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2.
Vet Microbiol ; 182: 18-27, 2016.
Article in English | MEDLINE | ID: mdl-26711024

ABSTRACT

To improve our understanding of squirrelpox virus (SQPV) infection in the susceptible host, three red squirrels were challenged with wild-type SQPV via scarification of the hind-limb skin. All squirrels seroconverted to the infection by the end of the experiment (17 days post-challenge). Challenged animals suffered disease characterised by the development of multiple skin and oral lesions with rapid progression of skin lesions at the infection site by day 10 post-challenge. No internal pathological changes were found at post-mortem examination. A novel SQPV Taqman(®) Real-time PCR detected viral DNA from multiple organs, with the largest amounts consistently associated with the primary and secondary skin and oral lesions where viral replication was most likely occurring. Immunohistochemistry clearly detected viral antigen in the stratified squamous epithelium of the epidermis, tongue and the oropharyngeal mucosa-associated lymphoid tissue and was consistently associated with histological changes resulting from viral replication. The lack of internal pathological changes and the detection of relatively low levels of viral DNA when compared with primary and secondary skin lesions argue against systemic disease, although systemic spread of the virus cannot be ruled out. This study allowed a comprehensive investigation of the clinical manifestation and progression of SQPV infection with a quantitative and qualitative analysis of virus dissemination and shedding. These findings suggest two separate routes of SQPV transmission under natural conditions, with both skin and saliva playing key roles in infected red squirrels.


Subject(s)
Poxviridae Infections/veterinary , Poxviridae/physiology , Sciuridae/virology , Animals , DNA, Viral , Female , Host-Pathogen Interactions , Male , Poxviridae/classification , Poxviridae Infections/virology , Virus Shedding
3.
Vet Rec ; 167(8): 297-302, 2010 Aug 21.
Article in English | MEDLINE | ID: mdl-20729517

ABSTRACT

Postmortem and virological examinations for squirrelpox virus (SQPV) were carried out on 262 red squirrels (Sciurus vulgaris) found dead or moribund in Scotland between September 2005 and July 2009, to determine the likely causes of death and highlight factors that might be threats to the red squirrel population. Most of the squirrels were submitted from Dumfries and Galloway, and 71 per cent of them were adults. Road traffic accidents, squirrelpox, trauma or starvation were responsible for death in a large proportion (73 per cent) of the squirrels. Thin or emaciated body condition was associated with deaths resulting from pneumonia SQPV infection and starvation, and with the presence of external parasites. There were differences between age groups with regard to the cause of death; a large proportion of juveniles died of starvation, whereas a large proportion of subadults and adults died in road traffic accidents. SQPV infection was associated with the presence of external parasites, but was not associated with the sex of the animals.


Subject(s)
Poxviridae Infections/veterinary , Rodent Diseases/epidemiology , Rodent Diseases/pathology , Sciuridae , Sentinel Surveillance/veterinary , Wounds and Injuries/veterinary , Accidents, Traffic , Age Factors , Animals , Disease Outbreaks/veterinary , Disease Reservoirs/veterinary , Disease Reservoirs/virology , Female , Male , Poxviridae Infections/epidemiology , Poxviridae Infections/mortality , Poxviridae Infections/pathology , Rodent Diseases/mortality , Scotland/epidemiology , Sex Factors , Wounds and Injuries/epidemiology , Wounds and Injuries/mortality , Wounds and Injuries/pathology
4.
Epidemiol Infect ; 138(7): 941-50, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20412610

ABSTRACT

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.


Subject(s)
Parapoxvirus , Poxviridae Infections/veterinary , Rodent Diseases/epidemiology , Sciuridae/virology , Animals , Body Weight , England/epidemiology , Female , Male , Population Surveillance , Poxviridae Infections/epidemiology , Scotland/epidemiology , Seasons , Sex Factors , Weather
5.
Vet Immunol Immunopathol ; 136(1-2): 34-42, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20197203

