Genome re-arrangements associated with loss of pathogenicity of the gamma-herpesvirus alcelaphine herpesvirus-1.

The alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in ruminants. Previous work had shown that serial passage of AlHV-1 in culture resulted in genome alterations that are associated with a loss in pathogenicity. Here we have analysed the re-arrangements that occur in more detail. None of the observed re-arrangements was entirely consistent. However, they did all involve translocation of a similar region of DNA from around the centre of the genome to areas either next to or in between terminal repeat elements at either end of the genome. There was also a concomitant loss of the wild-type locus. These re-arrangements appeared to be associated with the loss of virulence and the appearance of cell-free virus.

Outbreak of malignant catarrhal fever in Welsh Black cattle in Carmarthenshire.

An outbreak of malignant catarrhal fever (MCF) resulted in the deaths of 12 cattle in a herd of 77 animals during seven weeks in 1999; in addition, one cow developed a milder disease which was confirmed as MCF by PCR for ovine herpesvirus 2 DNA and an immunofluorescent antibody test for antibodies to the virus, but recovered. Further PCR and serological testing revealed the infection in three other animals, none of which developed clinical disease. Hypocuprosis and the possibility of a genetic predisposition were identified as factors which may have contributed to the outbreak.

Gamma herpesvirus carrier status of captive artiodactyls.

Between 1998 and 2000, 103 individuals of 19 species of the order Artiodactyla at Whipsnade Wild Animal Park were tested for evidence of infection with gamma herpesviruses in order to distinguish between species which are susceptible to malignant catarrhal fever (MCF), caused by alcelaphine herpesvirus-1 (AlHV-1) of wildebeest (Connochaetes sp.) or ovine herpesvirus-2 (OvHV-2) of domestic sheep, and species which carry related viruses sub-clinically. Gamma herpesvirus DNA was detected in the known, or suspected, carrier species: roan antelope (Hippotragus equinus), scimitar-horned oryx (Oryx dammah), gemsbok (Oryx gazella), musk ox (Ovibos muschatus) and mouflon (Ovis musimon). In six other species: lowland anoa (Bubalus depressicornis) yak (Bos grunniens), sitatunga (Tragelaphus spekei), greater kudu (Tragelaphus strepsiceros), waterbuck (Kobus ellipsiprymnus) and Nile lechwe (Kobus megaceros), DNA was present in some newborn calves and over 30% of adults, strongly suggesting a carrier state. In contrast five Père David's deer (Elaphurus davidianus) and two swamp deer (Cervus duvauceli) died of MCF during the study. A virus isolated from scimitar-horned oryx calves produced cytopathic effects in scimitar-horned oryx kidney cell-culture and caused MCF in a rabbit.

The role of lambs in louping-ill virus amplification.

In some areas of Scotland, the prevalence of louping-ill virus has not decreased despite the vaccination of replacement ewes for over 30 years. The role of unvaccinated lambs in viral persistence was examined through a combination of an empirical study of infection rates of lambs and mathematical modelling. Serological sampling revealed that most lambs were protected by colostral immunity at turnout in May/June but were fully susceptible by the end of September. Between 8 and 83% of lambs were infected over the first season, with seroconversion rates greater in late rather than early summer. The proportion of lambs that could have amplified the louping-ill virus was low, however, because high initial titres of colostral antibody on farms with a high force of infection gave protection for several months. A simple mathematical model suggested that the relationship between the force of infection and the percentage of lambs that became viraemic was not linear and that the maximum percentage of viraemic lambs occurred at moderately high infection rates. Examination of the conditions required for louping-ill persistence suggested that the virus could theoretically persist in a sheep flock with over 370 lambs, if the grazing season was longer than 130 days. In practice, however, lamb viraemia is not a general explanation for louping-ill virus persistence as these conditions are not met in most management systems and because the widespread use of acaracides in most tick-affected hill farming systems reduces the number of ticks feeding successfully.

Malignant catarrhal fever caused by ovine herpesvirus-2 in pigs in Norway.

This paper describes the first cases of malignant catarrhal fever (MCF) in pigs in which the diagnosis was verified aetiologically by polymerase chain reaction (PCR) and DNA analysis and by the demonstration of antibodies. Three pigs on two separate premises showed clinical signs, gross pathological and histopathological lesions which were in many respects similar to those of MCF in ruminants. The pigs were housed adjacent to sheep and DNA of ovine herpesvirus-2 (OHV-2) was detected by PCR in tissues of all the pigs. In addition, antibody to alcelaphine herpesvirus-1 was detected in the serum of the pigs and in five in-contact sheep. It is concluded that the disease described is MCF of pigs caused by OHV-2.

