Biomedical subjects

C J McInnes

Publications and source records attributed to C J McInnes.

At least 19 recordsLinked to original sources

PubMed2006

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.

Administration, Topical↗

PubMed2005

Human sealpox resulting from a seal bite: confirmation that sealpox virus is zoonotic.

The case of a marine mammal technician who sustained a seal-bite to the hand that produced a lesion clinically very similar to orf is described. Sequence analysis of the viral DNA amplified from the lesion by the polymerase chain reaction indicated that it was sealpox virus in origin. This is the first report providing unequivocal evidence that sealpox may be transmitted to humans and causes lesions very similar to orf.

Adult↗

PubMed2005

Glycosylation, disulfide bond formation, and the presence of a WSXWS-like motif in the orf virus GIF protein are critical for maintaining the integrity of Binding to ovine granulocyte-macrophage colony-stimulating factor and interleukin-2.

Orf virus (ORFV), the type species of the family Parapoxviridae, encodes a protein (GIF) that binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). There is no obvious sequence homology between the ORFV protein and any known mammalian GM-CSF- or IL-2-binding proteins. We demonstrate here that many of the biochemical properties of mammalian GM-CSF receptors that are required for efficient binding of GM-CSF are also critical to the GIF protein for binding to ovine GM-CSF (ovGM-CSF). Site-directed mutagenesis of the GIF protein demonstrated, first, the importance of disulfide bonds, and second, that a sequence motif (WDPWV), related to the WSXWS motif of the type 1 cytokine receptor superfamily, was necessary for biological activity. Finally, glycosylation of the GIF protein was also critical for binding to GM-CSF.

Amino Acid Motifs↗

PubMed2004

Variability in cytokine production and cell proliferation by mitogen-activated ovine peripheral blood mononuclear cells: modulation by interleukin (IL)-10 and IL-12.

T-cell reactivity is typically measured by cell proliferation and/or production of cytokines in response to antigenic/mitogenic stimulation. The choice of assays is more limited in ruminants than rodents, and complicated by the variability inherent in outbred populations. We have measured proliferation and production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells (PBMC) from 24 sheep, and compared the responses between sheep, within sheep over several sample points, and also drawn comparisons between the two assays. PBMC derived from different sheep varied by as much as ten-fold in both proliferation and IFN-gamma production, though not necessarily at the same sample time. Thus, there was a poor correlation between the two assays and also considerable variation in the responses from the same animal at different time points. Both parameters could be modulated by exogenous recombinant ovine interleukin (IL)-10 and IL-12, but we were unable to correlate IFN-gamma production with endogenous cytokine production in the assays. These data highlight the importance of assay selection for the measurement of immune responsiveness and also demonstrate the variation that can be expected between sheep and over time.

Animals↗

PubMed2002

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure.

The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41.1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

Amino Acid Motifs↗

PubMed2002

Orf virus encodes a functional dUTPase gene.

The present study is the first report on the functional activity of a parapoxvirus-encoded dUTPase. The dUTPase gene of the attenuated orf virus (ORFV), strain D1701, was expressed as a bacterial thioredoxin fusion protein. In vitro assays showed that ORFV dUTPase was highly specific for dUTP as substrate. The enzyme was active over a broad pH range (pH 6.0-9.0), with maximal enzymatic activity at pH 7.0 in the presence of Mg(2+) cations. Kinetic studies of the recombinant ORFV dUTPase revealed an apparent K(m) of 4.0 microM, which is more similar to that of the mammalian or African swine fever virus enzyme than to the K(m) of vaccinia virus dUTPase. Enzyme activity was also found with purified ORFV particles, indicating its virion association.

Deoxyuracil Nucleotides↗

PubMed2001

Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions.

In sheep infected with the parapoxvirus orf virus, primary infection orf skin lesions developed and resolved within 8 weeks. Reinfection lesions were smaller and resolved within 3 weeks. The host response in the skin was characterized by an accumulation of neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, B cells and T19+ gammadelta T cells. The magnitude of this accumulation paralleled orf virus replication in the skin. In situ hybridization was used to detect cells expressing interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) mRNAs in orf skin. Cells expressing IL-4 mRNA were not detected at any time after infection. Cells expressing IFN-gamma mRNA were detected after reinfection but not after primary infection. Cells expressing TNF-alpha mRNA included epidermal cells, vascular endothelium and uncharacterized cells that increased more rapidly in the skin after reinfection compared to primary infection. The results are consistent with a prominent role for IFN-gamma in the host immune response controlling the severity of the disease.

Animals↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

The cloning and expression of the cDNA for ovine stem cell factor (kit-ligand) and characterization of its in vitro haematopoietic activity.

