Robert G. Dalziel

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.

Epithelial tumour cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus

Sheep pulmonary adenomatosis (SPA) is a naturally occurring contagious lung tumour of sheep which has been associated aetiologically with a type D- and B-related retrovirus (Jaagsiekte retrovirus; JSRV). To improve understanding of the aetio-pathogenesis of SPA, the distribution and the sites of JSRV replication in sheep with naturally or experimentally induced SPA or in unaffected controls were identified. New immunological reagents were produced and a blocking enzyme-linked immunosorbent assay (B-ELISA) and an immunohistochemical technique for the detection of JSRV major capsid protein at the tissue and cellular levels were developed. JSRV was detected only in the respiratory tract of sheep affected by pulmonary adenomatosis and specifically in the transformed epithelial cells of the alveoli of SPA-affected sheep.

Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis

Jaagsiekte retrovirus (JSRV) is an exogenous type D-related retrovirus specifically associated with a contagious lung cancer of sheep (sheep pulmonary adenomatosis; SPA). Recently, epithelial tumour cells in the lungs of SPA-affected sheep were identified as major sites of JSRV replication by immunological techniques and RT-PCR amplification of part of JSRV gag. JSRV was not detected outside the lungs and their draining lymph nodes. However, low levels of JSRV expression in non-respiratory tissues could have been masked by co-amplification of endogenous JSRV-related sequences, which were differentiated from JSRV by the lack of a Scal restriction site in the PCR product. To further investigate the pathogenesis of SPA, an exogenous virus-specific hemi-nested PCR was developed utilizing primers in the U3 region of JSRV LTR, where major differences between endogenous and exogenous sequences exist. This technique was shown to be > or = 10(5)-fold more sensitive than the previous gag PCR/ScaI digestion method. Using this new assay the tissue distribution of JSRV in sheep with natural and experimentally induced SPA was analysed. Proviral DNA and JSRV transcripts were found in all tumours and lung secretions of SPA-affected sheep (n = 22) and in several lymphoid tissues. The mediastinal lymph nodes draining the lungs were consistently demonstrated to be infected by JSRV (10/10). JSRV transcripts were also detected in spleen (7/9), thymus (2/4), bone marrow (4/8) and peripheral blood mononuclear cells (3/7). Proviral DNA was also detected in these tissues although in a much lower proportion of cases. JSRV was not detected in 27 samples from unaffected control animals (n = 15).

Behavioural changes in the rat following infection with varicella-zoster virus

Following the establishment of a chronic varicella-zoster virus infection in the rat, behavioural allodynia and hyperalgesia were observed in the injected, but not the contralateral hind limb up to 33 days post-infection. This model may prove useful in investigating mechanisms involved in the establishment of post-herpetic neuralgia.

Novel histamine H3 receptor antagonists GSK189254 and GSK334429 are efficacious in surgically-induced and virally-induced rat models of neuropathic pain.

&NA; Several studies have implicated a potential role for histamine H3 receptors in pain processing, although the data are somewhat conflicting. In the present study we investigated the effects of the novel potent and highly selective H3 receptor antagonists GSK189254 (6‐[(3‐cyclobutyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐7‐yl)oxy]‐N‐methyl‐3‐pyridinecarboxamide hydrochloride) and GSK334429 (1‐(1‐methylethyl)‐4‐({1‐[6‐(trifluoromethyl)‐3‐pyridinyl]‐4‐piperidinyl}carbonyl)hexahydro‐1H‐1,4‐diazepine) in two rat models of neuropathic pain, namely the chronic constriction injury (CCI) model and the varicella‐zoster virus (VZV) model. Both GSK189254 (0.3, 3 and/or 10 mg/kg p.o.) and GSK334429 (1, 3 and 10 mg/kg p.o.) significantly reversed the CCI‐induced decrease in paw withdrawal threshold (PWT) measured using an analgesymeter and/or von Frey hairs. In addition, GSK189254 (3 mg/kg p.o.) and GSK334429 (10 mg/kg p.o.) both reversed the VZV‐induced decrease in PWT using von Frey hairs. We also investigated the potential site of action of this analgesic effect of H3 antagonists using autoradiography. Specific binding to H3 receptors was demonstrated with [3H]‐GSK189254 in the dorsal horn of the human and rat spinal cord, and in human dorsal root ganglion (DRG), consistent with the potential involvement of H3 receptors in pain processing. In conclusion, we have shown for the first time that chronic oral administration of selective H3 antagonists is effective in reversing neuropathic hypersensitivity in disease‐related models, and that specific H3 receptor binding sites are present in the human DRG and dorsal horn of the spinal cord. These data suggest that H3 antagonists such as GSK189254 and GSK334429 may be useful for the treatment of neuropathic pain.