ABSTRACT

Interferon-gamma (IFN-gamma) and interleukin (IL)-10 are cross-regulatory cytokines capable of driving and controlling the adaptive host immune response. The inter-relationship between IFN-gamma and IL-10 expression has not been defined in sheep despite biological evidence suggesting that they perform similar functions to their orthologues described in other species. To address this, we have developed a quantitative (q)PCR method to assess relative levels of IFN-gamma and IL-10 mRNA expression in activated ovine peripheral blood mononuclear cells (PBMC) and compared the kinetics of mRNA expression with amounts of cytokine secreted by the cells over a 96h period. PBMC were collected from sheep immunised with the nominal antigen ovalbumin (Ova) and re-stimulated in vitro with antigen and the T cell mitogen concanavalin A (ConA). The recall response to antigen was characterised by a single peak in IFN-gamma mRNA expression at 48h of culture (13-fold increase over unstimulated cells) and relatively lower expression of IL-10 mRNA (average 2-3-fold increase over the 96h culture period). Antigen-driven IFN-gamma protein concentration was greatest at the end of the culture period (96h) whereas IL-10 protein level was not elevated above that observed in unstimulated cells. The typical response to ConA was greater for both cytokines, with IFN-gamma mRNA expression peaking at 6h of culture (133-fold increase) then declining rapidly whereas IL-10 mRNA expression peaked at 24h (16-fold increase) and declined more gradually. Despite these differences in the relative kinetics of mRNA expression in mitogen-activated PBMC, the typical pattern of protein expression of the two cytokines was similar. Both showed a gradual rise in protein concentration starting from 12h of culture which was still rising at the end of the culture period (96h). These data demonstrate that the kinetics of mRNA expression for IFN-gamma and IL-10 in activated ovine PBMC do not necessarily correlate with detectable protein in culture.


Subject(s)
Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-10/biosynthesis , Interleukin-10/genetics , Sheep/genetics , Sheep/immunology , Animals , Base Sequence , DNA Primers/genetics , Female , In Vitro Techniques , Kinetics , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sheep/blood
6.
Vet Rec ; 164(17): 528-31, 2009 Apr 25.
Article in English | MEDLINE | ID: mdl-19395718

ABSTRACT

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.


Subject(s)
Poxviridae Infections/veterinary , Sciuridae/virology , Animals , Poxviridae Infections/blood , Poxviridae Infections/epidemiology , Scotland/epidemiology
7.
J Gen Virol ; 90(Pt 4): 970-977, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19264672

ABSTRACT

The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein. However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the BPSV protein.


Subject(s)
Conserved Sequence , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-2/metabolism , Parapoxvirus , Viral Proteins , Amino Acid Sequence , Animals , Cattle , Cloning, Molecular , Genetic Variation , Molecular Sequence Data , Orf virus/genetics , Orf virus/metabolism , Parapoxvirus/classification , Parapoxvirus/genetics , Parapoxvirus/metabolism , Pseudocowpox Virus/genetics , Pseudocowpox Virus/metabolism , Sequence Analysis, DNA , Sheep , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
9.
Antiviral Res ; 73(3): 169-74, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17049627

ABSTRACT

(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.


Subject(s)
Antiviral Agents/administration & dosage , Cytosine/analogs & derivatives , Ecthyma, Contagious/drug therapy , Orf virus/growth & development , Organophosphonates/administration & dosage , Administration, Topical , Animals , Cidofovir , Cytosine/administration & dosage , Ecthyma, Contagious/virology , Paraffin/administration & dosage , Sheep , Silicic Acid/administration & dosage
10.
J Virol ; 79(17): 11205-13, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16103172

ABSTRACT

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.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-2/metabolism , Orf virus/physiology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Disulfides , Glycosylation , Molecular Sequence Data , Protein Binding , Sequence Alignment , Structure-Activity Relationship , Viral Proteins/genetics , Virus Replication
11.
Br J Dermatol ; 152(4): 791-3, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15840117

ABSTRACT

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.


Subject(s)
Bites and Stings/virology , Chordopoxvirinae/isolation & purification , Hand Injuries/virology , Poxviridae Infections/virology , Seals, Earless/virology , Zoonoses , Adult , Amino Acid Sequence , DNA, Viral/analysis , Humans , Male , Sequence Alignment
12.
Vet Immunol Immunopathol ; 102(1-2): 67-76, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15451616

ABSTRACT

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.


Subject(s)
Interleukin-10/pharmacology , Interleukin-12/pharmacology , Leukocytes, Mononuclear/immunology , Lymphocyte Activation/immunology , Sheep/immunology , Animals , Concanavalin A/immunology , Concanavalin A/pharmacology , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Interferon-gamma/immunology , Interleukin-10/immunology , Interleukin-12/immunology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Lymphocyte Activation/drug effects , Recombinant Proteins/pharmacology , Sheep/blood
13.
J Gen Virol ; 83(Pt 11): 2845-2855, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12388821

ABSTRACT

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.


