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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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Histopathological characterization of the lesions induced by CyHV-3 ORF134 recombinants in the kidney. The fish infection methods are described in the legend of Figure 12.
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Figure 13: Histopathological characterization of the lesions induced by CyHV-3 ORF134 recombinants in the kidney. The fish infection methods are described in the legend of Figure 12.

Mentions: Finally, to investigate further the effect of ORF134 in CyHV-3 pathogenesis, the lesions induced by the FL BAC revertant, FL BAC revertant ORF134 Del and FL BAC revertant ORF134 Rev strains were compared in the gills and the kidney at various time points after infection (Figures 12 and 13). Histopathological preparations were grouped according to the virus genotype used for the infection and the time point of sampling. The groups of slides were observed by two independent examiners using a double-blind test. The principal histopathological changes were observed in gill filaments. Gills from mock-infected fish exhibited a normal structure. However, a weak lymphocytic hyperplasia was observed for the three mock-infected fish at the basis of the secondary lamellae, leading to their fusion. Few eosinophilic granulocytes were also observed along the primary lamella. As early as 2 days post-infection, both examiners were able to discriminate the three groups of infected fish from the mock-infected group. For all three infected groups, we observed congestion of the secondary lamellae, infiltration of lymphocytes and histiocytes at the basis of secondary lamellae further increasing their fusion. With the exception of one fish from the FL BAC revertant ORF134 Del group that exhibited weaker histopathological changes (see Figure 12, Day 2), the two other fish from this group expressed changes comparable to those observed in the two other infected groups. The absence of differences between the three viral groups was confirmed at the latter time points. At day 4 post-infection, all fish expressed comparable increased lymphocytic and histocytic infiltrate at the basis of the secondary lamellae. In some fish, an increase of eosinophilic granulocytes was observed (FL BAC revertant: 2 out of 3 fish; FL BAC revertant ORF134 Del: 2 out of 3 fish and FL BAC revertant ORF134 Rev strains 1 out of 3 fish). In comparison to day 2 post-infection, the infiltrate was more pronounced and the congestion was associated with edema of the secondary lamellae. The intensity of the lesions increased comparably in all three groups at latter time-points (Day 6 and Day 8). The infiltrate mainly lymphocytic induced the fusion of the lamellae on approximately 2/3 of their length. The respiratory epithelium exhibited hyperplasia and necrosis, associated in few cells with intranuclear inclusion bodies. Compared to day 6 post-infection, the infiltrate observed on day 8 was slightly reduced while the edema and the necrosis were increased. The lesions induced by the three recombinant strains were also compared in the kidney (Figure 13). The lesions observed in this organ were less obvious than in the gills. On day 2 post-infection, infected groups could not be differentiated from the mock-infected one. The diversity and the abundance of hematopoietic cells were normal. However, a slight increase of eosinophilic cells was observed in nearly all groups. Vacuolization of the epithelium was observed in all preparations, and was considered to be a preparation artifact. Starting on day 4 post-infection, both examiners were able to discriminate the three infected groups from the mock-infected one. However, they could not differentiate the three infected groups. Comparable proliferation of the hematopoietic cells, mainly lymphocytic and eosinophilic, was observed in all infected groups. The proliferation increased further on day 6 and 8. Intranuclear inclusion bodies were observed in a few hematopoietic cells on days 6 and 8, and in few epithelial cells on day 8. The absence of cross-contamination between tanks was controlled using the approach described in Figure 9 (data not shown).


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)

Histopathological characterization of the lesions induced by CyHV-3 ORF134 recombinants in the kidney. The fish infection methods are described in the legend of Figure 12.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 13: Histopathological characterization of the lesions induced by CyHV-3 ORF134 recombinants in the kidney. The fish infection methods are described in the legend of Figure 12.
Mentions: Finally, to investigate further the effect of ORF134 in CyHV-3 pathogenesis, the lesions induced by the FL BAC revertant, FL BAC revertant ORF134 Del and FL BAC revertant ORF134 Rev strains were compared in the gills and the kidney at various time points after infection (Figures 12 and 13). Histopathological preparations were grouped according to the virus genotype used for the infection and the time point of sampling. The groups of slides were observed by two independent examiners using a double-blind test. The principal histopathological changes were observed in gill filaments. Gills from mock-infected fish exhibited a normal structure. However, a weak lymphocytic hyperplasia was observed for the three mock-infected fish at the basis of the secondary lamellae, leading to their fusion. Few eosinophilic granulocytes were also observed along the primary lamella. As early as 2 days post-infection, both examiners were able to discriminate the three groups of infected fish from the mock-infected group. For all three infected groups, we observed congestion of the secondary lamellae, infiltration of lymphocytes and histiocytes at the basis of secondary lamellae further increasing their fusion. With the exception of one fish from the FL BAC revertant ORF134 Del group that exhibited weaker histopathological changes (see Figure 12, Day 2), the two other fish from this group expressed changes comparable to those observed in the two other infected groups. The absence of differences between the three viral groups was confirmed at the latter time points. At day 4 post-infection, all fish expressed comparable increased lymphocytic and histocytic infiltrate at the basis of the secondary lamellae. In some fish, an increase of eosinophilic granulocytes was observed (FL BAC revertant: 2 out of 3 fish; FL BAC revertant ORF134 Del: 2 out of 3 fish and FL BAC revertant ORF134 Rev strains 1 out of 3 fish). In comparison to day 2 post-infection, the infiltrate was more pronounced and the congestion was associated with edema of the secondary lamellae. The intensity of the lesions increased comparably in all three groups at latter time-points (Day 6 and Day 8). The infiltrate mainly lymphocytic induced the fusion of the lamellae on approximately 2/3 of their length. The respiratory epithelium exhibited hyperplasia and necrosis, associated in few cells with intranuclear inclusion bodies. Compared to day 6 post-infection, the infiltrate observed on day 8 was slightly reduced while the edema and the necrosis were increased. The lesions induced by the three recombinant strains were also compared in the kidney (Figure 13). The lesions observed in this organ were less obvious than in the gills. On day 2 post-infection, infected groups could not be differentiated from the mock-infected one. The diversity and the abundance of hematopoietic cells were normal. However, a slight increase of eosinophilic cells was observed in nearly all groups. Vacuolization of the epithelium was observed in all preparations, and was considered to be a preparation artifact. Starting on day 4 post-infection, both examiners were able to discriminate the three infected groups from the mock-infected one. However, they could not differentiate the three infected groups. Comparable proliferation of the hematopoietic cells, mainly lymphocytic and eosinophilic, was observed in all infected groups. The proliferation increased further on day 6 and 8. Intranuclear inclusion bodies were observed in a few hematopoietic cells on days 6 and 8, and in few epithelial cells on day 8. The absence of cross-contamination between tanks was controlled using the approach described in Figure 9 (data not shown).

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