Neil J. Watt

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.

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.

Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages

Viral load may be an important indicator of disease progression in sheep infected with maedi-visna virus (MVV). To assess this variable accurately in MVV-infected sheep, a quantitative competitive-polymerase chain reaction (QC-PCR) was developed. A conserved region of the MVV pol gene was selected. The RT-PCR MVV pol product was cloned and mutagenised in vitro by PCR to produce a competitor template reduced in length from 217 to 192 bp, but which retained the original flanking MVV pol PCR primers. The competitor template was quantified accurately and in an optimised QC-PCR protocol serial dilutions of this template were co-amplified with known amounts of sample DNA. MVV DNA levels in peripheral blood monocytes and alveolar macrophages from MVV-infected sheep (n=12) were assessed by QC-PCR. Viral DNA load in alveolar macrophages was significantly higher than that in peripheral blood monocytes when the animals were compared overall. A comparison was also made between alveolar macrophages from the lungs of seropositive animals with or without histopathological evidence of pulmonary lesions. The load of MVV DNA in alveolar macrophages was low in sheep without histopathological evidence of lesions in the lung. In contrast, in alveolar macrophages from sheep with histopathological lesions in the lung, there was a significantly higher level of MVV DNA. The correlation of MVV load with pulmonary lesions suggests that infected alveolar macrophages play a key role in the pathogenesis of this lymphoid interstitial pneumonia.

Phenotypic analysis of lymphocyte populations in the lungs and regional lymphoid tissue of sheep naturally infected with maedi visna virus

We have analysed the phenotype of lymphocytes in lung and regional lymph node of symptomatic and asymptomatic sheep infected with the ovine lentivirus, maedi visna virus (MVV). Compared to equilavent tissues from age‐matched, non‐infected controls, MVV‐infected sheep show increased numbers of lymphocytes in the lung, both in the bronchus‐associated lymphoid tissue (BALT) and in the alveolar septae. Both CD8+ and CD4+ T lymphocyte numbers in alveolar septae were increased, particularly in animals with clinical respiratory disease. The ratio of CD8+ to CD4+ lymphocytes was similar to that in normal lung. In both MVV‐infected and uninfected animals a high proportion of pulmonary lymphocytes, particularly in the alveolar septae, did not express the CD5 antigen, suggesting that they were activated. The number of activated cells was higher in infected sheep. Variable numbers of alveolar macrophages containing MVV‐core protein were present in alveolar lumina, the majority of positive cells showing morphological evidence of activation. In regional lymphoid tissue there were increased numbers of CD8 + and γδ expressing T cells in lymphoid follicles and germinal centres of infected animals. The specificity of these cells is unknown and we could find no evidence for the presence of cells productively infected with the virus in these structures. This study shows that activated T lymphocytes, particularly of the CD8 subset, play a major part in the pathogenesis of MVV‐induced pulmonary and regional lymph node lesions.

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.

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.

Phenotypic analysis of cells in bronchoalveolar lavage fluid and peripheral blood of maedi visna-infected sheep

A phenolypic analysis of bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) cells in maedi visna virus (MVVVinlecled sheep has been performed. The differential cell eount in BALF from MVV‐infeeted animals was characterized by a significant increase (P < 0±05) i n lymphocytes and neutrophils. Lymphocyte phenotyping in BALF from MVV‐infeeted sheep showed a significant decrease (P < 0·05) or CD4+ cells, a significant increase (P < 0·05) of CD8+ eells and a significant inversion (P<0·001) of the CD4+/CD8+ ratio. CD5+ lymphocytes were also significantly decreased (P < 0·05). γδ T cells and B cells did not differ significantly when compared with the controls. No correlation was observed between BALF and PB lymphocyte phenotypes. BALF macrophages from MVV‐infeeted animals showed inereased MHC class II expression and BALF lymphocytes from the same animals demonstrated up‐regulation of LFA‐1 and LFA‐3 expression. These findings and their relationship with lentiviral pathogenesis are discussed.

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.

A technique for the sequential isolation of RNA and DNA from embryos developed for screening for viruses.

A technique has been developed to detect RNA or DNA viruses in single embryos. This technique is based on sequential RNA and DNA isolation from the embryo and analysis of this nucleic acid by polymerase chain reaction. A constitutively expressed embryo gene (alpha subunit ATPase) is also analysed as an internal standard. Extraction of RNA was more efficient than that of DNA, however, positive results were obtained for the internal standard gene for 84% (RNA) and 30% (DNA) of embryos tested.

Retroviral arthritis: phenotypic analysis of cells in the synovial fluid of sheep with inflammatory synovitis associated with visna virus infection.

A phenotypic analysis on synovial fluid cells from the carpal and tarsal joints of sheep with visna virus-induced inflammatory synovitis was performed. The results showed increased representation of cells bearing lymphocyte, macrophage, and dendritic cell markers compared to equivalent synovial fluid cells from normal uninfected age-matched controls. In infected sheep, CD8+ T cells tended to predominate over CD4+ cells, while the numbers of gamma delta T cells varied from being absent in some samples to constituting the major T cell subset in others. B cells were found in relatively smaller numbers. Analysis of the large mononuclear cells showed that they stained with monoclonal antibodies that recognize macrophages and afferent lymph dendritic cells. Major histocompatibility complex (MHC) class II+ macrophage/dendritic cells were found in normal joints, but significantly elevated proportions of such cells were present in the carpal joints of infected sheep. The intensity of MHC class II staining was also significantly elevated in infected animals compared to control animals. A high proportion of these cells also stained for CD1 in both normal and infected animals, but were significantly elevated in number in the carpal joints of infected sheep. The elevated proportion of cells expressing molecules associated with accessary cell function and the increase in the numbers of accessory molecules per cell suggests an enhanced capacity for presenting antigen to a variety of T cell subsets within the joints of infected sheep, which could initiate or perpetuate potentially damaging local synovial inflammatory responses.