- Volume 83, Issue 5, 2002

The present study is the first report on the functional activity of a parapoxvirus-encoded dUTPase. The dUTPase gene of the attenuated orf virus (ORFV), strain D1701, was expressed as a bacterial thioredoxin fusion protein. In vitro assays showed that ORFV dUTPase was highly specific for dUTP as substrate. The enzyme was active over a broad pH range (pH 6·0–9·0), with maximal enzymatic activity at pH 7·0 in the presence of Mg2+ cations. Kinetic studies of the recombinant ORFV dUTPase revealed an apparent K m of 4·0 μM, which is more similar to that of the mammalian or African swine fever virus enzyme than to the K m of vaccinia virus dUTPase. Enzyme activity was also found with purified ORFV particles, indicating its virion association.

Orf virus (ORFV) is the type species of the parapoxvirus genus and produces cutaneous pustular lesions in sheep, goats and humans. The genome encodes a polypeptide with remarkable homology to interleukin-10 (IL-10), particularly ovine IL-10, and also to IL-10-like proteins encoded by Epstein–Barr virus (EBV) and equine herpesvirus. IL-10 is a pleiotropic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. We have expressed and purified C-terminal FLAG and His6-tagged versions of ORFV-IL-10 and shown that ORFV-IL-10 costimulates murine mast cells (MC/9) and inhibits tumour necrosis factor-α synthesis in activated mouse peritoneal macrophages. Our results demonstrate that although ORFV-IL-10 is structurally similar to EBV-IL-10 it has evolved a different spectrum of activities. EBV-IL-10 does not stimulate the proliferation of thymocytes or mast cells whereas ORFV-IL-10 has both of these activities. Recent studies show that the critical difference in molecular structure of human IL-10 and EBV-IL-10, which may be the basis of their functional differences, is linked to a single amino acid substitution. Consistent with the activity spectrum reported here for ORFV-IL-10, the viral gene encodes the critical amino acid seen in human IL-10. Although the ORFV-IL-10 gene has clearly undergone significant evolutionary change at the nucleotide level compared with ovine IL-10, it has largely retained the polypeptide structure and functional characteristics of its ovine counterpart, suggesting that mutations of the gene to a potentially more potent immunosuppressive form may compromise the co-existence of host and virus.

The WHO smallpox eradication program was concluded 21 years ago and the non-vaccinated population is now at risk of poxvirus infections, either by contact with monkeypox or through bioterrorism. Since drugs specific against poxvirus infections are limited, neutralizing monoclonal antibodies (mAbs) that are effective in vivo may be an important tool in controlling poxvirus infections. To this end, we studied the efficacy of the mAb C3, reactive against the trimeric 14-kDa protein of vaccinia virus (VV) localized in the membrane of the intracellular form of mature virus, for its ability to neutralize VV infection in mice. The results show that prophylactic as well as therapeutic administration of mAb C3 can be an effective means of control of VV replication within the host. The interval of antibody efficacy following a single administration, before and after VV inoculation, has been defined. This study reinforces the notion that neutralizing mAbs should be developed to control health-related human infections by poxviruses.

Among the important challenges to shrimp aquaculture worldwide are the diseases caused by viruses, in particular by white spot syndrome virus (WSSV), which has a genome estimated to contain 305 kb. By analysis and comparison of the WSSV genomic DNA and cDNA libraries, an ORF (vp28 gene) was identified. The gene, encoding a novel 204-amino-acid protein, was expressed in Escherichia coli and purified. A specific antibody was raised using the purified VP28 protein. After inoculation of healthy adult Penaeus monodon shrimp with WSSV, the gene transcript and VP28 protein were first detected at low levels at 6 and 18 h post-infection, respectively. These experiments suggest that it might be a late gene. Immuno-electron microscopy with gold-labelled antibody revealed that the gold particles were distributed in the outer envelope of WSSV virions and showed that vp28 encodes a virus envelope protein.

Chelonus inanitus (Braconidae, Hymenoptera) is a solitary egg-larval parasitoid of Spodoptera littoralis. Along with the egg the female wasp injects polydnaviruses, which are prerequisites for successful parasitoid development. The polydnavirus genome is segmented and consists of double-stranded circular DNA. Proviral DNA is integrated in the wasp’s genome; virus replication is restricted to the wasp’s ovary and does not occur in the parasitized host. The polydnavirus of C. inanitus (CiV) protects the parasitoid larva from encapsulation by the host’s immune system and causes a developmental arrest of the host in the prepupal stage. Here we report on the first two cloned CiV genes, which are named CiV14g1 and CiV14g2 because of their localization on segment CiV14. The cDNA of CiV14g1 has a size of 2036 bp; the gene contains seven exons interrupted by six introns of similar size and encodes a putative polypeptide of 548 amino acids. The cDNA of CiV14g2 has a size of 618 bp; the gene consists of three exons and encodes a putative peptide of 77 amino acids. Transcript quantities of both genes are very low up to the penultimate larval instar of the host. In the last instar, at the stage of pupal cell formation, CiV14g1 expression increases about 5-fold and CiV14g2 expression about a 1000-fold. These are the first data to show strong upregulation of polydnavirus genes towards the end of parasitization. These two genes might be involved in the reduction of host ecdysteroids observed at this stage.

Processing of the polyprotein encoded by Potato virus A (PVA; genus Potyvirus) was studied using expression of the complete PVA polyprotein or its mutants from recombinant baculoviruses in insect cells. The time-course of polyprotein processing by the main viral proteinase (NIaPro) was examined with the pulse–chase method. The sites at the P3/6K1, CI-6K2 and VPg/NIaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate. The CI-6K2 polyprotein was observed in the baculovirus system and in infected plant cells. In both cell types the majority of CI-6K2 was found in the membrane fraction, in contrast to fully processed CI. Deletion of the genomic region encoding the 6K1 protein prevented proper proteolytic separation of P3 from CI, but did not affect processing of VPg, NIaPro, NIb or CP from the polyprotein. The 6K2-encoding sequence could be removed without any detectable effect on polyprotein processing. However, deletion of either the 6K1 or 6K2 protein-encoding regions rendered PVA non-infectious. Mutations at the 6K2/VPg cleavage site reduced virus infectivity in plants, but had a less pronounced, albeit detectable, effect on proteolytic processing in the baculovirus system. The results of this study indicate that NIaPro catalyses proteolytic cleavages preferentially in cis, and that the 6K1/CI and NIb/CP sites can also be processed in trans. Both 6K peptides are indispensable for virus replication, and proteolytic separation of the 6K2 protein from the adjacent proteins by NIaPro is important for the rate of virus replication and movement.

Scrapie does not occur in New Zealand (NZ), although PrP gene alleles associated with susceptibility to the disease are found at relatively high frequencies in NZ sheep. The hypothesis that scrapie is a genetic disease of sheep is thus unlikely to be true. To confirm that NZ sheep are actually susceptible to scrapie infection, NZ sheep of various PrP genotypes were challenged by subcutaneous inoculation with a sheep-passaged scrapie isolate (SSBP/1). Showing similar PrP genetics to that seen in UK sheep, all NZ sheep carrying the VRQ PrP allele developed clinical signs typical of scrapie, with characteristic neurodegenerative changes and PrPSc evident on histopathological examination of their brains and lymphoid tissues. The incubation periods recorded in NZ sheep were generally shorter than those found in UK sheep. The results confirm that New Zealand sheep are as susceptible as their UK counterparts to experimental scrapie infection by subcutaneous inoculation.