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The nucleotide sequence around the capripoxvirus thymidine kinase gene reveals a gene shared specifically with leporipoxvirus.

We have extended previous comparisons of genetic organization between poxvirus genera by sequencing a 2.5K genomic fragment from isolate KS-1 (Kenya sheep-1) of the genus capripoxvirus. The fragment is located in the central region of the capripoxvirus genome and contains three complete and two incomplete open reading frames (ORFs). One of the complete ORFs is a gene for thymidine kinase (TK). This gene, with one of the other two complete ORFs and both the incomplete ORFs, are homologous to four contiguous ORFs from the central region of vaccinia virus (VV) DNA. They also match four ORFs of fowlpox virus (FPV) DNA, three of which are contiguous and the fourth, the FPV TK gene, is located elsewhere on the FPV genome. The third complete ORF of the capripoxvirus DNA fragment is located between the TK gene and the capripoxvirus homologue of the ORF immediately downstream of the VV TK gene. We show that a homologue to this third ORF is absent from VV and FPV DNAs, but is present downstream of the TK gene on Shope fibroma virus DNA. The sequence immediately upstream of the capripoxvirus homologue of a VV late gene contains a motif which is required for VV late gene expression. The motif required for VV early gene transcription termination is present in eight positions in the capripoxvirus sequence, and five of these positions are consistent with the motif having an equivalent function in capripoxvirus to that in VV.

Amino Acid Sequence↗

A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene.

Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.

Amino Acid Sequence↗

An antigen trapping ELISA for the detection of capripoxvirus in tissue culture supernatant and biopsy samples.

A trapping ELISA for the detection of capripoxvirus antigen in tissue culture supernatant and biopsy material was developed, using a guinea-pig polyclonal detector antiserum raised against a recombinant capripoxvirus specific antigen, expressed in Escherichia coli using the plasmid vector pGEX-2T. The ELISA detected antigen in tissue culture samples that on virus titration contained equal to or in excess of 10(2.8) TCID50/ml. Virus isolation and ELISA were compared for the detection of capripoxvirus in skin biopsy samples from sheep, goats and cattle. The ELISA compared well with virus isolation, and has applications as a diagnostic test. This assay reduces the reliance of diagnostic laboratories on tissue culture facilities, and can be used to confirm the presence of capripoxvirus in tissue culture.

Animals↗

Transmission of capripoxvirus.

The transmission of capripoxvirus to sheep, using an aerosol suspension of a Yemen isolate of the virus, was demonstrated. Capripoxvirus was also transmitted by contact to sheep and goats kept with animals infected with virus isolates from the Yemen, Sudan, India and Nigeria. The incubation period for capripoxvirus infection in sheep and goats was approximately eight to 12 days. Animals that had well developed clinical signs transmitted capripoxvirus more rapidly than animals which died of peracute disease or animals that had only mild clinical signs.

Aerosols↗

Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus.

The gene coding for the capripoxvirus structural protein P32 was cloned, expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and purified on glutathione Sepharose. An indirect enzyme linked immunosorbent assay (ELISA) using this antigen was developed to screen bovine sera for antibodies to capripoxvirus. Sequential serum samples from experimentally infected animals tested by ELISA and by virus neutralisation test (VNT) showed that the ELISA was more sensitive and detected antibodies to capripoxvirus earlier post-infection than the VNT.

Animals↗

A comparison of the genomes of capripoxvirus isolates of sheep, goats, and cattle.

HindIII, PstI, AvaI, and SalI sites were mapped on the genomes of six isolates of capripoxvirus from sheep, goats, and cattle. Genome pairs were aligned by the alignment of cross-hybridizing HindIII fragments and the introduction of padding fragments (pads) at specific locations. The majority of these pads represent the approximate positions of relative deletions or insertions. Three possible phylogenetic networks were generated for seven capripoxvirus isolates by Wagner parsimony analysis of the nonconserved HindIII sites on their genomes, and confidence limits were calculated for the network nodes. Nucleotide sequence divergence values, calculated from the numbers of nonconserved HindIII, PstI, AvaI, and SalI sites on the genomes of typical sheep, goat, and cattle isolates, indicated that the typical sheep and cattle isolates are more closely related to one another than to the typical goat isolate. Nonconserved HindIII, PstI, AvaI, and SalI sites were shown to be distributed throughout the genomes. Evidence that isolates YG-1 and OS-1 are descended from an isolate whose genome arose by recombination is discussed. Terminally repeated regions were identified on each of the capripoxvirus genomes mapped here. By mapping BamHI, ClaI, EcoRI, HindII, HindIII, and SalI sites present within the terminal 10 kb of the genome of isolate InS-1, the terminal repeats of this genome were shown to be between 2.25 and 3.40 kb in length and inverted with respect to one another.