The cDNA encoding the soluble form of ovine stem cell factor (SCF) has been cloned and expressed. The soluble protein is predicted to be 165/166 amino acids in length, one more than the human and murine SCFs with which it shares 87% and 81% identity respectively. Ovine SCF has 98.5%, 95% and 91% identity with cattle, pig and dog SCF, respectively. The recombinant ovine (rov) SCF protein has been expressed in Chinese hamster ovary (CHO) cells, purified, and its biological activity on ovine bone marrow cells compared with that of interleukin 3 (rovIL-3), granulocyte-macrophage colony-stimulating factor (rovGM-CSF), interleukin 5 (rovIL-5), human macrophage colony-stimulating factor (M-CSF) and human erythropoietin (epo). On its own rovSCF supported the development of small numbers of neutrophil, macrophage, eosinophil, granulocyte-macrophage, mixed cell phenotype, haemopoietic blast cell and basophilic granular cell colonies in a soft agar clonogenic assay. In combination with each of the above cytokines rovSCF supported an increase in the number and size of the lineage-specific colony types that were stimulated by the other cytokines on their own. In an assay for precursors of multipotential colony-forming cells (multi-CFC), rovSCF in combination with rovIL-3 (but neither cytokine alone) supported the development of these early haematopoietic progenitor cells.

Amino Acid Sequence↗

PubMed1998

Eosinophil-specific biological activity of recombinant ovine interleukin-5.

Recombinant ovine interleukin-5 (rovIL-5) expressed from Chinese hamster ovary (CHO) cells was tested for cell-specific bioreactivity, in vitro, in a soft agar clonogenic assay and in an enzyme-based microassay for eosinophil potentiating activity (EPA). In soft agar assays, colony and cluster formation from sheep bone marrow cells (SBMC) incubated with rovIL-5 was significantly enhanced compared with SBMC incubated with control supernatants from mock-transfected CHO cells. Colony analysis at 14 days demonstrated that for three separate rovIL-5 preparations 45%, 61% and 66% of colonies were eosinophilic, as were 55%-71% of clusters. In contrast, no eosinophil colonies were detected in parallel control cultures. RovIL-5 was also shown to possess potent and dose-responsive EPA, on the basis of eosinophil peroxidase (EPO) and arylsulphatase (EAS) assay in 7 day SBMC cultures. This activity was inhibited in a dose-responsive manner by TRFK-5, a rat anti-murine IL-5 monoclonal antibody (MAb) previously shown to have cross-reactivity in the ovine EPA assay. The results demonstrate that rovIL-5 exhibited eosinophil-specific properties similar to those of IL-5 derived from other mammalian species.

Agar↗

PubMed1998

Early immunopathological events in experimental ovine paratuberculosis.

An experimental oral infection of neonatal (< 2 weeks old) lambs with a cervine isolate of Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis), the causal agent of ruminant paratuberculosis (Johne's disease) was used to investigate bacteriological, histopathological and immunological changes during the early (up to 8 weeks) post-infection phase. In vitro culture for mycobacteria was positive in one faecal and three mesenteric lymph node (MLN) samples from the eight infected lambs. All mycobacterial isolates from MLN were identified as M.a. paratuberculosis by polymerase chain reaction (PCR). Small-to-medium sized focal granulomata were observed in jejunal (JPP) and ileal Peyer's patches (IPP) from four of the eight infected lambs. Compared with controls, JPP from all infected lambs had significantly (p < 0.05) higher proportions of CD8+ and CD2+ lymphocytes, and there were significantly (p < 0.05) fewer cells expressing B lymphocyte-associated markers in IPP and MLN. The T/B cell ratio was significantly (p < 0.05) increased in both JPP and MLN from infected lambs. The expression of a range of genes for cytokines was examined using specific reverse transcriptase PCR (RT-PCR) amplification of messenger RNA (mRNA) template isolated from MLN, JPP and IPP from both groups of animals. Densitometric analyses indicated that, in infected animals, MLN expressed significantly (p < 0.05) more mRNA for TNF-alpha: JPP had significantly increased (p < 0.05) mRNA for GM-CSF and significantly decreased (p < 0.05) mRNA for IL-4 and IFN-gamma. Infected lambs had significantly (p < 0.05) decreased titres of both circulating IgG and gut mycobacteria-associated IgG antibody. Infection was not associated with any consistent changes in lymphocyte reactivity to specific mycobacterial antigens, IFN-gamma release into supernatants from in vitro intestinal lymphocyte cultures or gut IgA antibody levels.

Animals↗

PubMed1998

Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L.

A homolog of the vaccinia virus (VAC) interferon resistance gene E3L has been discovered in orf virus strain NZ-2, a parapoxvirus that infects sheep, goats and humans. The gene is located 20 kb from the left terminus of the orf virus genome and is transcribed towards this terminus. RNase protection studies have been used to define the limits of the gene and Northern analysis revealed that it is expressed early in infection. The predicted amino acid sequence of the orf virus protein shares 31% identity (57% similarity) with the VAC E3L protein. Four of the six residues identified as being essential to dsRNA binding in the vaccinia virus protein are conserved in the orf virus protein whilst the other two amino acid changes are conservative substitutions. The orf virus gene has been sequenced in two other orf virus strains which vary markedly in their ability to produce experimental lesions in vivo. Their predicted protein sequences vary by less than 3% from the NZ-2 protein. The recombinant orf virus protein, expressed as a fusion protein in E. coli, bound double-stranded (ds)RNA but not dsDNA, single-stranded (ss)DNA or ssRNA . This is the first demonstration of a VAC E3L-like gene encoded by a parapoxvirus.

