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D M Stone

Publications and source records attributed to D M Stone.

At least 19 recordsLinked to original sources
PubMed2003

Emerging vesiculo-type virus infections of freshwater fishes in Europe.

Rhabdoviruses were isolated from perch Perca fluviatilis and largemouth bass Micropterus salmoides exhibiting clinical signs of disease. Preliminary studies indicated that these viruses could be neutralised by antisera to perch rhabdovirus (Dorson et al. 1984) and may be similar to those previously isolated from grayling Thymallus thymallus and pike-perch Stizostedion stizostedion. The relationship between these viruses and the previously characterised fish rhabdoviruses, pike fry rhabdovirus (PFRV), spring viraemia of carp virus (SVCV) and lake trout rhabdovirus, was investigated. Viruses were propagated in bluegill fry (BF-2) cells and were characterised using electron microscopy, serum neutralisation tests, immunofluorescence tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nucleotide sequence analysis. The bullet-shaped viral particles appeared to be compact, with spikes visible at the surface, a morphology similar to that of the vesiculovirus group of rhabdoviruses. Serum neutralisation tests showed that the viruses were antigenically closely related to the previously characterised perch rhabdovirus, but were not significantly neutralised by antisera to PFRV, SVCV or viral haemorrhagic septicaemia virus (VHSV). In immunofluorescence tests with perch rhabdovirus antisera, strong specific fluorescence was observed in cell cultures infected with the new rhabdovirus isolates, but no fluorescence was observed with antisera to PFRV, SVCV or VHSV. SDS-PAGE analysis revealed a polypeptide profile typical of vesiculoviruses, but the novel virus isolates had different relative mobilities of their P and M proteins compared to PFRV and SVCV. Nucleotide sequence analysis was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing of a 439 base-pair region of the viral L gene. The novel rhabdovirus isolates had <76% nucleotide sequence identity to PFRV, SVCV and lake trout rhabdovirus and >95% identity to perch rhabdovirus. Phylogenetic analysis using both maximum parsimony and neighbour-joining methods assigned the perch rhaboviruses to a separate group to that of PFRV, SVCV and lake trout rhabdovirus. These data are the initial characterisation of a group of emerging fish vesiculo-type viruses that are biochemically and genetically distinct from the PFRV, SVCV and lake trout rhabdoviruses.

Animals↗
PubMed2003

Isolation of a rhabdovirus during outbreaks of disease in cyprinid fish species at fishery sites in England.

A virus was isolated during disease outbreaks in bream Abramis brama, tench Tinca tinca, roach Rutilis rutilis and crucian carp Carassius carassius populations at 6 fishery sites in England in 1999. Mortalities at the sites were primarily among recently introduced fish and the predominant fish species affected was bream. The bream stocked at 5 of the 6 English fishery sites were found to have originated from the River Bann, Northern Ireland. Most fish presented few consistent external signs of disease but some exhibited clinical signs similar to those of spring viraemia of carp (SVC), with extensive skin haemorrhages, ulceration on the flanks and internal signs including ascites and petechial haemorrhages. The most prominent histopathological changes were hepatocellular necrosis, interstitial nephritis and splenitis. The virus induced a cytopathic effect in tissue cultures (Epithelioma papulosum cyprini [EPC] cells) at 20 degrees C and produced moderate signals in an enzyme immunoassay (EIA) for the detection of SVC virus. The virus showed a close serological relationship to pike fry rhabdovirus in both EIA and serum neutralisation assays and to a rhabdovirus isolated during a disease outbreak in a bream population in the River Bann in 1998. A high degree of sequence similarity (> or = 99.5% nucleotide identity) was observed between the English isolates and those from the River Bann. Experimental infection of juvenile bream, tench and carp with EPC cell-grown rhabdovirus by bath and intraperitoneal injection resulted in a 40% mortality of bream in the injection group only. The virus was re-isolated from pooled kidney, liver and spleen tissue samples from moribund bream. The field observations together with the experimental results indicate that this rhabdovirus is of low virulence but may have the potential to cause significant mortality in fishes under stress.

Animals↗
PubMed2003

Four years of monitoring for viral haemorrhagic septicaemia virus in marine waters around the United Kingdom.

Between 1995 and 1998, marine fish from around the coast of the UK were collected and samples analysed for viral haemorrhagic septicaemia virus (VHSV) using cell culture isolation methods. In 1997 and 1998 the samples were also analysed for VHSV by reverse transcription PCR (RT-PCR). A total of 1867 fish of 11 species were tested, but VHSV was isolated on only 1 occasion, from herring Clupea harengus, in 1996. However, despite VHSV not being isolated in 1997 and 1998, in both years samples of herring from the west and south coasts of England produced positive signals in the RT-PCR, and in 1997 cod from the east coast of England also produced positive signals in the RT-PCR. These results are believed to be true indications of the presence of VHSV nucleic acid in the fish. In 1997, birnaviruses from Serogroup B1 were isolated from herring (a previously unrecorded host for the virus) and cod Gadus morhua, and a birnavirus from Serogroup A2 was also isolated from cod. In 1998, an aquareovirus was isolated from haddock Melanogrammus aeglefinus, a previously unrecorded host for the virus.

