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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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Determination of ORF134 kinetic class of transcription. CCB cells were infected with CyHV-3 FL strain, in absence (Untreated) or presence of CHX or PAA as described in Materials and methods. At the indicated time post-infection, expression of IE ORF3, E ORF55, L ORF78, ORF134 and carp β-actin was studied by a RT-PCR approach. On the left RT-PCR or PCR represent PCR products generated when the RT was performed or omitted from the reactions, respectively. On the right, control PCR reactions were performed using genomic DNA as template (Ctrl+) or no template (Ctrl-).
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Figure 2: Determination of ORF134 kinetic class of transcription. CCB cells were infected with CyHV-3 FL strain, in absence (Untreated) or presence of CHX or PAA as described in Materials and methods. At the indicated time post-infection, expression of IE ORF3, E ORF55, L ORF78, ORF134 and carp β-actin was studied by a RT-PCR approach. On the left RT-PCR or PCR represent PCR products generated when the RT was performed or omitted from the reactions, respectively. On the right, control PCR reactions were performed using genomic DNA as template (Ctrl+) or no template (Ctrl-).

Mentions: Two independent studies have demonstrated that CyHV-3 ORF134 is transcribed during viral replication in vitro thereby meeting the criteria for being a gene [26,29]. It has been predicted to contain an 84 bp intron flanked by 2 exons encoding a 179 amino acid product (GenBank accession number DQ657948). Here, we used CHX and PAA to identify the transcriptional class of ORF134 (Figure 2). This experiment revealed that ORF134 expression is prevented by CHX and reduced but not prevented by PAA treatments, suggesting that ORF134 is an E-L gene. ORF3, ORF55 and ORF78 were used as controls in this experiment; the results presented in Figure 2 confirmed that they are IE, E and L genes, respectively. The absence of contaminant viral DNA in the mRNA preparations was confirmed by the absence of a PCR product when the reverse transcriptase was omitted from the reactions. Furthermore, the estimated molecular size of the major ORF134 RT-PCR product revealed that it was derived, from the amplification of cDNA (540 bp) rather than from the viral genome (624 bp). This observation is consistent with the earlier description of the ORF134 as a spliced gene [40,41]. However, a minor product corresponding to the unspliced transcript of ORF134 was also observed (see the faint 624 bp band in Figure 2). The classification of ORF134 as an E-L gene is consistent with the results published recently by Ilouze et al. who concluded that ORF134 is an E gene [29]. It is also consistent with the E expression reported for other vIL-10s [40,41].


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)

Determination of ORF134 kinetic class of transcription. CCB cells were infected with CyHV-3 FL strain, in absence (Untreated) or presence of CHX or PAA as described in Materials and methods. At the indicated time post-infection, expression of IE ORF3, E ORF55, L ORF78, ORF134 and carp β-actin was studied by a RT-PCR approach. On the left RT-PCR or PCR represent PCR products generated when the RT was performed or omitted from the reactions, respectively. On the right, control PCR reactions were performed using genomic DNA as template (Ctrl+) or no template (Ctrl-).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Determination of ORF134 kinetic class of transcription. CCB cells were infected with CyHV-3 FL strain, in absence (Untreated) or presence of CHX or PAA as described in Materials and methods. At the indicated time post-infection, expression of IE ORF3, E ORF55, L ORF78, ORF134 and carp β-actin was studied by a RT-PCR approach. On the left RT-PCR or PCR represent PCR products generated when the RT was performed or omitted from the reactions, respectively. On the right, control PCR reactions were performed using genomic DNA as template (Ctrl+) or no template (Ctrl-).
Mentions: Two independent studies have demonstrated that CyHV-3 ORF134 is transcribed during viral replication in vitro thereby meeting the criteria for being a gene [26,29]. It has been predicted to contain an 84 bp intron flanked by 2 exons encoding a 179 amino acid product (GenBank accession number DQ657948). Here, we used CHX and PAA to identify the transcriptional class of ORF134 (Figure 2). This experiment revealed that ORF134 expression is prevented by CHX and reduced but not prevented by PAA treatments, suggesting that ORF134 is an E-L gene. ORF3, ORF55 and ORF78 were used as controls in this experiment; the results presented in Figure 2 confirmed that they are IE, E and L genes, respectively. The absence of contaminant viral DNA in the mRNA preparations was confirmed by the absence of a PCR product when the reverse transcriptase was omitted from the reactions. Furthermore, the estimated molecular size of the major ORF134 RT-PCR product revealed that it was derived, from the amplification of cDNA (540 bp) rather than from the viral genome (624 bp). This observation is consistent with the earlier description of the ORF134 as a spliced gene [40,41]. However, a minor product corresponding to the unspliced transcript of ORF134 was also observed (see the faint 624 bp band in Figure 2). The classification of ORF134 as an E-L gene is consistent with the results published recently by Ilouze et al. who concluded that ORF134 is an E gene [29]. It is also consistent with the E expression reported for other vIL-10s [40,41].

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