virulent than VP1-145G viruses in

  14. Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

    SciTech Connect

    Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.

    2007-05-10

    The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associatedmore » with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.« less

  15. Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit.

    PubMed

    Mather, Stuart T; Wright, Edward; Scott, Simon D; Temperton, Nigel J

    2014-12-15

    Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

    PubMed

    Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

    2016-12-01

    Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

  17. Single-Injection Vaccine Protects Nonhuman Primates against Infection with Marburg Virus and Three Species of Ebola Virus▿

    PubMed Central

    Geisbert, Thomas W.; Geisbert, Joan B.; Leung, Anders; Daddario-DiCaprio, Kathleen M.; Hensley, Lisa E.; Grolla, Allen; Feldmann, Heinz

    2009-01-01

    The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (CIEBOV), and Marburgvirus (MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVΔG/SEBOVGP, VSVΔG/ZEBOVGP, and VSVΔG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVΔG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic filovirus species

  18. Development of a Sensitive and Specific Serological Assay Based on Luminex Technology for Detection of Antibodies to Zaire Ebola Virus

    PubMed Central

    Touré, Abdoulaye; Butel, Christelle; Keita, Alpha Kabinet; Binetruy, Florian; Sow, Mamadou S.; Foulongne, Vincent; Delaporte, Eric; Peeters, Martine

    2016-01-01

    ABSTRACT The recent Zaire Ebola virus (EBOV) outbreak in West Africa illustrates clearly the need for additional studies with humans and animals to elucidate the ecology of Ebola viruses (EBVs). In this study, we developed a serological assay based on the Luminex technology. Nine recombinant proteins representing different viral regions (nucleoprotein [NP], 40-kDa viral protein [VP40], and glycoprotein [GP]) from four of the five EBV lineages were used. Samples from 94 survivors of the EBOV outbreak in Guinea and negative samples from 108 patients in France were used to calculate test performance for EBOV detection and cross-reaction with other Ebola virus lineages. For EBOV antibody detection, sensitivities of 95.7%, 96.8%, and 92.5% and specificities of 94.4%, 95.4%, and 96.3% for NP, GP, and VP40, respectively, were observed. All EBOV-negative samples that presented a reaction, except for one, interacted with a single antigen, whereas almost all samples from EBOV survivors were simultaneously reactive with NP and GP (90/94) or with NP, GP, and VP40 (87/94). Considering as positive for past EBOV infection only samples that reacted with EBOV NP and GP, sensitivity was 95.7% and specificity increased to 99.1%. Comparing results with commercial EBOV NP and GP enzyme-linked immunosorbent assays (ELISAs; Alpha Diagnostic, San Antonio, TX), lower sensitivity (92.5%) and high specificity (100%) were observed with the same positivity criteria. Samples from EBOV survivors cross-reacted with GP from Sudan Ebola virus (GP-SUDV) (81.9%), GP from Bundibugyo Ebola virus (GP-BDBV) (51.1%), GP from Reston Ebola virus (GP-RESTV) (9.6%), VP40-SUDV (76.6%), and VP40-BDBV (38.3%). Overall, we developed a sensitive and specific high-throughput serological assay, and defined an algorithm, for epidemiological surveys with humans. PMID:27795350

  19. Development of a Sensitive and Specific Serological Assay Based on Luminex Technology for Detection of Antibodies to Zaire Ebola Virus.

    PubMed

    Ayouba, Ahidjo; Touré, Abdoulaye; Butel, Christelle; Keita, Alpha Kabinet; Binetruy, Florian; Sow, Mamadou S; Foulongne, Vincent; Delaporte, Eric; Peeters, Martine

    2017-01-01

    The recent Zaire Ebola virus (EBOV) outbreak in West Africa illustrates clearly the need for additional studies with humans and animals to elucidate the ecology of Ebola viruses (EBVs). In this study, we developed a serological assay based on the Luminex technology. Nine recombinant proteins representing different viral regions (nucleoprotein [NP], 40-kDa viral protein [VP40], and glycoprotein [GP]) from four of the five EBV lineages were used. Samples from 94 survivors of the EBOV outbreak in Guinea and negative samples from 108 patients in France were used to calculate test performance for EBOV detection and cross-reaction with other Ebola virus lineages. For EBOV antibody detection, sensitivities of 95.7%, 96.8%, and 92.5% and specificities of 94.4%, 95.4%, and 96.3% for NP, GP, and VP40, respectively, were observed. All EBOV-negative samples that presented a reaction, except for one, interacted with a single antigen, whereas almost all samples from EBOV survivors were simultaneously reactive with NP and GP (90/94) or with NP, GP, and VP40 (87/94). Considering as positive for past EBOV infection only samples that reacted with EBOV NP and GP, sensitivity was 95.7% and specificity increased to 99.1%. Comparing results with commercial EBOV NP and GP enzyme-linked immunosorbent assays (ELISAs; Alpha Diagnostic, San Antonio, TX), lower sensitivity (92.5%) and high specificity (100%) were observed with the same positivity criteria. Samples from EBOV survivors cross-reacted with GP from Sudan Ebola virus (GP-SUDV) (81.9%), GP from Bundibugyo Ebola virus (GP-BDBV) (51.1%), GP from Reston Ebola virus (GP-RESTV) (9.6%), VP40-SUDV (76.6%), and VP40-BDBV (38.3%). Overall, we developed a sensitive and specific high-throughput serological assay, and defined an algorithm, for epidemiological surveys with humans. Copyright © 2016 American Society for Microbiology.

  20. Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

    PubMed

    Becker, S; Klenk, H D; Mühlberger, E

    1996-11-01

    The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

  1. Identification of novel Ebola virus (EBOV) VP24 inhibitor from Indonesian natural products through in silico drug design approach

    NASA Astrophysics Data System (ADS)

    Tambunan, U. S. F.; Nasution, M. A. F.

    2017-07-01

    Ebola remains as one of the deadliest diseases in the world, with almost 29,000 cases were reported and kill 11,000 of them, and yet neither treatment nor vaccine that can combat this disease effectively. This disease is caused by ebolavirus (EBOV), a primary member of Filoviridae family. The life cycle of this virus has been operated by several key proteins, one of them is VP24 protein, which has been known for its crucial role in the transcription and replication of EBOV. Therefore, targeting VP24 protein can be a solution for treating this pathogenic disease. In this study, virtual screening of Indonesian natural products as EBOV VP24 inhibitor was performed. About 2,020 ligands from many sources, including HerbalDB database, were obtained and screened by using DataWarrior software to measure its molecular and pharmacological properties, resulting 301 ligands in the process. Then, the molecular docking simulation was performed to check the ligand's binding interaction and affinity with EBOV VP24 protein; this simulation was done by using MOE 2014.09 software. This study resulted that cycloartocarpin was the best ligand to inhibit the EBOV VP24 protein. Therefore, this ligand should be checked its stability through molecular dynamics simulation and performed in vitro test to verify its bioactivity against the EBOV VP24 protein.

  2. Molecular analysis of partial VP-2 gene amplified from rectal swab samples of diarrheic dogs in Pakistan confirms the circulation of canine parvovirus genetic variant CPV-2a and detects sequences of feline panleukopenia virus (FPV).

