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See detailDe novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis.
Seumois, Gregory; Fillet, Marianne ULg; Gillet, Laurent ULg et al

in Journal of Leukocyte Biology (2007), 81(6), 1477-1486

Neutrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly ... [more ▼]

Neutrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly understood. We show here that apoptosis of cultured neutrophils is preceded by a substantial increase in the intracellular levels of 16 and 24 carbon atom (C(16)- and C(24))-ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonisin B(2), a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C(16)- and C(24)-ceramides. Moreover, fumonisin B(2) significantly reduced caspase-3, -8, and -9 activation and apoptosis in these cells. Conversely, 3-O-methylsphingomyelin and fantofarone, which are specific inhibitors of neutral and acid sphingomyelinases, respectively, neither inhibited C(16)- and C(24)-ceramide production nor decreased the apoptosis rate in neutrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyelin. Further experiments showed that increasing endogenous C(16)- and C(24)-ceramide levels by using DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol and (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, two inhibitors of ceramide metabolism, enhances caspase-3, -8, and -9 activity and increases neutrophil apoptosis. Similarly, apoptosis was induced rapidly when synthetic C(16)- and/or C(24)-ceramides were added to neutrophil cultures. Finally, GM-CSF, a cytokine that delays neutrophil apoptosis, abrogated C(16)- and C(24)-ceramide accumulation totally in cultured neutrophils, whereas Fas ligation accelerated apoptosis in these cells without affecting de novo ceramide production. We conclude that de novo generation of C(16)- and C(24)-ceramides contributes to spontaneous neutrophil apoptosis via caspase activation and that GM-CSF exerts its antiapoptotic effects on neutrophils, at least partly through inhibition of ceramide accumulation. [less ▲]

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See detailSTAT5 is an Ambivalent Regulator of Neutrophil Homeostasis
Fievez, Laurence ULg; Desmet, Christophe ULg; Henry, E. et al

Poster (2007)

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See detailEvidence for a multiprotein gamma-2 herpesvirus entry complex.
Gillet, Laurent ULg; Stevenson, Philip G

in Journal of Virology (2007), 81(23), 13082-91

Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV ... [more ▼]

Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV-68) gB, gH, gL, and gp150 all participate in entry. gB and gL are involved in binding, gB and gH are conserved fusion proteins, and gp150 inhibits cell binding until glycosaminoglycans are engaged. Here we show that a gH-specific antibody coprecipitates gB and thus that gH and gB are associated in the virion membrane. A gH/gL-specific antibody also coprecipitated gB, implying a tripartite complex of gL/gH/gB, although the gH/gB association did not require gL. The association was also independent of gp150, and gp150 was not demonstrably bound to gB or gH. However, gp150 incorporation into virions was partly gL dependent, suggesting that it too contributes to a single entry complex. gp150- and gL- gp150- mutants bound better than the wild type to B cells and readily colonized B cells in vivo. Thus, gp150 and gL appear to be epithelial cell-adapted accessories of a core gB/gH entry complex. The cell binding revealed by gp150 disruption did not require gL and therefore seemed most likely to involve gB. [less ▲]

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See detailGeneration of a transposon insertion mutant library for bovine herpesvirus 4 cloned as a bacterial artificial chromosome by in vitro MuA based DNA transposition system.
Donofrio, Gaetano; Martignani, Eugenio; Sartori, Chiara et al

in Journal of Virological Methods (2007), 141(1), 63-70

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Although the BoHV-4 genome has been sequenced, the function of the majority of putative genes is elusive. Several ... [more ▼]

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Although the BoHV-4 genome has been sequenced, the function of the majority of putative genes is elusive. Several features make BoHV-4 attractive as a backbone for use as a viral expression vector and/or as a model to study gamma herpesvirus biology and determining which genes are essential for its replication is a very important task. Starting from BoHV-4 genome cloned as infectious bacterial artificial chromosome (BAC-BoHV-4) in Escherichia coli. A random insertion mutant library for BoHV4 was generated by the use of MuA transposase-catalyzed in vitro transposition reaction. Viral mutant transfection and direct sequencing allow the rapid determination of which BoHV-4 genes are essential for viral growth in a permissive eukaryotic cell line. BoHV-4 functional analysis information is fundamental when the BoHV-4 genome is modified for vector purposes. [less ▲]

