David R. Sargan

Nucleotide Sequence of EV1, a British Isolate Of Maedi-Visna Virus

We have isolated a maedi-visna-like virus from the peripheral blood mononuclear cells of a British sheep displaying symptoms of arthritis and pneumonia. After brief passage in fibroblasts this virus (designated EV1) was used to infect choroid plexus cells. cDNA clones of the virus were prepared from these cells and sequenced. Gaps between non-overlapping clones were filled using gene amplification by the polymerase chain reaction. The genome structure is similar to that described for visna virus strain 1514, and differs from that described for visna virus strain SA-OMVV in not having a W reading frame. Overall the genome differs by about 20% between each of these strains, but there is fivefold variation in the amount of divergence of derived amino acid sequences of different open reading frames. Two sequenced EV1 clones each contain only one copy of the 43 bp repeat, with paired AP-1 sites, which is a feature of other ruminant lentiviral long terminal repeats (LTRs). However, analysis of viral DNA in infected cells by gene amplification shows that LTRs with two repeats do occur, albeit at a relatively low frequency.

Confirmation of the rod cGMP phosphodiesterase β subunit (PDEβ) nonsense mutation in affected rcd-1 Irish setters in the UK and development of a diagnostic test

Rodkone dysplasia type one (rcd-1) is an early onset inherited retinal dystrophy segregating in the Irish setter breed. It is classed as one of the autosomal recessive canine generalised Progressive Retinal Atrophies (PRA). The disease results in complete loss of photoreceptors by approximately one year of age. Levels of retinal cGMP are markedly elevated and of abnormal distribution in rod photoreceptors. Rod phosphodiesterase activity is absent and mRNA encoding the beta subunit (PDEB) of the holoenzyme is uniquely reduced in predegenerate retinae. Using retinae from normal, unrelated adult dogs we have PCR-amplified and sequenced the cDNA for PDEβ. The cDNA is almost identical to that recently described for the Irish setter in the USA apart from two translationally silent single nucleotide changes. Using carrier and affected setters from a UK breeding colony we have screened genomic DNA and can confirm the G to A transition in rcd-1 affected dogs at position 2420, creating an amber mutation in codon 80...

Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay

The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.

Sequence of the cDNA encoding ovine tumor necrosis factor-α : problems with cloning by inverse PCR

Abstract We have cloned and sequenced the ovine tumor necrosis factor-α (TNF-α)-encoding cDNA, using gene amplification by polymerase chain reaction (PCR) technology, to aid studies of assorted diseases in this species. We used primers selected from published TnfA sequences of other species of on a cDNA template prepared from lipopolysaccharide-stimulated ovine alveolar macrophages, to generate a product representing the central region of the molecule. We then used a novel method based on ‘inverse PCR’ to generate a product containing the 5′ and 3′ ends of the molecule. Here, we present the complete sequence of the ovine TNF-α cDNA and compare it with other published TNF sequences. The cloned cDNA has a leader sequence of 156 bp followed by a protein-coding sequence of 702 bp and a 3′-untranslated region of 800 bp. The protein product of the gene is a protein of M r = 25 586 , 79% homologous to human TNF-α. An mRNA produced by alveolar macrophages, which hybridises to the cloned gene, is induced greatly, with a peak induction time of approx. 135 min, in response to stimulation by lipopolysaccharide and to plating on plastic. We also discuss the resolution of some artefacts of the inverse PCR technique.

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.

Identification of a region of the Alcelaphine Herpesvirus-1 genome associated with virulence for rabbits

The gammaherpesvirus Alcelaphine Herpesvirus 1 (AHV-1) causes the fatal lymphoproliferative disease known as malignant catarrhal fever (MCF), in susceptible hosts. The virulent C500 isolate of AHV-1 became attenuated for the laboratory model, the rabbit, as a result of serial passage in cells of bovine origin. This work describes the identification of a region of the central unique sequence of the C500 genome, located close to the terminal repeat units of the molecule, which is altered on attenuation. The virulent C500 genome contains two copies of a sequence of approximately 2 kbp, contained within a 7 kbp region of the unique DNA located adjacent to the terminal repeats at the left end of the molecule. In the genome of the attenuated virus, there are also two copies of the 2 kbp sequence but they are located at the ends of the attenuated genome unique region, adjacent to the terminally repeated sequences. One open reading frame (ORF), designated putative polypeptide 5, was altered on attenuation such that the 3 sequence was lost. The location of this ORF, coupled with the loss of its 3 sequence, suggests that this ORF may encode a gene involved in the virulent mechanisms of this virus, in a manner similar to that of the transforming proteins of Herpesvirus saimiri (HSV).

The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein

Maedi‐visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV), infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgGi subclass‐restricted MVV‐neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti‐Orf antibody is readily detected. By ELISA, the IgGi antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti‐MVV gag p. 25 can be detected in both normal and MVV‐infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freunds complete adjuvant. The failure to make an IgG2 MVV‐specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.

Characteristics of the T cell-mediated immune response to Maedi-Visna virus

Virus-specific T cell-mediated immunity was investigated in healthy sheep persistently infected with the ruminant lentivirus maedi-visna. Visna-specific lymphocyte proliferation was demonstrated in response to both purified virions and recombinant p25, the major core protein of maedi-visna virus. The responding T cell population in this assay was mainly of the CD4+ phenotype, although in some individuals CD8+ T cells were also shown to respond to visna antigen in this system.

The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-α and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-γ

Abstract An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-α whereas none were detected after 24 h in rov GM-CSF, rov IFN-γ or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-α, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50–200 units ml−1 of rov IFN-γ were added to cultures of ALDC along with TNF-α or rov TNF-α plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-α or rov TNF-α plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa.

Nucleotide sequence of the canine rod-opsin-encoding gene

The major parts of two canine rod-specific opsin (Ops) transcripts have been cloned by polymerase chain reaction from retinal mRNA. Both transcripts are derived from the same gene. The 5 leader sequence of the transcripts was cloned from canine peripheral blood DNA. The transcripts code for a protein of 348 amino acids (aa), M(r) 38,962 (prior to any protein modification). The aa sequence suggests that in common with other sequenced Ops, canine rod Ops contains seven transmembrane domains, and residues believed essential for retinal pigment binding and for palmitate binding are conserved in the canine protein. Northern blotting using the central part of the ops gene as probe suggested that mature transcripts of three different sizes (about 1900, 2600 and 5500 bases) were found in retina. Of these, the 2600-base transcript was the most abundant. RACE cloning of the 3 end of ops showed that at least two of these size classes originate from differential transcript termination.