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Orf virus (ORFV) strain D1701-V, a Parapoxvirus belonging to the family Poxviridae, became attractive as a novel virus vector system that we successfully used for the generation of recombinant vaccines. Therefore, the identification of viral genes involved in host tropisms or immune modulation is of great interest, as for instance the ORFV-encoded ankyrin-repeat (AR) containing proteins. The present study shows for the first time that the ANK-1 designated gene product of ORFV126 is targeted to mitochondria of ORFV-infected and in ANK-1 transiently expressing cells. Taking advantage of ANK-1 EGFP fusion proteins and confocal fluorescence microscopy mutational and deletion analyses indicated the importance of AR8 and AR9, which may contain a novel class of mitochondria-targeting sequence (MTS) in the central to C-terminal part of this AR-containing protein. The fluorescent findings were corroborated by cell fractionation and Western blotting experiments. The presented results open the avenue for more detailed investigations on cellular binding partners and the function of ANK-1 in viral replication or virulence.

The orf virus (OV) strain D1701 belongs to the genetically heterogenous 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 possible genetic mechanisms of PPV attenuation. The present study demonstrates a genomic map of the approximately 158 kbp DNA of OV D1701 established by hybridization studies of cloned restriction fragments covering the complete viral genome. The results show an enlargement of the inverted terminal repeats (ITR) to up to 18 kbp due to recombination between nonhomologous sequences during cell culture adaptation. DNA sequencing of the region adjacent to the ITR junction revealed the absence of one open reading frame designated E2L. In contrast to a transposition-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 presence of E2L gene in wild-type OV isolated directly from scab material. The data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and may represent one important viral gene in determining virulence and pathogenesis of OV.

Restriction enzyme analysis of DNA of a number of Pseudorabies virus (PRV) single plaque isolates revealed in several cases the existence of a unique EcoRI cleavage site, which has not been observed in PRV DNA before. This EcoRI site was mapped to the right end of the unique long region of the PRV genome, in BamHI-fragment 6. Sequence analysis of this region demonstrated the presence of an 11 bp tandem repeat in variable copy numbers in different PRV strains, suggesting the creation of the EcoRI recognition site by a recombinational event. The occurrence of variable reiterations and Northern blot analysis indicated an intergenic region. We therefore, used this site for integration and expression of heterologous DNA (the multiple cloning site of phage M13 and the E. coli lacZ gene). Viable PRV recombinants could be obtained which showed no detectable differences in virus growth in vitro compared to wild-type PRV. The novel insertion site can be used for the construction of PRV recombinants expressing foreign genes without apparent impairment of PRV genes.

Twenty-four calves were immunised four times with gE-deleted infectious bovine rhinotracheitis marker vaccines before being challenged with small doses of wild-type bovine herpesvirus type 1 (BHV-1). The repeated vaccinations induced strong immunity that prevented detectable virus replication and gE-seroconversion after the challenge infection in most of the calves. The hypervaccinated calves that shed virus after the challenge infection showed no delay in gE-seroconversion compared with unvaccinated control calves. Using a sensitive nested PCR, BHV-1 gE sequences could be detected in the trigeminal ganglia of several of the gE-seronegative, challenge-infected calves, possibly indicating the presence of wild-type BHV-1 DNA.

The present study provides for the first time an extended investigation of individual genes located at the near-terminal right end of the genome of parapoxvirus bovis 1, Bovine papular stomatitis virus (BPSV) strain B177 and Orf virus (ORFV). Comparison of the respective DNA sequences of ORFV strain D1701 (9.9 kbp) and BPSV B177 (7.7 kbp) revealed a very similar organization of closely related genes transcribed in a rightward orientation. The most salient findings of this study were: (i) the absence of the ORFV-specific vascular endothelial growth factor (VEGF-E) gene in the BPSV isolate; (ii) the presence of an interleukin-10 (IL-10) orthologue; and (iii) the detection of three new genes encoding ankyrin-repeat-containing polypeptides. These results not only contribute to potential improvements of future molecular differentiation between the parapoxvirus species, but also shed new light on different pathobiologies among parapoxviruses.