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Geoffrey L Smith - One of the best experts on this subject based on the ideXlab platform.
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golgi anti apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration
Journal of Biological Chemistry, 2015Co-Authors: Guia Carrara, Maddy Parsons, Bernadette Byrne, David L Prole, Colin W Taylor, Nuno Saraiva, Geoffrey L SmithAbstract:Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxViruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from Camelpox Virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.
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The vaccinia Virus soluble interferon-c receptor is a homodimer
2015Co-Authors: Antonio Alcami, Geoffrey L SmithAbstract:The vaccinia Virus (VV) interferon (IFN)-c receptor (IFN-cR) is a 43 kDa soluble glycoprotein that is secreted from infected cells early during infection. Here we demonstrate that the IFN-cR from VV, cowpox Virus and Camelpox Virus exists naturally as a homodimer, whereas the cellular IFN-cR dimerizes only upon binding the homodimeric IFN-c. The existence of the Virus protein as a dimer in the absence of ligand may provide an advantage to the Virus in efficient binding and inhibition of IFN-c in solution. Vaccinia Virus (VV) is an orthopoxVirus that expresses many proteins that modulate the host response to infection (Smith et al., 1997b). One strategy to suppress the host response to infection employed by VV and other poxViruses is to express proteins that are secreted from the infected cell and which bind host cytokines, chemokines, complement factors or interferons (IFNs). These secreted Virus proteins often share amino acid similarity with host proteins that bind the same ligands, although this is not always the case. To date, VV has been shown to express soluble proteins that bind tumour necrosis factor (TNF) (Alcamı! et al., 1999), interleukin (IL)-1
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and cell-surface tumour necrosis factor receptors
2014Co-Authors: Antonio Alcami, Anu Khanna, Nina L. Paul, Geoffrey L SmithAbstract:PoxViruses encode a broad range of proteins that interfere with host immune functions such as soluble versions of cytokine receptors. Soluble Virus tumour necrosis factor receptors (vTNFRs) were described originally in myxoma and Shope fibroma Viruses. Cowpox Virus (CPV) encodes three vTNFRs (CrmB, CrmC and CrmD). The genes equivalent to CrmB and CrmC in vaccinia Virus (VV) Copenhagen are mutated and are named B28R/C22L and A53R, respectively. CrmD was identified recently in CPV and ectromelia Virus but the gene is absent in VV Copenhagen. We have tested for expression of soluble binding activity for human TNF in cultures infected with 18 orthopoxViruses and have found that TNFRs are mostly absent but are produced by VV strains Lister, USSR and Evans, by the CPV elephantpox and by Camelpox Virus. Interestingly, we also found TNFR activity on the surface of cells infected with VV Lister, USSR and Evans. Sequence analysis of the relevant regions in VV Lister identified an intact A53R gene and an inactive B28R gene. Expression of VV Lister A53R in baculoVirus and VV Western Reserve demonstrated that gene A53R encodes an active soluble vTNFR of 22 kDa. Expression and characterization of recombinant vTNFRs from VV Lister (A53R) and CPV (CrmB and CrmC) showed a similar binding specificity, with each receptor binding TNF from man, mouse and rat, but not human lymphotoxin-a. Lastly, the VV Lister and CPV vTNFRs bind human TNF with high affinity and prevent the binding of TNF to cellular receptors
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related to variola Virus, the cause
2013Co-Authors: Caroline Gubser, Geoffrey L SmithAbstract:The sequence of Camelpox Virus shows it is most closel
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six transmembrane topology for golgi anti apoptotic protein gaap and bax inhibitor 1 bi 1 provides model for the transmembrane bax inhibitor containing motif tmbim family
Journal of Biological Chemistry, 2012Co-Authors: Guia Carrara, Caroline Gubser, Nuno Saraiva, Benjamin F Johnson, Geoffrey L SmithAbstract:Abstract The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia Virus (VACV) and Camelpox Virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins.
Christian Drosten - One of the best experts on this subject based on the ideXlab platform.
