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See detailPost-exposure vaccination improves gammaherpesvirus neutralization.
Gillet, Laurent ULg; May, Janet S; Stevenson, Philip G

in PLoS ONE (2007), 2(9), 899

Herpesvirus carriers transmit infection despite making virus-specific antibodies. Thus, their antibody responses are not necessarily optimal. An important question for infection control is whether ... [more ▼]

Herpesvirus carriers transmit infection despite making virus-specific antibodies. Thus, their antibody responses are not necessarily optimal. An important question for infection control is whether vaccinating carriers might improve virus neutralization. The antibody response to murine gamma-herpesvirus-68 (MHV-68) blocks cell binding, but fails to block and even enhances an IgG Fc receptor-dependent infection of myeloid cells. Viral membrane fusion therefore remains intact. Although gH/gL-specific monoclonal antibodies can block infection at a post-binding step close to membrane fusion, gH/gL is a relatively minor antibody target in virus carriers. We show here that gH/gL-specific antibodies can block both Fc receptor-independent and Fc receptor-dependent infections, and that vaccinating virus carriers with a gH/gL fusion protein improves their capacity for virus neutralization both in vitro and in vivo. This approach has the potential to reduce herpesvirus transmission. [less ▲]

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See detailThe murine gammaherpesvirus-68 gp150 acts as an immunogenic decoy to limit virion neutralization.
Gillet, Laurent ULg; May, Janet S; Colaco, Susanna et al

in PLoS ONE (2007), 2(1), 705

Herpesviruses maintain long-term infectivity without marked antigenic variation. They must therefore evade neutralization by other means. Immune sera block murine gammaherpesvirus-68 (MHV-68) infection of ... [more ▼]

Herpesviruses maintain long-term infectivity without marked antigenic variation. They must therefore evade neutralization by other means. Immune sera block murine gammaherpesvirus-68 (MHV-68) infection of fibroblasts, but fail to block and even enhance its infection of IgG Fc receptor-bearing cells, suggesting that the antibody response to infection is actually poor at ablating virion infectivity completely. Here we analyzed this effect further by quantitating the glycoprotein-specific antibody response of MHV-68 carrier mice. Gp150 was much the commonest glycoprotein target and played a predominant role in driving Fc receptor-dependent infection: when gp150-specific antibodies were boosted, Fc receptor-dependent infection increased; and when gp150-specific antibodies were removed, Fc receptor-dependent infection was largely lost. Neither gp150-specific monoclonal antibodies nor gp150-specific polyclonal sera gave significant virion neutralization. Gp150 therefore acts as an immunogenic decoy, distorting the MHV-68-specific antibody response to promote Fc receptor-dependent infection and so compromise virion neutralization. This immune evasion mechanism may be common to many non-essential herpesvirus glycoproteins. [less ▲]

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See detailThe paralogous salivary anti-complement proteins IRAC I and IRAC II encoded by Ixodes ricinus ticks have broad and complementary inhibitory activities against the complement of different host species.
Schroeder, Hélène ULg; Daix, Virginie; Gillet, Laurent ULg et al

in Microbes & Infection (2007), 9(2), 247-50

Several observations suggest that inhibition of the host complement alternative pathway by Ixodes tick saliva is crucial to achieve blood feeding. We recently described two paralogous anti-complement ... [more ▼]

Several observations suggest that inhibition of the host complement alternative pathway by Ixodes tick saliva is crucial to achieve blood feeding. We recently described two paralogous anti-complement proteins called Ixodes ricinus anti-complement (IRAC) proteins I and II co-expressed in I. ricinus salivary glands. Phylogenetic analyses suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the present study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. IRAC I and IRAC II were tested against the complement of eight I. ricinus natural host species (six mammals and two birds). The results demonstrate that IRAC I and IRAC II have broad and complementary inhibition activities against the complement of different host species. This report is the first description of paralogous anti-complement molecules encoded by a pathogen with broad and complementary inhibitory activities against the complement of different host species. [less ▲]

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See detailStat5 Is an Ambivalent Regulator of Neutrophil Homeostasis
Fievez, Laurence ULg; Desmet, Christophe ULg; Henry, Emmanuelle et al

in PLoS ONE (2007), 2(1), 727

Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs ... [more ▼]

Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils. CONCLUSION: We conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis. [less ▲]

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See detailIxodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.
Daix, Virginie ULg; Schroeder, Hélène ULg; Praet, N. et al

in Insect Molecular Biology (2007), 16(2), 155-66

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures ... [more ▼]

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717-18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection. [less ▲]

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See detailNatural antibody--complement dependent neutralization of bovine herpesvirus 4 by human serum
Machiels, Bénédicte ULg; Gillet, Laurent ULg; Brito, Sieberth Do Nascimento et al

in Microbes & Infection (2007), 9(14-15), 1530-1537

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are ... [more ▼]

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by naïve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from naïve individuals, in contrast to the sera of naïve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galalpha1-3Galbeta1-4GlcNAc-R epitope expressed by bovine cells [less ▲]

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

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

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

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

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

Poster (2007)

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

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

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

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

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

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

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

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

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

in BMC Biotechnology (2007), 7

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Poster (2006)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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