Pierre Drion

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.

Influence of the Ixodes ricinus tick blood-feeding on the antigen-specific antibody response in vivo

The blood meal of hard ticks such as Ixodes ricinus lasts several days. This is made possible by tick salivary factors that inhibit inflammation, haemostasis and the host immune response. We assessed the latter on a model of immune response in vivo. A significant reduction of specific IgM and IgG levels was observed in BALB/c mice infested 5 days before injection with bovine serum albumin (BSA) and QuilA but not in mice infested 5 days after the immunization. This effect was not observed in mock-infested mice and could not be attributed to the use of anesthetics. The antibody response was not merely delayed and the Th(1)/Th(2) balance appeared not altered. T-dependent zones and germinal centers in lymph nodes draining the tick bite site showed no apparent morphological alterations or shift in T cell subpopulations. However, the spleens of tick-infested mice had also an enlarged red pulp, indicating an increased extramedullary haematopoietic activity.

In vivo administration of a PKA type I inhibitor (Rp-8-Br-cAMPS) restores T-cell responses in retrovirus-infected mice

Murine AIDS (MAIDS) is caused by infection with the murine leukemia retrovirus RadLV-Rs and is character- ized by T-cell anergy and severe immunodeficiency with increased susceptibilty to several experimental opportunistic in- fections as observed in HIV infection. T cell anergy is associated with an increase of intracellular cAMP level, triggering a multistep pathway involving activation of PKA type I and resulting in inhibition of proximal TCR signaling. We have previously demonstrated that blocking PKA type I using the selective inhibitor Rp-8-Br-cAMPS, restores T-cell function in vitro in MAIDS as well as in HIV infection. In the present report, we investigated the effect of parenteral administra- tion of Rp-8-Br-cAMPS in mice with MAIDS. We show that the compound is not toxic and partially restores the ex vivo proliferative responses to anti-CD3 mAb, but that it has no effect on the lymphadenopathy and splenomegaly characteriz- ing the MAIDS syndrome.