[Foot-and-mouth disease:: an old disease--new solutions]. / Fièvre aphteuse: anciens défis--nouvelles solutions.

The recent outbreak of foot-and-mouth disease in United Kingdom provoked a crisis in the European Union with deleterious consequences not only for livestock industry. Public opinion is more and more concerned about stamping out measures used to control the disease even with previously vaccinated animals. Presently the trend is to "vaccinate for life". This policy change requires to improve vaccines and diagnostic tools. It is not foreseen nevertheless to come back to a generalized vaccination of cattle as it was the case previously in continental Europe, despite its efficacy. According to the new policy, it will only be emergency vaccination to control outbreaks; it will compulsorily use inactivated vaccines. The vaccines will have to confer quickly a strong (sterile) protection against several serotypes during the same outbreak. Several serotypes could be involved in case of agroterrorism. Another feature of foot-and-mouth virus infection is the generation of asymptomatic carriers of wild virus after infection even in previously vaccinated animals. In order to get round this problem, so-called "marker vaccines" associated with a companion diagnostic test are developed: it aims to be able to differentiate simply vaccinated animals from infected ones, whether they were previously vaccinated or not. These vaccines are presently highly purified inactivated vaccines and the companion diagnostic test is based upon the detection of specific antibodies directed against virus-induced non-structural proteins. These antibodies should not be detected in simply vaccinated animals. This technology takes into account the fact that foot-and-mouth disease virus multiplication implies the synthesis of a polyprotein subsequently cleaved. It allows to certify the absence of infection at a herd level not yet at an individual level. Another research trend is to identify virus receptors in animals in order to better understand the pathogenesis of the infection and the reasons why some animals become asymptomatic carriers of wild virus after infection.

Salivary excretion of rabies virus by healthy vampire bats.

Salivary excretion of rabies virus was evaluated in 14 adult vampire bats (Desmodus rotundus) intramuscularly injected with a large dose (10(6) MICLD50) of vampire rabies virus variant CASS88. Saliva samples were obtained from surviving bats every other day for 30 days, then weekly for 2 months, and finally 1 and 2 years later. Rabies virus was isolated in murine neuroblastoma cells and in randomly selected cases by PCR. Rabies virus was not detected in the saliva of any of the 11 animals that succumbed (somewhat early) to rabies challenge, nor in the control bats. In contrast, virus was detected early, and only once (days 6, 6 and 21) in each of the three animals that survived rabies challenge and remained healthy for at least 2 years after challenge. At that time even vigorous dexamethasone and cyclosporine administration failed to provoke further viral excretion.

Investigation of the susceptibility of human cell lines to bovine herpesvirus 4 infection: demonstration that human cells can support a nonpermissive persistent infection which protects them against tumor necrosis factor alpha-induced apoptosis.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.

Prevalence of Echinococcus multilocularis in the red fox (Vulpes vulpes) in southern Belgium.

Between June 1998 and February 2002, 709 red foxes killed in Wallonia (south of Belgium) were available for parasitological examination of the gut. The identification of Echinococcus multilocularis was based on morphological data. E. multilocularis adults were observed in 20.2% of the animals. The analysis of data revealed marked differences between the geological areas of Wallonia; the highest prevalence (33%) was found in the Ardenne and the lowest (0%) on the Plateau de Herve. Host gender and the collection season had no effect on the prevalence. However, the latter was significantly higher in juveniles (<8 months of age). The geographical distribution of E. multilocularis in Belgium is much wider than originally thought.

Genetic and antigenic variability in bovine viral diarrhea virus (BVDV) isolates from Belgium.