    PubMed

    Ahmed, Nisar; Riaz, Adeel; Zubair, Zahra; Saqib, Muhammad; Ijaz, Sehrish; Nawaz-Ul-Rehman, Muhammad Shah; Al-Qahtani, Ahmed; Mubin, Muhammad

    2018-03-15

    The infection in dogs due to canine parvovirus (CPV), is a highly contagious one with high mortality rate. The present study was undertaken for a detailed genetic analysis of partial VP2 gene i.e., 630 bp isolated from rectal swab samples of infected domestic and stray dogs from all areas of district Faisalabad. Monitoring of viruses is important, as continuous prevalence of viral infection might be associated with emergence of new virulent strains. In the present study, 40 rectal swab samples were collected from diarrheic dogs from different areas of district Faisalabad, Pakistan, in 2014-15 and screened for the presence of CPV by immunochromatography. Most of these dogs were stray dogs showing symptoms of diarrhea. Viral DNA was isolated and partial VP2 gene was amplified using gene specific primer pair Hfor/Hrev through PCR. Amplified fragments were cloned in pTZ57R/T (Fermentas) and completely sequenced. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA). The results with immunochromatography showed that 33/40 (82%) of dogs were positive for CPV. We were able to amplify a fragment of 630 bp from 25 samples. In 25 samples the sequences of CPV-2a were detected showing the amino acid substitution Ser297Ala and presence of amino acid (426-Asn) in partial VP2 protein. Interestingly the BLAST analysis showed the of feline panleukopenia virus (FPV) sequences in 3 samples which were already positive for new CPV-2a, with 99% sequence homology to other FPV sequences present in GenBank. Phylogenetic analysis showed clustering of partial CPV-VP-2 gene with viruses from China, India, Japan and Uruguay identifying a new variant, whereas the 3 FPV sequences showed immediate ancestral relationship with viruses from Portugal, South Africa and USA. Interesting observation was that CPV are clustering away from the commercial vaccine strains. In this work we provide a better understanding of CPV prevailing in Pakistan

  3. The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

    PubMed Central

    Xue, Qiao

    2017-01-01

    Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects domestic and wild cloven-hoofed animals. The structural protein VP1 plays an important role in FMDV pathogenesis. However, the interacting partners of VP1 in host cells and the effects of these interactions in FMDV replication remain incompletely elucidated. Here, we identified a porcine cell protein, serine/threonine kinase 3 (STK3), which interacts with FMDV VP1 using the yeast two-hybrid system. The VP1-STK3 interaction was further confirmed by coimmunoprecipitation experiments in human embryonic kidney 293T and porcine kidney 15 (PK-15) cells. The carboxyl-terminal region (amino acids 180–214) of VP1 was essential for its interaction with STK3. The effects of overexpression and underexpressing of STK3 in PK-15 cells were assessed, and the results indicated that STK3 significantly inhibited FMDV replication. Our data expand the role of STK3 during viral infection, provide new information regarding the host cell kinases that are involved in viral replication, and identify potential targets for future antiviral strategies. PMID:29226127

  4. The ebolavirus VP24 interferon antagonist

    PubMed Central

    Zhang, Adrianna P.P.; Abelson, Dafna M.; Bornholdt, Zachary A.; Liu, Tong; Woods, Jr, Virgil L.; Saphire, Erica Ollmann

    2012-01-01

    Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways.1,2 The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation.3 A more recent discovery is that VP24 also binds STAT1 directly,4 suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses. PMID:23076242

  5. Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

    PubMed

    Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

    2016-02-01

    Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Spatiotemporal Phylogenetic Analysis and Molecular Characterisation of Infectious Bursal Disease Viruses Based on the VP2 Hyper-Variable Region.

    PubMed

    Alfonso-Morales, Abdulahi; Martínez-Pérez, Orlando; Dolz, Roser; Valle, Rosa; Perera, Carmen L; Bertran, Kateri; Frías, Maria T; Majó, Natàlia; Ganges, Llilianne; Pérez, Lester J

    2013-01-01

    Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains. Sequences of the hyper-variable region of the VP2 (HVR-VP2) gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank database; Cuban sequences were obtained in the current work. All sequences were analysed by Bayesian phylogeographic analysis, implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST), Bayesian Tip-association Significance testing (BaTS) and Spatial Phylogenetic Reconstruction of Evolutionary Dynamics (SPREAD) software packages. Selection pressure on the HVR-VP2 was also assessed. The phylogeographic association-trait analysis showed that viruses sampled from individual countries tend to cluster together, suggesting a geographic pattern for IBDV strains. Spatial analysis from this study revealed that strains carrying sequences that were linked to increased virulence of IBDV appeared in Iran in 1981 and spread to Western Europe (Belgium) in 1987, Africa (Egypt) around 1990, East Asia (China and Japan) in 1993, the Caribbean Region (Cuba) by 1995 and South America (Brazil) around 2000. Selection pressure analysis showed that several codons in the HVR-VP2 region were under purifying selection. To our knowledge, this work is the first study applying the Bayesian phylogeographic reconstruction approach to analyse the emergence and spread of vvIBDV strains worldwide.

  7. Spatiotemporal Phylogenetic Analysis and Molecular Characterisation of Infectious Bursal Disease Viruses Based on the VP2 Hyper-Variable Region

    PubMed Central

    Dolz, Roser; Valle, Rosa; Perera, Carmen L.; Bertran, Kateri; Frías, Maria T.; Majó, Natàlia; Ganges, Llilianne; Pérez, Lester J.

    2013-01-01

    Background Infectious bursal disease is a highly contagious and acute viral disease caused by the infectious bursal disease virus (IBDV); it affects all major poultry producing areas of the world. The current study was designed to rigorously measure the global phylogeographic dynamics of IBDV strains to gain insight into viral population expansion as well as the emergence, spread and pattern of the geographical structure of very virulent IBDV (vvIBDV) strains. Methodology/Principal Findings Sequences of the hyper-variable region of the VP2 (HVR-VP2) gene from IBDV strains isolated from diverse geographic locations were obtained from the GenBank database; Cuban sequences were obtained in the current work. All sequences were analysed by Bayesian phylogeographic analysis, implemented in the Bayesian Evolutionary Analysis Sampling Trees (BEAST), Bayesian Tip-association Significance testing (BaTS) and Spatial Phylogenetic Reconstruction of Evolutionary Dynamics (SPREAD) software packages. Selection pressure on the HVR-VP2 was also assessed. The phylogeographic association-trait analysis showed that viruses sampled from individual countries tend to cluster together, suggesting a geographic pattern for IBDV strains. Spatial analysis from this study revealed that strains carrying sequences that were linked to increased virulence of IBDV appeared in Iran in 1981 and spread to Western Europe (Belgium) in 1987, Africa (Egypt) around 1990, East Asia (China and Japan) in 1993, the Caribbean Region (Cuba) by 1995 and South America (Brazil) around 2000. Selection pressure analysis showed that several codons in the HVR-VP2 region were under purifying selection. Conclusions/Significance To our knowledge, this work is the first study applying the Bayesian phylogeographic reconstruction approach to analyse the emergence and spread of vvIBDV strains worldwide. PMID:23805195

  8. Safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single-ascending-dose studies.

    PubMed

    Heald, Alison E; Iversen, Patrick L; Saoud, Jay B; Sazani, Peter; Charleston, Jay S; Axtelle, Tim; Wong, Michael; Smith, William B; Vutikullird, Apinya; Kaye, Edward

    2014-11-01

    Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

    PubMed

    Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

    2017-12-01

    Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Ebolavirus VP35 is a multifunctional virulence factor.

    PubMed

    Leung, Daisy W; Prins, Kathleen C; Basler, Christopher F; Amarasinghe, Gaya K

    2010-01-01

    Ebola virus (EBOV) is a member of the filoviridae family that causes severe hemorrhagic fever during sporadic outbreaks, and no approved treatments are currently available. The multifunctional EBOV VP35 protein facilitates immune evasion by antagonizing antiviral signaling pathways and is important for viral RNA synthesis. In order to elucidate regulatory mechanisms and to develop countermeasures, we recently solved the structures of the Zaire and Reston EBOV VP35 interferon inhibitory domain (IID) in the free form and of the Zaire EBOV VP35 IID bound to dsRNA. Together with biochemical, cell biological, and virological studies, our structural work revealed that distinct regions within EBOV VP35 IID contribute to virulence through host immune evasion and viral RNA synthesis. Here we summarize our recent structural and functional studies and discuss the potential of multifunctional Ebola VP35 as a therapeutic target.

