Limits...
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.

Show MeSH

Related in: MedlinePlus

Structural analysis of the FL BAC ORF134 recombinant plasmids and derived CyHV-3 recombinant strains. The CyHV-3 FL BAC, FL BAC ORF134 Del and FL BAC ORF134 Rev plasmids and the genome of the FL BAC revertant, FL BAC revertant ORF134 Del and FL BAC revertant ORF134 Rev strains were analyzed by SacI restriction (left panel) and further tested by southern blotting using probes corresponding to ORF55 (central panel) and ORF134 (right panel). White-outlined black arrowheads and white arrows indicate restriction fragments containing ORF55 and ORF134 loci, respectively. Marker sizes (MS) are indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750702&req=5

Figure 4: Structural analysis of the FL BAC ORF134 recombinant plasmids and derived CyHV-3 recombinant strains. The CyHV-3 FL BAC, FL BAC ORF134 Del and FL BAC ORF134 Rev plasmids and the genome of the FL BAC revertant, FL BAC revertant ORF134 Del and FL BAC revertant ORF134 Rev strains were analyzed by SacI restriction (left panel) and further tested by southern blotting using probes corresponding to ORF55 (central panel) and ORF134 (right panel). White-outlined black arrowheads and white arrows indicate restriction fragments containing ORF55 and ORF134 loci, respectively. Marker sizes (MS) are indicated on the left.

Mentions: In order to investigate subsequently the importance of ORF134 in virus replication in vitro and pathogenesis in vivo, a CyHV-3 strain deleted for ORF134 (FL BAC revertant ORF134 Del strain) and a revertant strain (FL BAC revertant ORF134 Rev strain) were produced using BAC cloning and prokaryotic recombination technologies as described in the Materials and methods (Figure 1). The FL BAC plasmid was used as parental plasmid. A wild type strain (FL BAC revertant strain) was also reconstituted from the FL BAC plasmid. The molecular structures of the recombinant strains produced were confirmed by a combined SacI restriction endonuclease and Southern blot approach targeting both ORF55 (the BAC cassette is inserted into the ORF55 locus) and ORF134 loci (Figure 4). In the three reconstituted strains, the ORF55 probe led to a single band corresponding to a 5.2 kb restriction fragment, demonstrating the reversion of ORF55 to wild type sequence (removal of the BAC cassette) [27]. In the FL BAC revertant and the FL BAC revertant ORF134 Rev, the ORF134Del probe led to a single band corresponding to a 6.3 kb restriction fragment consistent with the sequence of this region (6333kb). The absence of signal for the FL BAC revertant ORF134 Del demonstrated the deletion of ORF134. The molecular structure of the recombinants and the absence of contamination between strains was also controlled by PCR (Figure 5) and sequencing of the regions used to target recombination (data not shown). All approaches confirmed that the resulting recombinants have the correct molecular structure. Finally, using a RT-PCR approach, we controlled the process so that the deletion did not markedly affect the transcription of the ORFs located upstream and downstream of ORF134: ORF132, ORF133 and ORF135 (Figures 1a and 6). In these experiments, transcription of ORF55 was used as reference. For the three recombinants tested, transcripts of 602 bp, 264 bp, 238 bp and 293 bp were observed in infected cells for ORF55, ORF132, ORF133 and ORF135, respectively. No transcript was detected in mock-infected cells. When RT was omitted from the reactions, the product seen in infected cells was not detected, indicating that this product did not result from amplification of contaminant viral DNA. The three strains tested led to comparable signals for the four ORFs. Transcription analysis of ORF134 revealed that the FL BAC revertant and the FL BAC revertant ORF134 Rev expressed this ORF comparably, while no signal was detected for the FL BAC revertant ORF134 Del. Together, the results presented above demonstrate that the recombinants produced have the correct molecular structure and that the deletion of ORF134 has no marked polar effect on neighbor genes.


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)

Structural analysis of the FL BAC ORF134 recombinant plasmids and derived CyHV-3 recombinant strains. The CyHV-3 FL BAC, FL BAC ORF134 Del and FL BAC ORF134 Rev plasmids and the genome of the FL BAC revertant, FL BAC revertant ORF134 Del and FL BAC revertant ORF134 Rev strains were analyzed by SacI restriction (left panel) and further tested by southern blotting using probes corresponding to ORF55 (central panel) and ORF134 (right panel). White-outlined black arrowheads and white arrows indicate restriction fragments containing ORF55 and ORF134 loci, respectively. Marker sizes (MS) are indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Structural analysis of the FL BAC ORF134 recombinant plasmids and derived CyHV-3 recombinant strains. The CyHV-3 FL BAC, FL BAC ORF134 Del and FL BAC ORF134 Rev plasmids and the genome of the FL BAC revertant, FL BAC revertant ORF134 Del and FL BAC revertant ORF134 Rev strains were analyzed by SacI restriction (left panel) and further tested by southern blotting using probes corresponding to ORF55 (central panel) and ORF134 (right panel). White-outlined black arrowheads and white arrows indicate restriction fragments containing ORF55 and ORF134 loci, respectively. Marker sizes (MS) are indicated on the left.
Mentions: In order to investigate subsequently the importance of ORF134 in virus replication in vitro and pathogenesis in vivo, a CyHV-3 strain deleted for ORF134 (FL BAC revertant ORF134 Del strain) and a revertant strain (FL BAC revertant ORF134 Rev strain) were produced using BAC cloning and prokaryotic recombination technologies as described in the Materials and methods (Figure 1). The FL BAC plasmid was used as parental plasmid. A wild type strain (FL BAC revertant strain) was also reconstituted from the FL BAC plasmid. The molecular structures of the recombinant strains produced were confirmed by a combined SacI restriction endonuclease and Southern blot approach targeting both ORF55 (the BAC cassette is inserted into the ORF55 locus) and ORF134 loci (Figure 4). In the three reconstituted strains, the ORF55 probe led to a single band corresponding to a 5.2 kb restriction fragment, demonstrating the reversion of ORF55 to wild type sequence (removal of the BAC cassette) [27]. In the FL BAC revertant and the FL BAC revertant ORF134 Rev, the ORF134Del probe led to a single band corresponding to a 6.3 kb restriction fragment consistent with the sequence of this region (6333kb). The absence of signal for the FL BAC revertant ORF134 Del demonstrated the deletion of ORF134. The molecular structure of the recombinants and the absence of contamination between strains was also controlled by PCR (Figure 5) and sequencing of the regions used to target recombination (data not shown). All approaches confirmed that the resulting recombinants have the correct molecular structure. Finally, using a RT-PCR approach, we controlled the process so that the deletion did not markedly affect the transcription of the ORFs located upstream and downstream of ORF134: ORF132, ORF133 and ORF135 (Figures 1a and 6). In these experiments, transcription of ORF55 was used as reference. For the three recombinants tested, transcripts of 602 bp, 264 bp, 238 bp and 293 bp were observed in infected cells for ORF55, ORF132, ORF133 and ORF135, respectively. No transcript was detected in mock-infected cells. When RT was omitted from the reactions, the product seen in infected cells was not detected, indicating that this product did not result from amplification of contaminant viral DNA. The three strains tested led to comparable signals for the four ORFs. Transcription analysis of ORF134 revealed that the FL BAC revertant and the FL BAC revertant ORF134 Rev expressed this ORF comparably, while no signal was detected for the FL BAC revertant ORF134 Del. Together, the results presented above demonstrate that the recombinants produced have the correct molecular structure and that the deletion of ORF134 has no marked polar effect on neighbor genes.

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