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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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Identification of CyHV-3 ORF134 by 2D-LC MS/MS in the supernatant of CyHV-3 infected CCB cells. (A) Data collected on ORF134 through 2D-LC MS/MS analysis of cell culture supernatant. (B) Sequential alignment of CyHV-3 ORF134 and Cyprinus carpio IL-10. Sequence coverage: detected peptides are presented in rectangles.
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Figure 3: Identification of CyHV-3 ORF134 by 2D-LC MS/MS in the supernatant of CyHV-3 infected CCB cells. (A) Data collected on ORF134 through 2D-LC MS/MS analysis of cell culture supernatant. (B) Sequential alignment of CyHV-3 ORF134 and Cyprinus carpio IL-10. Sequence coverage: detected peptides are presented in rectangles.

Mentions: While two independent studies have previously shown that ORF134 is transcribed during viral replication [26,29], it is still to be determined whether ORF134 encodes a protein secreted from infected cells. To address this question, concentrated supernatant was produced from CyHV-3 infected CCB cultures and analyzed by 2D-LC MS/MS. Viral and cellular proteins identified by this approach are listed in Table 2. This list was restricted to proteins identified with p value lower than 0.05 as determined by the MASCOT program. Five viral and 46 cellular proteins were detected. CyHV-3 ORF12 and ORF134 were amongst the most abundant proteins in the sample as revealed by their relatively high emPAI scores (1.49 and 1.02, respectively). Only two cellular proteins had comparable scores (Beta-2-microglobulin and FK506 binding protein 1A, with emPAI scores of 1.79 and 1.39, respectively). ORF12 encodes a soluble TNF receptor superfamily homologue which, like ORF134, was expected to be secreted from infected cells. Three unique peptides covering 16% of the ORF134 sequence were sequenced (Figure 3a). These peptides were distributed throughout ORF134 sequence (Figure 3b). The divergence existing between CyHV-3 IL-10 and carp IL-10 excludes the hypothesis that the peptides detected could be derived from carp IL-10 rather than from CyHV-3 ORF134. In addition to CyHV-3 ORF12 and ORF134, three additional viral proteins (ORF52, ORF116 and ORF119) were detected in the CyHV-3 secretome. All three proteins are potential membrane proteins (Table 2). The presence of these putative membrane proteins in the CyHV-3 secretome cannot be explained by remaining viral particles in the prepared concentrated extracellular medium, as none of these proteins is structural [31]. It is also unlikely that the presence of these proteins reflects cell lysis resulting from the viral infection. Indeed, in such a case, a higher number of viral proteins would be expected, in particular the most abundant ones [31]. Several viral proteins are expressed as two different forms, a membrane-anchored form and a secreted form, the latter generated by proteolytic cleavage of the former [42,43]. Further experiments are required to determine whether this phenomenon applies to the putative CyHV-3 membrane proteins detected in the secretome.


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)

Identification of CyHV-3 ORF134 by 2D-LC MS/MS in the supernatant of CyHV-3 infected CCB cells. (A) Data collected on ORF134 through 2D-LC MS/MS analysis of cell culture supernatant. (B) Sequential alignment of CyHV-3 ORF134 and Cyprinus carpio IL-10. Sequence coverage: detected peptides are presented in rectangles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Identification of CyHV-3 ORF134 by 2D-LC MS/MS in the supernatant of CyHV-3 infected CCB cells. (A) Data collected on ORF134 through 2D-LC MS/MS analysis of cell culture supernatant. (B) Sequential alignment of CyHV-3 ORF134 and Cyprinus carpio IL-10. Sequence coverage: detected peptides are presented in rectangles.
Mentions: While two independent studies have previously shown that ORF134 is transcribed during viral replication [26,29], it is still to be determined whether ORF134 encodes a protein secreted from infected cells. To address this question, concentrated supernatant was produced from CyHV-3 infected CCB cultures and analyzed by 2D-LC MS/MS. Viral and cellular proteins identified by this approach are listed in Table 2. This list was restricted to proteins identified with p value lower than 0.05 as determined by the MASCOT program. Five viral and 46 cellular proteins were detected. CyHV-3 ORF12 and ORF134 were amongst the most abundant proteins in the sample as revealed by their relatively high emPAI scores (1.49 and 1.02, respectively). Only two cellular proteins had comparable scores (Beta-2-microglobulin and FK506 binding protein 1A, with emPAI scores of 1.79 and 1.39, respectively). ORF12 encodes a soluble TNF receptor superfamily homologue which, like ORF134, was expected to be secreted from infected cells. Three unique peptides covering 16% of the ORF134 sequence were sequenced (Figure 3a). These peptides were distributed throughout ORF134 sequence (Figure 3b). The divergence existing between CyHV-3 IL-10 and carp IL-10 excludes the hypothesis that the peptides detected could be derived from carp IL-10 rather than from CyHV-3 ORF134. In addition to CyHV-3 ORF12 and ORF134, three additional viral proteins (ORF52, ORF116 and ORF119) were detected in the CyHV-3 secretome. All three proteins are potential membrane proteins (Table 2). The presence of these putative membrane proteins in the CyHV-3 secretome cannot be explained by remaining viral particles in the prepared concentrated extracellular medium, as none of these proteins is structural [31]. It is also unlikely that the presence of these proteins reflects cell lysis resulting from the viral infection. Indeed, in such a case, a higher number of viral proteins would be expected, in particular the most abundant ones [31]. Several viral proteins are expressed as two different forms, a membrane-anchored form and a secreted form, the latter generated by proteolytic cleavage of the former [42,43]. Further experiments are required to determine whether this phenomenon applies to the putative CyHV-3 membrane proteins detected in the secretome.

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