Epidemiology of squirrelpox virus in grey squirrels in the UK.

The dramatic decline of the native red squirrel in the UK has been attributed to both direct and disease-mediated competition with the grey squirrel where the competitor acts as a reservoir host of squirrelpox virus (SQPV). SQPV is threatening red squirrel conservation efforts, yet little is known about its epidemiology. We analysed seroprevalence of antibody against SQPV in grey squirrels from northern England and the Scottish Borders in relation to season, weather, sex, and body weight using Generalized Linear Models in conjunction with Structural Equation Modelling. Results indicated a heterogeneous prevalence pattern which is male-biased, increases with weight and varies seasonally. Seroprevalence rose during the autumn and peaked in spring. Weather parameters had an indirect effect on SQPV antibody status. Our findings point towards a direct disease transmission route, which includes environmental contamination. Red squirrel conservation management options should therefore seek to minimize squirrel contact points.

First cases of squirrelpox in red squirrels (Sciurus vulgaris) in Scotland.

Squirrelpox, caused by a poxvirus, is a major threat to the remaining UK red squirrel population. The spread of antibody-positive grey squirrels has been monitored in the UK for the past decade. In 2005 grey squirrels that had been exposed to the virus appeared in the south of Scotland for the first time, followed approximately two years later by the appearance of squirrelpox disease in the local red squirrels. Four squirrels were examined. They all had gross external lesions and histological lesions typical of squirrelpox disease, but no significant internal lesions. The diagnosis was confirmed by PCR, electron microscopy and serology.

Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [corrected] (HPMPC, cidofovir, CDV, Vistide) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of codofovir [corrected] and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved in milder lesions that resolved [corrected] more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.

Recent isolates of bovine respiratory syncytial virus from Britain are more closely related to isolates from USA than to earlier British and current mainland European isolates.

Eight isolates of Bovine respiratory syncytial virus (BRSV) were made from calves with severe respiratory disease on seven farms in one region of Britain. Genetic analysis of the viruses showed that seven, which were isolated between 1997 and 1999 were almost identical and were distinguishable from an earlier 1991 isolate. When compared with the available sequences of BRSVs from other countries, the recent British isolates were more closely related to US isolates than to earlier British and current mainland European isolates.

Genomic comparison of an avirulent strain of Orf virus with that of a virulent wild type isolate reveals that the Orf virus G2L gene is non-essential for replication.

The genomic structure of two strains of orf virus (OV), a field isolate (MRI-Scab) which has never been passaged in cell culture, and a multiple-passage cell culture-adapted strain (Orf-11) were compared. The Orf-11 genome is approximately 8.0 kb longer than that of the MRI-Scab due to a duplication of the right-hand end. The duplicated region has been translocated to the left-hand end of the genome with a loss of sequence from that end. The lost sequence contains three complete genes, namely E2L, E3L and G1L and 80% of a fourth gene, namely G2L. The sequence lost from G2L in Orf-11 has been replaced by a region of unrelated sequence, encoding 98 amino acids. Northern analysis shows that mRNA is expressed from this "new" gene. The two viruses were also compared for in vivo virulence and ability to protect against subsequent OV challenge. In vivo, the field isolate was fully virulent and conferred good protection against challenge, whereas the cell culture-adapted virus produced only mild lesions and reduced protection against challenge.

Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2.

The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a K(d) of 369 pM and ovine IL-2 with a K(d) of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

Parapoxviruses are strongly inhibited in vitro by cidofovir.

Three parapoxviruses which cause orf or related diseases in humans and animals and the orthopoxvirus, vaccinia virus, were tested for their in vitro sensitivity to cidofovir. The 50% inhibitory concentration for the three parapoxviruses was between 0.21 and 0.27 microg/ml and for vaccinia was 1.32 microg/ml. The selectivity index varied from 198 to 264 for the parapoxviruses and was 42 for vaccinia virus. Virus yield assays confirmed the ability of cidofovir to reduce ortho- and parapoxvirus replication. The efficacy of cidofovir against parapoxviruses justifies its evaluation as a candidate drug for the treatment of parapoxvirus infections in humans and animals.

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.

Border disease of sheep and goats.

Border disease (BD) is a congenital virus disease of sheep and goats first reported in 1959 from the border region of England and Wales. BD virus (BDV) is a pestivirus in the genus Flaviviridae and is closely related to classical swine fever virus and bovine virus diarrhoea virus (BVDV). Nearly all isolates of BDV are non-cytopathogenic (ncp) in cell culture. There are no defined serotypes but pestiviruses isolated from sheep exhibit considerable antigenic diversity and three distinct antigenic groups have been identified. Distribution of the virus is worldwide. Prevalence rates vary in sheep from 5 to 50% between countries and from region-to-region within countries. The disease in goats is rare and characterized by abortion. Clinical signs in sheep include barren ewes, abortions, stillbirths and the birth of small weak lambs. Affected lambs can show tremor, abnormal body conformation and hairy fleeces (so-called 'hairy-shaker' or 'fuzzy' lambs). Vertical transmission plays an important role in the epidemiology of the disease. Infection of fetuses can result in the birth of persistently infected (PI) lambs. These PI lambs are viraemic, antibody negative and constantly excrete virus. The virus spreads from sheep to sheep with PI animals being the most potent source of infection. Apparently healthy PI sheep resulting from congenital infection can be identified by direct detection of viral antigen or viral RNA in leukocytes or by isolation of ncp virus from blood or serum in laboratory cell cultures. Isolation of virus is unreliable in lambs younger than 2 months old that have received colostral antibody. The isolation of virus from tissues of aborted or stillborn lambs is difficult but tissues from PI sheep contain easily detectable levels of virus. To detect the growth of virus in cell cultures it is essential to use an immune-labelling method. Acute infection is usually subclinical and viraemia is transient and difficult to detect. Sheep may also be infected following close contact with cattle excreting the closely related BVDV.

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.

Natural transmission of orf virus from clinically normal ewes to orf-naive sheep.

The apparent natural transmission of orf virus from clinically normal ewes to susceptible sheep was observed during a border disease vaccine experiment. The 14 susceptible sheep were persistently infected with border disease virus and had been reared indoors in isolation from other sheep since birth. Their ages ranged from two to four years and they were housed in two groups; group 1 consisted of four sheep persistently infected with the Moredun strain of border disease virus and group 2 consisted of 10 sheep persistently infected with the Oban strain of the virus. On day 0, six sheep were removed from group 2 and rehoused. To the remaining four sheep in each group were added eight four- to six-year-old pregnant conventionally reared ewes at 48 days gestation. Fourteen days later the four sheep in group 1 were moved to another pen housing eight similar five-year-old pregnant ewes at 48 days' gestation, and the four sheep from group 2 were rehoused with their original stallmates. Twenty-one days later lip lesions typical of orf were first observed on the sheep from both groups and the disease spread to all the sheep persistently infected with border disease virus over the next four weeks. Virological and serological evidence demonstrated that the source of infection for the sheep was almost certainly the conventionally reared ewes, on which no lesions resembling orf were observed at any time during the study.

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.

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.