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See detailEstablishment of a bovine herpesvirus 4 based vector expressing a secreted form of the bovine viral diarrhoea virus structural glycoprotein E2 for immunization purposes.
Donofrio, Gaetano; Sartori, Chiara; Ravanetti, Lara et al

in BMC Biotechnology (2007), 7

BACKGROUND: The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely ... [more ▼]

BACKGROUND: The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. RESULTS: A recombinant bovine herpesvirus 4 (BoHV-4CMV-IgKE2-14 Delta TK) expressing an enhanced secreted form of the bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering (recombination -mediated genetic engineering) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase - based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14 Delta TK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14 Delta TK, high levels of serum neutralized antibodies against BVDV were generated. CONCLUSION: This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2. [less ▲]

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See detailAntibody evasion by the N terminus of murid herpesvirus-4 glycoprotein B.
Gillet, Laurent ULg; Stevenson, Philip G

in EMBO Journal (2007), 26(24), 5131-42

Herpesviruses characteristically transmit infection from immune hosts. Although their success in escaping neutralization by pre-formed antibody is indisputable, the underlying molecular mechanisms remain ... [more ▼]

Herpesviruses characteristically transmit infection from immune hosts. Although their success in escaping neutralization by pre-formed antibody is indisputable, the underlying molecular mechanisms remain largely unknown. Glycoprotein B (gB) is the most conserved component of the herpesvirus entry machinery and its N terminus (gB-NT) is a common neutralization target. We used murid herpesvirus-4 to determine how gB-NT contributes to the virus-antibody interaction. Deleting gB-NT had no obvious impact on virus replication, but paradoxically increased virion neutralization by immune sera. This reflected greater antibody access to neutralization epitopes on gH/gL, with which gB was associated. gB-NT itself was variably protected against antibody by O-linked glycans; on virions from epithelial cells it was protected almost completely. gB-NT therefore provides a protective and largely protected cover for a vulnerable part of gH/gL. The conservation of predicted glycosylation sites in other mammalian herpesvirus gB-NTs suggests that this evasion mechanism is widespread. Interestingly, the gB-NT glycans that blocked antibody binding could be targeted for neutralization instead by a lectin, suggesting a means of therapeutic counterattack. [less ▲]

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See detailGlycoprotein L disruption reveals two functional forms of the murine gammaherpesvirus 68 glycoprotein H.
Gillet, Laurent ULg; May, Janet S; Colaco, Susanna et al

in Journal of Virology (2007), 81(1), 280-91

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and ... [more ▼]

The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells. [less ▲]

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See detailCloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome.
Dewals, Benjamin G ULg; Boudry, Christel ULg; Gillet, Laurent ULg et al

in Journal of General Virology (The) (2006), 87(Pt 3), 509-17

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order Artiodactyla. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, mainly due to the fact that AlHV-1 becomes attenuated during passage in culture. In this study, these difficulties were overcome by cloning the entire AlHV-1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC). A modified loxP-flanked BAC cassette was inserted in one of the two large non-coding regions of the AlHV-1 genome. This insertion allowed the production of an AlHV-1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. The loxP-flanked BAC cassette was excised from the genome of reconstituted virions by growing them in permissive cells stably expressing Cre recombinase. Importantly, BAC-derived AlHV-1 virions replicated comparably to the virulent (low-passage) AlHV-1 parental strain and induced MCF in rabbits that was indistinguishable from that of the virulent parental strain. The availability of the AlHV-1 BAC is an important advance for the study of MCF that will allow the identification of viral genes involved in MCF pathogenesis, as well as the production of attenuated recombinant candidate vaccines. [less ▲]

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See detailFelid herpesvirus 1 glycoprotein G is a structural protein that mediates the binding of chemokines on the viral envelope.
Costes, Bérénice ULg; Thirion, Muriel ULg; Dewals, Benjamin G ULg et al

in Microbes & Infection (2006), 8(11), 2657-67

Glycoprotein G (gG) orthologues have been described in several alphaherpesviruses. gG is expressed both as a membrane-anchored form on infected cells and as a secreted form. Recently, we reported that ... [more ▼]