This report describes the genetic and antigenic variability of bovine viral diarrhea virus strains isolated in Belgium. Part of the 5' untranslated region and the 5' end of the gp53 (E2) coding sequence were amplified by PCR and sequenced. Phylogenetic analysis showed that most field isolates segregated into genotypes Ib or II. Only one out of 28 field isolates belonged to genotype Ia. Interestingly, some type I strains were equally divergent from types Ia and Ib strains and clustered into additional subtypes within genotype I. Immune sera from young calves experimentally inoculated with field isolates first identified on the basis of their sequences were used in two-way neutralisation experiments. The results clearly differentiated type I from type II strains although some degree of cross-neutralisation was observed. Within type I, the new clusters could not be antigenically differentiated from the more prevalent type Ib strains or from type Ia strain NADL, suggesting that BVDV genotype I is antigenically homogeneous. The isolation of BVDV types I and II strains from cell lines and from a bovine vaccine suggest that molecular epidemiology surveillance is warranted for BVDV.

Humoral and cell-mediated immune responses of foxes (Vulpes vulpes) after experimental primary and secondary oral vaccination using SAG2 and V-RG vaccines.

Humoral and cell-mediated immune responses of 36 captive foxes to two oral vaccines against rabies currently used for foxes in Europe were studied. The Street Alabama Dufferin (SAD) mutant Gif (SAG2) vaccine has been selected by double mutation from the SAD virus. The vaccinia recombinant virus (V-RG) expresses the rabies glycoprotein. Both vaccines induce similar humoral and cell-mediated responses after primary and secondary oral administration. We observed a typical anamnestic response, although of a limited duration, after the booster vaccination. Therefore, our results suggested that two successive oral vaccination campaigns should not significantly improve the immunisation of foxes. Lymphocyte in vitro proliferative response to the SAD antigen highlighted the presence in blood of a T-cell specific memory 6 months after vaccination. The synthesis of several vulpine cytokines was detected in peripheral blood mononuclear cells (PBMC) stimulated by SAD antigen via reverse transcription polymerase chain amplification. The data showed a concomitant expression of interleukin (IL)-4 and interferon-gamma in PBMC of vaccinated foxes. No change was detected in the level of IL-2, IL-10 and IL-12 synthesis, whereas the pro-inflammatory cytokine tumour necrosis factor-alpha seemed involved in the activation of naive T lymphocytes.

A multipotential beta -1,6-N-acetylglucosaminyl-transferase is encoded by bovine herpesvirus type 4.

The beta-1,6-N-acetylglucosaminyltransferase (beta1,6GnT) gene family encodes enzymes playing crucial roles in glycan synthesis. Important changes in beta1,6GnT expression are observed during development, oncogenesis, and immunodeficiency. The most characterized beta1,6GnTs in this gene family are the human (h) C2GnT-L and h-IGnT, which have core 2 [Galbeta1-->3(GlcNAcbeta1-->6)GalNAc] and I branching [GlcNAcbeta1-->3(GlcNAcbeta1-->6)Gal] activities, respectively. Recently, h-C2GnT-M was shown to be unique in forming core 2, core 4 [GlcNAcbeta1-->3(GlcNAcbeta1-->6)GalNAc], and I structures. To date, the beta1,6GnT gene family has been characterized only in mammals. Here, we describe that bovine herpesvirus type 4 (BHV-4) encodes a beta1,6GnT expressed during viral replication and exhibiting all of the core 2, core 4, and I branching activities. Sequencing of the BHV-4 genome revealed an ORF, hereafter called BORFF3-4, encoding a protein (pBORFF3-4) exhibiting 81.1%, 50.7%, and 36.6% amino acid identity with h-C2GnT-M, h-C2GnT-L, and h-IGnT, respectively. Reverse transcriptase-PCR analysis revealed that BORFF3-4 is expressed during BHV-4 replication. Expression of BORFF3-4 in Chinese hamster ovary cells directed the expression of core 2 branched oligosaccharides and I antigenic structures on the cell surface. Moreover, a soluble form of pBORFF3-4 had core 4 branching activity in addition to core 2 and I branching activities. Finally, infection of a C2GnT-negative cell line with BHV-4 induced expression of core 2 branched oligosaccharides. This study extends the beta1,6GnT gene family to a viral gene and provides a model to study the biological functions of a beta1,6GnT in the context of viral infection.