Restriction endonuclease profiles of orf virus isolates from the British Isles.

A comparison of DNA profiles of representative isolates of orf virus, obtained using four different restriction endonucleases (RE), showed that the enzyme EcoRI could be used to discriminate between wild-type virus isolates and vaccine strains. The enzyme was used to compare the RE profiles of orf virus isolates from 43 outbreaks of orf that occurred in vaccinated flocks between 1988 and 1993; 21 outbreaks yielded wild-type virus, 10 yielded vaccine viruses, three produced both vaccine and wild-type viruses and no clear result was obtained from nine of the outbreaks. From the 21 outbreaks yielding wild-type viruses, 28 orf virus isolates had clear RE profiles and 15 distinct RE profiles were recorded. Usually only one virus type was associated with each outbreak but from two farms, two different wild-type viruses were recovered. No predominant genotype was identified, with four RE profile types being recovered for more than one outbreak. From the more severe form of orf involving the buccal cavities of lambs only wild-type viruses were recovered, with at least four different genotypes being represented.

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein.

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.

A novel strategy for determining protective antigens of the parapoxvirus, orf virus.

We investigated the feasibility of using vaccinia virus (VAC) recombinants containing large multigene fragments of orf virus DNA to identify protective antigens of orf virus (OV). Sixteen OV strain NZ2 DNA fragments with an average size of 11.4 kb were recombined into VAC strain Lister. Each fragment was mapped relative to OV restriction endonuclease maps but was otherwise uncharacterized. Together the recombinants represent 95% of the OV genome in an overlapping manner. Immunofluorescence showed all 16 constructs expressed products recognized by OV antiserum and radioimmune precipitation with the same antiserum allowed the localization of the major antigens of OV to specific recombinants. These data indicated the approximate genomic locations of the genes encoding the OV major antigens and showed that their expression was authentic rather than resulting from read through from VAC sequences adjacent to the site of recombination. Vaccination of OV-naive sheep with the recombinant library provided protection against a subsequent challenge with virulent OV. These data confirm the feasibility of the proposed strategy.

Identification of ovine herpesvirus-2 infection in sheep.

A polymerase chain reaction test for the detection of ovine herpesvirus-2 (OHV-2) DNA was used to identify sites of OHV-2 infection in peri-natal lambs and in adult sheep. OHV-2 was detected in the nasal secretions from all lambs within a period of two months following birth. Subsequently, OHV-2 DNA was identified in a number of epithelial tissues including the cornea, turbinates and pharynx. In addition, OHV-2 DNA was detected exclusively in B-lymphocytes from six of ten adult sheep tested. An infection cycle for OHV-2 in sheep is proposed which bears similarities with the gammaherpesviruses Epstein-Barr virus and mouse herpesvirus-68.

Tissue culture-propagated orf virus vaccine protects lambs from orf virus challenge.

Twenty, eight-day-old specific pathogen-free (SPF) lambs were vaccinated by a single scarification approximately 4 cm in length on the inner right thigh with a double-pronged applicator. The titre of live virus in the vaccine was 10(7.2) TCID50/ml and the estimated dose per lamb was 0.04 ml. Three months and six months later 10 of the vaccinated lambs and five age-matched unvaccinated control specific pathogen free lambs were challenged by a single scarification with virulent virus on the inner left thigh in the same way. After the vaccination all 20 lambs developed lesions characteristic of orf virus infection that had largely resolved four weeks later, when they all had reciprocal ELISA antibody titres > or = 3200 that persisted in all but one of them until they were challenged. After the challenge, the development of lesions in the vaccinated and unvaccinated sheep was compared daily for four weeks by means of a clinical scoring system. Both groups of vaccinated lambs had significantly lower (P < 0.01) total clinical scores after challenge at three months and six months than the unvaccinated lambs.

Antigenic similarity of central European encephalitis and louping-ill viruses.

Twenty isolates of Central European encephalitis (CEE) virus were compared with 20 isolates of louping-ill (LI) virus in indirect immunofluorescence test (IIFT), using a panel of 17 monoclonal antibodies (MoAbs) prepared against the prototype LI virus. Three Asian members of the tick-borne encephalitis (TBE) complex were also included in the comparison: Turkish sheep encephalitis (TSE), Russian spring-summer encephalitis (RSSE) and Langat (LGT) viruses. Antigenic relationships of the viruses were evaluated by Dice similarity coefficient and cluster analysis. The results revealed antigenic heterogeneity of LI isolates, antigenic homogeneity of CEE isolates, and indicated that CEE and LI are related varieties of Eurasian TBE flavivirus that also includes TSE and RSSE strains.

Isolation of a parapoxvirus from a grey seal (Halichoerus grypus).

