ANALYSIS OF GENOMIC REARRANGEMENT AND SUBSEQUENT GENE DELETION OF THEATTENUATED ORF VIRUS-STRAIN D1701

The orf virus (OV) strain D1701 belongs to the genetically heterogenou s parapoxvirus (PPV) genus of the family Poxviridae. The attenuated OV D1701 has been licensed as a live vaccine against contagious ecthyma in sheep. Detailed knowledge on the genetic structure and organization of this PPV vaccine strain is an important prerequisite to reveal pos sible genetic mechanisms of PPV attenuation. The present study demonst rates a genomic map of the approximately 158 kbp DNA of OV D1701 estab lished by hybridization studies of cloned restriction fragments coveri ng the complete viral genome. The results show an enlargement of the i nverted terminal repeats (ITR) to up to 18 kbp due to recombination be tween nonhomologous sequences during cell culture adaptation. DNA sequ encing of the region adjacent to the ITR junction revealed the absence of one open reading frame designated E2L. In contrast to a transposit ion-deletion variant of the New Zealand OV strain NZ2 (Fleming et al., 1995) the two genes E3L (a homologue of dUTPase) and G1L neighbouring E2L are retained in OV D1701. DNA and RNA analyses proved the presenc e of E2L gene in wild-type OV isolated directly from scab material. Th e data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and may represent one important vira l gene in determining virulence and pathogenesis of OV. (C) 1998 Elsev ier Science B.V. All rights reserved.