Amino Acid Sequence↗

PubMed1998

The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon-inducible, double-stranded RNA-dependent kinase.

The parapoxvirus orf virus was resistant to type 1 (IFN-alpha) and type 2 (IFN-gamma) interferons in cultures of ovine cells. The recently identified orf virus OV20.0L gene exhibits 31% predicted amino acid identity to the vaccinia virus E3L interferon-resistance gene, and is referred to as the (putative) orf virus interferon-resistance gene (OVIFNR). The objective of this study was to determine whether OVIFNR was involved in interferon resistance. Recombinant OVIFNR as a thioredoxin fusion protein (OVIFNR-Tx) inhibited the activation (by autophosphorylation) of an interferon-inducible, double-stranded (ds) RNA-dependent kinase (PKR) of sheep, which was shown to bind dsRNA (poly I:C). PKR in other species is involved in the inhibition of protein synthesis as part of the antiviral state in infected cells. Virus-infected cell lysates, but not control lysates, from cells grown in the presence of cytosine arabinoside also contained PKR inhibitory activity, which indicated that the inhibitory activity was associated with early viral gene expression. Significantly, the OVIFNR gene expressed in interferon-treated ovine fibroblasts protected the unrelated Semliki Forest virus from the antiviral effect of both type 1 and type 2 interferons. Taken together, the results indicate that the OVIFNR gene functions as an interferon-resistance gene, the product of which inhibits PKR in a similar way to the vaccinia virus E3L gene product.

Animals↗

PubMed1998

Cloning of a cDNA encoding ovine keratinocyte growth factor.

A cDNA encoding the ovine keratinocyte growth factor (KGF) has been cloned. A 622-bp cDNA, containing the entire protein coding region, was amplified from an ovine adenocarcinoma fibroblast cell line. The cDNA was found to share 95% nucleotide identity with dog KGF, 94% identity with human KGF, 90% identity with mouse KGF and 88% identity with rat KGF. The predicted ovine amino-acid sequence shares 98.5, 98, 96 and 94% identity, respectively, with the corresponding dog, human, mouse and rat proteins. The KGF gene is present as a single copy in the ovine genome.

Amino Acid Sequence↗

PubMed1996

Cyclosporin A abrogates the acquired immunity to cutaneous reinfection with the parapoxvirus orf virus.

The effect of cyclosporin A (CsA) on host immunity to cutaneous reinfection with the parapoxvirus orf virus was studied in 6-month-old lambs. In control reinfected animals, clinical lesions and viral replication (measured by the presence of vesicular/pustular lesions and viral antigen) in regenerating epidermal cells were at a maximum on day 4 with resolution by day 9. Lesion histology revealed recruitment of T cells, B cells and dermal dendritic cells (DDC) which increased and decreased in parallel with the clinical course of the reinfection. In animals treated with CsA (25 mg/kg/day) 1 day before and for 8 days after reinfection, more severe clinical lesions and viral replication typical of primary infections were recorded and had not resolved by 28 days following reinfection. During CsA treatment, the recruitment of T cells, B cells and DDC was inhibited. With cessation of CsA treatment there was dramatic recruitment of CD4+ T cells followed by DDC then B cells to the lesion site but rapid onset of acquired immunity was not recorded. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of cytokine mRNAs from lesion biopsies showed individual sheep variations. However, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) mRNAs were detected in the control reinfected animals on days 3 and/or 9 after reinfection but not on these days in animals undergoing treatment with CsA. In the untreated lambs there was an inexplicable lack of IL-2 and IFN-gamma mRNAs on day 6 after reinfection. Tumour necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF) mRNAs were unaffected by CsA treatment. The data suggest that CsA abrogates acquired immunity to orf virus reinfection by targetting T-cell lymphokine production.

Animals↗

PubMed1994

Cloning of a cDNA encoding ovine interleukin-3.

A cDNA encoding the ovine hematopoietic growth factor interleukin-3 (IL-3), has been cloned. A 560-bp cDNA, containing the entire protein coding region, was amplified from activated mesenteric lymph node cells. The cDNA was found to share 58% nucleotide identity with hIL-3 and 48% identity with mIL-3. The predicted ovine amino-acid sequence shares 36 and 30% identity, respectively, with the corresponding human and murine proteins.

Amino Acid Sequence↗

PubMed1992

Kinetics of ovine interferon-gamma production: detection of mRNA and characterisation of biological activity.

The kinetics of interferon-gamma (IFN-gamma) production were studied in sheep mesenteric lymph node (MLN) cells at the molecular level using an ovine IFN-gamma cDNA probe and by bioassay which was verified by blocking antiviral activity with a monoclonal antibody (Mab) against recombinant bovine IFN-gamma IFN-gamma mRNA appeared in MLN cells within 4 h of stimulation with phorbol ester and Concanavalin A and was not detectable by 72 h after stimulation. Biologically active IFN-gamma appeared in the culture supernatants 8 h after stimulation and was still present 96 h later when de novo synthesis had terminated. Acid dialysis and Mab neutralisation demonstrated conclusively that native ovine IFN-gamma is a pH 2 labile cytokine.

Animals↗