References of "Gillet, Laurent"
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See detailProteomic characterization of bovine herpesvirus 4 extracellular virions.
Lété, Céline ULg; Palmeira, Leonor ULg; Leroy, Baptiste et al

in Journal of Virology (2012), 86(21), 11567-80

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful ... [more ▼]

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host's antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi's sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses. [less ▲]

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See detailVirion endocytosis is a major target for Murid Herpesvirus-4 neutralization.
Glauser, D; Gillet, Laurent ULg; Stevenson, PG

in Journal of General Virology (The) (2012)

Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid Herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to ... [more ▼]

Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid Herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless neutralization remains possible by targeting the virion glycoprotein H (gH) / gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH/gL. We analysed here how gH/gL-directed neutralization works. The MuHV-4 gH/gL binds to heparan sulfate. However most gH/gL-specific neutralizing antibodies did not block this interaction. Nor did they act directly on fusion. Instead they blocked virion endocytosis and transport to the late endosomes where membrane fusion normally occurs. The poor endocytosis of gH/gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore driving virion uptake appears to be an important function of gH/gL that provides a major target for antibody-mediated neutralization. [less ▲]

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See detailGenital re-excretion of Murid gammaherpesvirus 4 following intranasal infection
François, Sylvie ULg; Vidick, Sarah ULg; Sarlet, Michaël ULg et al

in Proceedings of the 1st Scientific Meeting of the Faculty of Veterinary Medicine (2011, December 09)

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model ... [more ▼]

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4) in inbred laboratory mouse strains which are commonly accepted as a good model for studying gammaherpesviruses in vivo. To date, it has however never been possible to monitor viral reexcretion and virus transmission in this species. In order to identify potential re-excretion sites, intranasally infected mice were followed through global luciferase imaging for up to six months after infection. Surprisingly, we detected transient viral replication in mice genital tract at various times after latency establishment. Ex vivo imaging, quantitative PCR and immunohistochemistry revealed that virus genomes were present in high quantity in the vaginal tissue and that viral replication occurred mainly at the vaginal external border. Moreover, we highlighted the presence of free infectious viruses in the vaginal cavity at the moment of the observation of viral replication. As this ephemeral viral reexcretion could reveal a link with reproductive cycle, we compared reexcretion in normal and ovariectomized mice. Interestingly, no viral reactivation was observed in absence of hormonal cycle. In conclusion, we experimentally indentified for the first time a reexcretion site for MuHV-4 in mice that had been intranasaly infected. In the future, these results could help us to better understand the biology of gammaherpesviruses but should also allow us to develop strategies that could prevent the spread of these viruses in natural populations. [less ▲]

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See detailGenital re-excretion of Murid gammaherpesvirus 4 following intranasal infection
François, Sylvie ULg; Vidick, Sarah ULg; Sarlet, Michaël ULg et al

Poster (2011, November 16)

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model ... [more ▼]

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4) in inbred laboratory mouse strains which are commonly accepted as a good model for studying gammaherpesviruses in vivo. To date, it has however never been possible to monitor viral reexcretion and virus transmission in this species. In order to identify potential re-excretion sites, intranasally infected mice were followed through global luciferase imaging for up to six months after infection. Surprisingly, we detected transient viral replication in mice genital tract at various times after latency establishment. Ex vivo imaging, quantitative PCR and immunohistochemistry revealed that virus genomes were present in high quantity in the vaginal tissue and that viral replication occurred mainly at the vaginal external border. Moreover, we highlighted the presence of free infectious viruses in the vaginal cavity at the moment of the observation of viral replication. As this ephemeral viral reexcretion could reveal a link with reproductive cycle, we compared reexcretion in normal and ovariectomized mice. Interestingly, no viral reactivation was observed in absence of hormonal cycle. In conclusion, we experimentally indentified for the first time a reexcretion site for MuHV-4 in mice that had been intranasaly infected. In the future, these results could help us to better understand the biology of gammaherpesviruses but should also allow us to develop strategies that could prevent the spread of these viruses in natural populations. [less ▲]

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See detailAntibody evasion by a gammaherpesvirus O-glycan shield.
Machiels, Bénédicte ULg; Gillet, Laurent ULg

Conference (2011, November)

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See detailAntibody evasion by a gammaherpesvirus o-glycan shield.
Machiels, Bénédicte ULg; Lété, Céline ULg; Guillaume, Antoine ULg et al

in PLoS Pathogens (2011), 7(11), 1002387

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the ... [more ▼]

