References of "Vanderplasschen, Alain"
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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 detailA recombinant koi herpesvirus (KHV) or Cyprinid herpesvirus 3 (CyHV-3) and a vaccine for the prevention of a desease caused by KHV/CyHV-3 in Cyprinus carpio or Cyprinus carpio koi
Costes, Bérénice ULg; Lieffrig, F.; Vanderplasschen, Alain ULg

in EP 07115093.2 (2007)

The present invention refers to a recombinant koi herpesvirus (KHV) or Cyprinid herpesvirus 3 (CyHV-3), which is immunogenic in fish, preferably in carps, more preferably in Cyprinus carpio, and to a ... [more ▼]

The present invention refers to a recombinant koi herpesvirus (KHV) or Cyprinid herpesvirus 3 (CyHV-3), which is immunogenic in fish, preferably in carps, more preferably in Cyprinus carpio, and to a vaccine for preventive and/or therapeutic treatment of a disease caused by koi herpesvirus (KHV) or CyHV-3. The 5 recombinant herpesvirus is used to confer immunity on fish, preferably on carps, more preferably on Cyprinus carpio, against a disease caused by koi herpesvirus (KHV) or Cyprinid herpesvirus 3 (CyHV-3). [less ▲]

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See detailFeline herpesvirus 1, the causative agent of feline viral rhinotracheitis
Costes, Bérénice ULg; Van Den Brande, A.; Thiry, Etienne ULg et al

in Annales de Médecine Vétérinaire (2007), 73

Infectious respiratory diseases also called ‘cat flu’ are nowadays one of the most relevant areas of feline medicine. Epidemiologic surveys revealed that 80% of the cases are due to feline calicivirus and ... [more ▼]

Infectious respiratory diseases also called ‘cat flu’ are nowadays one of the most relevant areas of feline medicine. Epidemiologic surveys revealed that 80% of the cases are due to feline calicivirus and Felid herpesvirus 1 (FeHV-1). FeHV-1 is an alphaherpesvirus that has a worldwide distribution in cat population. It is responsible for feline viral rhinotracheitis. This disease can be acute, chronic or latent. It is characterized by fever and respiratory or ocular signs among which conjunctivitis and keratitis are the most common. Severe cases can cause complete blindness of the cat mostly following repetitive reactivations. Latent viral carriers are epidemiologically important because they are the main source of infection to susceptible cats. Nowadays no vaccine can prevent infection. At best, available vaccines help to reduce clinical signs but fail to prevent establishment of latency or reactivation. Consequently, feline viral rhinotracheitis still represents a major problem in domestic cats. This review focuses on the current knowledge about feline viral rhinotracheitis and its etiologic agent, FeHV-1. [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 detailIntraspecific bovine herpesvirus 1 recombinants carrying glycoprotein E deletion as a vaccine marker are virulent in cattle
Muylkens, Benoît ULg; Meurens, F.; Schynts, F. et al

in Journal of General Virology (2006), 87(Pt 8), 2149-2154

Vaccines used in control programmes of Bovine herpesvirus 1 (BoHV-1) utilize highly attenuated BoHV-1 strains marked by a deletion of the glycoprotein E (gE) gene. Since BoHV-1 recombinants are obtained ... [more ▼]

Vaccines used in control programmes of Bovine herpesvirus 1 (BoHV-1) utilize highly attenuated BoHV-1 strains marked by a deletion of the glycoprotein E (gE) gene. Since BoHV-1 recombinants are obtained at high frequency in experimentally coinfected cattle, the consequences of recombination on the virulence of gE-negative BoHV-1 were investigated. Thus, gE-negative BoHV-1 recombinants were generated in vitro from several virulent BoHV-1 and one mutant BoHV-1 deleted in the gC and gE genes. Four gE-negative recombinants were tested in the natural host. All the recombinants were more virulent than the gE-negative BoHV-1 vaccine and the gC- and gE-negative parental BoHV-1. The gE-negative recombinant isolated from a BoHV-1 field strain induced the highest severe clinical score. Latency and reactivation studies showed that three of the recombinants were reexcreted. Recombination can therefore restore virulence of gE-negative BoHV-1 by introducing the gE deletion into a different virulence background. [less ▲]

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See detailRecombination in the alphaherpesvirus bovine herpesvirus 1
Thiry, Etienne ULg; Muylkens, Benoît ULg; Meurens, F. et al

in Veterinary Microbiology (2006), 113(3-4), 171-177

Herpesviruses are DNA viruses characterized by a low rate of nucleotide substitution. Therefore. other mechanisms must be involved to their evolution, like recombination that can be seen as an essential ... [more ▼]

Herpesviruses are DNA viruses characterized by a low rate of nucleotide substitution. Therefore. other mechanisms must be involved to their evolution, like recombination that can be seen as an essential evolutionary driving force of these viruses. Recombination contributes to the long-term evolution of alphaherpes viruses. It acts also to continuously create new alphaherpesvirus strains. We have used bovine herpesvirus 1 to investigate recombination both within DNA concatemers in infected cells and in vitro and in vivo at the end of the lytic cycle. The following results have been obtained: (i) intramolecular recombination occurs at the level of concatemers and gives rise to genomic segment inversions: (ii) intraspecific recombination occurs frequently both in vitro and in vivo; (iii) interspecific recombination is possible and requires two highly genetically related viruses (iv) only simultaneous or closely separated infections lead to the production of recombinant viruses: (v) recombination between wild-type and glycoprotein defective vaccine virus can produce a glycoprotein defective virus keeping part of the virulence of parental wild-type virus. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines. (c) 2005 Elsevier B.V. All rights reserved. [less ▲]