  11. Productive Replication of Ebola Virus Is Regulated by the c-Abl1 Tyrosine Kinase

    PubMed Central

    García, Mayra; Cooper, Arik; Shi, Wei; Bornmann, William; Carrion, Ricardo; Kalman, Daniel; Nabel, Gary J.

    2016-01-01

    Ebola virus causes a fulminant infection in humans resulting in diffuse bleeding, vascular instability, hypotensive shock, and often death. Because of its high mortality and ease of transmission from human to human, Ebola virus remains a biological threat for which effective preventive and therapeutic interventions are needed. An understanding of the mechanisms of Ebola virus pathogenesis is critical for developing antiviral therapeutics. Here, we report that productive replication of Ebola virus is modulated by the c-Abl1 tyrosine kinase. Release of Ebola virus–like particles (VLPs) in a cell culture cotransfection system was inhibited by c-Abl1–specific small interfering RNA (siRNA) or by Abl-specific kinase inhibitors and required tyrosine phosphorylation of the Ebola matrix protein VP40. Expression of c-Abl1 stimulated an increase in phosphorylation of tyrosine 13 (Y13) of VP40, and mutation of Y13 to alanine decreased the release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain was inhibited by c-Abl1–specific siRNAs or by the Abl-family inhibitor nilotinib by up to four orders of magnitude. These data indicate that c-Abl1 regulates budding or release of filoviruses through a mechanism involving phosphorylation of VP40. This step of the virus life cycle therefore may represent a target for antiviral therapy. PMID:22378924

  12. Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

    PubMed Central

    Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto

    2018-01-01

    The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance. PMID:29495311

  13. Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling.

    PubMed

    Fanunza, Elisa; Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto; Corona, Angela; Tramontano, Enzo

    2018-02-24

    The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z'- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

  14. Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2.

    PubMed

    Patial, Sonika; Chaturvedi, V K; Rai, A; Saini, M; Chandra, Rajesh; Saini, Y; Gupta, Praveen K

    2007-05-16

    A bicistronic DNA vaccine against rabies and parvovirus infection of dogs was developed by subcloning rabies glycoprotein and canine parvovirus (CPV) VP2 genes into a bicistronic vector. After characterizing the expression of both the proteins in vitro, the bicistronic DNA vaccine was injected in mice and induced immune response was compared with monocistronic DNA vaccines. There was no significant difference in ELISA and virus neutralizing (VN) antibody responses against rabies and CPV in mice immunized with either bicistronic or monocistronic DNA vaccine. Further, there was significantly similar protection in mice immunized with either bicistronic or monocistronic rabies DNA vaccine on rabies virus challenge. Similarly, dogs immunized with monocistronic and bicistronic DNA vaccines developed comparable VN antibodies against rabies and CPV. This study indicated that bicistronic DNA vaccine can be used in dogs to induce virus neutralizing immune responses against both rabies and CPV.

  15. Ebolavirus VP35 is a multifunctional virulence factor

    PubMed Central

    Leung, Daisy W; Prins, Kathleen C; Basler, Christopher F

    2010-01-01

    Ebola virus (EBOV) is a member of the filoviridae family that causes severe hemorrhagic fever during sporadic outbreaks, and no approved treatments are currently available. The multifunctional EBOV VP35 protein facilitates immune evasion by antagonizing antiviral signaling pathways and is important for viral RNA synthesis. In order to elucidate regulatory mechanisms and to develop countermeasures, we recently solved the structures of the Zaire and Reston EBOV VP35 interferon inhibitory domain (IID) in the free form and of the Zaire EBOV VP35 IID bound to dsRNA. Together with biochemical, cell biological and virological studies, our structural work revealed that distinct regions within EBOV VP35 IID contribute to virulence through host immune evasion and viral RNA synthesis. Here we summarize our recent structural and functional studies and discuss the potential of multifunctional Ebola VP35 as a therapeutic target. PMID:21178490

  16. Eukaryotic translational initiation factor 4AII reduces the replication of infectious bursal disease virus by inhibiting VP1 polymerase activity.

    PubMed

    Gao, Li; Li, Kai; Zhong, Li; Zhang, Lizhou; Qi, Xiaole; Wang, Yongqiang; Gao, Yulong; Wang, Xiaomei

    2017-03-01

    Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although an interaction between eukaryotic translational initiation factor 4AII (eIF4AII) of the host and viral protein 1 (VP1), the RNA-dependent RNA polymerase (RdRp) of IBDV, has been established, the underlying effects of this interaction on IBDV and the molecular mechanism remain unclear. We here report that interaction of the host eIF4AII with VP1 inhibits the RNA polymerase activity of IBDV to reduce its replication in host cells. We found that ectopically expressed eIF4AII markedly inhibited IBDV growth in DF1 cells, and knockdown of eIF4AII by small interfering RNA significantly enhanced viral replication in CEF cells. Furthermore, IBDV infection led to an increase in host eIF4AII expression, suggesting a feedback mechanism between the host and virus infection both in vitro and in vivo, which further confirmed the involvement of the host eIF4AII in the IBDV life cycle. Thus, via the interaction with VP1, eIF4AII plays a critical role in the IBDV life cycle, by inhibiting viral RNA polymerase activity, leading to a reduction of IBDV replication in cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

    SciTech Connect

    Ye Ling; Lin Jianguo; Sun Yuliang

    2006-08-01

    Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity ofmore » Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.« less

  18. The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

    PubMed Central

    Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

    2004-01-01

    The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

  19. Screening of binding proteins that interact with Chinese sacbrood virus VP3 capsid protein in Apis cerana larvae cDNA library by the yeast two-hybrid method.

    PubMed

    Fei, Dongliang; Wei, Dong; Yu, Xiaolei; Yue, Jinjin; Li, Ming; Sun, Li; Jiang, Lili; Li, Yijing; Diao, Qingyun; Ma, Mingxiao

    2018-03-15

    Chinese sacbrood virus (CSBV) causes larval death and apiary collapse of Apis cerana. VP3 is a capsid protein of CSBV but its function is poorly understood. To determine the function of VP3 and screen for novel binding proteins that interact with VP3, we conducted yeast two-hybrid screening, glutathione S-transferase pull-down, and co-immunoprecipitation assays. Galectin (GAL) is a protein involved in immune regulation and host-pathogen interactions. The yeast two-hybrid screen implicated GAL as a major VP3-binding candidate. The assays showed that the VP3 interacted with GAL. Identification of these cellular targets and clarifying their contributions to the host-pathogen interaction may be useful for the development of novel therapeutic and prevention strategies against CSBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Discovery of simian virus 40 (SV40) and its relationship to poliomyelitis virus vaccines.

    PubMed

    Hilleman, M R

    1998-01-01

    Simian Virus 40 (SV40) was discovered in 1959 as a covert contaminant of poliovirus vaccines prepared using Macacus monkey renal cell cultures. This inapparent polyoma virus of monkeys was detected using Cercopithecus renal cell cultures and was eliminated from poliovaccines. There has been no evidence to implicate SV40 virus of vaccine origin in long- or short-term consequences in human subjects. Of importance, SV40 virus provided a new model for basic studies of viral pathogenesis and for cell transformation and neoplasia. Neoplastic transformation is fixed on the promiscuous binding of SV40 large T antigen to anti-oncogene cellular protein elements. SV40 also served as a valuable model for defining the immunology of virus-induced cancer and in its prevention and cure. Further, it has been a prime tool for elucidating the molecular details of eukaryotic cell processes. Numerous techniques now used in molecular biology were pioneered in the SV40 system. The SV40 promoter is commonly used in vector expression constructs and it has continued to be a model to develop new tools for site-specific mutagenesis. The virus has been critically important to studies in modern genetics and in molecular biology.

