References of "Gillet, Laurent"
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See detailLong term culture, cryopreservation and genetic modification of chicken primordial germ cells
Tonus, Céline ULg; Garcia Gil, Francisco José ULg; Cloquette, Karine et al

Poster (2015, October 16)

Avian primordial germ cells (PGCs) are precursor of gametes and appear during early stages of embryonic development. Under appropriate culture conditions, these cells can keep their germ cells properties ... [more ▼]

Avian primordial germ cells (PGCs) are precursor of gametes and appear during early stages of embryonic development. Under appropriate culture conditions, these cells can keep their germ cells properties in vitro and are foreseen as promising tools for developing efficient avian genetic engineering and preservation of germplasm. We propose original methods that allow long term expansion, efficient cryopreservation and genetic modification of primary cultures of undifferentiated PGCs. PGCs are collected from embryonic blood during their migratory period and grown in cell-culture insert in the presence of feeder cells (BRL). This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of PGCs lines. Forty percent of blood samples gave rise to lines originating from three commercial layer and two Belgian endangered breeds. PGCs lines were characterized for the expression of the stem cells and PGCs marker SSEA-1 by FACS. RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. All lines were male although isolated from pooled male and female blood samples. Two cryopreservation methods were developed based upon slow-freezing and aseptic vitrification. Both have shown a similar effectiveness in allowing storage without phenotype drift. Stably expressing lines were obtained by Lipofectamine® mediated transfection of a GFP plasmid. PGCs were subsequently injected in recipient embryos. Persistence of exogenous PGCs in the developing gonad of recipient embryos confirmed that PGCs retain their gonadal colonisation ability, both after long-term culture and after cryopreservation. [less ▲]

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See detailCanine idopathic pulmonary fibrosis is not associated with herpes virus infection
Roels, Elodie ULg; Dourcy, Mickael ULg; Holopainen, S. et al

Poster (2015, October)

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See detailHost entry by gamma-herpesviruses-lessons from animal viruses?
Gillet, Laurent ULg; Frederico, Bruno; Stevenson, Philip G.

in Current Opinion in Virology (2015), 15

The oncogenicity of gamma-herpesviruses (gammaHVs) motivates efforts to control them and their persistence makes early events key targets for intervention. Human gammaHVs are often assumed to enter naive ... [more ▼]

The oncogenicity of gamma-herpesviruses (gammaHVs) motivates efforts to control them and their persistence makes early events key targets for intervention. Human gammaHVs are often assumed to enter naive hosts orally and infect B cells directly. However, neither assumption is supported by direct evidence, and vaccination with the Epstein-Barr virus (EBV) gp350, to block virion binding to B cells, failed to reduce infection rates. Thus, there is a need to re-evaluate assumptions about gammaHV host entry. Given the difficulty of analysing early human infections, potentially much can be learned from animal models. Genomic comparisons argue that gammaHVs colonized mammals long before humans speciation, and so that human gammaHVs are unlikely to differ dramatically in behaviour from those of other mammals. Murid Herpesvirus-4 (MuHV-4), which like EBV and the Kaposi's Sarcoma-associated Herpesvirus (KSHV) persists in memory B cells, enters new hosts via olfactory neurons and exploits myeloid cells to spread. Integrating these data with existing knowledge of human and veterinary gammaHVs suggests a new model of host entry, with potentially important implications for infection control. [less ▲]

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See detailCanine idiopathic pulmonary fibrosis is not associated with herpes virus infection
Roels, Elodie ULg; Dourcy, Mickael ULg; Holopainen, S. et al

Poster (2015)

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See detailThe alpha2,3-Sialyltransferase Encoded by Myxoma Virus Is a Virulence Factor that Contributes to Immunosuppression.
Boutard, Berengere; Vankerckhove, Sophie; Markine-Goriaynoff, Nicolas et al

in PloS one (2015), 10(2), 0118806

Myxoma virus (MYXV) induces a lethal disease called Myxomatosis in European rabbits. MYXV is one of the rare viruses that encodes an alpha2,3-sialyltransferase through its M138L gene. In this study, we ... [more ▼]

Myxoma virus (MYXV) induces a lethal disease called Myxomatosis in European rabbits. MYXV is one of the rare viruses that encodes an alpha2,3-sialyltransferase through its M138L gene. In this study, we showed that although the absence of the enzyme was not associated with any in vitro deficit, the M138L deficient strains are highly attenuated in vivo. Indeed, while all rabbits infected with the parental and the revertant strains died within 9 days post-infection from severe myxomatosis, all but one rabbit inoculated with the M138L deficient strains survived the infection. In primary lesions, this resistance to the infection was associated with an increased ability of innate immune cells, mostly neutrophils, to migrate to the site of virus replication at 4 days post-infection. This was followed by the development of a better specific immune response against MYXV. Indeed, at day 9 post-infection, we observed an important proliferation of lymphocytes and an intense congestion of blood vessels in lymph nodes after M138L knockouts infection. Accordingly, in these rabbits, we observed an intense mononuclear cell infiltration throughout the dermis in primary lesions and higher titers of neutralizing antibodies. Finally, this adaptive immune response provided protection to these surviving rabbits against a challenge with the MYXV WT strain. Altogether, these results show that expression of the M138L gene contributes directly or indirectly to immune evasion by MYXV. In the future, these results could help us to better understand the pathogenesis of myxomatosis but also the importance of glycans in regulation of immune responses. [less ▲]