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See detailBiological characterization of bovine herpesvirus 1 recombinants possessing the vaccine glycoprotein E negative phenotype
Muylkens, Benoît ULg; Meurens, F.; Schynts, F. et al

in Veterinary Microbiology (2006), 113(3-4), 283-291

Intramolecular recombination is a frequent event during the replication cycle of bovine herpesvirus 1 (BoHV-1). Recombinant viruses frequently arise and survive in cattle after concomitant nasal ... [more ▼]

Intramolecular recombination is a frequent event during the replication cycle of bovine herpesvirus 1 (BoHV-1). Recombinant viruses frequently arise and survive in cattle after concomitant nasal infections with two BoHV-1 mutants. The consequences of this process, related to herpesvirus evolution, have to be assessed in the context of large use of live marker vaccines based on glycoprotein E (gE) gene deletion. In natural conditions, double nasal infections by vaccine and wild-type strains are likely to occur. This situation might generate virulent recombinant viruses inducing a serological response indistinguishable from the vaccine one. This question was addressed by generating in vitro BoHV-1 recombinants deleted in the gE gene from seven wild-type BoHV-1 strains and one mutant strain deleted in the genes encoding gC and gE. In vitro growth properties were assessed by virus production, one step growth kinetics and plaque size assay. Heterogeneity in the biological properties was shown among the investigated recombinant viruses. The results demonstrated that some recombinants. in spite of their gE minus phenotype, have biological characteristics close to wild-type BoHV-1. (c) 2005 Elsevier B.V. All rights reserved. [less ▲]

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See detailCharacterization of caprine herpesvirus 1 glycoprotein D gene and its translation product
Keuser, Véronique; Detry, Bruno; Thiry, Julien ULg et al

in Virus Research (2006), 115(2), 112-121

Caprine herpesvirus 1 (CpHV- 1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 ... [more ▼]

Caprine herpesvirus 1 (CpHV- 1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main inummogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD, shares most characteristics with other alphaherpesviruses and especially BoHV-1. (c) 2005 Elsevier B.V. All rights reserved. [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 detailRuminant alphaherpesviruses related to bovine herpesvirus 1.
Thiry, Julien ULg; Keuser, Veronique; Muylkens, Benoît ULg et al

in Veterinary Research (2006), 37(2), 169-90

Herpesviruses have mainly co-evolved with their hosts for millions of years. Consequently, different related host species may have been infected by various genetically related herpesviruses. Illustrating ... [more ▼]

Herpesviruses have mainly co-evolved with their hosts for millions of years. Consequently, different related host species may have been infected by various genetically related herpesviruses. Illustrating this concept, several ruminant alphaherpesviruses have been shown to form a cluster of viruses closely related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5, bubaline herpesvirus 1, caprine herpesvirus 1, cervid herpesviruses 1 and 2 and elk herpesvirus 1. These viruses share common antigenic properties and the serological relationships between them can be considered as a threat to BoHV-1 eradication programmes. BoHV-1 is a herpesvirus responsible for infectious bovine rhinotracheitis, which is a disease of major economic concern. In this article, the genetic properties of these ruminant alphaherpesviruses are reviewed on a comparative basis and the issue of interspecific recombination is assessed. The pathogenesis of these infections is described with emphasis on the host range and crossing of the host species barrier. Indeed, the non bovine ruminant species susceptible to these ruminant alphaherpesviruses may be potential BoHV-1 reservoirs. The differential diagnosis of these related infections is also discussed. In addition, available epidemiological data are used to assess the potential of cross-infection in ruminant populations. A better knowledge of these ruminant alphaherpesvirus infections is essential to successfully control infectious bovine rhinotracheitis. [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 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 detailVaricella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex
Di Valentin, Emmanuel ULg; Bontems, Sébastien ULg; Habran, Lionel ULg et al

in Biological Chemistry (2005), 386(3), 255-267

Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67 ... [more ▼]

Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67) promoters. IE63 was shown to repress the basal activity of most of the promoters tested in epithelial (Vero) and neuronal (ND7) cells to various extents. Trans -repressing activities were also observed on heterologous viral and cellular promoters. Since a construct carrying only a TATA box sequence and a series of wild-type or mutated interleukin (IL)-8 promoters was also repressed by IE63, the role of upstream regulatory elements was ruled out. Importantly, the basal activity of a TATA-less promoter was not affected by IE63. Using a series of IE63 deletion constructs, amino acids 151-213 were shown to be essential to the transrepressing activity in Vero cells, while in ND7 cells the essential region extended to a much larger carboxy-terminal part of the protein. We also demonstrate that IE63 is capable of disrupting the transcriptional pre-initiation complex and of interacting with several general transcription factors. The central and carboxy-terminal domains of IE63 are important for these effects. Altogether, these results demonstrate that IE63 protein is a transcriptional repressor whose activity is directed towards general transcription factors. [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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