Animals↗

Genomic relationship between capripoxviruses.

Capripoxvirus DNAs from field isolates and vaccine samples were analysed by digestion with the restriction enzyme Hind III. The patterns of fragments generated by digestion with Hind III are sufficiently similar to show that all capripoxviruses are closely related, although patterns of different isolates can be grouped in a way which correlates with the animal of origin. The close relatedness was also demonstrated by the high level of sequence homology detected using the Southern Cross hybridization system. Despite the sequence homology, the molecular weights of the genomes of different isolates varied from 73 to 91 MDa. The presence of two rapidly reannealing restriction fragments in the Hind III digests of capripoxvirus DNA indicated the presence of terminal cross-links.

Animals↗

The characterization of African strains of capripoxvirus.

Isolates of capripoxvirus collected from sub-Saharan Africa were compared in sheep, goats and cattle and by restriction endonuclease digestion of their purified DNA. Biochemical techniques were used to precisely identify strains of capripoxvirus for epidemiological investigations. Strains of capripoxvirus infecting cattle have remained very stable over a 30-year period and are closely related to strains recovered from sheep in Africa.

Africa↗

A comparison of the genome organization of capripoxvirus with that of the orthopoxviruses.

Comprehensive comparisons of genome organizations for poxviruses of different genera have not previously been reported. Here we have made such a comparison by cross-hybridizing genome fragments from capripoxvirus KS-1 and vaccinia virus WR (VV). This showed that a 100- to 115-kilobase (kb) centrally placed section is essentially colinear in organization in the two viruses and that a small region has translocated between the ends of one or other of the genomes during their divergence. No cross-hybridization could be detected between VV DNA and the respective left- and right-hand terminal 8 and 25 kb of capripoxvirus DNA or between capripoxvirus DNA and the respective left- and right-hand terminal 38 and 35 kb of VV DNA. By using the cross-hybridization data, a 4-kb fragment of KS-1 DNA was identified, which corresponds to the regions of the cowpox virus and VV genomes containing genes for the orthopoxvirus A-type inclusion body protein ("ATI"). The sequence of the KS-1 DNA fragment contains homologs of genes which are on either side of the orthopoxvirus ATI genes but contains no homolog of the ATI gene itself. Overall, these results show that the pattern of genomic conservation and variation between two poxvirus genera reflects the pattern within the orthopoxvirus genus but that, as observed previously, individual genes may not be present in genomic regions which are otherwise conserved in organization.

Amino Acid Sequence↗

Insect transmission of capripoxvirus.

Capripoxvirus was transmitted between sheep using Stomoxys calcitrans as a vector. Attempts to transmit capripoxvirus between sheep and between goats using biting lice (Mallophaga species), sucking lice (Damalinia species), sheep head flies (Hydrotaea irritans) and midges (Culicoides nubeculosus) were unsuccessful, although capripoxvirus was isolated from sheep head flies that had previously fed on infected sheep.

Animals↗

Sequence analysis of HindIII Q2 fragment of capripoxvirus reveals a putative gene encoding a G-protein-coupled chemokine receptor homologue.

The DNA sequence of the HindIII Q2 fragment near the left terminus of the capripoxvirus (KS-1 strain) genome was determined. The sequence contains two complete open reading frames (ORFs) and a part of a third. Analysis of the deduced amino acid sequence of one of these ORFs, Q2/3L, revealed that this gene has the capacity to encode a protein which is related to members of the G-protein coupled chemokine receptor subfamily, the swinepoxvirus K2R and the human cytomegalovirus US28 ORFs. It has the key structural characteristics of the G-protein-coupled receptor superfamily, e.g., seven hydrophobic regions, predicted to span the cell membrane, and the cysteine residues in the first and second extracellular loops that are implicated in formation of a disulfide bond. Southern blot analysis showed that all three species of the Capripoxvirus genus, i.e., sheep pox, goat pox, and lumpy skin disease of cattle, contain copies of this putative G-protein-coupled chemokine receptor homologue.

Amino Acid Sequence↗

Control of capripoxvirus infections.

The capripoxviruses cause the most severe pox diseases of animals. Epidemiologically the diseases of sheep pox, goat pox and lumpy skin disease differ, but all three viruses may be mechanically transmitted by biting insects, and control without vaccination is extremely difficult in endemic areas. Recently developed live attenuated vaccines provide good, virtually lifelong, protection, which is dependent on stimulating cell-mediated immunity. Lumpy skin disease currently threatens to extend beyond its existing boundaries, causing concern and renewed interest in vaccine development. With the capripoxviruses showing great promise as vector vaccines, widespread use of a recombinant vaccine seems increasingly likely.