Glycoprotein G (gG) orthologues have been described in several alphaherpesviruses. gG is expressed both as a membrane-anchored form on infected cells and as a secreted form. Recently, we reported that both forms of gG encoded by alphaherpesviruses infecting large herbivores and by Felid herpesvirus 1 (FeHV-1) bind with high affinity to a broad range of CXC, CC and C-chemokines. Based on the viral species, gG has been reported either as a structural or a non-structural protein. To date, the incorporation of FeHV-1 gG into virions has never been tested, nor the property of alphaherpesvirus structural gG to bind chemokines on the virion surface. In the present study, to address these questions, various FeHV-1 gG recombinant strains were produced using an original technique based on an infectious FeHV-1 BAC clone and restriction endonuclease mediated recombination. Using the recombinants produced, we were able to determine that FeHV-1 gG is a structural protein that acts as a chemokine-binding protein on the virion surface. In the light of these results, putative roles of gG in alphaherpesvirus infections are discussed, and an evolutionary scenario is proposed to explain the structural versus non-structural property of gG amongst alphaherpesviruses. [less ▲]

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See detailCloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome
Dewals, Benjamin G ULg; Boudry, Christel; Gillet, Laurent ULg et al

Poster (2006)

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order Artiodactyla. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, mainly due to the fact that AlHV-1 becomes attenuated during passage in culture. In this study, these difficulties were overcome by cloning the entire AlHV-1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC). A modified loxP-flanked BAC cassette was inserted in one of the two large non-coding regions of the AlHV-1 genome. This insertion allowed the production of an AlHV-1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. The loxP-flanked BAC cassette was excised from the genome of reconstituted virions by growing them in permissive cells stably expressing Cre recombinase. Importantly, BAC-derived AlHV-1 virions replicated comparably to the virulent (low-passage) AlHV-1 parental strain and induced MCF in rabbits that was indistinguishable from that of the virulent parental strain. The availability of the AlHV-1 BAC is an important advance for the study of MCF that will allow the identification of viral genes involved in MCF pathogenesis, as well as the production of attenuated recombinant candidate vaccines. [less ▲]

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See detailMurine gammaherpesvirus-68 glycoprotein H-glycoprotein L complex is a major target for neutralizing monoclonal antibodies.
Gill, Michael B.; Gillet, Laurent ULg; Colaco, Susanna et al

in Journal of General Virology (The) (2006), 87(Pt 6), 1465-75

Herpesviruses characteristically persist in immune hosts as latent genomes, but to transmit infection they must reactivate and replicate lytically. The interaction between newly formed virions and pre ... [more ▼]

Herpesviruses characteristically persist in immune hosts as latent genomes, but to transmit infection they must reactivate and replicate lytically. The interaction between newly formed virions and pre-existing antibody is therefore likely to be a crucial determinant of viral fitness. Murine gammaherpesvirus-68 (MHV-68) behaves as a natural pathogen of conventional, inbred mice and consequently allows such interactions to be analysed experimentally in a relatively realistic setting. Here, monoclonal antibodies (mAbs) were derived from MHV-68-infected mice and all those recognizing infected-cell surfaces were tested for their capacity to neutralize MHV-68 virions. All of the neutralizing mAbs identified were specific for the viral glycoprotein H (gH)-gL heterodimer and required both gH and gL to reproduce their cognate epitopes. Based on antibody interference, there appeared to be two major neutralization epitopes on gH-gL. Analysis of a representative mAb indicated that it blocked infection at a post-binding step--either virion endocytosis or membrane fusion. [less ▲]

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See detailEvolution of Bovine herpesvirus 4: recombination and transmission between African buffalo and cattle
Dewals, Benjamin G ULg; Thirion, Muriel ULg; Markine-Goriaynoff, N. et al

in Journal of General Virology (2006), 87(Pt 6), 1509-1519

Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle throughout the world, but virological and serological studies have suggested that the African buffalo is also a natural host for this virus. It ... [more ▼]

Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle throughout the world, but virological and serological studies have suggested that the African buffalo is also a natural host for this virus. It has previously been found that the Bo17 gene of BoHV-4 was acquired from an ancestor of the African buffalo, probably around 1.5 million years ago. Analysis of the variation of the Bo17 gene sequence among BoHV-4 strains suggested a relatively ancient transmission of BoHV-4 from the buffalo to the Bos primigenius lineage, followed by a host-dependent split between zebu and taurine BoHV-4 strains. In the present study, the evolutionary history of BoHV-4 was investigated by analysis of five gene sequences from each of nine strains representative of the viral species: three isolated from African buffalo in Kenya and six from cattle from Europe, North America and India. No two gene sequences had the same evolutionary tree, indicating that recombination has occurred between divergent lineages; six recombination events were delineated for these sequences. Nevertheless, exchange has been infrequent enough that a clonal evolutionary history of the strains could be discerned, upon which the recombination events were superimposed. The dates of divergence among BoHV-4 lineages were estimated from synonymous nucleotide-substitution rates. The inferred evolutionary history suggests that African buffalo were the original natural reservoir of BoHV-4 and that there have been at least three independent transmissions from buffalo to cattle, probably via intermediate hosts and - at least in the case of North American strains - within the last 500 years. [less ▲]