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog - gp180 - contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target. [less ▲]

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See detailAlternative attachment factors and internalization pathways for GIII.2 bovine noroviruses.
Mauroy, Axel ULg; Gillet, Laurent ULg; Mathijs, Elisabeth ULg et al

in Journal of General Virology (The) (2011)

Bovine noroviruses belong to the family Caliciviridae, genus Norovirus. Two genotypes are described and viruses genetically related to the Jena and Newbury-2 strains are classified into genotypes 1 and 2 ... [more ▼]

Bovine noroviruses belong to the family Caliciviridae, genus Norovirus. Two genotypes are described and viruses genetically related to the Jena and Newbury-2 strains are classified into genotypes 1 and 2 respectively. In this study, virus-like particles (VLP) of the previously detected B309 Belgian strain, genetically related to genotype 2 bovine noroviruses, were used to investigate virus-host interactions in vitro. B309 VLP were shown to bind to several bovine cell lines. This binding was not affected by heparinase or chondroitinase treatment but was significantly inhibited by both sodium periodate, alpha-galactosidase, trypsin and phospholipase C treatment. Cell treatment by neuraminidase also moderately affected this binding. Taken together, these results show that, in addition to a galactosyl residue, sialic acid could also be involved in binding to susceptible cells. In addition, both the cholesterol-dependent pathway and macropinocytosis are used for B309 VLP internalisation by Madin-Darby Bovine Kidney cells. The data increase the knowledge on bovine norovirus cell interactions. [less ▲]

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See detailEx vivo bioluminescent detection of Alcelaphine herpesvirus 1 infection during malignant catarrhal fever.
Dewals, Benjamin G ULg; Myster, Françoise ULg; Palmeira, Leonor ULg et al

in Journal of Virology (2011), 85(14), 6941-54

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

Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and non-lymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to the parental wild-type strain with hyperthermia and increase of both CD8(+) T cells frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in non-lymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF. [less ▲]

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See detailSequencing of Bovine herpesvirus 4 V.test strain reveals important genome features
Palmeira, Leonor ULg; Machiels, Bénédicte ULg; Lété, Céline ULg et al

in Virology Journal (2011), 8(1), 406

Background Bovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this ... [more ▼]

Background Bovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. Conclusions In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses. [less ▲]

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See detailAntibody production by injection of living cells expressing non self antigens as cell surface type II transmembrane fusion protein.
Nizet, Yannick; Gillet, Laurent ULg; SCHROEDER, Hélène ULg et al

in Journal of immunological methods (2011)

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps ... [more ▼]

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens. [less ▲]

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See detailBovine herpesvirus 4 immediate early 2 (Rta) gene is an essential gene and is duplicated in bovine herpesvirus 4 isolate U.
Franceschi, V.; Capocefalo, A.; Ravanetti, L. et al

in Veterinary Microbiology (2011)

The ORF50/Rta gene has been shown to be an essential gene for many gammaherpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early ... [more ▼]

The ORF50/Rta gene has been shown to be an essential gene for many gammaherpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U. [less ▲]

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See detailBovine Herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects.
Machiels, Bénédicte ULg; Lété, Céline ULg; Defays, Katalin et al

in Journal of Virology (2011), 85(2), 1011-1024

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's Sarcoma associated herpesvirus (KSHV) K8.1. In this study, we characterized that of ... [more ▼]

All gammaherpesviruses encode a glycoprotein positionally homologous to the Epstein-Barr virus gp350 and the Kaposi's Sarcoma associated herpesvirus (KSHV) K8.1. In this study, we characterized that of Bovine Herpesvirus-4 (BoHV-4), encoded by the Bo10 gene. We identified a 180 kDa gene product, gp180, which was incorporated into the virion envelope. A Bo10 deletion virus was viable, but showed a growth deficit associated with reduced binding to epithelial cells. This seemed to reflect an interaction of gp180 with glycosaminoglycans (GAGs), since the Bo10 mutant was both less infectious for GAG(+) cells than the wild-type and more infectious for GAG(-) cells. However, we could not identify a direct interaction between gp180 and GAGs, implying that any direct interaction must be of low affinity. This function of gp180 was very similar to that previously identified for the Murid Herpesvirus 4 gp150, and also to the Epstein-Barr virus gp350 that promotes CD21(+) cell infection and inhibits CD21(-) cell infection. We propose that such proteins generally regulate virion attachment both by binding to cells and by covering another receptor-binding protein until they are displaced. Thus they regulate viral tropism both positively and negatively depending upon the presence or absence of their receptor. [less ▲]

