The genome of a tortoise herpesvirus (testudinid herpesvirus 3) has a novel structure and contains a large region that is not required for replication in vitro or virulence in vivo.
Gandar, Frederic ; ; et al
in Journal of Virology (2015), 89(22), 11438-11456
Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on ... [more ▼]
Testudinid herpesvirus 3 (TeHV-3) is the causative agent of a lethal disease affecting several tortoise species. The threat that this virus poses to endangered animals is focusing efforts on characterizing its properties, in order to enable the development of prophylactic methods. We have sequenced the genomes of the two most studied TeHV-3 strains (1976 and 4295). TeHV-3 strain 1976 has a novel genome structure and is most closely related to a turtle herpesvirus, thus supporting its classification into genus Scutavirus, subfamily Alphaherpesvirinae, family Herpesviridae. The sequence of strain 1976 also revealed viral counterparts of cellular interleukin-10 and semaphorin, which have not been described previously in members of subfamily Alphaherpesvirinae. TeHV-3 strain 4295 is a mixture of three forms (m1, m2, and M), in which, in comparison to strain 1976, the genomes exhibit large, partially overlapping deletions of 12.5 to 22.4 kb. Viral subclones representing these forms were isolated by limiting dilution assays, and each replicated in cell culture comparably to strain 1976. With the goal of testing the potential of the three forms as attenuated vaccine candidates, strain 4295 was inoculated intranasally into Hermann's tortoises (Testudo hermanni). All inoculated subjects died, and PCR analyses demonstrated the ability of the m2 and M forms to spread and invade the brain. In contrast, the m1 form was detected in none of the organs tested, suggesting its potential as the basis of an attenuated vaccine candidate. Our findings represent a major step toward characterizing TeHV-3 and developing prophylactic methods against it. [less ▲]Detailed reference viewed: 27 (8 ULg)
Proteomic characterization of murid herpesvirus 4 extracellular virions.
Vidick, Sarah ; ; Gonon Rodrigues Palmeira, Leonor et al
in PloS one (2013), 8(12), 83842
Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic ... [more ▼]
Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi's sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology. [less ▲]Detailed reference viewed: 22 (6 ULg)
Feeding Cyprinus carpio with infectious materials mediates cyprinid herpesvirus 3 entry through infection of pharyngeal periodontal mucosa
Fournier, Guillaume ; Boutier, Maxime ; et al
in Veterinary Research (2012), 43(6),
Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using ... [more ▼]
Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is the etiological agent of a mortal disease in common and koi carp. Recently, we investigated the entry of CyHV-3 in carp using bioluminescence imaging and a CyHV-3 recombinant strain expressing luciferase (LUC). We demonstrated that the skin is the major portal of entry after inoculation of carp by immersion in water containing CyHV-3. While this model of infection mimics some natural conditions in which infection takes place, other epidemiological conditions could favour entry of virus through the digestive tract. Here, we investigated whether ingestion of infectious materials mediates CyHV-3 entry through the digestive tract. Carp were fed with materials contaminated with the CyHV-3 LUC recombinant (oral contamination) or immersed in water containing the virus (contamination by immersion). Bioluminescence imaging analyses performed at different times post-infection led to the following observations: (i) the pharyngeal periodontal mucosa is the major portal of entry after oral contamination, while the skin is the major portal of entry after contamination by immersion. (ii) Both modes of inoculation led to the spreading of the infection to the various organs tested. However, the timing and the sequence in which some of the organs turned positive were different between the two modes of inoculation. Finally, we compared the disease induced by the two inoculation modes. They led to comparable clinical signs and mortality rate. The results of the present study suggest that, based on epidemiological conditions, CyHV-3 can enter carp either by skin or periodontal pharyngeal mucosal infection. [less ▲]Detailed reference viewed: 48 (10 ULg)
Proteomic characterization of bovine herpesvirus 4 extracellular virions.
Lété, Céline ; Palmeira, Leonor ; 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 ▲]Detailed reference viewed: 44 (9 ULg)
Antibody evasion by a gammaherpesvirus o-glycan shield.
Machiels, Bénédicte ; Lété, Céline ; Guillaume, Antoine 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 ▲]Detailed reference viewed: 78 (29 ULg)
Bovine Herpesvirus 4 Bo10 gene encodes a nonessential viral envelope protein that regulates viral tropism through both positive and negative effects.