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Real-Time PCR System for Detection of OrthopoxViruses and Simultaneous Identification of Smallpox Virus
Journal of clinical microbiology, 2004Co-Authors: Victoria A. Olson, Thomas Laue, Miriam Laker, I. V. Babkin, Christian Drosten, Sergei N. Shchelkunov, Matthias Niedrig, Hermann MeyerAbstract:A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox Virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox Virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola Virus isolates of the Russian World Health Organization smallpox Virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus OrthopoxVirus. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox Virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox Virus from other orthopoxViruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxVirus species variola, vaccinia, Camelpox, mousepox, cowpox, and monkeypox Virus. The melting temperatures (Tms) determined for 46 strains of variola Virus (Tms, 55.9 to 57.8°C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia Virus (Tms, 61.7 to 62.7°C), 15 strains of monkeypox Virus (Tms, 61.9 to 62.2°C), 40 strains of cowpox Virus (Tms, 61.3 to 63.7°C), 8 strains of mousepox Virus (Tm, 61.9°C), and 8 strains of Camelpox Virus (Tms, 64.0 to 65.0°C). As most of the smallpox Virus samples were derived from infected cell cultures and tissues, smallpox Virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox Virus target DNA per sample. The DNAs of several human herpesViruses as well as poxViruses other than orthopoxViruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxViruses, with the advantage that it can simultaneously identify variola Virus.
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rapid detection and differentiation of human pathogenic orthopox Viruses by a fluorescence resonance energy transfer real time pcr assay
Clinical Chemistry, 2004Co-Authors: Marcus Panning, Marcel Asper, Stefanie Kramme, Herbert Schmitz, Christian DrostenAbstract:Background: The orthopox Viruses that are pathogenic for humans include variola major Virus (VAR), monkeypox Virus (MPV), cowpox Virus (CPV), and to a lesser extent, Camelpox Virus (CML) and vaccinia Virus (VAC). PCR is a powerful tool to detect and differentiate orthopox Viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. Results: Strain differentiation results showed little interassay variability (CV, 0.4–0.6%), and the test was 100-fold more sensitive than Virus culture on Vero cells. Low copy numbers of DNA could be detected with ≥95% probability (235–849 genome copies/mL of plasma). Conclusions: The real-time PCR assay can detect and differentiate human pathogenic orthopox Viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.
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Rapid detection and differentiation of human pathogenic orthopox Viruses by a fluorescence resonance energy transfer real-time PCR assay. Clin. Chem
2004Co-Authors: Marcus Panning, Marcel Asper, Stefanie Kramme, Herbert Schmitz, Christian DrostenAbstract:Background: The orthopox Viruses that are pathogenic for humans include variola major Virus (VAR), monkey-pox Virus (MPV), cowpox Virus (CPV), and to a lesser extent, Camelpox Virus (CML) and vaccinia Virus (VAC). PCR is a powerful tool to detect and differentiate orthopox Viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of con-tamination, capability of strain differentiation, and use of multiplexed probes. Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction
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Rapid detection and differentiation of human pathogenic orthopox Viruses by fluorescence resonance energy. Clin. Chem
2004Co-Authors: Marcus Panning, Marcel Asper, Stefanie Kramme, Herbert Schmitz, Christian DrostenAbstract:Background: The orthopox Viruses that are pathogenic for humans include variola major Virus (VAR), monkey-pox Virus (MPV), cowpox Virus (CPV), and to a lesser extent, Camelpox Virus (CML) and vaccinia Virus (VAC). PCR is a powerful tool to detect and differentiate orthopox Viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of con-tamination, capability of strain differentiation, and use of multiplexed probes. Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction
Graciela Andrei - One of the best experts on this subject based on the ideXlab platform.
