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See detailCyprinid Herpesvirus 3 ORF57 is an essential virulence factor
Boutier, Maxime ULiege; Gao, Yuan; Vancsok, Catherine et al

Poster (2017)

The genus Cyprinivirus comprises phylogenetically related viruses causing important economic losses in aquaculture. Its archetype species, the cyprinid herpesvirus 3 (CyHV 3) is the causative agent of ... [more ▼]

The genus Cyprinivirus comprises phylogenetically related viruses causing important economic losses in aquaculture. Its archetype species, the cyprinid herpesvirus 3 (CyHV 3) is the causative agent of mass mortalities in koi and common carp worldwide. Recently, we reported the development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. This attenuated vaccine encodes a deletion encompassing ORF56 and ORF57. In the present study, we investigated the relative contribution of ORF56 and ORF57 in the safety observed for the ORF56-57 double deleted genotype. Single deleted recombinants were produced based on deletion-insertion of a galK cassette, inserting stop codons in frame with the sequence upstream of the deletion and disrupting the frame of the remaining sequence downstream of the deletion. Inoculation of these recombinants to carp demonstrated that the absence of ORF56 protein expression did not affect virulence; while ORF57 deletion led to an attenuation comparable to the one observed for the ORF56-57 deletion. To further demonstrate the role of ORF57 as a key virulence factor, a recombinant unable to express ORF57 protein was produced by insertion of multiple stop codons in the ORF. In vivo testing of this recombinant revealed a safety-efficacy profile comparable to the one observed for the vaccine candidate deleted for ORF56 and ORF57. Altogether, the present study demonstrates the role of ORF57 as a key virulence factor of CyHV 3 and identifies ORF57 orthologues encoded by other pathogenic cypriniviruses as gene candidates for production of attenuated recombinant vaccines. [less ▲]

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See detailIdentification of an essential virulence gene of cyprinid herpesvirus 3.
Boutier, Maxime ULiege; Gao, Yuan; Vancsok, Catherine ULiege et al

in Antiviral Research (2017), 145

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 ... [more ▼]

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 (CyHV-3) causes mass mortalities worldwide in koi and common carp. A CyHV-3 mutant was described previously that is attenuated in vivo by a deletion affecting two genes (ORF56 and ORF57). The relative contributions of ORF56 and ORF57 to the safety and efficacy profile of this vaccine candidate have now been assessed by analysing viruses individually deleted for ORF56 or ORF57. Inoculation of these viruses into carp demonstrated that the absence of ORF56 did not affect virulence, whereas the absence of ORF57 led to an attenuation comparable to, though slightly less than, that of the doubly deleted virus. To demonstrate further the role of ORF57 as a key virulence factor, a mutant retaining the ORF57 region but unable to express the ORF57 protein was produced by inserting multiple in-frame stop codons into the coding region. Analysis of this virus in vivo revealed a safety and efficacy profile comparable to that of the doubly deleted virus. These findings show that ORF57 encodes an essential CyHV-3 virulence factor. They also indicate that ORF57 orthologues in other cypriniviruses may offer promising targets for the rational design of attenuated recombinant vaccines. [less ▲]

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See detailConserved Fever Pathways across Vertebrates: A Herpesvirus Expressed Decoy TNF-alpha Receptor Delays Behavioral Fever in Fish.
Rakus, Krzysztof; Ronsmans, Maygane; Forlenza, Maria et al

in Cell Host & Microbe (2017), 21(2), 244-253

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the ... [more ▼]

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the manifestation of behavioral fever in the common carp infected by cyprinid herpesvirus 3, a native carp pathogen. Carp maintained at 24 degrees C died from the infection, whereas those housed in multi-chamber tanks encompassing a 24 degrees C-32 degrees C gradient migrated transiently to the warmest compartment and survived as a consequence. Behavioral fever manifested only at advanced stages of infection. Consistent with this, expression of CyHV-3 ORF12, encoding a soluble decoy receptor for TNF-alpha, delayed the manifestation of behavioral fever and promoted CyHV-3 replication in the context of a temperature gradient. Injection of anti-TNF-alpha neutralizing antibodies suppressed behavioral fever, and decreased fish survival in response to infection. This study provides a unique example of how viruses have evolved to alter host behavior to increase fitness. [less ▲]