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See detailRecombinant bovine herpesvirus 4 (BoHV-4) expressing glycoprotein D of BoHV-1 is immunogenic and elicits serum-neutralizing antibodies against BoHV-1 in a rabbit model.
Donofrio, Gaetano; Cavirani, Sandro; Vanderplasschen, Alain ULg et al

in Clinical and Vaccine Immunology (2006), 13(11), 1246-54

Several biological characteristics of bovine herpesvirus 4 (BoHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity ... [more ▼]

Several biological characteristics of bovine herpesvirus 4 (BoHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types coming from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Starting from BoHV-4 cloned as a bacterial artificial chromosome (BAC), we used MuA transposase-mediated in vitro transposition to generate recombinant BoHV-4 expressing the immunodominant glycoprotein D (gD) of BoHV-1, one of the most important pathogens of cattle. Although a cis-acting element from woodchuck hepatitis virus (the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) in the 3' end of the gD expression cassette was required for maximal gD expression from plasmids in transient transfection assays, this element was not necessary for efficient expression of gD from recombinant BoHV-4 genomes. BoHV-4 recombinants containing gD expression cassettes with or without the WPRE expressed gD at similarly high levels. Several cell lines originating from different animal species expressed gD when infected with BoHV-4 recombinants. When rabbits were immunized with one of the recombinants, high levels of serum neutralizing antibodies against BoHV-1 were generated. This work is one of the first demonstrations of the use BoHV-4 as a vector for vaccine purposes and may provide the basis for BoHV-1 vaccination of cattle with recombinant BoHV-4. [less ▲]

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See detailMurine gammaherpesvirus-68 glycoprotein B presents a difficult neutralization target to monoclonal antibodies derived from infected mice.
Gillet, Laurent ULg; Gill, Michael B; Colaco, Susanna et al

in Journal of General Virology (The) (2006), 87(Pt 12), 3515-27

Persistent viruses disseminate from immune hosts. They must therefore resist neutralization by antibody. Murine gammaherpesvirus-68 (MHV-68) represents an accessible model with which to address how ... [more ▼]

Persistent viruses disseminate from immune hosts. They must therefore resist neutralization by antibody. Murine gammaherpesvirus-68 (MHV-68) represents an accessible model with which to address how resistance to neutralization is achieved and how overcoming it might improve infection control. The MHV-68 glycoprotein B (gB), like that of other herpesviruses, is a virion protein that is essential for infectivity. As such, it presents a potential neutralization target. In order to test whether virus-induced antibodies reduce virion infectivity by binding to gB, monoclonal antibodies (mAbs) were derived from MHV-68-infected mice. gB-specific mAbs were common, but only an IgM specific for the gB N terminus reduced virion infectivity significantly. It inhibited MHV-68 entry into BHK-21 cells at a post-binding step that was linked closely to membrane fusion. Reducing the mAb to IgM monomers compromised neutralization severely, suggesting that a pentameric structure was crucial to its function. Antibody treatment never blocked BHK-21 cell infection completely and blocked the infection of NMuMG epithelial cells hardly at all. Virions saturated with antibody also remained infectious to mice. Thus, the MHV-68 gB presents at best a very difficult target for antibody-mediated neutralization. [less ▲]

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See detailDemonstration by flow cytometry that CD5+CD8+ cells carry alcelaphine herpesvirus 1 in inoculated rabbits developing malignant catarrhal fever
Dewals, Benjamin G ULg; Gillet, Laurent ULg; Vanderplasschen, Alain ULg

Poster (2005, November 18)

Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest (Connochaetes taurinus) asymptomatically, causes malignant catarrhal fever (MCF) when cross species transmitted to a variety of susceptible ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest (Connochaetes taurinus) asymptomatically, causes malignant catarrhal fever (MCF) when cross species transmitted to a variety of susceptible species of the Artiodactyla order. MCF is a fascinating disease described as a combination of lymphoproliferative and degenerative lesions. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, due mainly to the fact that AlHV 1 becomes attenuated during passage in culture. Here, we have overcome these difficulties by (i) cloning the entire AlHV 1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC), and (ii) by using prokaryotic recombination technology for the production of an AlHV 1 recombinant. Firstly, the AlHV 1 genome was BAC cloned using one insertion site in a region containing no open reading frame. This insertion allowed the production of an AlHV 1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. BAC derived AlHV 1 virions induced MCF in rabbits comparably to the AlHV 1 wild type (WT) strain. Secondly, a two step mutagenesis procedure in E. coli was used to generate a recombinant strain expressing enhanced-green fluorescent protein (EGFP) as a reporter gene. After reconstitution of recombinant virions into permissive cells and excision of the BAC cassette, flow cytometry analyses were performed to validate the recombinant strain and to investigate the pathogenesis of MCF. The results of these analyses can be summarized as follows: (i) the validity of the EGFP expression cassette as a reporter gene has been demonstrated by in vitro infections; (ii) inoculation of rabbits revealed that the recombinant strain has retained the pathogenicity of its parental strain and that the cell types carrying AlHV-1 in peripheral blood mononuclear cells, lymph nodes and the spleen are mainly CD5+ CD8+ cells. [less ▲]

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See detailClonage de l’herpèsvirus alcélaphin 1 sous forme d’un chromosome artificiel bactérien infectieux
Dewals, Benjamin G ULg; Boudry, Christel; Markine-Goriaynoff, Nicolas et al

Poster (2005, April)

L’herpèsvirus alcélaphin 1 (AlHV-1) est un Gammaherpesvirinae du genre Rhadinovirus ayant pour hôte naturel le gnou (Connochaetes taurinus). Apathogène pour son hôte naturel, ce virus induit par ailleurs ... [more ▼]

L’herpèsvirus alcélaphin 1 (AlHV-1) est un Gammaherpesvirinae du genre Rhadinovirus ayant pour hôte naturel le gnou (Connochaetes taurinus). Apathogène pour son hôte naturel, ce virus induit par ailleurs une pathologie mortelle lorsqu’il est transmis à un grand nombre d’espèces de ruminants sensibles. Cette pathologie, appelée forme africaine du coryza gangreneux (FACG) est associée à une lymphoprolifération et une destruction des tissus de l’hôte infecté. Pour étudier le rôle de l’AlHV-1 dans la pathogenèse de la FACG, il est nécessaire de pouvoir manipuler le génome viral afin de générer des virus recombinants et révertants pour certains gènes. A ce jour, aucune souche d’AlHV-1 recombinante n’a pu être produite. Cette carence résulte du fait que ce virus est strictement associé aux cellules et qu’il possède la caractéristique de s’atténuer spontanément lors de sa multiplication in vitro. De ce fait, la réalisation de virus mutants se révèle impossible par une approche classique de recombinaison homologue en cellules eucaryotes. Récemment, le développement des technologies de chromosome artificiel bactérien (BAC) a permis de cloner le génome entier de plusieurs gammaherpèsvirus de manière stable en bactérie ainsi que la production rapide de nombreuses souches recombinantes. Dans la présente étude, nous avons cloné le génome entier de la souche C500 de l’AlHV-1 sous forme d’un BAC appelé ci-après BAC-AlHV-1. Les résultats obtenus peuvent se résumer comme suit : (i) le BAC-AlHV-1 permet une propagation stable du génome de l’AlHV-1 en bactérie ; (ii) la transfection du BAC-AlHV-1 en cellules eucaryotes permet de régénérer des particules virales infectieuses ; (iii) la cassette BAC insérée initialement dans le génome de l’AlHV-1 étant flanquée de séquences loxP, la multiplication des virions générés à partir du BAC-AlHV-1 en cellules EBL-Cre (embryonic bovine lung ; exprimant la Cre recombinase) permet l’excision de la cassette BAC ; (iv) enfin, la capacité des virions générés à partir du BAC à induire la FACG en modèle lapin a été démontrée. En conclusion, le clone BAC-AlHV-1 généré dans cette étude va enfin permettre l’étude des rôles des différents gènes de l’AlHV-1 dans la genèse de la FACG. [less ▲]

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See detailGynogenesis induction and sex determination in the Eurasian perch, Perca fluviatilis
Rougeot, Carole ULg; Ngingo, J. V.; Gillet, Laurent ULg et al

in Aquaculture (2005), 243(1-4), 411-415

In the present study, we used meiotic gynogenesis, widely used in studies on sex determination, to confirm female homogamety in Eurasian perch, Perca fluviatilis. Sperm irradiated with UV for 400 s was ... [more ▼]