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st 246 is a key antiviral to inhibit the viral f13l phospholipase one of the essential proteins for orthopoxVirus wrapping
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Sophie Duraffour, Hermann Meyer, Graciela Andrei, Dimitrios Topalis, Maria M Lorenzo, Gudrun Zoller, Doug Grosenbach, Dennis E Hruby, Rafael Blasco, Robert SnoeckAbstract:OBJECTIVES ST-246 is one of the key antivirals being developed to fight orthopoxVirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. METHODS Vaccinia Virus (VACV), cowpox Virus (CPXV) and Camelpox Virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. RESULTS Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant Viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked Virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. CONCLUSIONS Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.
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Study of Camelpox Virus Pathogenesis in Athymic Nude Mice
2013Co-Authors: Sophie Duraffour, Patrick Matthys, Joost J. Van Den Oord, Tim De Schutter, Tania Mitera, Graciela AndreiAbstract:Camelpox Virus (CMLV) is the closest known orthopoxVirus genetically related to variola Virus. So far, CMLV was restricted to camelids but, recently, three human cases of Camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b + F4/80 + macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c + CD8a + lymphoid and CD11c + CD11b + myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100 % protection from morbidity. It appeared that bot
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mutations conferring resistance to viral dna polymerase inhibitors in Camelpox Virus give different drug susceptibility profiles in vaccinia Virus
Journal of Virology, 2012Co-Authors: Sophie Duraffour, Graciela Andrei, Marcela Krečmerová, Jeanmarc Crance, Daniel Garin, Dimitrios Topalis, Robert SnoeckAbstract:Cidofovir or (S)-HPMPC is one of the three antiviral drugs that might be used for the treatment of orthopoxVirus infections. (S)-HPMPC and its 2,6-diaminopurine counterpart, (S)-HPMPDAP, have been described to select, in vitro, for drug resistance mutations in the viral DNA polymerase (E9L) gene of vaccinia Virus (VACV). Here, to extend our knowledge of drug resistance development among orthopoxViruses, we selected, in vitro, Camelpox Viruses (CMLV) resistant to (S)-HPMPDAP and identified a single amino acid change, T831I, and a double mutation, A314V+A684V, within E9L. The production of recombinant CMLV and VACV carrying these amino acid substitutions (T831I, A314V, or A314V+A684V) demonstrated clearly their involvement in conferring reduced sensitivity to viral DNA polymerase inhibitors, including (S)-HPMPDAP. Both CMLV and VACV harboring the A314V change showed comparable drug-susceptibility profiles to various antivirals and similar impairments in viral growth. In contrast, the single change T831I and the double change A314V+A684V in VACV were responsible for increased levels of drug resistance and for cross-resistance to viral DNA polymerase antivirals that were not observed with their CMLV counterparts. Each amino acid change accounted for an attenuated phenotype of VACV in vivo. Modeling of E9L suggested that the T→I change at position 831 might abolish hydrogen bonds between E9L and the DNA backbone and have a direct impact on the incorporation of the acyclic nucleoside phosphonates. Our findings demonstrate that drug-resistance development in two related orthopoxVirus species may impact drug-susceptibility profiles and viral fitness differently.
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study of Camelpox Virus pathogenesis in athymic nude mice
PLOS ONE, 2011Co-Authors: Sophie Duraffour, Robert Snoeck, Joost Van Den Oord, Patrick Matthys, Tim De Schutter, Tania Mitera, Graciela AndreiAbstract:Camelpox Virus (CMLV) is the closest known orthopoxVirus genetically related to variola Virus. So far, CMLV was restricted to camelids but, recently, three human cases of Camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b(+)F4/80(+) macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c(+)CD8α(+) lymphoid and CD11c(+)CD11b(+) myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100% protection from morbidity. It appeared that both treatments did not affect immune cell responses or cytokine expression. In conclusion, we demonstrated that immunodeficient mice are permissive for CMLV propagation. These results provide a basis for studying the pathogenesis of CMLV, as well as for evaluating potential antiviral therapies in an immunodeficiency context.