A grey seal (Halichoerus grypus) developed cutaneous pocks which progressed to involve the skin extensively, necessitating euthanasia. Macroscopically and histologically the lesions resembled previous descriptions of parapoxvirus infections of seals and virus particles were observed in preparations of a scab and a skin lesion. Suspensions of the scab and skin lesion were prepared and inoculated on to monolayer cultures of grey seal kidney cells. After 25 days in culture and three passages, cytopathic effects were observed and parapoxvirus particles were detected by electron microscopy in the supernatant fluid. Both isolates were adapted to cultures of fetal lamb muscle cells and shown to be antigenically related to orf virus.

Ultrastructural Studies of Orf Virus Infection and Replication in Fetal Lamb Fibrocytes.

Résumé- Le cycle de réplication du virus de l'ecthyma contagieux a été identifié dans des études in vitro et un modèle hypothétique a été développé. Pendant la premiére phase, qui dure à peu près 5 heures, le virus pénètre la cellule par le biais d'un processus de phagocytose, et perd ses enveloppes. La phase d'éclipse, pendant laquelle le virus est apparemment intégréà l'ADN de l'hôte, dure environ 8 à 10 heures. Pendant la phase finale, les virions se développent dans des zones biens définies du viroplasme à partir desquelles les viriojns matures vont migrer jusqu'aux bords de la cellule. Là, ils sortent soit par exocytose, soit à l'intérieur de projections microvilleuses qui sont pincées à leur base, soit encore par désintégration de la cellule hôte. [Onwuka, S.K., Jenkinson, D. Mc, Inglis, L., Pow, I., GRAY, E.W., Reid, H.W. Ultrastructural studies of orf virus infection and replication in fetal lamb fibrocytes (Etudes ultrasturcturales de l'infection par le virus de l'ecthyma contagieux et de sa réplication dans les fibrocytes de foetus d'agneau). Resumen- Se identificó el ciclo de replicación del virus del ectima contagioso en estudios temporales in vitro y se desarroló un posible modelo experimental. Durante la primera fase, que dura unas 5 h, el virus penetra en la células por fagocitosis y se libera de la cubierta. La fase de "eclipse", con el virus presentándose como hebras de DNA, dura aproximadamente de 8 a 10 h. En la fase final los viriones se desarrollan dentro de zonas bien definidas en el viroplasma desde las cuales los viriones maduros migran hasta los limites celulares. A partir de alii parecen salir por exocitosis o en proyecciones de microvellosidades "pinzadas" hacia el exterior; también pueden ser liberados como consecuencia de la desintegración de la célula huésped. [Onwuka, S.K., Jenkinson, D. Mc, Inglis, L., Pow, I., GRAY, E.W., Reid, H.W. Ultrastructural studies of orf virus infection and replication in fetal lamb fibrocytes (Estudios ultraestructurales de la infección por el virus del ectima contagioso y replicación en fibrocitos fetales de carnero). Abstract- The cycle of replication of orf virus was identified in temporal in vitro studies and a putative model was developed. During the first phase, which lasts about 5 h, the virus enters the cells by a phagocytic process and uncoats. The "eclipse" phase, with the virus apparently present as strands of DNA, lasts for approximately the next 8-10 h. In the final phase virions develop within well-defined zones of viroplasm from which mature virions migrate to the margins of the cell. There they apparently exit either by exocytosis or within microvillous projections which are "pinched off"; they can also be released by disintegration of the host cell.

PCR detection of the sheep-associated agent of malignant catarrhal fever.

From a genomic library previously constructed from a lymphoblastoid cell line (LCL) propagated from a bovine case of sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine herpesvirus-2 (OHV-2), several OHV-2 clones were identified and characterised by hybridisation using probes from the unique region of the Alcelaphine herpesvirus-1 (AVH-1) genome. Nucleotide sequence from one clone was generated and the predicted amino acid sequence was found to contain regions of homology with the 140 and 160 kDa tegument proteins of Epstein-Barr virus and herpesvirus saimiri respectively. Oligonucleotide primers were constructed and a polymerase chain reaction (PCR) test was developed for the detection of OHV-2 viral DNA. Amplified product was identified by restriction with RsaI and BmyI. The primers were highly specific for OHV-2 DNA with a limit of detection of 6.4 pg of genomic DNA derived from the parent LCL. This was estimated to correspond to one diploid bovine cell. The PCR was successfully applied to detect OHV-2 DNA in peripheral blood leucocytes (pbl) from clinical cases of SA-MCF and normal sheep.

Antibody to alcelaphine herpesvirus-1 (AHV-1) in hamsters experimentally infected with AHV-1 and the 'sheep-associated' agent of malignant catarrhal fever.