Animals↗

Improved detection of capripoxvirus in biopsy samples by PCR.

A simple test based on the polymerase chain reaction (PCR) was used to detect capripoxvirus DNA in tissue culture supernatants and biopsy samples. The identity of the PCR products was confirmed by restriction enzyme analysis. The test has greater sensitivity and good specificity compared to an antigen trapping enzyme-linked immunosorbent assay which uses a detector antibody raised against a recombinant capripoxvirus-specific antigen. The reagents for the PCR-based test are all available commercially and the test provides a valuable addition to the current methods of virus detection.

Animals↗

Capripoxvirus disease in an Arabian oryx (Oryx leucoryx) from Saudi Arabia.

Lumpy skin disease caused by a capripoxvirus was observed in a captive-bred female Arabian oryx (Oryx leucoryx) at the National Wildlife Research Center, Taif, Saudi Arabia. Clinical signs included severe general depression with fever, anorexia, greater than 1,000 nodular cutaneous lesions and gradual recovery over 2 mo. The virus was found by electron microscopy and paired sera showed an increasing virus neutralization antibody titer against capripoxvirus. A serologic survey of the herd of 90 oryx showed a low prevalence (2%) of this infection. This report describes the first case of lumpy skin disease in an Arabian oryx.

Abortion, Veterinary↗

Recombinant capripoxvirus expressing the hemagglutinin protein gene of rinderpest virus: protection of cattle against rinderpest and lumpy skin disease viruses.

A cDNA clone containing the complete coding sequence of the hemagglutinin (H) protein gene of the RBOK vaccine strain of rinderpest virus, under the control of the vaccinia late promoter p11, was inserted by homologous recombination into the thymidine kinase gene of the KS-1 strain of capripoxvirus. The recombinant virus produced authentic H protein as judged by its electrophoretic mobility, transport to the cell surface of infected lamb testis cells, and reactivity with monoclonal antibodies specific for the H protein of rinderpest virus. The recombinant virus induced significant levels of rinderpest virus neutralizing antibodies in vaccinated cattle and protected them from clinical rinderpest after challenge with a lethal dose of a highly virulent heterologous strain of the virus. Protection was achieved using vaccine doses lower than those used with a similar recombinant expressing the fusion protein gene of rinderpest. The parental KS-1 virus is widely used as a vaccine against capripox viruses and so the rinderpest recombinant acts as a dual vaccine to protect cattle against both rinderpest and lumpy skin disease.

Animals↗

Protection of goats against peste des petits ruminants with recombinant capripoxviruses expressing the fusion and haemagglutinin protein genes of rinderpest virus.

Goats were protected against a lethal challenge of peste des petits ruminants (PPR) virus following vaccination with a recombinant capripoxvirus containing either the fusion (F) gene of rinderpest virus or the haemagglutinin (H) gene of rinderpest virus. The H gene recombinant produced high titres of neutralizing antibody to rinderpest virus in the vaccinated goats, whereas the F gene recombinant failed to stimulate detectable levels of neutralizing antibody. A similar response to the two recombinant vaccines has previously been reported for cattle. Neither recombinant produced detectable levels of specific antibodies to PPR virus.

Animals↗

Protection of cattle against rinderpest and lumpy skin disease with a recombinant capripoxvirus expressing the fusion protein gene of rinderpest virus.

Cattle were protected against challenge with rinderpest and lumpy skin disease viruses by vaccination with a recombinant capripoxvirus containing the fusion protein (F) gene of rinderpest virus. The minimum protective immunising doses for rinderpest and lumpy skin disease were 5.5 x 10(4) plaque forming units (pfu) and 1.5 x 10(3) pfu, respectively.

Animals↗

Physical characterization of the genome of a cattle isolate of capripoxvirus.

HindIII, Pstl, Aval, and SalI site maps have been determined for the genome of a cattle isolate of capripoxvirus, KC-1. The length of the genome was estimated, by summation of the lengths of individual HindIII fragments, to be 145.6 kb, and the genome was shown to possess terminally repeated regions 1.13-6.23 kb in length. The restriction site maps demonstrate that the genome of KC-1 does not possess a high degree of nucleotide sequence homology with the genomes of isolates of orthopoxvirus, parapoxvirus, leporipoxvirus, or African swine fever virus.

Chromosome Mapping↗