Anthony J. Robinson

The structure and cloning of orf virus DNA

A map of cleavage sites for the restriction endonucleases EcoRI, HindIII, BamHI, HpaI, and KpnI for a New Zealand strain of orf virus (NZ2) DNA has been deduced. Also, the entire genome, apart from approximately 0.1 kbp at each end, has been cloned into various vectors. The genome is 139 kbp in length and, in common with other poxviruses, has inverted terminal repetitions and crosslinked ends.

Molecular genetic analyses of parapoxviruses pathogenic for humans

The current members of the genus parapoxvirus are orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV) and parapoxvirus of red deer in New Zealand (PVNZ). BPSV and PCPV are maintained in cattle while ORFV is maintained in sheep and goats, but all three are zoonoses. Only the recently reported PVNZ has yet to be recorded as infecting humans. Tentative members of the genus are camel contagious ecthyma virus, chamois contagious ecthyma virus and sealpoxvirus. The separation of the parapoxviruses into 4 distinct groups has been based on natural host range, pathology and, more recently, on restriction endonuclease and DNA/DNA hybridisation analyses. The latter studies have shown that the parapoxviruses share extensive homology between central regions of their genomes, but much lower levels of relatedness within the genome termini. The high G + C content of parapoxvirus DNA is in contrast to most other poxviruses and suggests that a significant genetic divergence from other genera of this family has occurred. DNA sequencing of portions of the genome of ORFV, the type species of the genus, has allowed a detailed comparison with the fully sequenced genome of the orthopoxvirus, vaccinia virus (VACV). These studies have provided a genetic map of ORFV and revealed a central core of 88 kbp within which the genomic content was strikingly similar to that of VACV. This conservation is not maintained in the genome termini where insertions, deletions and translocations have occurred. The characterisation of specific ORFV genes may lead to the construction of attenuated vaccine strains in which genes such as those with the potential to interfere with the immune response of the host have been deleted. The current ORFV vaccines are living unattenuated virus and vaccination lesions produce virus which contaminates the environment in a manner similar to natural infection. The virus in scab material is relatively resistant to inactivation and this virus both perpetuates the disease in sheep and provides the most likely source of human infections. A vaccine which immunises animals without perpetuating the disease could be the best way of reducing the incidence of ORFV infection of humans. It is likely that protection against infection by ORFV is cell mediated and will require the endogenous production of relevant antigens. We have recently constructed a series of VACV recombinants each of which contains a large multigene fragment of ORFV DNA. Together the recombinants represent essentially all of the ORFV genome in an overlapping manner. Vaccination of sheep with the recombinant library provided protection against challenge with virulent ORFV. Further studies with this library may enable dominant protective antigens of ORFV to be identified and lead to their incorporation into a subunit vaccine.

Conservation and variation in orf virus genomes

The genomes of several orf virus strains were analyzed with the restriction endonucleases EcoRI, HindIII, BamHI, and KpnI, and cleavage site maps were deduced. In general, the right half of the genome showed conservation of restriction sites compared with the left half. Variations in size of up to 0.5 kbp were found within an inverted terminal repetition, and a 1-kbp deletion was detected in some strains in a subterminal fragment at the left end. A region of approximately 20 kbp, some 12 kbp in from the left end, showed the greatest cleavage site variability although there was no evidence of large deletions in this region. A 1.55-kbp cloned DNA fragment from the internal variable region of NZ2 failed to hybridize to the DNA from three other strains. A fragment in the variable region of strain NZ7 was cloned and compared by hybridization and restriction endonuclease analysis with cloned NZ2 fragments from the same region. The region of nonhomology extended for at least 2.75 kbp. It is suggested that this internal variable region may provide sites for the insertion of foreign genes.

Genomic analysis of a transposition-deletion variant of orf virus reveals a 3.3 kbp region of non-essential DNA

Restriction endonuclease analysis of the DNA extracted orf virus strain NZ2, which had been serially passaged in primary bovine testis cells, revealed a population of variants that had over-grown the wild-type virus. At least three distinct mutant forms were identified in which the right end of the genome had been duplicated and translocated to the left end, accompanied by deletions of sequences at the left end. Sequencing of a single variant isolated from the heterogeneous population revealed that recombination had occurred between non-homologous sequences. In this case, 6.6 kb of DNA at the left end of the genome had been replaced by 19.3 kb from the right end. The transposition resulted in the deletion at the left end of 3.3 kb of DNA encoding three genes and the terminal sequences of a fourth gene. The three genes completely deleted were a homologue of dUTPase, a gene that encodes a protein containing ankyrin-like repeats and a homologue of the 5K gene of the vaccinia virus WR strain. Experimental inoculation of sheep showed that the genes are also non-essential in vivo, but that the size of the lesion was reduced, compared with that induced by the wild-type, and resolved more rapidly.