Malignant catarrhal fever was induced in four groups of hamsters by the inoculation of cells infected with either the C/500 isolate of alcelaphine herpes-virus-1 (AHV-1) or the sheep-associated agent derived from cattle, red deer or Père David's deer. Using an indirect immunofluorescence assay, antibody to AHV-1 was detected in sera of clinically affected animals of all four groups. The reaction of sera from hamsters affected with malignant catarrhal fever induced by AHV-1 caused diffuse cytoplasmic staining while that from sera of hamsters with the sheep-associated form of the disease stained particulate nuclear antigens. Tests employing three other bovid herpesviruses were negative and no reaction was found with sera from normal hamsters. These studies provide convincing evidence that a virus antigenically related to AHV-1 is the cause of sheep-associated malignant catarrhal fever and that the same virus probably causes this form of the disease in both cattle and deer.

Actions of bovine skin washings and sera on the motile zoospores of Dermatophilus congolensis.

Concentrated skin washings, even from vaccinated animals, failed to inhibit the motility of the infective zoospores of Dermatophilus congolensis, or to prevent them from germinating or infecting cattle; their constituent immunoglobulins did not attach to the flagella although IgA and IgG2 did bind to the cell bodies. It is concluded that the specific antibodies at the skin surface of ruminants are unlikely to have a role in zoospore immobilisation. Post vaccination sera rapidly immobilised and clumped the zoospores by means of a coat around the flagella, in which immunoglobulins, particularly IgM, were detected. IgM and IgG1 also attached to the cell bodies of the zoospores.

Isolation and characterisation of lymphoblastoid cells from cattle and deer affected with 'sheep-associated' malignant catarrhal fever.

Cells with the histological and ultrastructural characteristics of large granular lymphocytes (LGL) have been obtained in culture from both cattle and red deer (Cervus elaphus) reacting with 'sheep-associated' malignant catarrhal fever (MCF). Such cells have been derived from thymus, lymph node and spleen suspensions as well as from cerebrospinal fluid cells and cultured cornea. On most occasions their presence was observed only transitorily but by providing the cells with feeder monolayers and, or, interleukin-2, several lines were maintained indefinitely, and some became independent of these factors after prolonged culture. A similar cell line was also derived from a Père David's deer affected with MCF at Whipsnade zoological park. Functionally, cultured LGL were cytotoxic to both primary cell cultures and cell lines and their cytotoxicity was not restricted to histocompatible target cells. These findings suggest that the cultured cells have natural killer cell-like activity and that they are important targets for the agent of MCF in cattle and deer. One cell line derived from a red deer transmitted the disease but none of the cells generated from cattle did.

Immunoblotting analysis of the reaction of wildebeest, sheep and cattle sera with the structural antigens of alcelaphine herpesvirus-1 (malignant catarrhal fever virus).

Malignant catarrhal fever (MCF) is a disease of cattle and some other ruminants caused by alcelaphine herpesvirus-1 (AHV-1), a virus of wildebeest. The disease also occurs in the absence of wildebeest and is then thought to be caused by a viral agent harboured by the sheep. The structural proteins of AHV-1 have been used as antigens for the immunoblotting analysis of sera from wildebeest, sheep and cattle infected by either AHV-1 or the "sheep-associated" form of the disease. Wildebeest sera showed a uniform response reacting strongly with six polypeptides. Sheep sera also gave positive results but individual sera reacted with varying subsets of the antigens recognized by wildebeest. These results support the earlier suggestion that sheep harbour a herpesvirus related to AHV-1. A bovine serum from a case of MCF caused by AHV-1 also reacted only with a subset of the six wildebeest-reactive polypeptides. Sera from cattle affected with the "sheep-associated" form of the disease gave reactions in only two of the eight cases tested; both positive sera reacted to a few polypeptides only.

Transmission of wildebeest-associated and sheep-associated malignant catarrhal fever to hamsters, rats and guinea-pigs.

Wildebeest-derived malignant catarrhal fever (WD-MCF) was transmitted to hamsters, rats and guinea-pigs by inoculation of rabbit lymphoid cells infected with alcelaphine herpesvirus-1, strain C-500. Sheep-associated MCF (SA-MCF) was transmitted to hamsters by inoculation of lymphoid cells from rabbits affected with SA-MCF derived from deer. Mice were refractory to both forms of the disease. With both forms of MCF, the incubation period during initial transmission varied from 21 to 90 days and disease was readily passaged in rodents by inoculation of live lymphoid cells. Clinical signs in hamsters most closely resembled those described for naturally occurring MCF. Results given here and in two following papers indicate that rodents are useful models to study the aetiology and pathogenesis of both forms of MCF.