Bovine herpesvirus 1-induced apoptotic cell death: role of glycoprotein D.

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in peripheral blood mononuclear cells and in bovine B lymphoma (BL-3) cells. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells, suggesting that one or more BHV-1 envelope glycoproteins could be involved in the activation of the apoptotic process. In the present study, we demonstrate that, although BHV-1 virions devoid of glycoprotein D (BHV-1 gD-/-) still bind to BL-3 cells, they are no longer able to induce apoptosis. In contrast, virions that contain glycoprotein D (gD) in the viral envelope but do not genetically encode gD (BHV-1 gD-/+) induce a level of apoptosis comparable to that produced by wild-type (wt) BHV-1. In addition, monoclonal antibodies directed against gD, but not against gB or gC, strongly reduced the high levels of apoptosis induced by BHV-1. These observations demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Interestingly, binding of affinity-purified gD to BL-3 cells did not induce apoptosis but inhibited the ability of wt BHV-1 to induce apoptosis. Altogether, these results provide evidence for the direct or indirect involvement of gD in the mechanism by which BHV-1 induces apoptosis.

Attachment but not penetration of bovine herpesvirus 1 is necessary to induce apoptosis in target cells.

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene.

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

Bovine herpesvirus 1-induced apoptosis: phenotypic characterization of susceptible peripheral blood mononuclear cells.

Bovine herpesvirus 1 (BHV-1), a member of the Alphaherpesvirinae, induces apoptotic cell death in peripheral blood mononuclear cells (PBMC). To investigate the process by which BHV-1 induces apoptosis, we determined the susceptibility of the three main PBMC subpopulations to BHV-1-induced apoptosis. This study shows that BHV-1 can induce apoptosis individually in T lymphocytes, B lymphocytes and monocytes. This conclusion is based on the following findings: (i) BHV-1 substantially reduces the percentages of viable T and B lymphocytes in PBMCs. (ii) Concomitant detection of cell phenotype and apoptosis indeed showed higher percentages of apoptotic T lymphocytes and B lymphocytes in BHV-1-infected PBMCs than in mock-infected cells. (iii) Each individual PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) undergo apoptosis when incubated with BHV-1. These data also suggest that BHV-1 does not require the recruitment of one or more individual PBMC subpopulations (e.g. cytotoxic cells) to induce apoptosis. Finally, we observed that BL-3 cells which have been characterized as bovine tumoral B lymphocytes also undergo apoptosis when incubated with BHV-1. Therefore, the use of the BL-3 cell line provides a new experimental model to investigate the apoptotic process induced by BHV-1 in vitro.

[Apoptosis induced by bovine herpesvirus 1]. / Apoptose induite par le bovine herpesvirus 1.

The mechanism by which Bovine herpesvirus 1 (BHV-1) induces apoptic cell death is not fully understood. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells suggesting that one or more BHV-1 envelope glycoprotein(s) could be involved in the activation of the apoptotic process. In this context, we show that BHV-1 virions devoid of glycoprotein D (gD) are no longer able to induce apoptosis. In contrast, virions which contain gD in the viral envelope but do not genetically encode gD (BHV-1 gD-/+) induce comparable level of apoptosis as wild type (wt) BHV-1. In addition, monoclonal antibodies directed against gD strongly reduced the high levels of apoptosis induced by wt BHV-1 and BHV-1 gD-/+ but not the background level of apoptosis induced by BHV-1 gD-/-. This demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Altogether, these results provide evidence for the involvement of gD in the mechanism by which BHV-1 induces apoptosis. This could have important implications in the development of new, more effective and safer vaccines and also help to better understand the pathogenesis of BHV-1.

Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the gamma2 herpesvirus bovine herpesvirus-4.

Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.

Glycoprotein B of bovine herpesvirus 4 is a major component of the virion, unlike that of two other gammaherpesviruses, Epstein-Barr virus and murine gammaherpesvirus 68.

This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.

Bovine herpesvirus 4: genomic organization and relationship with two other gammaherpesviruses, Epstein-Barr virus and herpesvirus saimiri.

Bovine herpesvirus 4 (BHV-4) belongs to the gammaherpesvirinae subfamily. Although the whole sequence of BHV-4 genome is not known it was possible, based on random sequencing, to assume that its genomic organization consists of genes clustered in blocks whose orientation and location in the genome are conserved within a herpesvirus subfamily. Between these blocks lie genes which are specific to either a particular virus or a virus subfamily. BHV-4 genome consists of 5 gene blocks conserved among the gammaherpesviruses and particularly within the Epstein-Barr virus (EBV) and the herpesvirus saimiri (HVS) genomes. Analysis of the regions located outside the gene blocks showed the presence of 12 open reading frames (ORFs). Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or beta-herpesviruses. Nevertheless, 5 ORFs were homologous in amino acid sequences to proteins encoded by HVS and one was similar to a protein encoded by both HVS and EBV. On the basis of the molecular data BHV-4 is more closely related to HVS than to EBV. Genes homologous to cellular genes have been described in both HVS and EBV genomes. No genes homologous to presently sequenced cellular genes were found among those found in the BHV-4 genome to date.

Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins.

This paper focuses on the structure and functions of bovine herpesvirus 1 minor glycoproteins gH, gE, gG and gp42. It reviews the progress which has been made in their identification and characterization, in the study of their temporal expression and processing in infected cells, and finally in the understanding of their biological activities. In addition, aspects discussed include a comparison with two other alphaherpesviruses, namely herpes simplex virus and pseudorabies virus.

The role of glycoproteins gC, gE, gI, and gG in the induction of cell-mediated immune responses to bovine herpesvirus 1.

Mutant viruses with deletions in genes encoding non-essential glycoproteins are considered as promising bovine herpesvirus 1 (BHV1) vaccine candidates. The present study compared the influence of various gene deletions (gC, gE, gI, gG) on the induction of cell-mediated immune responses against the virus. The highest BHV1 specific lymphoproliferative response was observed in the group of calves inoculated with the gC- mutant. However, in all groups of inoculated calves, limiting dilution analysis showed marked individual variability in the number of BHV1 specific T lymphocytes that were stimulated. The same animals were then challenged with wild-type BHV1. In these animals, limiting dilution analysis did not reveal gE, gI nor gG as a major T lymphocyte antigen. However, further analysis suggested the T cell antigenicity of gE in a low number of BHV1 hyperimmunized calves. Stimulation of MHC unrestricted cytotoxicity was also evaluated after inoculation with the various deletion mutants. Cytotoxicity in gC- inoculated calves was as high as in BHV1 inoculated calves. In conclusion, among the BHV1 deletion mutants that were tested, the gC- mutant stimulated the best cell-mediated immune responses.

The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle.

Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.

Analysis of bovine herpesvirus 4 genomic regions located outside the conserved gammaherpesvirus gene blocks.

Bovine herpesvirus 4 (BHV-4) DNA sequences located outside the gene blocks conserved among the gammaherpesviruses BHV-4, herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) were analysed. Twelve potential open reading frames (ORFs) were found. Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or betaherpesviruses. Nevertheless, six of the ORFs were homologous in amino acid sequences to proteins encoded by HVS but apparently not to those encoded by EBV. Furthermore, the location and orientation of these six ORFs in the BHV-4 genome were similar to the corresponding ORFs in the HVS genome. No genes homologous to known cellular genes were found in the BHV-4 genome; this feature is the major difference between the BHV-4 and HVS genomes with regards to the overall gene content.