Cloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome

Alcelaphine herpesvirus 1 (AIHV-1), carried asymptornatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order Artioclactyla. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, mainly due to the fact that AIHV-1 becomes attenuated during passage in culture. In this study, these difficulties were overcome by cloning the entire AIHV-1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC). A modified /oxP-flanked BAC cassette was inserted in one of the two large non-coding regions of the AIHV-1 genome. This insertion allowed the production of an AIHV-1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. The /oxP-flanked BAC cassette was excised from the genome of reconstituted virions by growing them in permissive cells stably expressing Cre recombinase. Importantly, BAC-derived AIHV-1 virions replicated comparably to the virulent (low-passage) AIHV-1 parental strain and induced MCF in rabbits that was indistinguishable from that of the virulent parental strain. The availability of the AIHV-1 BAC is an important advance for the study of MCF that will allow the identification of viral genes involved in MCF pathogenesis, as well as the production of attenuated recombinant candidate vaccines.