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activities of several classes of acyclic nucleoside phosphonates against Camelpox Virus replication in different cell culture models
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Sophie Duraffour, Antonín Holý, Robert Snoeck, Marcela Krečmerová, Erik De Clercq, Joost Van Den Oord, Rita Vos, Jeanmarc Crance, Daniel Garin, Graciela AndreiAbstract:Camelpox Virus (CMLV), the etiologic agent of Camelpox, belongs to the family Poxviridae, genus OrthopoxVirus. Camelpox occurs naturally in Old World camels including Camelus dromedarius and Camelus bactrianus (43). This viral infectious disease has been reported throughout areas of Africa, north of the Equator, the Middle East, and Asia, and outbreaks of CMLV infection in camelids can be responsible for severe economic losses in these countries (22, 27). Camelids may become infected via small abrasions of the skin, by aerosol infection of the respiratory tract, or by arthropod bites. Clinically, two distinct forms can be distinguished: the severe generalized form (mostly among young animals) and the milder localized form (mostly in old camels). In both forms, initial multiplication of the Virus occurs at the site of entry. In systemic disease, further viral multiplication in the draining lymph nodes is followed by a primary viremia and viral replication in organs and tissues (43). This results in a second viremia and subsequent infection of the skin. Eruptions over the entire body are found in generalized forms, in which the mortality rate can reach 28% (26). In the milder forms, pustules appear on the nostrils and eyelids, and on the oral and nasal mucosae. Camelpox is most probably not a zoonosis. Skin eruptions of camel herdsmen for whom CMLV was clinically highly suspected but not confirmed microbiologically have been reported (26). In 2002, a comparative study of the CMLV and variola Virus (VARV) genomes showed that of all poxViruses, CMLV is the orthopoxVirus most closely related to VARV, the causative agent of smallpox (23). These two Viruses not only share colinearity in their genomes but also have similar abilities to induce high morbidity and mortality in a single host species (20). Although smallpox has been eradicated, there are concerns about the potential use of VARV in bioterrorism. Moreover, most of the world's population has become susceptible to any potential infection with poxViruses since the end of the smallpox vaccination campaign in 1978 (6). Thus, human health may be threatened by the emergence or reemergence of orthopoxViruses such as VARV and monkeypox Virus (18). Several orthopoxViruses have been extensively studied as surrogate models of VARV. This research has been based on molecular biology, genomics, diagnostics, vaccines, and antiviral drugs. The cytosine derivative cidofovir [(S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC; Vistide)], belonging to the first class of acyclic nucleoside phosphonates (ANPs), has been licensed for the treatment of cytomegaloVirus retinitis in AIDS patients and is currently recommended by the U.S. Centers for Disease Control and Prevention for the treatment of severe adverse effects following smallpox vaccination (9, 14). Recently, vaccinia-immune globulins, cidofovir, and ST-246 have been used to treat a child suffering from eczema vaccinatum (31). Cidofovir is available for intravenous use as a solution, and the nephrotoxicity that may be associated with its use can be prevented by coadministration of probenecid (13). Cidofovir is a potent antiviral agent that has been shown to be active against poxViruses in cell cultures and in animal models (13). It improved the survival of mice infected with lethal doses of cowpox Virus or vaccinia Virus (8, 36, 38) and of monkeys with lethal respiratory monkeypox Virus infections (25, 42). Recently, two new classes of ANPs have been described: 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (DAPy) derivatives, which are considered to be the second class of ANPs, and a third class of ANPs, including HPMP derivatives with a 5-azacytosine moiety (4, 16, 24, 28; M. Krecmerova, A. Holý, R. Pohl, M. Masojidkova, G. Andrei, L. Naesens, J. Neyts, J. Balzarini, E. De Clercq, and R. Snoeck, submitted for publication). In this study we evaluated the activities of more than 40 compounds selected from the three classes of ANPs against the replication of CMLV strains Iran (33) and Dubai (32) in cell monolayers, as well as in 3-dimensional (3-D) epithelial raft cultures. We used both CMLV strains, isolated from two independent outbreaks, as surrogate models with which to identify compounds potentially active against infections caused by VARV and other poxViruses.
Sophie Duraffour - One of the best experts on this subject based on the ideXlab platform.