Identification of a putative cellular receptor for the lentivirus visna virus

One mechanism by which viral tropism may be controlled is by the expression of a specific virus receptor on the cell surface. This paper reports the identification of a putative cellular receptor for visna virus, the prototype virus of the family Lentiviridae. Using a virus overlay protein blot assay we identified a group of polypeptides of apparent Mr 30K to 33K which interacts with visna virus and is present on permissive but not non-permissive cells. A rat polyclonal anti-ovine major histocompatibility complex (MHC) class II antigen (Ag) serum raised to immunopurified MHC class II Ag, but not preimmune serum, blocked the interaction of visna virus with these polypeptides. In an ELISA, immunopurified MHC class II Ag bound to visna virus but not to bovine parainfluenza 3 virus. Preincubation of visna virus with immunopurified soluble MHC class II Ag resulted in a marked decrease in virus-induced syncytium formation, i.e. preincubation with class II Ag inhibited infection with visna virus, but we have been unable to inhibit infection using class II Ag-specific antisera. These results suggest that ovine MHC class II Ag acts as a component of a cellular receptor for visna virus. This is of particular interest owing to the close similarities between visna virus and human immunodeficiency virus (HIV), and the relationship between MHC class II and CD4, the cellular receptor for HIV. It is also of relevance to recent reports that a growing number of viruses utilize polypeptides of the Ig supergene family as receptors.

The product of gene US11 of herpes simplex virus type 1 is expressed as a true late gene

The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression.

The Nuclear Autoimmune Antigen Ku IS Also Present on the Cell Surface

Polyclonal antibodies were raised against the individual 85 and 70 kDa subunits of the Ku complex purified from nuclear extract prepared from the T cell line MLA144. They specifically recognise the appropriate subunits of the Ku complex from whole cell extract of HeLa cells using Western blot analysis. They are also able to identify the Ku proteins present in the cell membrane using FACS analysis.

Identification of the sheep homologue of the monocyte cell surface molecule--CD14

An ovine monocyte/macrophage cell surface antigen was recognized by three mouse monoclonal antibodies (mAbs) VPM65, VPM66 and VPM67. These mAbs also reacted with bovine cells. The antibodies immunoprecipitated a single, glycosyl-phosphatidylinositol-linked polypeptide of M(r) 55,000 which, when deglycosylated, was reduced to M(r) 53,000. They reacted strongly with peripheral blood monocytes, alveolar macrophages and peripheral blood granulocytes, and weakly with afferent lymph dendritic cells. They also reacted with macrophages in many different tissues but were non-reactive with lymphocytes. Competitive flow cytometry shows that these three mAbs recognize the same or a closely related epitope of a single antigen. An antigen-specific capture ELISA using the anti-human CD14 mAb (TUK4) revealed that all four mAbs associate with the same antigen. These data demonstrate that the mAbs react with the ovine homologue of the lipopolysaccharide (LPS)-LPS binding protein receptor, CD14.

Two B cell subpopulations have distinct recirculation characteristics

This report describes two subpopulations of B cells in sheep. These subpopulations have distinct recirculation characteristics and tissue distributions. Phenotypically the populations are distinguished by their differential expression of the complement receptors, CD21 (CR2) and CD11b/CD18 (CR3). CD11b+ B cells are surface (s)IgMhi , co‐express CD11c but are L‐selectin negative. They populate the splenic marginal zone but are absent from splenic and ileal Peyers patch (IPP) follicles and both afferent and efferent lymph compartments. Fluorescent tracing experiments showed that the CD11b+ B cells are non‐recirculating as they did not appear in lymph after intravenous inoculation but are restricted to the blood and spleen. The CD11b‐negative population expresses a conformational determinant of CD21 that is recognized by the monoclonal antibody Du 2 – 74. These cells are sIgMlo and co‐express L‐selectin. They populate the splenic and IPP follicles, are absent from the splenic marginal zone and are the only B cells in afferent lymph, efferent lymph and all lymph nodes. Fluorescence tracing experiments showed that the CD21+ B cells are recirculating cells with their entry into efferent lymph being detectable by 16 h and peaking at 24 – 30 h. These data suggest that there are at least two lineages of B cells in the sheep with different phenotypic, functional and recirculation characteristics.