Subject(s)
Conserved Sequence , Endothelial Growth Factors , Endothelial Growth Factors/genetics , Genetic Variation , Intercellular Signaling Peptides and Proteins , Lymphokines , Orf virus/genetics , Sheep Diseases/virology , Viral Proteins/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cattle , Cell Line , Crystallography, X-Ray , Endothelial Growth Factors/chemistry , Endothelial Growth Factors/metabolism , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Lymphokines/chemistry , Lymphokines/genetics , Lymphokines/metabolism , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Sheep , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth Factor Receptor-2 , Vascular Endothelial Growth Factor Receptor-3 , Vascular Endothelial Growth Factors , Viral Proteins/chemistry , Viral Proteins/metabolism
14.
J Gen Virol ; 83(Pt 5): 1043-1048, 2002 May.
Article in English | MEDLINE | ID: mdl-11961258

ABSTRACT

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.


Subject(s)
Orf virus/enzymology , Pyrophosphatases/genetics , Deoxyuracil Nucleotides/metabolism , Orf virus/genetics
15.
Vet Immunol Immunopathol ; 83(3-4): 161-76, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11730927

ABSTRACT

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.


Subject(s)
Cytokines/biosynthesis , Ecthyma, Contagious/immunology , Orf virus/immunology , RNA, Messenger/biosynthesis , Skin Diseases, Viral/veterinary , Animals , Biopsy/veterinary , Cytokines/genetics , Ecthyma, Contagious/pathology , Ecthyma, Contagious/virology , Immunohistochemistry/veterinary , In Situ Hybridization/veterinary , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Orf virus/growth & development , RNA Probes/chemistry , RNA, Messenger/genetics , Sheep , Skin Diseases, Viral/immunology , Skin Diseases, Viral/pathology , Skin Diseases, Viral/virology
16.
Virus Genes ; 22(2): 141-50, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11324750

ABSTRACT

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.


Subject(s)
Genes, Viral , Genome, Viral , Orf virus/genetics , Virus Replication/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , DNA Restriction Enzymes , Molecular Sequence Data , Orf virus/pathogenicity , Restriction Mapping , Sheep , Transcription, Genetic , Virulence
17.
J Virol ; 74(3): 1313-20, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10627542

ABSTRACT

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.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Interleukin-2/antagonists & inhibitors , Orf virus/metabolism , Viral Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cattle , Cells, Cultured , Chromatography, Gel , Cricetinae , DNA, Complementary/genetics , Dimerization , Ecthyma, Contagious/virology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interleukin-2/metabolism , Keratinocytes/virology , Lymph/chemistry , Molecular Sequence Data , Orf virus/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sheep , Viral Proteins/chemistry , Viral Proteins/metabolism , Viral Proteins/pharmacology
18.
Cytokine ; 11(4): 249-56, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10328863

ABSTRACT

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.


Subject(s)
DNA, Complementary/genetics , Hematopoiesis/drug effects , Stem Cell Factor/genetics , Stem Cell Factor/pharmacology , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cattle , Cloning, Molecular , Colony-Forming Units Assay , Cricetinae , Cytokines/pharmacology , DNA Primers/genetics , Dogs , Gene Expression , Humans , In Vitro Techniques , Mice , Molecular Sequence Data , Rats , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Sequence Homology, Amino Acid , Sheep , Species Specificity
19.
Virus Genes ; 17(2): 107-15, 1998.
Article in English | MEDLINE | ID: mdl-9857983

ABSTRACT

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.


Subject(s)
Antiviral Agents/pharmacology , Interferon-alpha/pharmacology , Orf virus/genetics , RNA-Binding Proteins/genetics , Viral Proteins/genetics , Amino Acid Sequence , Base Sequence , Drug Resistance, Microbial , Molecular Sequence Data , Orf virus/metabolism , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Recombinant Proteins , Sequence Analysis, DNA , Transcription, Genetic , Viral Proteins/chemistry , Viral Proteins/metabolism
20.
Vet Immunol Immunopathol ; 63(3): 265-87, 1998 May 29.
Article in English | MEDLINE | ID: mdl-9656460

ABSTRACT

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.


Subject(s)
Paratuberculosis/immunology , Paratuberculosis/pathology , Sheep Diseases/immunology , Sheep Diseases/pathology , Animals , Animals, Newborn , Antibodies, Bacterial/blood , Antibodies, Bacterial/metabolism , Base Sequence , Cytokines/biosynthesis , Cytokines/genetics , DNA Primers/genetics , Female , Gene Expression , In Vitro Techniques , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocyte Activation , Lymphocyte Subsets/immunology , Lymphocyte Subsets/pathology , Male , Mycobacterium avium subsp. paratuberculosis/genetics , Mycobacterium avium subsp. paratuberculosis/immunology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/microbiology , Peyer's Patches/immunology , Peyer's Patches/pathology , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sheep , Sheep Diseases/microbiology
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