  1. Identification of VP1/2A and 2C as Virulence Genes of Hepatitis A Virus and Demonstration of Genetic Instability of 2C

    PubMed Central

    Emerson, Suzanne U.; Huang, Ying K.; Nguyen, Hanh; Brockington, Alicia; Govindarajan, Sugantha; St. Claire, Marisa; Shapiro, Max; Purcell, Robert H.

    2002-01-01

    Fourteen different chimeric virus genomes were constructed from two infectious cDNA clones encoding a virulent and an attenuated isolate, respectively, of the HM175 strain of hepatitis A virus. The ability of each recombinant virus to infect tamarins and to cause acute hepatitis was determined. Comparisons of the genotype and phenotype of each virus suggested that VP1/2A and 2C genes were responsible for virulence. The 2C gene derived from the attenuated parent virus was unstable, and one or more mutations arose in this gene during the first passage in tamarins. PMID:12163575

  2. Identification of a T-helper cell epitope on the rotavirus VP6 protein.

    PubMed Central

    Baños, D M; Lopez, S; Arias, C F; Esquivel, F R

    1997-01-01

    In this work, we have studied the T-helper (Th)-cell response against rotavirus, in a mouse model. Adult BALB/c mice were inoculated parenterally with porcine rotavirus YM, and the Th-cell response from spleen cells against the virus and two overlapping fragments of the major capsid protein VP6 (VP6(1-192) and VP6(171-397)) were evaluated in vitro. The Th cells recognized the YM virus and the two protein fragments, suggesting that there are at least two Th-cell epitopes on the VP6 molecule. To study the specificity of Th cells against VP6 at the clonal level, we established two Th-cell hybridomas cross-reactive for the VP6 protein of rotavirus strains YM and SA11. Both hybridomas recognized the VP6(171-397) polypeptide, and a synthetic peptide comprising the amino acids 289 to 302 (RLSFQLVRPPNMTP) of YM VP6 in the context of the major histocompatibility complex class II IEd molecule. The Th-cell hybridomas recognized rotavirus VP6 in a highly cross-reactive fashion, since they could be stimulated by eight different strains of rotavirus, including the murine rotavirus EDIM, that represent five G serotypes and at least two subgroups. The amino acid sequence of the VP6 epitope is highly conserved in most group A rotavirus strains sequenced so far. On the other hand, it was found that Th cells specific for the VP6 epitope may constitute an important proportion of the total polyclonal Th-cell response against rotavirus YM in spleen cells. These results demonstrate that VP6 can be a target for highly cross-reactive Th cells. PMID:8985366

  3. Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

    PubMed

    Dolnik, Olga; Stevermann, Lea; Kolesnikova, Larissa; Becker, Stephan

    2015-01-01

    Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. A Rapid Screen for Host-Encoded miRNAs with Inhibitory Effects against Ebola Virus Using a Transcription- and Replication-Competent Virus-Like Particle System.

    PubMed

    Wang, Zhongyi; Li, Jiaming; Fu, Yingying; Zhao, Zongzheng; Zhang, Chunmao; Li, Nan; Li, Jingjing; Cheng, Hongliang; Jin, Xiaojun; Lu, Bing; Guo, Zhendong; Qian, Jun; Liu, Linna

    2018-05-16

    MicroRNAs (miRNAs) may become efficient antiviral agents against the Ebola virus (EBOV) targeting viral genomic RNAs or transcripts. We previously conducted a genome-wide search for differentially expressed miRNAs during viral replication and transcription. In this study, we established a rapid screen for miRNAs with inhibitory effects against EBOV using a tetracistronic transcription- and replication-competent virus-like particle (trVLP) system. This system uses a minigenome comprising an EBOV leader region, luciferase reporter, VP40, GP, VP24, EBOV trailer region, and three noncoding regions from the EBOV genome and can be used to model the life cycle of EBOV under biosafety level (BSL) 2 conditions. Informatic analysis was performed to select up-regulated miRNAs targeting the coding regions of the minigenome with the highest binding energy to perform inhibitory effect screening. Among these miRNAs, miR-150-3p had the most significant inhibitory effect. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and double fluorescence reporter experiments demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40. This novel, rapid, and convenient screening method will efficiently facilitate the exploration of miRNAs against EBOV under BSL-2 conditions.

  5. Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

    PubMed

    Chung, Ivy Yeuk Wah; Paetzel, Mark

    2013-05-03

    Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.

  6. Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis

    USDA-ARS?s Scientific Manuscript database

    In this study, we generated a recombinant foot-and-mouth disease virus (FMDV) particle derived from A24 Cruzeiro with a FLAG tag (DYKDDDDK) substitution in the hypervariable antigenic site of the G-H loop of the VP1 capsid protein in an effort to expand the immunogenicity of the virus particle and t...

  7. VP2 (PTA motif) encoding DNA vaccine confers protection against lethal challenge with infectious pancreatic necrosis virus (IPNV) in trout.

    PubMed

    Ahmadivand, Sohrab; Soltani, Mehdi; Behdani, Mahdi; Evensen, Øystein; Alirahimi, Ehsan; Soltani, Elahe; Hassanzadeh, Reza; Ashrafi-Helan, Javad

    2018-02-01

    IPNV in Atlantic salmon is represented by various strains with different virulence and immunogenicity linked to various motifs of the VP2 capsid. IPNV variant with P 217 , T 221 , A 247 (PTA) motif is found to be avirulent in Atlantic salmon, but virulent in rainbow trout, and other salmonid species. This study describes a DNA vaccine delivered intramuscularly encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) with PTA motif that confers high protection in rainbow trout (Oncorhynchus mykiss). Intramuscular injection of 2, 5 and 10 μg of DNA (pcDNA3.1-VP2) in rainbow trout fry (4-5 g), confers relative protection of 75-83% in the different vaccine groups at 30 days post vaccination (450° days). The VP2 gene is expressed in spleen, kidney, muscle and liver at day 30 post-vaccination (RT-PCR), and IFN-1 and Mx-1 mRNA are upregulated at early time post vaccination, and so also for IgM, IgT, CD4 and CD8 in the head kidney of vaccinated fish compared to controls, 15 and 30 days post vaccination. Significant increase of serum anti-IPNV antibodies was found 30-90 days post-vaccination that was correlated with protection levels. Mortality corresponded with viral VP4 gene expression were significantly decreased in vaccinated and challenged fish. This shows for the first time that a VP2-encoding DNA vaccine delivered intramuscularly elicits a high level of protection alongside with high levels of circulating antibodies in rainbow trout and a lowered viral replication. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Recombinant cell lines expressing shRNA targeting herpes simplex virus 2 VP16 inhibit virus replication.

    PubMed

    Zhang, Rui; Wang, Yan; Song, Bo; Han, Zhi Qiang; Xu, Yu Ming

    2012-01-01

    To establish HSV2 VP16 targeting shRNA-expressing cell lines and investigate the antiviral effect of shRNA targeting HSV2 VP16. The cell lines Vero-shRNAs and negative-control Vero-shCON were established. Their inhibition effects on VP16 mRNA expression were tested by real-time fluorescent quantitative polymerase chain reaction (PCR) and their antiviral effects were evaluated by yield reduction assay. The influence of passage numbers on the inhibition ability of cell lines was researched. Vero-shRNA24 targeting the upper stream, Vero-shRNA642 targeting the lower stream and Vero-shCON were established. Vero-shRNA24, Vero-shRNA642 and Vero-shRNA24 + 642 could reduce the VP16 mRNA significantly. Vero-shRNA24 was the most efficient. The HSV2 titers in Vero and Vero-shCON were the highest at 72 h after infection, and started decreasing thereafter. The viral titers of the Vero-shRNA groups reached a peak after 84 h and the highest titers were lower than in the Vero group. The inhibiting effect on VP16 mRNA expression and viral replication of Vero-shRNA24 cell lines of passages 10 and 20 were not significantly different from the primary cell line. Although of no statistical significance, the passage 50 cell line showed decreased inhibiting ability. Recombinant cell lines expressing shRNA targeting HSV2 VP16 were established. They can stably inhibit HSV2 VP16 mRNA expression and viral replication within passage 50. Copyright © 2012 S. Karger AG, Basel.

