Study of the Roles of Cyprinid Herpesvirus 3 ORF134 in the Biology of the Infection

Abstract

The common carp is one of the most important freshwater fish species in aquaculture, and its colourful subspecies koi is grown for personal pleasure and competitive exhibitions. Both two subspecies are economically important. In the late 1990s, a highly contagious and lethal pathogen called koi herpesvirus (KHV) or cyprinid herpesvirus 3 (CyHV-3) began to cause severe financial losses in these two carp industries worldwide. In 2005, CyHV-3 has been classified in the Alloherpesviridae family of the order Herpesvirales. Because of its economic importance and its numerous original biological properties, CyHV 3 became rapidly an attractive subject for applied and fundamental research. However, to date, there is a little information on the roles of individual CyHV-3 genes in the biology of CyHV-3 infection or its pathogenesis. Moreover, there is a lack of safe and efficacious vaccine for the control of CyHV-3 disease. The goal of this thesis was to study the roles of CyHV-3 ORF134 encoding an IL-10 homologue in the biology of the infection.

CyHV-3 ORF134 has been predicted to contain an 84 bp intron flanked by 2 exons encoding together a 179 amino acid product. Transcriptomic analyses reveal that ORF134 is expressed as a spliced early-late gene. The identification of the CyHV-3 secretome was achieved using 2D-LC MS/MS proteomic approach. This method led to the identification of 5 viral and 46 cellular proteins in concentrated infected cell culture supernatant. CyHV-3 ORF12 and ORF134 were amongst the most abundant proteins detected. To investigate the roles of ORF134 in the biological of the infection, a strain deleted for ORF134 and a derived revertant strain were produced by using BAC cloning and prokaryotic recombination technologies. Comparison of these strains demonstrated that CyHV-3 ORF134 does not contribute significantly to viral growth in vitro or to virulence in vivo in the present laboratory setting. The present study addressed for the first time the in vivo role of a vIL-10 encoded by a member of the family Alloherpesviridae. This study has been published in VeterinaryResearch.

During the course of the first study, we obtained an unexpected recombination event while we were reconstituting infectious virus from mutated BAC plasmids. To generate a revertant ORF134 Del galK strain, CCB cells were co-transfected with the FL BAC ORF134 Del galK plasmid and the pGEMT-TK vector to remove the BAC cassette inserted in the ORF55 locus (encoding thymidine kinase). One of the clones obtained had an unexpected recombination leading to the deletion of ORF56 and ORF57 in addition to the expected deletion of ORF134. Unexpectedly, this triple deleted strain replicated efficiently in vitro, exhibited an attenuated phenotype in vivo and was proved to confer in a dose dependent manner an immune protection against a lethal challenge. The goal of the second experimental chapter was to investigate the role of the ORF56-57 and ORF134 deletions in the observed safety/efficacy profile of the triple deleted recombinant. To reach this goal, a collection of recombinant strains were produced using BAC cloning technologies, characterized and tested in vivofor their safety/efficacy profile. The results obtained demonstrated that the ORF56-57 deletion is responsible for the phenotype observed and that ORF134 deletion does not contribute to this phenotype significantly.