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Abstract :
[en] Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest (Connochaetes taurinus) asymptomatically, causes malignant catarrhal fever (MCF) when cross species transmitted to a variety of susceptible species of the Artiodactyla order. MCF is a fascinating disease described as a combination of lymphoproliferative and degenerative lesions. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, due mainly to the fact that AlHV 1 becomes attenuated during passage in culture. Here, we have overcome these difficulties by (i) cloning the entire AlHV 1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC), and (ii) by using prokaryotic recombination technology for the production of an AlHV 1 recombinant. Firstly, the AlHV 1 genome was BAC cloned using one insertion site in a region containing no open reading frame. This insertion allowed the production of an AlHV 1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. BAC derived AlHV 1 virions induced MCF in rabbits comparably to the AlHV 1 wild type (WT) strain. Secondly, a two step mutagenesis procedure in E. coli was used to generate a recombinant strain expressing enhanced-green fluorescent protein (EGFP) as a reporter gene. After reconstitution of recombinant virions into permissive cells and excision of the BAC cassette, flow cytometry analyses were performed to validate the recombinant strain and to investigate the pathogenesis of MCF. The results of these analyses can be summarized as follows: (i) the validity of the EGFP expression cassette as a reporter gene has been demonstrated by in vitro infections; (ii) inoculation of rabbits revealed that the recombinant strain has retained the pathogenicity of its parental strain and that the cell types carrying AlHV-1 in peripheral blood mononuclear cells, lymph nodes and the spleen are mainly CD5+ CD8+ cells.