  9. Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

    PubMed

    Ekström, Jens-Ola; Habayeb, Mazen S; Srivastava, Vaibhav; Kieselbach, Thomas; Wingsle, Gunnar; Hultmark, Dan

    2011-09-01

    The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

    DTIC Science & Technology

    2010-06-01

    viruses . N-glycosylation of viral envelopes is an important such target shared between in- fluenza, HIV, HCV, West Nile virus , SARS-CoV, Hendra virus ...host cells. Therefore, MBL preferentially recognizes glycosylated viruses including influenza virus , human immunodeficiency virus , severe acute...respiratory syndrome coronovirus (SARS-CoV), Ebola virus , and Marburg virus . It also recognizes many glycosylated gram- positive and gram-negative bacteria [1

  11. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

    PubMed

    Panjwani, Anusha; Strauss, Mike; Gold, Sarah; Wenham, Hannah; Jackson, Terry; Chou, James J; Rowlands, David J; Stonehouse, Nicola J; Hogle, James M; Tuthill, Tobias J

    2014-08-01

    Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

  12. Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

    PubMed

    Solís-Lucero, G; Manoutcharian, K; Hernández-López, J; Ascencio, F

    2016-08-01

    White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

    PubMed

    Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

    2000-04-01

    VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

  14. Molecular characterization of amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.

    PubMed

    Esmaelizad, Majid; Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

    2011-12-01

    The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

  15. Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

    PubMed

    Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

    2015-08-26

    The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

  16. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    PubMed

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

    PubMed

    Wen, X J; Cheng, A C; Wang, M S; Jia, R Y; Zhu, D K; Chen, S; Liu, M F; Liu, F; Chen, X Y

    2014-09-01

    Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV. © 2014 Poultry Science Association Inc.

  18. The ebolavirus VP24 interferon antagonist: know your enemy.

    PubMed

    Zhang, Adrianna P P; Abelson, Dafna M; Bornholdt, Zachary A; Liu, Tong; Woods, Virgil L; Saphire, Erica Ollmann

    2012-08-15

    Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways. The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation. A more recent discovery is that VP24 also binds STAT1 directly, suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses.

  19. Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

    PubMed

    Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

    2016-01-01

    Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

  20. Characterization of Nora Virus Structural Proteins via Western Blot Analysis

    PubMed Central

    Ericson, Brad L.; Carlson, Darby J.

    2016-01-01

    Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses. PMID:27298753

  1. From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein.

    PubMed

    Hempel, Franziska; Maurer, Michael; Brockmann, Björn; Mayer, Christian; Biedenkopf, Nadine; Kelterbaum, Anne; Becker, Stephan; Maier, Uwe G

    2017-07-27

    The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014-2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.

  2. Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

    PubMed

    Schweneker, Marc; Laimbacher, Andrea S; Zimmer, Gert; Wagner, Susanne; Schraner, Elisabeth M; Wolferstätter, Michael; Klingenberg, Marieken; Dirmeier, Ulrike; Steigerwald, Robin; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2017-06-01

    There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant. IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified

  3. Interferon-β therapy prolongs survival in rhesus macaque models of Ebola and Marburg hemorrhagic fever.

    PubMed

    Smith, Lauren M; Hensley, Lisa E; Geisbert, Thomas W; Johnson, Joshua; Stossel, Andrea; Honko, Anna; Yen, Judy Y; Geisbert, Joan; Paragas, Jason; Fritz, Elizabeth; Olinger, Gene; Young, Howard A; Rubins, Kathleen H; Karp, Christopher L

    2013-07-15

    There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust interferon (IFN)-α production, with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those seen in other viral infections, but little IFN-β production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-β therapy in filovirus infection. Here we show that early postexposure treatment with IFN-β significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-β also significantly increased survival time after Marburg virus infection. IFN-β may have promise as an adjunctive postexposure therapy in filovirus infection.

  4. Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp.

    PubMed

    Taengchaiyaphum, Suparat; Nakayama, Hideki; Srisala, Jiraporn; Khiev, Ratny; Aldama-Cano, Diva January; Thitamadee, Siripong; Sritunyalucksana, Kallaya

    2017-11-01

    To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Role of natural killer cells in innate protection against lethal ebola virus infection.

    PubMed

    Warfield, Kelly L; Perkins, Jeremy G; Swenson, Dana L; Deal, Emily M; Bosio, Catharine M; Aman, M Javad; Yokoyama, Wayne M; Young, Howard A; Bavari, Sina

    2004-07-19

    Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1-3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell-mediated protection clearly depended on perforin, but not interferon-gamma secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection.

  6. Characterization of an Extremely Basic Protein Derived from Granulosis Virus Nucleocapsids †

    PubMed Central

    Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

    1980-01-01

    Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex. Images PMID:16789190

  7. [Effect of white spot syndrome virus envelope protein Vp28 expressed in silkworm (Bombyx mori) pupae on disease resistence in Procambarus clarkii].

    PubMed

    Wei, Ke Qiang; Xu, Zi Rong

    2005-06-01

    The vaccine made of recombinant envelope protein (rVp28) of white spot syndrome virus (WSSV) expressed in silkworm (Bombyx mori) pupae using a baculovirus vector was used to investigate the efficacy of oral administration on WSSV disease resistance of Procambarus clarkii. Vaccine was mixed with diet at a ratio of 2% (w/w), and Procambarus clarkii were orally administered throughout 75 days. Vaccination with rVP28 showed the significantly higher cumulative survival compared with positive and negative control (P < 0.05) following an oral challenge on the 35th day post-vaccination (dpv), with PRP values 54.16% and 59.26%, respectively. rVP28 induced higher resistance via IM (intramuscular) injection challenge with WSSV stock, with PRP value of 46.12% and 49.99%, respectively. The survivors were subsequently re-challenged on the 55th dpv. rVP28 induced the significantly higher resistance to oral re-challenge (P < 0.05), with both PRP values 55.80% and 63.16%, respectively. rVP28 induced higher resistance to IM injection re-challenge, with both PRP values 31.25%. A DIG labeled WSSV DNA probe was used to detect WSSV by in situ hybridization. The positive cells were observed in epithelial cells of stomach, hepatopancreas and gut of the infected control crayfish, while negative reaction were observed in the tissues of survivors-vaccinated. These results indicated that vaccination of crayfish with recombinant protein had significant effect on oral infection, and had higher resistance against intramuscular injection challenge. This suggested the protection against WSSV could be induced in crayfish by recombinant protein rVp28 expressed in silkworm pupae.

  8. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    NASA Astrophysics Data System (ADS)

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-03-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

  9. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    PubMed Central

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-01-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research. PMID:26996514

  10. Molecular basis for ebolavirus VP35 suppression of human dendritic cell maturation.

    PubMed

    Yen, Benjamin; Mulder, Lubbertus C F; Martinez, Osvaldo; Basler, Christopher F

    2014-11-01

    Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA)-binding protein that inhibits RIG-I signaling and alpha/beta interferon (IFN-α/β) responses by both dsRNA-binding-dependent and -independent mechanisms. VP35 also suppresses dendritic cell (DC) maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) that independently ablate dsRNA binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs (MDDCs) using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/β but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA, or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I · C). The F239A and R322A mutants exhibited greatly reduced suppression of IFN-α/β and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the VP35 mutants impaired IFN-β production induced by Toll-like receptor 3 (TLR3) or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Furthermore, VP35 did not prevent lipopolysaccharide (LPS)-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. Therefore, it may be possible to counteract EBOV immune evasion by using treatments that bypass the VP35-imposed block to DC maturation. The VP35 protein, which is an

  11. Profiling the Native Specific Human Humoral Immune Response to Sudan Ebola Virus Strain Gulu by Chemiluminescence Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Sobarzo, Ariel; Perelman, Eddie; Groseth, Allison; Dolnik, Olga; Becker, Stephan; Lutwama, Julius Julian; Dye, John M.; Yavelsky, Victoria; Marks, Robert S.