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st 246 is a key antiviral to inhibit the viral f13l phospholipase one of the essential proteins for orthopoxVirus wrapping
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Sophie Duraffour, Hermann Meyer, Graciela Andrei, Dimitrios Topalis, Maria M Lorenzo, Gudrun Zoller, Doug Grosenbach, Dennis E Hruby, Rafael Blasco, Robert SnoeckAbstract:OBJECTIVES ST-246 is one of the key antivirals being developed to fight orthopoxVirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. METHODS Vaccinia Virus (VACV), cowpox Virus (CPXV) and Camelpox Virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. RESULTS Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant Viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked Virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. CONCLUSIONS Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.
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Study of Camelpox Virus Pathogenesis in Athymic Nude Mice
2013Co-Authors: Sophie Duraffour, Patrick Matthys, Joost J. Van Den Oord, Tim De Schutter, Tania Mitera, Graciela AndreiAbstract:Camelpox Virus (CMLV) is the closest known orthopoxVirus genetically related to variola Virus. So far, CMLV was restricted to camelids but, recently, three human cases of Camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b + F4/80 + macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c + CD8a + lymphoid and CD11c + CD11b + myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100 % protection from morbidity. It appeared that bot
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mutations conferring resistance to viral dna polymerase inhibitors in Camelpox Virus give different drug susceptibility profiles in vaccinia Virus
Journal of Virology, 2012Co-Authors: Sophie Duraffour, Graciela Andrei, Marcela Krečmerová, Jeanmarc Crance, Daniel Garin, Dimitrios Topalis, Robert SnoeckAbstract:Cidofovir or (S)-HPMPC is one of the three antiviral drugs that might be used for the treatment of orthopoxVirus infections. (S)-HPMPC and its 2,6-diaminopurine counterpart, (S)-HPMPDAP, have been described to select, in vitro, for drug resistance mutations in the viral DNA polymerase (E9L) gene of vaccinia Virus (VACV). Here, to extend our knowledge of drug resistance development among orthopoxViruses, we selected, in vitro, Camelpox Viruses (CMLV) resistant to (S)-HPMPDAP and identified a single amino acid change, T831I, and a double mutation, A314V+A684V, within E9L. The production of recombinant CMLV and VACV carrying these amino acid substitutions (T831I, A314V, or A314V+A684V) demonstrated clearly their involvement in conferring reduced sensitivity to viral DNA polymerase inhibitors, including (S)-HPMPDAP. Both CMLV and VACV harboring the A314V change showed comparable drug-susceptibility profiles to various antivirals and similar impairments in viral growth. In contrast, the single change T831I and the double change A314V+A684V in VACV were responsible for increased levels of drug resistance and for cross-resistance to viral DNA polymerase antivirals that were not observed with their CMLV counterparts. Each amino acid change accounted for an attenuated phenotype of VACV in vivo. Modeling of E9L suggested that the T→I change at position 831 might abolish hydrogen bonds between E9L and the DNA backbone and have a direct impact on the incorporation of the acyclic nucleoside phosphonates. Our findings demonstrate that drug-resistance development in two related orthopoxVirus species may impact drug-susceptibility profiles and viral fitness differently.
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study of Camelpox Virus pathogenesis in athymic nude mice
PLOS ONE, 2011Co-Authors: Sophie Duraffour, Robert Snoeck, Joost Van Den Oord, Patrick Matthys, Tim De Schutter, Tania Mitera, Graciela AndreiAbstract:Camelpox Virus (CMLV) is the closest known orthopoxVirus genetically related to variola Virus. So far, CMLV was restricted to camelids but, recently, three human cases of Camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b(+)F4/80(+) macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c(+)CD8α(+) lymphoid and CD11c(+)CD11b(+) myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100% protection from morbidity. It appeared that both treatments did not affect immune cell responses or cytokine expression. In conclusion, we demonstrated that immunodeficient mice are permissive for CMLV propagation. These results provide a basis for studying the pathogenesis of CMLV, as well as for evaluating potential antiviral therapies in an immunodeficiency context.
