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See detailRational Design of a Safe and Efficacious Attenuated Recombinant Vaccine Against Cyprinid Herpesvirus 3 Using Prokaryotic Mutagenesis and In Vivo Imaging System
Boutier, Maxime ULg

Conference (2014, October 02)

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease in common and koi carp. Since its emergence, in the late 1990s, CyHV 3 has caused severe economic losses worldwide creating a ... [more ▼]

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease in common and koi carp. Since its emergence, in the late 1990s, CyHV 3 has caused severe economic losses worldwide creating a need for a safe and efficacious vaccine. With this goal in mind, recombinant strains deleted for single gene were produced using prokaryotic recombination technologies. While producing such a recombinant for ORF134, we unexpectedly obtained a clone deleted for ORF56 and ORF57 in addition to the expected deletion of ORF134. Interestingly, this triple deleted recombinant replicated efficiently in vitro, exhibited an attenuated profile and induced an immune protection against a lethal challenge in vivo. To determine the role of each deleted locus in the phenotype of the triple deleted recombinant, a series of recombinant were produced and tested in vivo. Firstly, to investigate the role of ORF134 deletion, a double ORF56-57 deleted recombinant and an independent triple ORF56-57-ORF134 deleted recombinant were produced. These experiments revealed that ORF134 deletion did contribute significantly neither to the attenuation nor to the immune protection observed for the triple deleted recombinant. Secondly, to determine the relative importance of ORF56 and ORF57 deletion in the attenuation observed, two single deleted recombinants were produced for ORF56 and ORF57. These experiments demonstrated that ORF57 deletion, in contrast to ORF56 deletion, correlates with an attenuated phenotype. Based on its safety-efficacy profile, the ORF56-57 double deleted recombinant was selected as a candidate vaccine. Complementary experiments were performed to further investigate the potential of this strain as a safe and efficacious attenuated recombinant vaccine. The results obtained can be summarized as follows: (i) In vivo imaging (IVIS) of vaccinated fish challenged with a wild type strain expressing luciferase as a reporter gene, suggested that vaccination induces a sterile immunity at the portal of entry. (ii) Study of viral tropism by qPCR, IVIS, and histopathological analyses, demonstrated that in comparison to the wild type parental strain, the vaccine strain replicates at lower level, at later time post-infection and for a shorter period of time. (iii) Transmission studies demonstrated that there is no detectable spread of the vaccine from freshly vaccinated fish to naive fish located immediately down stream of vaccinated fish (water sharing). All together, the results of the present study demonstrate the potential of ORF56-ORF57 double deleted CyHV-3 recombinant strain for safe and efficacious mass vaccination of carp. [less ▲]

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See detailThe IL-10 homologue encoded by cyprinid herpesvirus 3 is essential neither for viral replication in vitro nor for virulence in vivo
Ouyang, Ping ULg; Rakus, Krzysztof ULg; Boutier, Maxime ULg et al

in Veterinary Research (2013)

Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae, is the causative agent of a lethal disease in common and koi carp. CyHV-3 ORF134 encodes an interleukin-10 (IL-10) homologue. The ... [more ▼]

Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae, is the causative agent of a lethal disease in common and koi carp. CyHV-3 ORF134 encodes an interleukin-10 (IL-10) homologue. The present study was devoted to this ORF. Transcriptomic analyses revealed that ORF134 is expressed as a spliced gene belonging to the early-late class. Proteomic analyses of CyHV-3 infected cell supernatant demonstrated that the ORF134 expression product is one of the most abundant proteins of the CyHV-3 secretome. To investigate the role of ORF134 in viral replication in vitro and in virulence in vivo, a deleted strain and a derived revertant strain were produced using BAC cloning technologies. The recombinant ORF134 deleted strain replicated in vitro comparably to the parental and the revertant strains. Infection of fish by immersion in water containing the virus induced comparable CyHV-3 disease for the three virus genotypes tested (wild type, deleted and revertant). Quantification of viral DNA by real time TaqMan PCR (in the gills and the kidney) and analysis of carp cytokine expression (in the spleen) by RT-qPCR at different times post-infection did not revealed any significant difference between the groups of fish infected with the three virus genotypes. Similarly, histological examination of the gills and the kidney of infected fish revealed no significant differences between fish infected with ORF134 deleted virus versus fish infected with the control parental or revertant strains. All together, the results of the present study demonstrate that the IL-10 homologue encoded by CyHV-3 is essential neither for viral replication in vitro nor for virulence in common carp. [less ▲]

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See detailCyprinid herpesvirus 3 : an intersting virus for applied and fundamental research
Rakus; Ouyang, Ping; Boutier, Maxime ULg et al

in Veterinary Research (2013), 44

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See detailLaboratory validation of a lateral flow device for the detection of CyHV-3 antigens in gill swabs
Vrancken, Robert; Boutier, Maxime ULg; Ronsmans, Maygane ULg et al

in Journal of Virological Methods (2013), 193

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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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