    2012-01-01

    Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP1-294 and GP1-649) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP1-649, followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP1-649, and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future. PMID:22993411

  12. Development of Prototype Filovirus Recombinant Antigen Immunoassays

    PubMed Central

    Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

    2015-01-01

    Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

  13. Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes.

    PubMed

    Wagner, Melany J; Smiley, James R

    2009-12-01

    Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus.

  14. A STAT-1 Knockout Mouse Model for Machupo Virus Pathogenesis

    DTIC Science & Technology

    2011-06-14

    hemorrhagic fever viruses, including Ebola, Marburg, Junín, and Crimean - Congo Hemorrhagic Fever viruses [11-14...Akerstrom S, Klingstrom J, Mirazimi A: Crimean - Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice. J...Shieh WJ, Camus G, Stroher U, Zaki S, Jones SM: Pathogenesis and immune response of Crimean - Congo hemorrhagic fever virus in a STAT-1 knockout

  15. A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate.

    PubMed

    Hong, Qi; Qian, Ping; Li, Xiang-Min; Yu, Xiao-Lan; Chen, Huan-Chun

    2007-11-01

    Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.

  16. Structure of the Ebola VP35 interferon inhibitory domain.

    PubMed

    Leung, Daisy W; Ginder, Nathaniel D; Fulton, D Bruce; Nix, Jay; Basler, Christopher F; Honzatko, Richard B; Amarasinghe, Gaya K

    2009-01-13

    Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 A and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.

  17. Experimental Inoculation of Egyptian Rousette Bats (Rousettus aegyptiacus) with Viruses of the Ebolavirus and Marburgvirus Genera.

    PubMed

    Jones, Megan E B; Schuh, Amy J; Amman, Brian R; Sealy, Tara K; Zaki, Sherif R; Nichol, Stuart T; Towner, Jonathan S

    2015-06-25

    The Egyptian rousette bat (Rousettus aegyptiacus) is a natural reservoir for marburgviruses and a consistent source of virus spillover to humans. Cumulative evidence suggests various bat species may also transmit ebolaviruses. We investigated the susceptibility of Egyptian rousettes to each of the five known ebolaviruses (Sudan, Ebola, Bundibugyo, Taï Forest, and Reston), and compared findings with Marburg virus. In a pilot study, groups of four juvenile bats were inoculated with one of the ebolaviruses or Marburg virus. In ebolavirus groups, viral RNA tissue distribution was limited, and no bat became viremic. Sudan viral RNA was slightly more widespread, spurring a second, 15-day Sudan virus serial euthanasia study. Low levels of Sudan viral RNA disseminated to multiple tissues at early time points, but there was no viremia or shedding. In contrast, Marburg virus RNA was widely disseminated, with viremia, oral and rectal shedding, and antigen in spleen and liver. This is the first experimental infection study comparing tissue tropism, viral shedding, and clinical and pathologic effects of six different filoviruses in the Egyptian rousette, a known marburgvirus reservoir. Our results suggest Egyptian rousettes are unlikely sources for ebolaviruses in nature, and support a possible single filovirus-single reservoir host relationship.

  18. Experimental Inoculation of Egyptian Rousette Bats (Rousettus aegyptiacus) with Viruses of the Ebolavirus and Marburgvirus Genera

    PubMed Central

    Jones, Megan E.B.; Schuh, Amy J.; Amman, Brian R.; Sealy, Tara K.; Zaki, Sherif R.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-01-01

    The Egyptian rousette bat (Rousettus aegyptiacus) is a natural reservoir for marburgviruses and a consistent source of virus spillover to humans. Cumulative evidence suggests various bat species may also transmit ebolaviruses. We investigated the susceptibility of Egyptian rousettes to each of the five known ebolaviruses (Sudan, Ebola, Bundibugyo, Taï Forest, and Reston), and compared findings with Marburg virus. In a pilot study, groups of four juvenile bats were inoculated with one of the ebolaviruses or Marburg virus. In ebolavirus groups, viral RNA tissue distribution was limited, and no bat became viremic. Sudan viral RNA was slightly more widespread, spurring a second, 15-day Sudan virus serial euthanasia study. Low levels of Sudan viral RNA disseminated to multiple tissues at early time points, but there was no viremia or shedding. In contrast, Marburg virus RNA was widely disseminated, with viremia, oral and rectal shedding, and antigen in spleen and liver. This is the first experimental infection study comparing tissue tropism, viral shedding, and clinical and pathologic effects of six different filoviruses in the Egyptian rousette, a known marburgvirus reservoir. Our results suggest Egyptian rousettes are unlikely sources for ebolaviruses in nature, and support a possible single filovirus—single reservoir host relationship. PMID:26120867

  19. The Globoside Receptor Triggers Structural Changes in the B19 Virus Capsid That Facilitate Virus Internalization▿

    PubMed Central

    Bönsch, Claudia; Zuercher, Christoph; Lieby, Patricia; Kempf, Christoph; Ros, Carlos

    2010-01-01

    Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections. PMID:20826697

  20. Development and Characterization of a Mouse Model for Marburg Hemorrhagic Fever

    DTIC Science & Technology

    2009-07-01

    Microbiology. All Rights Reserved. Development and Characterization of a Mouse Model for Marburg Hemorrhagic Fever Kelly L. Warfield,* Steven B...mouse model has hampered an understanding of the pathogenesis and immunity of Marburg hemorrhagic fever (MHF), the disease caused by marburgvirus (MARV...cause severe hemorrhagic fevers in humans and non- human primates (27). The incubation time is estimated to be 3 to 21 days, with human case fatality

  1. Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies

    PubMed Central

    Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol

    2012-01-01

    Summary VP22, encoded by the UL49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. PMID:22569534

  2. Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells.

    PubMed

    Li, Yang; Basavappa, Megha; Lu, Jinfeng; Dong, Shuwei; Cronkite, D Alexander; Prior, John T; Reinecker, Hans-Christian; Hertzog, Paul; Han, Yanhong; Li, Wan-Xiang; Cheloufi, Sihem; Karginov, Fedor V; Ding, Shou-Wei; Jeffrey, Kate L

    2016-12-05

    Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens 1 . However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago 2 , remains unknown 3 . Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence 4-7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs) 8,9 . Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice 10,11 . However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells 12-21 . Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

  3. Canine Parvovirus VP2 Protein Expressed in Silkworm Pupae Self-Assembles into Virus-Like Particles with High Immunogenicity

    PubMed Central

    Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4+ and CD8+ T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease. PMID:24465364

  4. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

    PubMed

    Feng, Hao; Hu, Gui-qiu; Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  5. Bioinformatic analysis suggests that the Orbivirus VP6 cistron encodes an overlapping gene