Animals↗
PubMed2003

Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups.

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of putative spring viraemia of carp viruses (SVCV) and pike fry rhabdoviruses (PFRV), including isolates from tench, grass carp, roach, bream and false harlequin, sheatfish and orfe. Phylogenetic analysis of a 550 nucleotide (nt) region of the glycoprotein gene identified 4 groups, I to IV. Significantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared <80% nucleotide identity with the PFRV reference strain (Genogroup III). The similarity between another PFRV-like virus isolated from grass carp and representatives of Genogroups III and IV was also <80%, indicating that this virus belonged to a third group (Genogroup II). All of the putative SVC viruses were assigned to a 4th group (Genogroup I), sharing <61% nucleotide identity with viruses in Genogroups II to IV.

Amino Acid Sequence↗
PubMed2003

Lethal and sublethal effects of a neem-based insecticide on balsam fir sawfly (Hymenoptera: Diprionidae).

Lethal and sublethal effects of Neemix 4.5 EC, a commercial neem preparation, on balsam fir sawfly, Neodiprion abietis (Harris), were determined in the laboratory. Larval mortality of N. abietis increased in a concentration-dependent manner, and lethal time decreased with increasing Neemix 4.5 EC concentration. Fifty percent of the larvae died after 4.6 d at a concentration of 90 ppm azadirachtin (AZA) and 12.3 d at a concentration of 0.08 ppm. Neemix 4.5 EC showed some deterrent effects to feeding site selection on N. abietis larvae at high concentrations, but not at low concentrations. Strong reduction of food intake by N. abietis larvae after exposure to Neemix 4.5 EC was demonstrated by significant reduction of frass production. Larvae fed on Neemix 4.5 EC-treated foliage at 90 ppm AZA produced only 16% as much frass as that produced by larvae fed on control foliage (0 ppm). Neemix 4.5 EC at a concentration of 0.08 ppm AZA retarded larval and pupal development. Sublethal doses significantly reduced pupal weight and adult emergence, although the sex ratio of N. abietis adults was not affected. Results indicate that sublethal effects of Neemix 4.5 EC on N. abietis may contribute greatly to the overall field efficacy.

Abies↗
PubMed2001

Isolation and characterisation of rhabdovirus from wild common bream Abramis brama, roach Rutilus rutilus, farmed brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss in Northern Ireland.

Rhabdovirus was isolated from wild common bream Abramis brama during a disease outbreak with high mortality in Northern Ireland during May 1998. Rhabdovirus was also isolated at the same time from healthy farmed rainbow Oncorhynchus mykiss and brown trout Salmo trutta on the same stretch of river and 11 mo later from healthy wild bream and roach Rutilus rutilus in the same river system. Experimental intra-peritoneal infection of bream and mirror carp Cyprinus carpio var specularis with 2 of these isolates produced low mortality rates of < or = 12%. Serological testing of these isolates by virus neutralisation indicated that they were antigenically closely related to pike fry rhabdovirus (PFRV) but not to spring viraemia of carp virus (SVCV), while testing by enzyme-linked immunosorbent assay indicated them to be antigenically different from both. Comparison of nucleotide sequence data of a 550 base pair segment of the viral glycoprotein generated by reverse transcription-polymerase chain reaction indicated a high (> or = 96.6%) degree of similarity between these isolates and a previous Northern Ireland isolate made in 1984, a 1997 isolate from bream in the Republic of Ireland and an earlier Dutch isolate from roach. In contrast, similarity between these isolates and PFRV was < 82.4%, indicating that these viruses belong to 2 distinct genogroups, while similarity to SVCV was even lower (< 67.4%).

Amino Acid Sequence↗
PubMed2001

Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus.

A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.

Animals↗
PubMed2001

Live-attenuated strains of improved genetic stability.

The current live-attenuated vaccine strains of poliovirus are genetically unstable and capable of rapid evolution in human hosts, resulting in reversion to neurovirulence and, occasionally, disease. They can also be shed by recipients for a considerable time after vaccination. This raises questions about how and when to stop vaccination after wild-type viruses have been eliminated. Persistence of vaccine revertant viruses in the population would present a risk to new cohorts of unvaccinated children and threaten the success of the eradication programme. A number of Sabin vaccine strain derivatives have been described that are, in theory, genetically more stable than the present vaccines and therefore less likely to revert to virulence. The approaches used in their derivation are outlined here and data presented for two strains showing a significant improvement in genetic stability. These strains were designed according to our understanding of the molecular basis of attenuation and incorporate changes in the sequence of an RNA structural domain that plays a key role in attenuation. They may also be less transmissible than the current type 3 vaccine strain and are potentially useful in the strategically difficult final stages of poliomyelitis eradication.

