Bérénice Costes

The Major Portal of Entry of Koi Herpesvirus in Cyprinus carpio Is the Skin

ABSTRACT Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed “U-tube,” to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions.

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the aetiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome and a tegument. This study identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry-based proteomic approaches. In addition, exponentially modified protein abundance index analyses were used to estimate the relative abundance of the identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins, which were classified based on bioinformatic analyses as capsid (three), envelope (13), tegument (two) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated into CyHV-3 virions and determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus.

Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry.

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.

ABSTRACT Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

Cyprinid herpesvirus 3.

TOC summary: This virus is useful for fundamental and applied research.

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

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.

Clinical, virological, and immunological parameters associated with superinfection of latently with FeHV-1 infected cats.

Infections with feline herpesvirus type 1 (FeHV-1) are frequently associated with recurrent ocular disease, which may occur even in vaccinated cats. The underlying pathogenesis is poorly understood. Specifically, the role of circulating, superinfecting virus strains is unknown. To begin addressing this complex question, we reconstituted a marker-tagged mutant FeHV-1 from a bacterial artificial chromosome (BAC) harboring the FeHV-1 genome. This mutant was deleted for the glycoprotein G gene (DeltagG) but carried instead a gene encoding the green fluorescent protein (GFP). Nine latently with wild-type (wt) FeHV-1-infected cats were superinfected with this mutant and monitored for clinical, virological, and immunological parameters. While the mutant virus replicated locally, induced a rise in neutralizing antibody titers, and stimulated the interferon system, no evidence for ocular illness or reactivation of the underlying wtFeHV-1-infection was detected. However, cyclophosphamide-dexamethasone (C-D) treatment, applied 16 months after the superinfection, was able to reactivate wtFeHV-1. Reactivation was accompanied by recrudescence of ocular disease signs. In contrast, reactivation of the superinfecting mutant virus was not detected. Since kittens are normally infected with wtFeHV-1 prior to the first immunization, the data described in this study may be valuable for designing future live attenuated FeHV-1 vaccines.