    PubMed Central

    Firth, Andrew E

    2008-01-01

    Background The genus Orbivirus includes several species that infect livestock – including Bluetongue virus (BTV) and African horse sickness virus (AHSV). These viruses have linear dsRNA genomes divided into ten segments, all of which have previously been assumed to be monocistronic. Results Bioinformatic evidence is presented for a short overlapping coding sequence (CDS) in the Orbivirus genome segment 9, overlapping the VP6 cistron in the +1 reading frame. In BTV, a 77–79 codon AUG-initiated open reading frame (hereafter ORFX) is present in all 48 segment 9 sequences analysed. The pattern of base variations across the 48-sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP6 reading frame are taken into account; MLOGD software). In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP6. The ORFX AUG codon has a strong Kozak context in all 48 sequences. Each has only one or two upstream AUG codons, always in the VP6 reading frame, and (with a single exception) always with weak or medium Kozak context. Thus, in BTV, ORFX may be translated via leaky scanning. A long (83–169 codon) ORF is present in a corresponding location and reading frame in all other Orbivirus species analysed except Saint Croix River virus (SCRV; the most divergent). Again, the pattern of base variations across sequence alignments indicates multiple coding in the VP6 and ORFX reading frames. Conclusion At ~9.5 kDa, the putative ORFX product in BTV is too small to appear on most published protein gels. Nonetheless, a review of past literature reveals a number of possible detections. We hope that presentation of this bioinformatic analysis will stimulate an attempt to experimentally verify the expression and functional role of ORFX, and hence lead to a greater understanding of the molecular biology of these important pathogens. PMID:18489030

  6. Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

    NASA Astrophysics Data System (ADS)

    Zhang, Minchuan; He, Peiming; Su, Jing; Singh, Dadabhai T.; Su, Hailei; Su, Haibin

    Ebola virus is a highly lethal filovirus, claimed thousands of people in its recent outbreak. Seven viral proteins constitute ebola viral structure, and four of them (nucleoprotein (NP), polymerase L, VP35 and VP30) participate majorly in viral replication and transcription. We have elucidated a conformation change of NP cleft by VP35 NP-binding protein domains through superimposing two experimental NP structure images and discussed the function of this conformation change in the replication and transcription with polymerase complex (L, VP35 and VP30). The important roles of VP30 in viral RNA synthesis have also been discussed. A “tapping” model has been proposed in this paper for a better understanding of the interplay among the four viral proteins (NP, polymerase L, VP35 and VP30). Moreover, we have pinpointed some key residue changes on NP (both NP N- and C-terminal) and L between Reston and Zaire by computational studies. Together, this paper provides a description of interactions among ebola viral proteins (NP, L, VP35, VP30 and VP40) in viral replication and transcription, and sheds light on the complex system of viral reproduction.

  7. Herpes Simplex Virus Requires VP11/12 To Induce Phosphorylation of the Activation Loop Tyrosine (Y394) of the Src Family Kinase Lck in T Lymphocytes ▿

    PubMed Central

    Wagner, Melany J.; Smiley, James R.

    2009-01-01

    Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. PMID:19776125

  8. An oral Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus delivered by Escherichia coli elicits immune responses in dogs.

    PubMed

    Dahiya, S S; Saini, M; Kumar, P; Gupta, P K

    2011-01-01

    A Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus (CPV) was delivered by Escherichia coli to elicit immune responses. The orally immunized dogs developed CPV-specific serum IgG and virus neutralizing antibody responses. The cellular immune responses analyzed using lymphocyte proliferation test and flow cytometry indicated CPV-specific sensitization of both CD3+CD4+ and CD3+CD8+ lymphocytes. This study demonstrated that the oral CPV DNA vaccine delivered by E. coli can be considered as a promising approach for vaccination of dogs against CPV.

  9. Evolutionary and genetic analysis of the VP2 gene of canine parvovirus.

    PubMed

    Li, Gairu; Ji, Senlin; Zhai, Xiaofeng; Zhang, Yuxiang; Liu, Jie; Zhu, Mengyan; Zhou, Jiyong; Su, Shuo

    2017-07-17

    Canine parvovirus (CPV) type 2 emerged in 1978 in the USA and quickly spread among dog populations all over the world with high morbidity. Although CPV is a DNA virus, its genomic substitution rate is similar to some RNA viruses. Therefore, it is important to trace the evolution of CPV to monitor the appearance of mutations that might affect vaccine effectiveness. Our analysis shows that the VP2 genes of CPV isolated from 1979 to 2016 are divided into six groups: GI, GII, GIII, GIV, GV, and GVI. Amino acid mutation analysis revealed several undiscovered important mutation sites: F267Y, Y324I, and T440A. Of note, the evolutionary rate of the CPV VP2 gene from Asia and Europe decreased. Codon usage analysis showed that the VP2 gene of CPV exhibits high bias with an ENC ranging from 34.93 to 36.7. Furthermore, we demonstrate that natural selection plays a major role compared to mutation pressure driving CPV evolution. There are few studies on the codon usage of CPV. Here, we comprehensively studied the genetic evolution, codon usage pattern, and evolutionary characterization of the VP2 gene of CPV. The novel findings revealing the evolutionary process of CPV will greatly serve future CPV research.

  10. Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35

    SciTech Connect

    Leung, Daisy W.; Prins, Kathleen C.; Borek, Dominika M.

    2010-03-12

    Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also importantmore » for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.« less

  11. Changes associated with Ebola virus adaptation to novel species.

    PubMed

    Pappalardo, Morena; Reddin, Ian G; Cantoni, Diego; Rossman, Jeremy S; Michaelis, Martin; Wass, Mark N

    2017-07-01

    Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity. We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24 and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin α5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses. m.n.wass@kent.ac.uk , m.michaelis@kent.ac.uk or j.s.rossman@kent.ac.uk. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  12. Targeting of rotavirus VP6 to DEC-205 induces protection against the infection in mice.

    PubMed

    Badillo-Godinez, O; Gutierrez-Xicotencatl, L; Plett-Torres, T; Pedroza-Saavedra, A; Gonzalez-Jaimes, A; Chihu-Amparan, L; Maldonado-Gama, M; Espino-Solis, G; Bonifaz, L C; Esquivel-Guadarrama, F

    2015-08-20

    Rotavirus (RV) is the primary etiologic agent of severe gastroenteritis in human infants. Although two attenuated RV-based vaccines have been licensed to be applied worldwide, they are not so effective in low-income countries, and the induced protection mechanisms have not been clearly established. Thus, it is important to develop new generation vaccines that induce long lasting heterotypic immunity. VP6 constitutes the middle layer protein of the RV virion. It is the most conserved protein and it is the target of protective T-cells; therefore, it is a potential candidate antigen for a new generation vaccine against the RV infection. We determined whether targeting the DEC-205 present in dendritic cells (DCs) with RV VP6 could induce protection at the intestinal level. VP6 was cross-linked to a monoclonal antibody (mAb) against murine DEC-205 (αDEC-205:VP6), and BALB/c mice were inoculated subcutaneously (s.c.) twice with the conjugated containing 1.5 μg of VP6 in the presence of polyinosinic-polycytidylic acid (Poly I:C) as adjuvant. As controls and following the same protocol, mice were immunized with ovalbumin (OVA) cross-linked to the mAb anti-DEC-205 (αDEC-205:OVA), VP6 cross-linked to a control isotype mAb (Isotype:VP6), 3 μg of VP6 alone, Poly I:C or PBS. Two weeks after the last inoculation, mice were orally challenged with a murine RV. Mice immunized with α-DEC-205:VP6 and VP6 alone presented similar levels of serum Abs to VP6 previous to the virus challenge. However, after the virus challenge, only α-DEC-205:VP6 induced up to a 45% IgA-independent protection. Memory T-helper (Th) cells from the spleen and the mesenteric lymph node (MLN) showed a Th1-type response upon antigen stimulation in vitro. These results show that when VP6 is administered parenterally targeting DEC-205, it can induce protection at the intestinal level at a very low dose, and this protection may be Th1-type cell dependent. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

    PubMed

    Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

    2016-07-01

    A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.

  14. The pH Stability of Foot-and-Mouth Disease Virus Particles Is Modulated by Residues Located at the Pentameric Interface and in the N Terminus of VP1.