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See detailA gammaherpesvirus infection protects against allergic asthma.
Machiels, Bénédicte ULg; Dourcy, Mickael ULg; Sabatel, Catherine ULg et al

Poster (2014, December 12)

The “hygiene hypothesis” proposes that the augmentation of allergic diseases in developed countries could be linked to a reduced exposure to infections during childhood. Surprisingly, the potential ... [more ▼]

The “hygiene hypothesis” proposes that the augmentation of allergic diseases in developed countries could be linked to a reduced exposure to infections during childhood. Surprisingly, the potential protective role of herpesvirus infections against allergy development has never been addressed directly. In this study, we used the Murid herpesvirus 4 (MuHV-4) to study the impact of a persistent gammaherpesvirus infection on the development of House Dust Mites (HDM)-induced allergic asthma. Our results revealed that MuHV-4 infection affects both the sensitization and the challenging phases of HDM-induced airway allergy. In particular, we highlighted that MuHV-4 infection strongly impacts the lung innate immune response. Indeed, while the dendritic cells remained competent to uptake antigens and to migrate to the draining lymph nodes, MuHV-4 infection impaired their ability to trigger HDM sensitization. In the future, these results could allow us to develop strategies to prevent the development of TH2-skewed responses against respiratory allergens. [less ▲]

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See detailLa virothérapie oncolytique médiée par le virus de la myxomatose
Krygier, David; Gillet, Laurent ULg; Marlier, Didier ULg

in Annales de Médecine Vétérinaire (2013), 157(1), 5-14

Le virus de la myxomatose est un poxvirus du genre Leporipoxvirus qui induit une pathologie spécifique, la myxomatose, chez le lapin européen (Oryctolagus cuniculus). Ce virus a la particularité d’être ... [more ▼]

Le virus de la myxomatose est un poxvirus du genre Leporipoxvirus qui induit une pathologie spécifique, la myxomatose, chez le lapin européen (Oryctolagus cuniculus). Ce virus a la particularité d’être non pathogène pour les autres espèces de vertébrés y compris l’homme. Le virus de la myxomatose (MYXV) présente aussi, de manière inattendue, un tropisme pour les cellules cancéreuses humaines in vitro ainsi qu’un potentiel oncolytique in vivo. La tolérance de ces cellules au MYXV est intimement liée au niveau intracellulaire d’Akt phosphorylée. Cette enzyme, est une serine/thréonine protéine kinase qui joue un rôle essentiel dans de nombreux processus cellulaires et fait partie de la voie de signalisation PI3k/Akt/mTOR (mammalian target of rapamycin), fréquemment amplifiée par l’oncogénèse. La protéine virale à répétitions ankyrines, M-T5, interagit avec Akt ce qui module le tropisme du MYXV pour les cellules tumorales humaines. Un régulateur de la croissance cellulaire et du métabolisme situé en aval d’Akt, mTOR, est spécifiquement inhibé par la rapamycine. Ainsi, l’utilisation de la rapamycine en combinaison avec le MYXV permet d’augmenter la concentration d’Akt phosphorylée, et par conséquent, d’amplifier l’oncolyse. Un meilleur contrôle chimique de la voie de signalisation d’Akt ou de la modification génétique de son génome constituera une étape décisive pour que le MYXV devienne l’un des nouveaux traitements des cancers chez l’homme [less ▲]

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See detailProteomic characterization of murid herpesvirus 4 extracellular virions.
Vidick, Sarah ULg; Leroy, Baptiste; Gonon Rodrigues Palmeira, Leonor ULg et al

in PloS one (2013), 8(12), 83842

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic ... [more ▼]

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology. [less ▲]

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See detailIllumination of murine gammaherpesvirus-68 cycle reveals a sexual transmission route from females to males in laboratory mice.
François, Sylvie ULg; Vidick, Sarah ULg; Sarlet, Mickael et al

in PLoS Pathogens (2013), 9(4), 1003292

Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which ... [more ▼]

Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naive males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these viruses in natural populations. [less ▲]

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See detailGlycoprotein B cleavage is important for murid herpesvirus 4 to infect myeloid cells.
Glauser, Daniel L.; Milho, Ricardo; Frederico, Bruno et al

in Journal of Virology (2013)

Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many - but not all - herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide ... [more ▼]

Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many - but not all - herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally, but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a post-endocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless. [less ▲]

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See detailAlternative splicing switches tropism of a gammaherpesvirus
Machiels, Bénédicte ULg; Gillet, Laurent ULg

