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.

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

“…The insertion was performed between predicted polyadenylation signals of the ORFs to reduce the risk that the insertion might affect the expression of the ORFs. The KHV FL BAC plasmid described previously was used as the parental plasmid (4). In this plasmid, the BAC cassette is inserted into the TK locus.…”

“…The LUC probe was produced by PCR using the forward primer LUCfw, the reverse primer LUCrev, and the pcDNA3-LUC plasmid as a template ( Table 1). The TK probe was described previously (4).…”

“…Second, prolonged KHV cultivation in vitro leads to spontaneous attenuation of the virus, making the production of KHV recombinants using classical homologous recombination in eukaryotic cells difficult (27). To circumvent this problem, we cloned the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) that could be used to produce KHV recombinant strains (4).…”

“…The KHV FL strain was isolated from the kidney of a fish that died from KHV infection (CER, Marloie, Belgium) (4). The KHV FL BAC strain was described previously (4). This recombinant strain carries a BAC cassette inserted in the thymidine kinase (TK) locus.…”

Herpesviruses of Fish, Amphibians and Invertebrates

“…Also, DNA vaccination with several different ORFs has been tested for IcHV-1 (Nusbaum et al, 2002). Later, an overlapping and a full BAC have been developed for IcHV-1 (Kunec et al, 2008) and CyHV-3 (Costes et al, 2008), respectively. The latter was used to test attenuation after disruption of the thymidine kinase locus and deletion of ORF16.…”