Sequence analysis of the inverted terminal repetition in the genome of the parapoxvirus orf virus

Two BamHI fragments from the right-hand terminal region of the orf virus genome have been sequenced. The bulk of the inverted terminal repetition (ITR) sequence is contained within these fragments and makes up 3388 bp of the 4425-bp sequence reported. The overall base composition of the larger sequence is 59.4% G + C and of the ITR, 60.2% G + C. An extremely G/C-rich (83.2%) block of sequence was found spanning the ITR/unique sequence junction. The bulk of the ITR could be divided into three blocks of directly repeated sequences. One block begins about 250 nucleotides from the terminus and is a direct repeat 15 bp long, repeated 14 times. The other blocks contain seven sequence sets ranging from 16 to 36 nucleotides which are repeated 2 to 4 times, interspersed with one another, interrelated in sequence, and sometimes separated by unique sequence. Eight open reading frames (ORFs), each with the potential to code for polypeptides of 50 residues or more, were identified. Three were found within the ITR, four spanned the ITR/unique sequence junction and one was found outside the ITR. A search for putative poxvirus transcriptional control signals indicated that three of the eight ORFs are likely to be transcribed early, all in the same direction toward the right end of the genome. Sequences of the type T(A)3-5T were found only twice in the sequence and only one preceded an ORF.

A homologue of retroviral pseudoproteases in the parapoxvirus, orf virus

The nucleotide sequence of a near-terminal region of orf virus DNA was determined. Examination of the sequence revealed an open reading frame encoding a peptide with significant amino acid homology to the pseudoprotease domains recently identified in a number of retroviruses including mouse mammary tumor virus, simian Mason-Pfizer virus, maedi-visna virus, and equine infectious anaemia virus. The orf virus pseudoprotease shares up to 28% amino acid homology with retroviral pseudoproteases and appears to be a discrete transcriptional unit rather than a subunit of a larger polypeptide as is the case in retroviruses. The sharing of amino acid composition across such wide taxonomic boundaries suggests that this polypeptide has a functional significance in both retroviruses and poxviruses.

Orf virus replication in bovine testis cells: kinetics of viral DNA, polypeptide, and infectious virus production and analysis of virion polypeptides.

SummaryThe replication of orf virus in bovine testis cells was analysed in one-step growth experiments. Newly replicated viral DNA was detected 4 to 6 hours post-infection (p.i.), accumulated rapidly between 8 and 16 hours p.i. and reached a plateau between 25 and 30 hours p.i. Most virus-induced polypeptides were first detected in a two hour period beginning 10 hours p.i., reached a peak rate of synthesis between 14 and 16 hours p.i., and continued at that rate for at least 10 hours. Host polypeptide synthesis declined to very low levels by 20 hours p.i. From these results, the transition between early and late events appears to occur between 8 and 10 hours p.i. Infectious virus was first detected between 16 and 18 hours p.i. and continued to be produced at a steady rate till 40 hours p.i.Up to 35 polypeptides were detected in SDS-polyacrylamide gels of purified orf virions disrupted in SDS/2-ME. Virions treated with NP40/2-ME were separable into soluble and insoluble components by centrifugation. Some 13 polypeptides were found in the soluble fraction and a polypeptide of molecular weight 38,500 believed to be the basic subunit of the virion surface tubule structure. Little difference was found between polypeptide profiles of five independently isolated NZ orf virus strains.

Sequence and transcriptional analysis of an orf virus gene encoding ankyrinlike repeat sequences

A 1608 bp region located approximately 5.0 kb from the left end of the orf virus (OV) genome (strain NZ2) was sequenced. The sequence revealed a single open reading frame designated G1L. The predicted amino acid sequence of G1L contained eight ankyrinlike repeat sequences. Transcriptional analysis of G1L showed it was transcribed towards the genome terminus during the early phase of infection. S1 nuclease and primer extension analyses showed that the transcriptional start site of the gene was located a short distance downstream from an A+T-rich sequence similar to a vaccinia virus early promoter.

Lack of cross-protection between vaccinia virus and orf virus in hysterectomy-procured, barrier-maintained lambs.

Hysterectomy-procured, barrier maintained lambs were immunised with either of virus or vaccinia virus and subsequently challenged with both viruses. Under these conditions lambs were protected from challenge with the homologous virus but no cross-protection was observed. The feeding of colostrum that contained antibodies to orf virus had no effect on the duration of viral lesions. Immunoblotting analysis and ELISA of serum samples taken during the course of the experiment indicated that the animals mounted antibody responses to both viruses. The cross recognition of 3 vaccinia virus antigens by the hyperimmune anti-orf virus serum was revealed by immunoblotting.

Sequence and transcriptional analysis of a near-terminal region of the orf virus genome

A 3605 bp region located approximately 6.6 kb from the left end of the orf virus genome (strain NZ2) was sequenced. The sequence revealed two open reading frames, which we have designated G2L and B1L. The predicted amino acid sequences of G2L and B1L were found to be homologous to the vaccinia virus (VAC) F11L and F12L gene products, respectively, and were found to be arranged on the genome in the same orientation and relative position as their VAC counterparts. Transcriptional analysis of both G2L and B1L showed they were transcribed toward the genome terminus during the early phase of infection. S1 nuclease and primer-extension analyses showed that the transcriptional start sites of both genes were located a short distance downstream from A + T-rich sequences, similar to vac virus early promoters.