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See detailA mechanistic basis for potent, glycoprotein B-directed gammaherpesvirus neutralization.
Glauser, Daniel L; Kratz, Anne*-Sophie; Gillet, Laurent ULg et al

in Journal of General Virology (The) (2011), 92(Pt 9), 2020-33

Glycoprotein B (gB) is a conserved, essential component of gammaherpes virions and so potentially vulnerable to neutralization. However, few good gB-specific neutralizing antibodies have been identified ... [more ▼]

Glycoprotein B (gB) is a conserved, essential component of gammaherpes virions and so potentially vulnerable to neutralization. However, few good gB-specific neutralizing antibodies have been identified. Here, we show that murid herpesvirus 4 is strongly neutralized by mAbs that recognize an epitope close to one of the gB fusion loops. Antibody binding did not stop gB interacting with its cellular ligands or initiating its fusion-associated conformation change, but did stop gB resolving stably to its post-fusion form, and so blocked membrane fusion to leave virions stranded in late endosomes. The conservation of gB makes this mechanism a possible general route to gammaherpesvirus neutralization. [less ▲]

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See detailGenital re-excretion of Murid gammaherpesvirus 4 following intranasal infection
François, Sylvie ULg; Vidick, Sarah ULg; Sarlet, Michaël ULg et al

Poster (2010, November 18)

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections ... [more ▼]

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections of immunocompetent hosts. Thus, infected individuals simultaneously both elicit antiviral protective immune response and secrete infectious virions. The best studied gammaherpesviruses are Human herpesvirus 4 and Human herpesvirus 8. As these viruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4), a virus that has originally been isolated from bank voles (Myodes glareolus). Although MuHV-4 has not been isolated from house mice (Mus musculus), infection of inbred laboratory mouse strains is commonly accepted as a good model for studying gammaherpesviruses in vivo. To date, it has however never been possible to monitor viral reexcretion and virus transmission in this species suggesting that this model could be imperfect. In order to identify potential re-excretion sites, intranasally infected mice were followed through global luciferase imaging for up to six months after infection. By this technique, we were able to detect appearance of viral replication in mice genital tract at various times post-infection. Typically, it firstly occurred between days 20 to 30 after infection, a period at which it is admitted that latency is established. Ex vivo imaging, quantitative PCR and immunohistochemistry helped us to determine that virus genomes were present in high quantity in the vaginal tissue and that viral replication occurred mainly at the vaginal external border. Finally, we highlighted the presence of free infectious viruses in the vaginal cavity at the moment of the observation of viral replication. In conclusion, we experimentally indentified for the first time a reexcretion site for MuHV-4 in mice that had been intranasaly infected. It therefore suggests potential genital transmission, either horizontal or vertical, of this virus in mice populations. In the future, these results could help us to better understand the biology of gammaherpesviruses but should also allow us to develop vaccinal strategies that could prevent the spread of these viruses in natural populations. [less ▲]

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See detailComparative study of Murid gamma-herpesvirus 4 infection in mice and in a natural host, the bank voles.
François, Sylvie ULg; Vidick, Sarah ULg; Sarlet, Michaël ULg et al

in Journal of General Virology (The) (2010)

Gamma-herpesviruses are archetypal pathogenic persistent viruses. The known human gamma-herpesviruses (Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus) are host-specific and therefore lack ... [more ▼]

