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See detailStudy of the portals of entry of Cyprinid herpesvirus 3 in Cyprinus carpio
Fournier, Guillaume ULg

Doctoral thesis (2012)

The common carp is one of the most important freshwater species in aquaculture and its colourful subspecies koi is grown for personal pleasure and exhibitions. Both subspecies are economically important ... [more ▼]

The common carp is one of the most important freshwater species in aquaculture and its colourful subspecies koi is grown for personal pleasure and exhibitions. Both subspecies are economically important. In the 1990s, a highly contagious and lethal pathogen called koi herpesvirus (KHV) or cyprinid herpesvirus 3 (CyHV-3) began to cause severe financial losses in these two carp industries worldwide. Because of its economic importance and its numerous original biological properties, CyHV 3 became rapidly an attractive subject for applied and fundamental research. The goal of this thesis was to identify the portals of entry of CyHV-3 in carp. This information is essential to understand the pathogenesis and the epidemiology of the infection, but also to develop efficacious vaccines. Prolonged CyHV-3 cultivation in vitro leads to the spontaneous attenuation of the virus. To circumvent this problem, the entire viral genome was cloned as a bacterial artificial chromosome (BAC). Then to test the usefulness of the BAC clone, several recombinants strains were generated as described in the first chapter. In the second chapter, we took profit of the CyHV-3 BAC clone to produce a recombinant strain encoding a firefly luciferase (LUC) expression cassette. Infection of carp by immersion in water containing the CyHV-3 LUC strain demonstrated, using bioluminescent in vivo imaging system (IVIS), that the skin, and not the gills, is the major portal of entry for CyHV-3. Fish skin provides mechanical, chemical and immune protection against injury and pathogenic microorganisms. Its mucus layer confers an innate immune protection against pathogen entry. However, there is little in vivo evidence on the role of skin mucus as a first line of innate immune protection against bacterial and viral infections. In the third chapter, we used the CyHV-3 LUC strain and IVIS to investigate the roles of epidermal mucus as an innate immune barrier against CyHV-3 entry. Our results demonstrate that the mucus of the skin inhibits CyHV-3 binding to epidermal cells and contains soluble molecules able to neutralize CyHV-3 infectivity. The skin is the major portal of entry after inoculation 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 favor entry of virus through the digestive tract. Consequently, in the fourth and last chapter, we investigated the role of the carp digestive tract as a viral portal of entry using bioluminescence imaging. We found that feeding carp with infectious materials induces CyHV-3 entry through infection of the pharyngeal periodontal mucosa. In conclusion, this study demonstrated that according to epidemiological conditions, CyHV-3 can enter carp either through infection of the skin (immersion in infectious water) or through infection of the pharyngeal periodontal mucosa (feeding on infectious materials). The existence of these two portal of entry adapted to different epidemiological conditions most probably contributes to the high contagious nature of the virus. [less ▲]

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

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

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

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

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See detailSkin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells
RAJ, Victor; Fournier, Guillaume ULg; Rakus, Krzysztof ULg et al

in Veterinary Research (2011), 42(92),

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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 detailClonage du génome l'herpesvirus cyprin-3 en tant que chromosome artificiel bactérien
Fournier, Guillaume ULg

Master's dissertation (2008)

Koi herpesvirus (KHV) or Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease in koi and common carp. In the present study, we described the cloning of the KHV genome as a stable and ... [more ▼]

Koi herpesvirus (KHV) or Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease in koi and common carp. In the present study, we described the cloning of the KHV genome as a stable and infectious BAC clone that can be used to produce KHV recombinant strains. This goal was achieved by insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when transfected into permissive cells. Reconstituted virions free of the BAC cassette but with a disrupted TK locus were produced. Similarly, virions with a wild type revertant TK sequence were produced. The FL BAC excised strain and the FL BAC revertant strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV in koi carp that was indistinguishable from that of the parental strain, while FL BAC excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombinant studies was demonstrated by production of an ORF16 deleted strain using prokaryotic recombination technology. The availability of KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines. [less ▲]

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See detailCloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.
Costes, Bérénice ULg; Fournier, Guillaume ULg; Michel, Benjamin ULg et al

in Journal of Virology (2008), 82(10), 4955-4964

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial ... [more ▼]

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines. [less ▲]

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See detailLes homologues viraux des récepteurs cellulaires couplés aux protéines G
Boudry, Christel; Costes, Bérénice ULg; Fournier, Guillaume ULg et al

in Virologie (2007), 11(6), 457-70

G-protein-coupled receptors (GPCR) are seven transmembrane proteins that convert extracellular stimuli to cell signaling.Viral genes homologous to cellular GPCR have been described in the genome of ... [more ▼]

G-protein-coupled receptors (GPCR) are seven transmembrane proteins that convert extracellular stimuli to cell signaling.Viral genes homologous to cellular GPCR have been described in the genome of Betaherpesvirinae, Gammaherpesvirinae and Poxviridae. The goal of this review is to summarize the knowledge available on viral GPCR (vGPCR) with a special interest for their roles in the biology and the pathogenesis of the infection. This review highlights some properties of vGPCR that are not shared by their cellular homologues and stresses the diversity of their functions in the biology of the infection. [less ▲]

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