Conference (2012, November)

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See detailMethod for producing antibody using "naked" expression vector expressing type II transmembrane fusion protein
Renauld, Jean Christophe; Lemaire, Muriel; Dumoutier, Laurie et al

Patent (2012)

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See detailFeeding Cyprinus carpio with infectious materials mediates cyprinid herpesvirus 3 entry through infection of pharyngeal periodontal mucosa
Fournier, Guillaume ULg; Boutier, Maxime ULg; Victor, Stalin Raj et al

in Veterinary Research (2012), 43(6),

Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using ... [more ▼]

Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using bioluminescence imaging and a CyHV-3 recombinant strain expressing luciferase (LUC). We demonstrated that the skin is the major portal of entry after inoculation of carp by immersion in water containing CyHV-3. While this model of infection mimics some natural conditions in which infection takes place, other epidemiological conditions could favour entry of virus through the digestive tract. Here, we investigated whether ingestion of infectious materials mediates CyHV-3 entry through the digestive tract. Carp were fed with materials contaminated with the CyHV-3 LUC recombinant (oral contamination) or immersed in water containing the virus (contamination by immersion). Bioluminescence imaging analyses performed at different times post-infection led to the following observations: (i) the pharyngeal periodontal mucosa is the major portal of entry after oral contamination, while the skin is the major portal of entry after contamination by immersion. (ii) Both modes of inoculation led to the spreading of the infection to the various organs tested. However, the timing and the sequence in which some of the organs turned positive were different between the two modes of inoculation. Finally, we compared the disease induced by the two inoculation modes. They led to comparable clinical signs and mortality rate. The results of the present study suggest that, based on epidemiological conditions, CyHV-3 can enter carp either by skin or periodontal pharyngeal mucosal infection. [less ▲]

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See detailMyeloid infection links epithelial and B cell tropisms of murid herpesvirus-4.
Frederico, Bruno; Milho, Ricardo; May, Janet S. et al

in PLoS Pathogens (2012), 8(9), 1002935

Gamma-herpesviruses persist in lymphocytes and cause disease by driving their proliferation. Lymphocyte infection is therefore a key pathogenetic event. Murid Herpesvirus-4 (MuHV-4) is a rhadinovirus that ... [more ▼]

Gamma-herpesviruses persist in lymphocytes and cause disease by driving their proliferation. Lymphocyte infection is therefore a key pathogenetic event. Murid Herpesvirus-4 (MuHV-4) is a rhadinovirus that like the related Kaposi's Sarcoma-associated Herpesvirus persists in B cells in vivo yet infects them poorly in vitro. Here we used MuHV-4 to understand how virion tropism sets the path to lymphocyte colonization. Virions that were highly infectious in vivo showed a severe post-binding block to B cell infection. Host entry was accordingly an epithelial infection and B cell infection a secondary event. Macrophage infection by cell-free virions was also poor, but improved markedly when virion binding improved or when macrophages were co-cultured with infected fibroblasts. Under the same conditions B cell infection remained poor; it improved only when virions came from macrophages. This reflected better cell penetration and correlated with antigenic changes in the virion fusion complex. Macrophages were seen to contact acutely infected epithelial cells, and cre/lox-based virus tagging showed that almost all the virus recovered from lymphoid tissue had passed through lysM(+) and CD11c(+) myeloid cells. Thus MuHV-4 reached B cells in 3 distinct stages: incoming virions infected epithelial cells; infection then passed to myeloid cells; glycoprotein changes then allowed B cell infection. These data identify new complexity in rhadinovirus infection and potentially also new vulnerability to intervention. [less ▲]

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See detailBovine Herpesvirus Type 4 Glycoprotein L Is Nonessential for Infectivity but Triggers Virion Endocytosis during Entry
Lété, Céline ULg; Machiels, Bénédicte ULg; Stevenson, P. G. et al

in Journal of Virology (2012)

The core entry machinery of mammalian herpesviruses comprises glycoproteins B, H and L (gB, gH and gL). gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or ... [more ▼]

The core entry machinery of mammalian herpesviruses comprises glycoproteins B, H and L (gB, gH and gL). gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or beta-herpesviruses lack gL, gH misfolds and progeny virions are non-infectious. However, the gL of the rhadinovirus Murid herpesvirus 4 (MuHV-4) is non-essential for infection. In order to define more generally what role gL plays in rhadinovirus infections, we disrupted its coding sequence in Bovine herpesvirus-4 (BoHV-4). BoHV-4 lacking gL showed altered gH glycosylation and incorporated somewhat less gH into virions but remained infectious. However, gL- virions showed poor growth associated with an entry deficit. Moreover a major part of their entry defect appeared to reflect impaired endocytosis, which occurs upstream of membrane fusion itself. Thus, the rhadinovirus gL may be more important for driving virion endocytosis than for incorporating gH into virions, and is non-essential for membrane fusion. [less ▲]

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