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activities of several classes of acyclic nucleoside phosphonates against Camelpox Virus replication in different cell culture models
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Sophie Duraffour, Antonín Holý, Robert Snoeck, Marcela Krečmerová, Erik De Clercq, Joost Van Den Oord, Rita Vos, Jeanmarc Crance, Daniel Garin, Graciela AndreiAbstract:Camelpox Virus (CMLV), the etiologic agent of Camelpox, belongs to the family Poxviridae, genus OrthopoxVirus. Camelpox occurs naturally in Old World camels including Camelus dromedarius and Camelus bactrianus (43). This viral infectious disease has been reported throughout areas of Africa, north of the Equator, the Middle East, and Asia, and outbreaks of CMLV infection in camelids can be responsible for severe economic losses in these countries (22, 27). Camelids may become infected via small abrasions of the skin, by aerosol infection of the respiratory tract, or by arthropod bites. Clinically, two distinct forms can be distinguished: the severe generalized form (mostly among young animals) and the milder localized form (mostly in old camels). In both forms, initial multiplication of the Virus occurs at the site of entry. In systemic disease, further viral multiplication in the draining lymph nodes is followed by a primary viremia and viral replication in organs and tissues (43). This results in a second viremia and subsequent infection of the skin. Eruptions over the entire body are found in generalized forms, in which the mortality rate can reach 28% (26). In the milder forms, pustules appear on the nostrils and eyelids, and on the oral and nasal mucosae. Camelpox is most probably not a zoonosis. Skin eruptions of camel herdsmen for whom CMLV was clinically highly suspected but not confirmed microbiologically have been reported (26). In 2002, a comparative study of the CMLV and variola Virus (VARV) genomes showed that of all poxViruses, CMLV is the orthopoxVirus most closely related to VARV, the causative agent of smallpox (23). These two Viruses not only share colinearity in their genomes but also have similar abilities to induce high morbidity and mortality in a single host species (20). Although smallpox has been eradicated, there are concerns about the potential use of VARV in bioterrorism. Moreover, most of the world's population has become susceptible to any potential infection with poxViruses since the end of the smallpox vaccination campaign in 1978 (6). Thus, human health may be threatened by the emergence or reemergence of orthopoxViruses such as VARV and monkeypox Virus (18). Several orthopoxViruses have been extensively studied as surrogate models of VARV. This research has been based on molecular biology, genomics, diagnostics, vaccines, and antiviral drugs. The cytosine derivative cidofovir [(S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC; Vistide)], belonging to the first class of acyclic nucleoside phosphonates (ANPs), has been licensed for the treatment of cytomegaloVirus retinitis in AIDS patients and is currently recommended by the U.S. Centers for Disease Control and Prevention for the treatment of severe adverse effects following smallpox vaccination (9, 14). Recently, vaccinia-immune globulins, cidofovir, and ST-246 have been used to treat a child suffering from eczema vaccinatum (31). Cidofovir is available for intravenous use as a solution, and the nephrotoxicity that may be associated with its use can be prevented by coadministration of probenecid (13). Cidofovir is a potent antiviral agent that has been shown to be active against poxViruses in cell cultures and in animal models (13). It improved the survival of mice infected with lethal doses of cowpox Virus or vaccinia Virus (8, 36, 38) and of monkeys with lethal respiratory monkeypox Virus infections (25, 42). Recently, two new classes of ANPs have been described: 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (DAPy) derivatives, which are considered to be the second class of ANPs, and a third class of ANPs, including HPMP derivatives with a 5-azacytosine moiety (4, 16, 24, 28; M. Krecmerova, A. Holý, R. Pohl, M. Masojidkova, G. Andrei, L. Naesens, J. Neyts, J. Balzarini, E. De Clercq, and R. Snoeck, submitted for publication). In this study we evaluated the activities of more than 40 compounds selected from the three classes of ANPs against the replication of CMLV strains Iran (33) and Dubai (32) in cell monolayers, as well as in 3-dimensional (3-D) epithelial raft cultures. We used both CMLV strains, isolated from two independent outbreaks, as surrogate models with which to identify compounds potentially active against infections caused by VARV and other poxViruses.
