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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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Schematic representation of the strategy used to produce CyHV-3 FL BAC ORF134 recombinants. (A) The region of CyHV-3 genome encoding ORF134 is illustrated for wild type (WT), ORF134 Del galK and ORF134 Del genotypes. (B) Flow chart of stages performed to produce FL BAC ORF134 recombinant plasmids and to reconstitute virus strains.
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Figure 1: Schematic representation of the strategy used to produce CyHV-3 FL BAC ORF134 recombinants. (A) The region of CyHV-3 genome encoding ORF134 is illustrated for wild type (WT), ORF134 Del galK and ORF134 Del genotypes. (B) Flow chart of stages performed to produce FL BAC ORF134 recombinant plasmids and to reconstitute virus strains.

Mentions: CyHV-3 recombinants were produced using prokaryotic recombination technologies (Figure 1). The FL BAC plasmid was used as parental plasmid [27]. In this plasmid, the BAC cassette is inserted in ORF55 encoding thymidine kinase (TK). ORF134 recombinant plasmids were produced using two-steps galactokinase gene (galK) positive/negative selection in bacteria as described previously [34]. The first recombination process (galK positive selection) consisted to replace ORF134 by galK resulting in the FL BAC ORF134 Del galK plasmid. Recombination was achieved using the H1-galK-H2 recombination cassette (Figure 1b) which consisted of the galK gene flanked by 50-bp sequences homologous to CyHV-3 genome regions flanking ORF134 deletion (Figure 1a). H1-galK-H2 recombination cassette was produced by PCR (primers 134 galK F and 134 galK R) using the pgalK vector as template. Primer 134 galK F consisted of nucleotides 229836–229885 (50bp) of CyHV-3 genome and 1–24 (24bp) of the pgalK vector. Primer 134 galK R consisted of nucleotides 229262–229311 (50bp) of the CyHV-3 genome and nucleotides 1212–1231 (20bp) of the pgalK vector (Table 1). The 50-bp sequences of the H1-galK-H2 corresponding to CyHV-3 genome were used to target homologous recombination in bacteria. The second recombination process (galK negative selection) consisted to remove the galK gene (FL BAC ORF134 Del plasmid) or to replace the galK gene by CyHV-3 wild type ORF134 sequence (FL BAC ORF134 Rev plasmid) (Figure 1). The FL BAC ORF134 Del plasmid was obtained by recombination with the H1-H2 cassette (Figure 1b). This cassette was synthesized and consisted of 200 bp of CyHV-3 genome upstream and downstream of ORF134 deletion, respectively. The FL BAC ORF134 Rev plasmid was produced by recombination with the H1-ORF134-H2 cassette. This cassette was produced by PCR (primers H1F and H2R) using CyHV-3 FL DNA as template corresponding to nucleotides 229057–229076 and nucleotides 230056–230075 of CyHV-3 genome, respectively. To reconstitute infectious virus encoding a wild type TK locus (removal of the BAC cassette), the BAC plasmids (FL BAC, FL BAC ORF134 Del and FL BAC ORF134 Rev) were co-transfected with the pGEMT-TK plasmid (molecular ratio, 1:75) into CCB cells [27]. Plaque negative for enhanced green fluorescent protein (EGFP) expression (the BAC cassette encodes an EGFP expression cassette) were picked and amplified.


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)

Schematic representation of the strategy used to produce CyHV-3 FL BAC ORF134 recombinants. (A) The region of CyHV-3 genome encoding ORF134 is illustrated for wild type (WT), ORF134 Del galK and ORF134 Del genotypes. (B) Flow chart of stages performed to produce FL BAC ORF134 recombinant plasmids and to reconstitute virus strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Schematic representation of the strategy used to produce CyHV-3 FL BAC ORF134 recombinants. (A) The region of CyHV-3 genome encoding ORF134 is illustrated for wild type (WT), ORF134 Del galK and ORF134 Del genotypes. (B) Flow chart of stages performed to produce FL BAC ORF134 recombinant plasmids and to reconstitute virus strains.
Mentions: CyHV-3 recombinants were produced using prokaryotic recombination technologies (Figure 1). The FL BAC plasmid was used as parental plasmid [27]. In this plasmid, the BAC cassette is inserted in ORF55 encoding thymidine kinase (TK). ORF134 recombinant plasmids were produced using two-steps galactokinase gene (galK) positive/negative selection in bacteria as described previously [34]. The first recombination process (galK positive selection) consisted to replace ORF134 by galK resulting in the FL BAC ORF134 Del galK plasmid. Recombination was achieved using the H1-galK-H2 recombination cassette (Figure 1b) which consisted of the galK gene flanked by 50-bp sequences homologous to CyHV-3 genome regions flanking ORF134 deletion (Figure 1a). H1-galK-H2 recombination cassette was produced by PCR (primers 134 galK F and 134 galK R) using the pgalK vector as template. Primer 134 galK F consisted of nucleotides 229836–229885 (50bp) of CyHV-3 genome and 1–24 (24bp) of the pgalK vector. Primer 134 galK R consisted of nucleotides 229262–229311 (50bp) of the CyHV-3 genome and nucleotides 1212–1231 (20bp) of the pgalK vector (Table 1). The 50-bp sequences of the H1-galK-H2 corresponding to CyHV-3 genome were used to target homologous recombination in bacteria. The second recombination process (galK negative selection) consisted to remove the galK gene (FL BAC ORF134 Del plasmid) or to replace the galK gene by CyHV-3 wild type ORF134 sequence (FL BAC ORF134 Rev plasmid) (Figure 1). The FL BAC ORF134 Del plasmid was obtained by recombination with the H1-H2 cassette (Figure 1b). This cassette was synthesized and consisted of 200 bp of CyHV-3 genome upstream and downstream of ORF134 deletion, respectively. The FL BAC ORF134 Rev plasmid was produced by recombination with the H1-ORF134-H2 cassette. This cassette was produced by PCR (primers H1F and H2R) using CyHV-3 FL DNA as template corresponding to nucleotides 229057–229076 and nucleotides 230056–230075 of CyHV-3 genome, respectively. To reconstitute infectious virus encoding a wild type TK locus (removal of the BAC cassette), the BAC plasmids (FL BAC, FL BAC ORF134 Del and FL BAC ORF134 Rev) were co-transfected with the pGEMT-TK plasmid (molecular ratio, 1:75) into CCB cells [27]. Plaque negative for enhanced green fluorescent protein (EGFP) expression (the BAC cassette encodes an EGFP expression cassette) were picked and amplified.

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