Gamma-herpesviruses are archetypal pathogenic persistent viruses. The known human gamma-herpesviruses (Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus) are host-specific and therefore lack a convenient in vivo infection model. This makes related animal gamma-herpesviruses an important source of information. We are studying Murid herpesvirus 4 (MuHV-4), a virus originally isolated from bank voles (Myodes glareolus). MuHV-4 infection of inbred laboratory mouse strains (Mus musculus) is commonly used as a general model of gamma-herpesvirus pathogenesis. However, MuHV-4 has not been isolated from house mice, and no systematic comparison has been made between experimental MuHV-4 infections of mice and bank voles. We have therefore characterized MuHV-4 (strain MHV-68) infection of bank voles, both through global luciferase imaging and through classical virological methods. As in mice, intranasal virus inoculation led to productive replication in bank vole lungs, accompanied by massive cellular infiltrates. However, the extent of lytic virus replication was ~1000 fold lower in bank voles than in mice. Peak latency titers in lymphoid tissue were also lower, although latency was still established. Finally, we tested viral transmission between animals maintained in captivity. However, as observed in mice, MuHV-4 did not transmit between voles in these conditions. In conclusion, this study revealed that despite quantitative differences, replication and latency sites of MuHV-4 are comparable in bank voles and in mice. It appears therefore so far that Mus musculus represents a suitable host for studying gamma-herpesvirus pathogenesis with MuHV-4. Establishing transmission conditions in captivity will be a vital step for further research in that field. [less ▲]

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See detailBovine herpesvirus 4 ORF73 is dispensable for viral growth in vitro but is essential for viral persistence in vivo.
Thirion, M.; Machiels, Bénédicte ULg; Farnir, Frédéric ULg et al

in Journal of General Virology (The) (2010), 91(10), 2574-84

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency ... [more ▼]

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue with a size equivalent to only 22% of the largest orthologues. The present study focused on determining if BoHV 4 ORF73 is a bona fide gene and investigating whether it is essential for latency as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early bicistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by wild-type and revertant recombinants. Together, these results demonstrate that despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology. [less ▲]

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See detailComparative study of Murid gammaherpesvirus 4 infection in mice and in its natural host, the bank voles.
François, Sylvie ULg; Vidick, Sarah ULg; Koteja, Pawel et al

Poster (2009, December 11)

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections ... [more ▼]

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections of immunocompetent hosts. Thus, infected individuals simultaneously both elicit antiviral protective immune response and secrete infectious virions. The best studied gammaherpesviruses are Human herpesvirus 4 and Human herpesvirus 8. As these viruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4), a virus that has originally been isolated from bank voles (Myodes glareolus). Although MuHV-4 has not been isolated from house mice (Mus musculus), infection of inbred laboratory mouse strains is commonly accepted as a good model for studying gammaherpesviruses in vivo. It has however never been possible to monitor viral reexcretion and virus transmission in this species suggesting that this model could be imperfect. In this study, we therefore characterized MuHV-4 infection in its natural host, the bank voles, through classical virological methods but also through global luciferase imaging for an anatomical complete view of the infection. Results obtained show that, after intra-nasal infection, the natural route of infection is similar in mice and voles. Following nasal productive infection, the virus spreads to the lung where the infection is accompanied by massive cellular infiltrates. By opposition to extensive viral replication observed in mice, the different analyses indicated that the viral replication was ~1000 fold lower in bank voles. This lower replication did however not affect colonization of latency sites in superficial cervical lymph nodes and spleen as measured by real-time PCR quantification of viral genomes in these organs. In conclusion, this study revealed that MuHV-4 can experimentally infect bank voles, the supposed natural host, but with a lower replicative power. As, gammaherpesvirus epidemiology indicates that transmission correlates with the latent load, our results suggest that gammaherpesviruses may have evolved to infect their hosts without extensive lytic spread. In the future, establishment of experimental transmission in a population of Myodes glareolus should help us to better understand mechanisms used by gammaherpesviruses to evade immune response. [less ▲]

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See detailThe major portal of entry of koi herpesvirus in cyprinus carpio is the skin.
Costes, Bérénice ULg; Stalin Raj, V.; Michel, Benjamin ULg et al

in Journal of Virology (2009)

Koi herpesvirus (KHV), recently designated in the species Cyprinid Herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of ... [more ▼]

Koi herpesvirus (KHV), recently designated in the species Cyprinid Herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp using bioluminescence imaging. Taking profit of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between ORF 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid: the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain encoding a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 hours post-infection; while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different time post-infection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish, covering the fins and also the body, is the major portal of entry of KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system nicknamed "U-tube" to perform per-cutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin and not the gills is the major portal of entry of KHV in carp. [less ▲]

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See detailIn vivo imaging of murid herpesvirus-4 infection.
Milho, Ricardo; Smith, Christopher M; Marques, Sofia et al

in Journal of General Virology (The) (2009), 90(Pt 1), 21-32

Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine ... [more ▼]

Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7-10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions. [less ▲]

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