Robert Snoeck - One of the best experts on this subject based on the ideXlab platform.
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st 246 is a key antiviral to inhibit the viral f13l phospholipase one of the essential proteins for orthopoxVirus wrapping
Journal of Antimicrobial Chemotherapy, 2015Co-Authors: Sophie Duraffour, Hermann Meyer, Graciela Andrei, Dimitrios Topalis, Maria M Lorenzo, Gudrun Zoller, Doug Grosenbach, Dennis E Hruby, Rafael Blasco, Robert SnoeckAbstract:OBJECTIVES ST-246 is one of the key antivirals being developed to fight orthopoxVirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. METHODS Vaccinia Virus (VACV), cowpox Virus (CPXV) and Camelpox Virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. RESULTS Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant Viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked Virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. CONCLUSIONS Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.
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mutations conferring resistance to viral dna polymerase inhibitors in Camelpox Virus give different drug susceptibility profiles in vaccinia Virus
Journal of Virology, 2012Co-Authors: Sophie Duraffour, Graciela Andrei, Marcela Krečmerová, Jeanmarc Crance, Daniel Garin, Dimitrios Topalis, Robert SnoeckAbstract:Cidofovir or (S)-HPMPC is one of the three antiviral drugs that might be used for the treatment of orthopoxVirus infections. (S)-HPMPC and its 2,6-diaminopurine counterpart, (S)-HPMPDAP, have been described to select, in vitro, for drug resistance mutations in the viral DNA polymerase (E9L) gene of vaccinia Virus (VACV). Here, to extend our knowledge of drug resistance development among orthopoxViruses, we selected, in vitro, Camelpox Viruses (CMLV) resistant to (S)-HPMPDAP and identified a single amino acid change, T831I, and a double mutation, A314V+A684V, within E9L. The production of recombinant CMLV and VACV carrying these amino acid substitutions (T831I, A314V, or A314V+A684V) demonstrated clearly their involvement in conferring reduced sensitivity to viral DNA polymerase inhibitors, including (S)-HPMPDAP. Both CMLV and VACV harboring the A314V change showed comparable drug-susceptibility profiles to various antivirals and similar impairments in viral growth. In contrast, the single change T831I and the double change A314V+A684V in VACV were responsible for increased levels of drug resistance and for cross-resistance to viral DNA polymerase antivirals that were not observed with their CMLV counterparts. Each amino acid change accounted for an attenuated phenotype of VACV in vivo. Modeling of E9L suggested that the T→I change at position 831 might abolish hydrogen bonds between E9L and the DNA backbone and have a direct impact on the incorporation of the acyclic nucleoside phosphonates. Our findings demonstrate that drug-resistance development in two related orthopoxVirus species may impact drug-susceptibility profiles and viral fitness differently.
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study of Camelpox Virus pathogenesis in athymic nude mice
PLOS ONE, 2011Co-Authors: Sophie Duraffour, Robert Snoeck, Joost Van Den Oord, Patrick Matthys, Tim De Schutter, Tania Mitera, Graciela AndreiAbstract:Camelpox Virus (CMLV) is the closest known orthopoxVirus genetically related to variola Virus. So far, CMLV was restricted to camelids but, recently, three human cases of Camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b(+)F4/80(+) macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c(+)CD8α(+) lymphoid and CD11c(+)CD11b(+) myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100% protection from morbidity. It appeared that both treatments did not affect immune cell responses or cytokine expression. In conclusion, we demonstrated that immunodeficient mice are permissive for CMLV propagation. These results provide a basis for studying the pathogenesis of CMLV, as well as for evaluating potential antiviral therapies in an immunodeficiency context.
