The IL-10 homologue encoded by cyprinid herpesvirus 3 is essential neither for viral replication in vitro nor for virulence in vivo

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.

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10

“…It is likely that cyhv3Il10 helps to avoid immune surveillance and elimination by specifically targeting components of the carp immune system. However, in a recent study using infection with Il10-deleted and Il10-revertant recombinant CyHV-3 strains, we could not show that cyhv3Il10 is essential for replication in vitro or for virulence in vivo (10), indicating that the full potential of this viral gene product could not be revealed under the conditions used, thereby leaving the exact immunobiological function of cyhv3Il10 unresolved. Also, a recent study in zebrafish embryos injected with CyHV-3 il10 mRNA showed more lysozyme-positive cells, the effect of which could be abrogated by morpholino-mediated downregulation of il10 receptor-1 (il10r1), providing only indirect evidence for a biological role of cyhv3Il10 (11).…”

“…In the present study, we investigated the biological activities of cyhv3Il10 on carp phagocytes and lymphocytes and show that the lack of a clear phenotype observed in our previous study (10) during infection with recombinant cyhv3Il10-deleted viruses cannot be ascribed to the lack of biological activity of this viral homolog. In fact, we demonstrate that cyhv3Il10 shares the signaling pathway and several prototypical activities with cIl10, its host homolog, including the ability to inhibit proinflammatory responses of macrophages and induce proliferation of Igm+ B cells and memory T cells.…”

“…A recombinant CyHV-3 FL strain deleted for the ORF134 (cyhv3Il10) and the derived revertant strain were produced using bacterial artificial chromosome cloning technologies as previously described (10). For clarity, throughout this study ORF134 is referred to as cyhv3Il10.…”

“…For clarity, throughout this study ORF134 is referred to as cyhv3Il10. To obtain cell culture supernatants enriched for, or depleted of, cyhv3Il10, CCB cells were infected with wild-type, cyhv3Il10-deleted, and cyhv3Il10-revertant recombinant CyHV-3 FL strains, or mock infected as described previously (10).…”

“…ORF134 of CyHV-3 encodes for an Il10-like protein and is the second most abundant of all proteins in the CyHV-3 secretome (10). Furthermore, ORF134, here referred to as cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]), is highly expressed in tissue of infected carp during the acute and reactivation phases of CyHV-3 infection and at lower levels during virus persistence at low temperature (11).…”