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See detailA protein conserved in cypriniviruses is a major virulence factor of Cyprinid herpesvirus 3
Boutier, Maxime ULiege; Ronsmans, Maygane; Vancsok, Catherine et al

Poster (2016)

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

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease in common and koi carp. Since its emergence, CyHV‑3 has caused severe economic losses worldwide creating a need for a safe and efficacious vaccine. In a previous study, we showed that a recombinant strain deleted for ORF56 and ORF57 exhibited a safety/efficacy profile compatible with its use as an attenuated recombinant vaccine. In the present study, we investigated the relative contribution of the two genes to the attenuated phenotype observed. To reach this goal, a series of recombinants deleted either for ORF56 or ORF57 were produced. These recombinants were characterized in vitro for their correct molecular structure. In addition, immunofluorescence staining showed that the deletion of ORF56 did not abrogate the expression of ORF57, and vice versa. In vivo experiments demonstrated that the deletion of ORF57 explains most of the attenuation observed for the ORF56-57 deletion. Furthermore, we observed that ORF57 deletion induced in vitro a growth defect (reduction of both the production of infectious particles and the size of viral plaque). Orthologue of CyHV-3 ORF57 in Anguillid herpesvirus 1 (AngHV-1) has been shown to be a tegumental protein. Interestingly, using both qPCR and western blot based approaches, we demonstrated that the particles produced by the ORF57 deleted mutant are less infectious than those of the wild type virus. In conclusion, this study demonstrates that ORF57 is a major virulence factor of CyHV-3. Importantly, as ORF57 is conserved in cypriniviruses, its orthologues could therefore represent a target for production of attenuated vaccine against several other major fish pathogens such as AngHV-1 and Cyprinid herpesvirus 2 (CyHV-2). [less ▲]

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See detailRational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging
Boutier, Maxime ULiege; Ronsmans, Maygane ULiege; Ouyang, Ping et al

in PLoS Pathogens (2015), 11(2), 1004690

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We ... [more ▼]

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin. [less ▲]

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See detailA conserved protein of cypriniviruses is a major virulence factor of Cyprinid herpesvirus 3
Boutier, Maxime ULiege; Ronsmans, Maygane; Ouyang, Ping et al

Conference (2015)

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 vaccine. In a previous study, we showed that the double deletion of ORF56 and ORF57 is associated with a safe and efficacious profile in vivo. This attenuated vaccine candidate is currently under development. In the present study, we investigated the contribution of each gene to the attenuated phenotype observed. To do so, a series of recombinant single deleted for ORF56 or ORF57 were produced. The deleted sequences were removed in between predicted eukaryotic promoters for neighbored ORFs and replaced by a galactokinase cassette. These recombinants were characterized in vitro for their correct molecular structure. In addition, immunofluorescence staining showed that the deletion of ORF56 did not abrogate the expression of ORF57, and vice versa. These recombinants were further tested in vivo and revealed that the attenuation resulted mostly from the single deletion of ORF57. In conclusion, this study demonstrates the role ORF57 as a major virulence factor of CyHV-3, responsible for most of the attenuation observed in the double deleted ORF56-57 vaccine candidate. Interestingly, ORF57 is conserved in cypriniviruses and could therefore represent a target for attenuated vaccine development in several other major fish pathogens. [less ▲]

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See detailThe Structure of the Cyprinid herpesvirus 3 ORF112-Zalpha.Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response.
Kus, Krzysztof; Rakus, Krzysztof; Boutier, Maxime ULiege et al

in Journal of Biological Chemistry (2015), 290(52), 30713-25

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In ... [more ▼]

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zalpha domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zalpha domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zalpha in complex with an 18-bp CpG DNA repeat, at 1.5 A. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zalpha domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI. [less ▲]

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See detailCyprinid herpesvirus 3 : an archetype of fish alloherpesviruses
Boutier, Maxime ULiege; Ronsmans, Maygane ULiege; Rakus, Krzysztof ULiege et al

in Advances in Virus Research (2015), 93

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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 ULiege; Ronsmans, Maygane; Ouyang, Ping et al

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

Conference (2013)

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See detailCyprinid herpesvirus 3 : an intersting virus for applied and fundamental research
Rakus; Ouyang, Ping; Boutier, Maxime ULiege 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 ULiege; Ronsmans, Maygane ULiege 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 ULiege; Boutier, Maxime ULiege; 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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