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activities of several classes of acyclic nucleoside phosphonates against Camelpox Virus replication in different cell culture models
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Sophie Duraffour, Antonín Holý, Robert Snoeck, Marcela Krečmerová, Erik De Clercq, Joost Van Den Oord, Rita Vos, Jeanmarc Crance, Daniel Garin, Graciela AndreiAbstract:Camelpox Virus (CMLV), the etiologic agent of Camelpox, belongs to the family Poxviridae, genus OrthopoxVirus. Camelpox occurs naturally in Old World camels including Camelus dromedarius and Camelus bactrianus (43). This viral infectious disease has been reported throughout areas of Africa, north of the Equator, the Middle East, and Asia, and outbreaks of CMLV infection in camelids can be responsible for severe economic losses in these countries (22, 27). Camelids may become infected via small abrasions of the skin, by aerosol infection of the respiratory tract, or by arthropod bites. Clinically, two distinct forms can be distinguished: the severe generalized form (mostly among young animals) and the milder localized form (mostly in old camels). In both forms, initial multiplication of the Virus occurs at the site of entry. In systemic disease, further viral multiplication in the draining lymph nodes is followed by a primary viremia and viral replication in organs and tissues (43). This results in a second viremia and subsequent infection of the skin. Eruptions over the entire body are found in generalized forms, in which the mortality rate can reach 28% (26). In the milder forms, pustules appear on the nostrils and eyelids, and on the oral and nasal mucosae. Camelpox is most probably not a zoonosis. Skin eruptions of camel herdsmen for whom CMLV was clinically highly suspected but not confirmed microbiologically have been reported (26). In 2002, a comparative study of the CMLV and variola Virus (VARV) genomes showed that of all poxViruses, CMLV is the orthopoxVirus most closely related to VARV, the causative agent of smallpox (23). These two Viruses not only share colinearity in their genomes but also have similar abilities to induce high morbidity and mortality in a single host species (20). Although smallpox has been eradicated, there are concerns about the potential use of VARV in bioterrorism. Moreover, most of the world's population has become susceptible to any potential infection with poxViruses since the end of the smallpox vaccination campaign in 1978 (6). Thus, human health may be threatened by the emergence or reemergence of orthopoxViruses such as VARV and monkeypox Virus (18). Several orthopoxViruses have been extensively studied as surrogate models of VARV. This research has been based on molecular biology, genomics, diagnostics, vaccines, and antiviral drugs. The cytosine derivative cidofovir [(S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC; Vistide)], belonging to the first class of acyclic nucleoside phosphonates (ANPs), has been licensed for the treatment of cytomegaloVirus retinitis in AIDS patients and is currently recommended by the U.S. Centers for Disease Control and Prevention for the treatment of severe adverse effects following smallpox vaccination (9, 14). Recently, vaccinia-immune globulins, cidofovir, and ST-246 have been used to treat a child suffering from eczema vaccinatum (31). Cidofovir is available for intravenous use as a solution, and the nephrotoxicity that may be associated with its use can be prevented by coadministration of probenecid (13). Cidofovir is a potent antiviral agent that has been shown to be active against poxViruses in cell cultures and in animal models (13). It improved the survival of mice infected with lethal doses of cowpox Virus or vaccinia Virus (8, 36, 38) and of monkeys with lethal respiratory monkeypox Virus infections (25, 42). Recently, two new classes of ANPs have been described: 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (DAPy) derivatives, which are considered to be the second class of ANPs, and a third class of ANPs, including HPMP derivatives with a 5-azacytosine moiety (4, 16, 24, 28; M. Krecmerova, A. Holý, R. Pohl, M. Masojidkova, G. Andrei, L. Naesens, J. Neyts, J. Balzarini, E. De Clercq, and R. Snoeck, submitted for publication). In this study we evaluated the activities of more than 40 compounds selected from the three classes of ANPs against the replication of CMLV strains Iran (33) and Dubai (32) in cell monolayers, as well as in 3-dimensional (3-D) epithelial raft cultures. We used both CMLV strains, isolated from two independent outbreaks, as surrogate models with which to identify compounds potentially active against infections caused by VARV and other poxViruses.