    PubMed

    Caridi, Flavia; Vázquez-Calvo, Angela; Sobrino, Francisco; Martín-Acebes, Miguel A

    2015-05-01

    The picornavirus foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects important livestock species. The FMDV capsid is highly acid labile, and viral particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid sensitivity is related to the mechanism of viral uncoating and genome penetration from endosomes. In this study, we have analyzed the molecular basis of FMDV acid-induced disassembly by isolating and characterizing a panel of novel FMDV mutants differing in acid sensitivity. Amino acid replacements altering virion stability were preferentially distributed in two different regions of the capsid: the N terminus of VP1 and the pentameric interface. Even more, the acid labile phenotype induced by a mutation located at the pentameric interface in VP3 could be compensated by introduction of an amino acid substitution in the N terminus of VP1. These results indicate that the acid sensitivity of FMDV can be considered a multifactorial trait and that virion stability is the fine-tuned product of the interaction between residues from different capsid proteins, in particular those located within the N terminus of VP1 or close to the pentameric interface. The viral capsid protects the viral genome from environmental factors and contributes to virus dissemination and infection. Thus, understanding of the molecular mechanisms that modulate capsid stability is of interest for the basic knowledge of the biology of viruses and as a tool to improve the stability of conventional vaccines based on inactivated virions or empty capsids. Using foot-and-mouth disease virus (FMDV), which displays a capsid with extreme acid sensitivity, we have performed a genetic study to identify the molecular determinants involved in capsid stability. A panel of FMDV mutants with differential sensitivity to acidic pH was generated and characterized, and the results showed that two different regions of FMDV

  15. Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

    PubMed

    Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

    2010-11-01

    In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

  16. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    SciTech Connect

    Xu, Juan; Department of Microbiology and Immunology, Nanjing Medical University; Wang, Shixia

    Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71more » (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.« less

  17. Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

    PubMed

    Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

    2015-03-01

    Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign

  18. Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT.

    PubMed

    van Leeuwen, Hans; Okuwaki, Mitsuru; Hong, Rui; Chakravarti, Debabrata; Nagata, Kyosuke; O'Hare, Peter

    2003-09-01

    Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

  19. Efficacy of Vesicular Stomatitis Virus-Ebola Virus Postexposure Treatment in Rhesus Macaques Infected With Ebola Virus Makona.

    PubMed

    Marzi, Andrea; Hanley, Patrick W; Haddock, Elaine; Martellaro, Cynthia; Kobinger, Gary; Feldmann, Heinz

    2016-10-15

    The Ebola virus (EBOV) epidemic in West Africa increased the focus on vaccine development against this hemorrhagic fever-causing pathogen, and as a consequence human clinical trials for a few selected platforms were accelerated. One of these vaccines is vesicular stomatitis virus (VSV)-EBOV, also known as rVSV-ZEBOV, a fast-acting vaccine against EBOV and so far the only vaccine with reported efficacy against EBOV infections in humans in phase III clinical trials. In this study, we analyzed the potential of VSV-EBOV for postexposure treatment of rhesus macaques infected with EBOV-Makona. We treated groups of animals with 1 dose of VSV-EBOV either in a single injection at 1 or 24 hours after EBOV exposure or with 2 injections, half the dose at each time point; 1 control group received the same dose of the VSV-based Marburg virus vaccine at both time points; another group remained untreated. Although all untreated animals succumbed to EBOV infection, 33%-67% of the animals in each treatment group survived the infection, including the group treated with the VSV-based Marburg virus vaccine. This result suggests that protection from postexposure vaccination may be antigen unspecific and due rather to an early activation of the innate immune system. In conclusion, VSV-EBOV remains a potent and fast-acting prophylactic vaccine but demonstrates only limited efficacy in postexposure treatment. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  20. The Disappearing Fourth Wall: John Marburger, Science Policy, and the SSC

    NASA Astrophysics Data System (ADS)

    Crease, Robert

    2015-04-01

    John H. Marburger (1941-2011) was a skilled science administrator who had a fresh and unique approach to science policy and science leadership. His posthumously published book Science Policy up Close contains recollections of key science policy episodes in which he participated or observed closely. One was the administration of the Superconducting Supercollider (SSC); Marburger was Chairman of the Universities Research Association, the group charged with managing the SSC, from 1988-1994. Many accounts of the SSC saga attribute its demise to a combination of transitory factors: poor management, rising cost estimates, the collapse of the Soviet Union and thus of the Cold War threat, complaints by ``small science'' that the SSC's ``big science'' was consuming their budget, Congress's desire to cut spending, unwarranted contract regulations imposed by the Department of Energy (DOE) in response to environmental lapses at nuclear weapons laboratories, and so forth. Marburger tells a subtler story whose implications for science policy are more significant and far-reaching. The story involves changes in the attitude of the government towards large scientific projects that reach back to management reforms introduced by the administration of Presidents Johnson, Nixon, and Carter in the 1960s and 1970s. This experience impressed Marburger with the inevitability of public oversight of large scientific projects, and with the need for planners of such projects to establish and make public a cost and schedule tracking system that would model the project's progress and expenditures.

  1. Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins

    NASA Technical Reports Server (NTRS)

    Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

  2. Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses

    PubMed Central

    Pappalardo, Morena; Juliá, Miguel; Howard, Mark J.; Rossman, Jeremy S.; Michaelis, Martin; Wass, Mark N.

    2016-01-01

    Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission. PMID:27009368

  3. Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses.

    PubMed

    Pappalardo, Morena; Juliá, Miguel; Howard, Mark J; Rossman, Jeremy S; Michaelis, Martin; Wass, Mark N

    2016-03-24

    Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission.

  4. A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy.

    PubMed

    Lupini, Caterina; Giovanardi, Davide; Pesente, Patrizia; Bonci, Michela; Felice, Viviana; Rossi, Giulia; Morandini, Emilio; Cecchinato, Mattia; Catelli, Elena

    2016-08-01

    A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.

  5. 75 FR 20828 - Availability for Non-Exclusive, Exclusive, or Partially Exclusive Licensing of U.S. Patent...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... Partially Exclusive Licensing of U.S. Patent Application Concerning a Chimeric Ebola and Marburg Virus Glycoproteins Virus Like Particle Vaccine To Protect Against Diverse Ebola and Marburg Viruses AGENCY... Ebola and Marburg Virus Glycoproteins Virus Like Particle Vaccine To Protect Against Diverse Ebola and...

  6. Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

    SciTech Connect

    Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.

    2005-06-01

    The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by themore » virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement.« less

  7. Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression.

    PubMed

    Gnirss, Kerstin; Kühl, Annika; Karsten, Christina; Glowacka, Ilona; Bertram, Stephanie; Kaup, Franziska; Hofmann, Heike; Pöhlmann, Stefan

    2012-03-01

    Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Hemorrhagic Fever Virus Budding Studies.

    PubMed

    Harty, Ronald N

    2018-01-01

    Independent expression of the VP40 or Z matrix proteins of filoviruses (marburgviruses and ebolaviruses) and arenaviruses (Lassa fever and Junín), respectively, gives rise to the production and release of virus-like particles (VLPs) that are morphologically identical to infectious virions. We can detect and quantify VLP production and egress in mammalian cells by transient transfection, SDS-PAGE, Western blotting, and live cell imaging techniques such as total internal reflection fluorescence (TIRF) microscopy. Since the VLP budding assay accurately mimics budding of infectious virus, this BSL-2 assay is safe and useful for the interrogation of both viral and host determinants required for budding and can be used as an initial screen to identify and validate small molecule inhibitors of virus release and spread.

  9. H1PVAT is a novel and potent early-stage inhibitor of poliovirus replication that targets VP1.

    PubMed

    Tijsma, Aloys; Thibaut, Hendrik Jan; Spieser, Stéphane A H; De Palma, Armando; Koukni, Mohamed; Rhoden, Eric; Oberste, Steve; Pürstinger, Gerhard; Volny-Luraghi, Antonia; Martin, Javier; Marchand, Arnaud; Chaltin, Patrick; Neyts, Johan; Leyssen, Pieter

    2014-10-01

    A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core β-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game. Copyright © 2014. Published by Elsevier B.V.