In the present study, we used meiotic gynogenesis, widely used in studies on sex determination, to confirm female homogamety in Eurasian perch, Perca fluviatilis. Sperm irradiated with UV for 400 s was used to artificially fertilized eggs. The diploid of the resulting embryos was restored by a heat shock (30 degreesC) applied to the eggs 5 min postfertilization, for 25 min. Fertilization (ranging between 45% and 75%) and survival rates at hatching (ranging between 3.4% and 46.6%) were not significantly different (P>0.05) between the diploid control and gynogenetics. The diploid controls and two batches of gynogenetics contained 100% diploid larvae, whereas two other batches of gynogenetics contained 6.7% and 10.0% triploid larvae. The sex ratios of the diploid controls were not significantly different from 1:1, whereas all gynogenetic families were 100% female. These results confirm female homogamety in Eurasian perch, demonstrated by the use of hormonally mascilinized breeders in a previous study. (C) 2004 Elsevier B.V. All rights reserved. [less ▲]

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See detailShort communication: Pasteurization of milk abolishes bovine herpesvirus 4 infectivity.
Bona, C.; Dewals, Benjamin G ULg; Wiggers, L. et al

in Journal of Dairy Science (2005), 88(9), 3079-83

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus highly prevalent in the cattle population that has been isolated from the milk and the serum of healthy infected cows. Several studies reported the ... [more ▼]

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus highly prevalent in the cattle population that has been isolated from the milk and the serum of healthy infected cows. Several studies reported the sensitivity and the permissiveness of some human cells to BoHV-4 infection. Moreover, our recent study demonstrated that some human cells sensitive but not permissive to BoHV-4 support a persistent infection protecting them from tumor necrosis factor-alpha-induced apoptosis. Together, these observations suggested that BoHV-4 could represent a danger for public health. To evaluate the risk of human infection by BoHV-4 through milk or serum derivatives, we investigated the resistance of BoHV-4 to the mildest thermal treatments usually applied to these products. The results demonstrated that milk pasteurization and thermal decomplementation of serum abolish BoHV-4 infectivity by inactivation of its property to enter permissive cells. Consequently, our results demonstrate that these treatments drastically reduce the risk of human infection by BoHV-4 through treated milk or serum derivatives. [less ▲]

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See detailBovine herpesvirus 4 induces apoptosis of human carcinoma cell lines in vitro and in vivo
Gillet, Laurent ULg; Dewals, Benjamin G ULg; Farnir, Frédéric ULg et al

in Cancer Research (2005), 65(20), 9463-9472

The idea of using oncolytic viruses for the treatment of cancers was proposed a century ago. During the last two decades, viruses able to replicate specifically in cancer cells and to induce their lysis ... [more ▼]

The idea of using oncolytic viruses for the treatment of cancers was proposed a century ago. During the last two decades, viruses able to replicate specifically in cancer cells and to induce their lysis were identified and were genetically modified to improve their viro-oncolytic properties. More recently, a new approach consisting of inducing selective apoptosis in cancer cells through viral infection has been proposed; this approach has been called viro-oncoapoptosis. In the present study, we report the property of bovine herpesvirus-4 (BoHV-4) to induce, in vitro and in vivo, apoptosis of some human carcinomas. This conclusion relies on the following observations: (a) In vitro, BoHV-4 infection induced apoptosis of A549 and OVCAR carcinoma cell lines in a time- and dose-dependent manner. (b) Apoptosis was induced by the expression of an immediate-early or an early BoHV-4 gene, but did not require viral replication. (c) Cell treatment with caspase inhibitors showed that apoptosis induced by BoHV-4 relied mainly on caspase-10 activation. (d) Infection of cocultures of A549 or OVCAR cells mixed with human 293 cells (in which BoHV-4 does not induce apoptosis) showed that BoHV-4 specifically eradicated A549 or OVCAR cancer cells from the cocultures. (e) Finally, in vivo experiments done with nude mice showed that BoHV-4 intratumoral injections reduced drastically the growth of preestablished A549 xenografts. Taken together, these results suggest that BoHV-4 may have potential as a viro-oncoapoptotic agent for the treatment of some human carcinomas. Moreover, further identification of BoHV-4 proapoptotic gene(s) and the cellular pathways targeted by this or these gene(s) could lead to the design of new cancer therapeutic strategies. [less ▲]

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