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The IL-10 homologue encoded by cyprinid herpesvirus 3 is essential neither for viral replication in vitro nor for virulence in vivo.

Ouyang P, Rakus K, Boutier M, Reschner A, Leroy B, Ronsmans M, Fournier G, Scohy S, Costes B, Wattiez R, Vanderplasschen A - Vet. Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology-Vaccinology (B43b), Department of Infectious and Parasitic Diseases (B43b), Faculty of Veterinary Medicine, University of Liège, Liège, B-4000, Belgium. a.vdplasschen@ulg.ac.be.

ABSTRACT
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.

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RT-qPCR analysis of cytokines. Kinetics of gene expression was measured in the spleen of mock-infected fish and fish infected with CyHV-3 ORF134 recombinants (see legend of Figure 10). Gene expression was normalized relative to the expression of the S11 protein of the 40S ribosomal subunit. Data are presented as mean ± SD (n = 6). Symbol (*) indicates statistical differences (p ≤ 0.05) observed between infected and mock-infected fish. Symbol (a) indicates statistical differences (p ≤ 0.05) observed for a specific time point between groups of fish infected with different CyHV-3 recombinants.
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Figure 11: RT-qPCR analysis of cytokines. Kinetics of gene expression was measured in the spleen of mock-infected fish and fish infected with CyHV-3 ORF134 recombinants (see legend of Figure 10). Gene expression was normalized relative to the expression of the S11 protein of the 40S ribosomal subunit. Data are presented as mean ± SD (n = 6). Symbol (*) indicates statistical differences (p ≤ 0.05) observed between infected and mock-infected fish. Symbol (a) indicates statistical differences (p ≤ 0.05) observed for a specific time point between groups of fish infected with different CyHV-3 recombinants.

Mentions: To further test these hypotheses, we investigated the effect of ORF134 deletion on viral load (Figure 10) and on cytokine expression (Figure 11) during CyHV-3 disease. Naïve common carp were inoculated by immersion in water containing the FL BAC revertant, FL BAC revertant ORF134 Del or FL BAC revertant ORF134 Rev strains (Figure 10). At different times after inoculation gill, kidney and spleen were collected from randomly selected fish. Viral loads were analyzed in gill and kidney by real-time TaqMan PCR (Figure 10) while cytokine expression was studied in spleen by RT-qPCR (Figure 11).


The IL-10 homologue encoded by cyprinid herpesvirus 3 is essential neither for viral replication in vitro nor for virulence in vivo.

Ouyang P, Rakus K, Boutier M, Reschner A, Leroy B, Ronsmans M, Fournier G, Scohy S, Costes B, Wattiez R, Vanderplasschen A - Vet. Res. (2013)

RT-qPCR analysis of cytokines. Kinetics of gene expression was measured in the spleen of mock-infected fish and fish infected with CyHV-3 ORF134 recombinants (see legend of Figure 10). Gene expression was normalized relative to the expression of the S11 protein of the 40S ribosomal subunit. Data are presented as mean ± SD (n = 6). Symbol (*) indicates statistical differences (p ≤ 0.05) observed between infected and mock-infected fish. Symbol (a) indicates statistical differences (p ≤ 0.05) observed for a specific time point between groups of fish infected with different CyHV-3 recombinants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750702&req=5

Figure 11: RT-qPCR analysis of cytokines. Kinetics of gene expression was measured in the spleen of mock-infected fish and fish infected with CyHV-3 ORF134 recombinants (see legend of Figure 10). Gene expression was normalized relative to the expression of the S11 protein of the 40S ribosomal subunit. Data are presented as mean ± SD (n = 6). Symbol (*) indicates statistical differences (p ≤ 0.05) observed between infected and mock-infected fish. Symbol (a) indicates statistical differences (p ≤ 0.05) observed for a specific time point between groups of fish infected with different CyHV-3 recombinants.
Mentions: To further test these hypotheses, we investigated the effect of ORF134 deletion on viral load (Figure 10) and on cytokine expression (Figure 11) during CyHV-3 disease. Naïve common carp were inoculated by immersion in water containing the FL BAC revertant, FL BAC revertant ORF134 Del or FL BAC revertant ORF134 Rev strains (Figure 10). At different times after inoculation gill, kidney and spleen were collected from randomly selected fish. Viral loads were analyzed in gill and kidney by real-time TaqMan PCR (Figure 10) while cytokine expression was studied in spleen by RT-qPCR (Figure 11).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology-Vaccinology (B43b), Department of Infectious and Parasitic Diseases (B43b), Faculty of Veterinary Medicine, University of Liège, Liège, B-4000, Belgium. a.vdplasschen@ulg.ac.be.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus