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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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PCR detection and characterization of CyHV-3 genomes recovered from infected carp. The analyses reported in this figure are the follow-up of the experiment described in Figure 8. Three mock-infected carp (selected randomly before the challenge) and three dead carp from each of the groups infected with the FL BAC Revertant, FL BAC Revertant ORF134 Del and FL BAC Revertant ORF134 Rev strains were dissected. DNA was extracted from the kidney. PCRs were performed with the ORF55InF/ORF55stopR and ORF134InF/ORF134InR pairs of primers (Table 1). FL strain DNA and distilled water were used as positive (Ctrl+) and negative (Ctrl-) controls, respectively.
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Figure 9: PCR detection and characterization of CyHV-3 genomes recovered from infected carp. The analyses reported in this figure are the follow-up of the experiment described in Figure 8. Three mock-infected carp (selected randomly before the challenge) and three dead carp from each of the groups infected with the FL BAC Revertant, FL BAC Revertant ORF134 Del and FL BAC Revertant ORF134 Rev strains were dissected. DNA was extracted from the kidney. PCRs were performed with the ORF55InF/ORF55stopR and ORF134InF/ORF134InR pairs of primers (Table 1). FL strain DNA and distilled water were used as positive (Ctrl+) and negative (Ctrl-) controls, respectively.

Mentions: To investigate the importance of ORF134 in the pathogenesis of 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 8). The three strains induced at comparable levels all the clinical signs associated with the disease, including apathy, folding of the dorsal fin, hyperemia, increased mucus secretions, skin lesions, suffocation, erratic swimming, and the loss of equilibrium. The mortality rate and the kinetics of mortality observed for the three strains were not significantly different. At necropsy, similar lesions were observed for the three strains including the discoloration of gill filaments, herpetic skin lesions, and necrotic nephritis. To control that the infection of all groups of fish was performed with the correct viral strain and to exclude any possibility of wild type virus spread among tanks, PCR assays were performed on three randomly selected dead fish from each infected group and three mock-infected fish randomly selected (Figure 9). The PCR results confirmed that each tank was infected with the correct strain and demonstrated the absence of viral spread between tanks. Next, to determine whether the ORF134 deletion affects the adaptive immune response developed by fish that survived primary infection; surviving fish were challenged by co-habitation with fish inoculated with the wild type FL strain (Figure 8). Independently of the viral strain used for the primary infection, none of the challenged fish developed CyHV-3 disease (Figure 8). In contrast, CyHV-3 disease developed in the two tanks that were initially mock-infected. Taken together, the results presented above suggest that ORF134 deletion does not affect CyHV-3 pathogenicity in common carp and the protective immune response developed by surviving fish.


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)

PCR detection and characterization of CyHV-3 genomes recovered from infected carp. The analyses reported in this figure are the follow-up of the experiment described in Figure 8. Three mock-infected carp (selected randomly before the challenge) and three dead carp from each of the groups infected with the FL BAC Revertant, FL BAC Revertant ORF134 Del and FL BAC Revertant ORF134 Rev strains were dissected. DNA was extracted from the kidney. PCRs were performed with the ORF55InF/ORF55stopR and ORF134InF/ORF134InR pairs of primers (Table 1). FL strain DNA and distilled water were used as positive (Ctrl+) and negative (Ctrl-) controls, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: PCR detection and characterization of CyHV-3 genomes recovered from infected carp. The analyses reported in this figure are the follow-up of the experiment described in Figure 8. Three mock-infected carp (selected randomly before the challenge) and three dead carp from each of the groups infected with the FL BAC Revertant, FL BAC Revertant ORF134 Del and FL BAC Revertant ORF134 Rev strains were dissected. DNA was extracted from the kidney. PCRs were performed with the ORF55InF/ORF55stopR and ORF134InF/ORF134InR pairs of primers (Table 1). FL strain DNA and distilled water were used as positive (Ctrl+) and negative (Ctrl-) controls, respectively.
Mentions: To investigate the importance of ORF134 in the pathogenesis of 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 8). The three strains induced at comparable levels all the clinical signs associated with the disease, including apathy, folding of the dorsal fin, hyperemia, increased mucus secretions, skin lesions, suffocation, erratic swimming, and the loss of equilibrium. The mortality rate and the kinetics of mortality observed for the three strains were not significantly different. At necropsy, similar lesions were observed for the three strains including the discoloration of gill filaments, herpetic skin lesions, and necrotic nephritis. To control that the infection of all groups of fish was performed with the correct viral strain and to exclude any possibility of wild type virus spread among tanks, PCR assays were performed on three randomly selected dead fish from each infected group and three mock-infected fish randomly selected (Figure 9). The PCR results confirmed that each tank was infected with the correct strain and demonstrated the absence of viral spread between tanks. Next, to determine whether the ORF134 deletion affects the adaptive immune response developed by fish that survived primary infection; surviving fish were challenged by co-habitation with fish inoculated with the wild type FL strain (Figure 8). Independently of the viral strain used for the primary infection, none of the challenged fish developed CyHV-3 disease (Figure 8). In contrast, CyHV-3 disease developed in the two tanks that were initially mock-infected. Taken together, the results presented above suggest that ORF134 deletion does not affect CyHV-3 pathogenicity in common carp and the protective immune response developed by surviving fish.

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