Machiels, Bénédicte ; Lété, Céline ; 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 ▲]Detailed reference viewed: 107 (48 ULg)
Brucella ceti infection in a harbor porpoise (Phocoena phocoena)
Jauniaux, Thierry ; ; et al
in Emerging Infectious Diseases (2010), 139(11), 254-7Detailed reference viewed: 45 (17 ULg)
Production de pseudoparticules de norovirus humains et bovins et leur utilisation diagnostique
Mauroy, Axel ; ; Saegerman, Claude et al
Poster (2007, April)
Les norovirus sont des agents majeurs de gastroentérite humaine d’origine alimentaire et ceci à travers le monde entier. La contamination est habituellement oro-fécale. Les norovirus sont non enveloppés ... [more ▼]
Les norovirus sont des agents majeurs de gastroentérite humaine d’origine alimentaire et ceci à travers le monde entier. La contamination est habituellement oro-fécale. Les norovirus sont non enveloppés et ont un génome composé d’ARN monocaténaire de polarité positive d’approximativement 7,5 kb. Au sein de ce génome, trois cadres ouverts de lecture (ORFs) sont décrits. L’ORF 1 code pour une polyprotéine qui sera par la suite clivée pour donner les différentes protéines non-structurales. L’ORF 2 encode pour l’unique protéine de capside. Une protéine structurale mineure dont le rôle est encore peu caractérisé est encodée par l’ORF 3. Ces virus sont très résistants dans l’environnement et une infection peut survenir même avec une très faible dose infectieuse. Appartenant à la famille des Caliciviridae, le genre norovirus est composé de cinq génogroupes (G) et contient également des virus infectant les animaux (bovins, porcins, murins). L’impact des norovirus animaux et plus particulièrement des norovirus bovins peut être envisagé à travers un possible caractère zoonotique de ces virus ; ainsi ils pourraient par exemple être transmis à l’homme via des eaux d’effluent contaminées. L’étude des norovirus humains et bovins est encore à l’heure actuelle entravée par l’absence d’un système de culture cellulaire. Cependant différents systèmes d’expression protéique ont été utilisés afin d’exprimer la protéine de capside, celle-ci s’assemblant spontanément avec d’autres pour reformer des pseudoparticules virales ou virus-like particles (VLPs). Ces VLPs sont morphologiquement et antigéniquement semblables aux virus natifs. Le but de ce travail était dans un premier temps d’obtenir des VLPs de souches humaines et bovines. Les gènes codant pour la protéine de capside d’une souche humaine (H384) et d’une souche bovine (B309) ont été amplifiés par RT-PCR à partir d’échantillons de matières fécales collectés dans des laboratoires de diagnostic humain et vétérinaire. Ces gènes ont été séquencés et comparés grâce à des outils informatiques (Basic Local Alignment Search Tool, BLAST) à la banque de séquences déjà disponibles sur le site du NCBI. Nous avons pu déterminer que la séquence de capside de la souche H384 était proche de celle de la souche HuNV/Altenkirchen 140/01/DE, une souche de GII et de génotype 4 tandis que la séquence de la souche B309 était proche de celles de GIII et de génotpe 2 (groupe Newbury2). Des plasmides contenant les séquences des ORFs 2 de deux souches de norovirus humains de référence, la souche de Norwalk (GI) et celle de Hawaii (GII), ont été aimablement fournies par le Docteur Jan Vinjé (CDC, Atlanta, USA). Le système d’expression protéique baculovirus (Invitrogen) a été utilisé pour obtenir des VLPs. Brièvement, des cellules d’insecte des lignées Sf9 et H5 ont été infectées avec des baculovirus recombinants pour la protéine de capside des norovirus. Les VLPs ont été purifiées à partir des surnageants par ultracentrifugation sur un coussin de sucrose à 30% suivie d’une ultracentrifugation en gradient de chlorure de césium. Ce gradient a été fractionné et les différentes fractions ont été analysées pour la présence de la protéine de capside des norovirus par SDS-PAGE. Pour vérifier que les fractions positives contenaient bien des VLPs, les surnageants ont été observés en microscopie électronique. Nous avons ainsi obtenu des VLPs de norovirus de trois des cinq génogroupes décrits (GI, II et III). Ces VLPs ont été utilisées pour immuniser des souris afin de produire des anticorps monoclonaux et pour immuniser des lapins afin d’obtenir des anticorps polyclonaux. Nous avons pu aussi tester une banque de sérums bovins par ELISA pour la présence d’anticorps dirigés contre les souches de norovirus de génogroupe III et de génotype 2, les plus fréquemment mises en évidence au sein de notre banque de matières fécales bovines. [less ▲]Detailed reference viewed: 21 (3 ULg)
Virus-like Particles of Human and Bovine Noroviruses
Mauroy, Axel ; ; et al
Poster (2006, September)
1.Introduction and Objectives Noroviruses have a positive-sense, single-stranded RNA genome of about 7.5 kb containing three open reading frames (ORFs). ORF1 encodes the non-structural proteins, ORF2 ... [more ▼]
1.Introduction and Objectives Noroviruses have a positive-sense, single-stranded RNA genome of about 7.5 kb containing three open reading frames (ORFs). ORF1 encodes the non-structural proteins, ORF2 encodes the single capsid protein with a molecular weight of 56 kD and ORF3 encodes a minor structural protein. These viruses are very stable and successful infection can be achieved even with very low infectious dose. Human noroviruses are the major cause of non bacterial, food-borne gastroenteritis worldwide. Contamination is usually oro-fecal (Green et al. 2001). Belonging to the Caliciviridae family, the genus Norovirus also contains viruses infecting animals, in particular bovines. This genus is divided into five genogroups (Ando et al. 2000). The existence of bovine noroviruses raises the question of their possible zoonotic character; the viruses could be transmitted to human by contaminated effluents for example (Scipioni et al., this congress). The study of norovirus is hampered by the lack of a cell culture system. However virus like particles (VLPs) can be produced and they are antigenically and morphologically similar to the wild virus (Jiang et al. 1992). The aim of this work was to obtain VLPs from human and bovine strains. Such VLPs will be used as antigens to produce specific polyclonal and monoclonal antibodies. 2.Material and Methods The capsid protein genes of a human norovirus strain (H384) and a bovine strain (B309) were amplified by RT PCR from stools collected from human and veterinary diagnostic laboratories. These sequences were analysed by the Basic Local Alignment Search Tool (BLAST) available on the NCBI website. Plasmids containing the ORF2 sequence of two reference human strains, the Norwalk (NV, belonging to the genogroup I) and the Hawaii (HV, belonging to the genogroup II) strains, were kindly received from Jan Vinje (University of North Carolina, USA). The baculovirus protein expression system (Invitrogen®) was used to obtain VLPs. Briefly, cells and supernatants were harvested after freezing/thawing cycles. Cells were pelleted. VLPs were purified from supernatants by ultracentrifugation through a 30% sucrose cushion and then by isopycnic ultracentrifugation in CsCl. Fractions resulting from the gradient were analysed by SDS-PAGE. Protein assembly into VLPs was checked by electron microscopy. 3.Results H384 strain ORF2 is closely related to the HuNV/Altenkirchen 140/01/DE strain (99% of identity). Thus, H384 most likely belongs to genogroup II genotype 4 (GII.4, the Lordsdale group). B309 capsid gene is closely related to genogroup III genotype 2 (Newbury2). Electron microscopy confirmed the presence of norovirus-like particles in the supernatant of recombinant baculovirus infected cell culture. By SDS PAGE applied on the fractions resulting of the CsCl gradient, a protein of 56 kD was detected and confirmed the expression of the capsid protein. Therefore, VLPs were obtained for all the 4 strains investigated. 4.Discussion and Conclusions Currently we dispose of representative surrogates of three norovirus genogroups (GI, GII and GIII). H384 (GII.4) is a representative of the most commonly diagnosed genotype from current outbreaks in many countries (Blanton et al. 2006). These VLPs will therefore serve as antigens to raise polyclonal and monoclonal antibodies. These tools will be used for the diagnosis of the related infections in human and domestic animals. [less ▲]Detailed reference viewed: 22 (1 ULg)
Felid herpesvirus 1 glycoprotein G is a structural protein that mediates the binding of chemokines on the viral envelope.
Costes, Bérénice ; Thirion, Muriel ; Dewals, Benjamin G 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 ▲]Detailed reference viewed: 26 (7 ULg)