5' Untranslated Regions↗
PubMed2000

CD4 T lymphocyte activation in BLV-induced persistent B lymphocytosis in cattle.

Bovine leukemia virus (BLV) is an oncogenic retrovirus in the human T cell leukemia virus family. BLV infects B lymphocytes and induces a nonmalignant persistent lymphocytosis (PL) and leukemia/lymphoma in cattle. There is evidence that CD4 T lymphocytes are activated during BLV infection and promote the development of PL. How CD4 T lymphocytes are activated by BLV infection is not known. We observed that CD4 T lymphocytes from PL cattle proliferated in the presence of autologous, irradiated peripheral blood mononuclear cells (PBMC), whereas no proliferation occurred in cell cultures from BLV-infected non-PL cattle. Proliferation required direct contact with metabolically active irradiated PBMC but was not associated with viral protein expression or inhibited by antibodies to BLV. Unexpectedly, B lymphocytes alone failed to account for the irradiated PBMC stimulation of CD4 T lymphocytes. These observations and the magnitude of the proliferative response suggest that activation is polyclonal and involves mechanisms other than BLV antigen-specific stimulation.

Animals↗
PubMed2000

Nucleotide sequence analysis of the entire coding regions of virulent and avirulent strains of viral haemorrhagic septicaemia virus.

The complete nucleotide sequences of the six open reading frames (N, P, M, G, NV and L) and the intergenic regions were determined for two virulent freshwater strains and two avirulent marine strains of viral haemorrhagic septicaemia virus (VHSV). Sequence analysis of 10,845 nucleotides revealed > 97.2% nucleotide sequence similarity and > 98.6% amino acid similarity, confirming the close genetic relationship between marine and freshwater strains of VHSV. Moreover, as few as 10 amino acid substitutions were identically substituted between the marine and freshwater strains investigated. The results of this study suggest that only a limited number of amino acid residues may be involved in the determination of VHSV virulence for salmonids and further highlights the potential risk that marine strains may pose to freshwater aquaculture.

Animals↗
PubMed2000

Spontaneously proliferating lymphocytes from bovine leukaemia virus-infected, lymphocytotic cattle are not the virus-expressing lymphocytes, as these cells are delayed in G(0)/G(1) of the cell cycle and are spared from apoptosis.

Bovine leukaemia virus (BLV) is in the family of oncogenic retroviruses which includes human T cell leukaemia virus (HTLV). BLV infects B lymphocytes and induces a non-neoplastic persistent lymphocytosis (PL) of B lymphocytes in cattle. A characteristic of BLV- and HTLV-induced disease is spontaneous lymphocyte proliferation of cultured peripheral blood mononuclear cells (PBMC). To investigate the role of virus expression on lymphocyte survival and proliferation, we evaluated cell cycle position, apoptosis and virus expression on a single-cell basis of cultured PBMC from BLV-infected PL cattle, BLV-infected non-PL cattle and uninfected cattle. Results demonstrated that the majority of bovine B lymphocytes spontaneously entered G(2)/M of the cell cycle and died by apoptosis by 24 h post-culture, regardless of BLV infection or PL status. The spontaneous proliferation that characterizes PL cattle was primarily due to a small population of surviving B lymphocytes, but T lymphocytes also contributed. Viral protein expression was detectable in only 5-15% of cultured PBMC from PL cattle and the majority of these lymphocytes were delayed in cell cycle and spared from apoptosis. Unexpectedly, we determined that only 3% of the spontaneously proliferating lymphocytes expressed viral proteins. Previous reports show that spontaneous proliferation decreases when virus expression is suppressed. Together with our results, this suggests that virus expression by one population of B lymphocytes promotes proliferation of another population of B lymphocytes that does not express virus. This may be due to an effect of virus on CD4 T lymphocytes, as depletion of CD4 T lymphocytes significantly decreased spontaneous proliferation.

Animals↗
PubMed1999

Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli.

Drosophila Suppressor of fused (Su(fu)) encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Here we describe the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) (hSu(fu)). Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms were expressed in multiple embryonic and adult tissues, and exhibited a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus mapped to chromosome 10q24-q25, a region which is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) was found to repress activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homologue. Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway.

Adult↗
PubMed1999

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Amino Acid Sequence↗
PubMed1998

Characterization of two patched receptors for the vertebrate hedgehog protein family.

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human PTCH2 gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.

Amino Acid Sequence↗
PubMed1998

B-lymphocyte proliferation during bovine leukemia virus-induced persistent lymphocytosis is enhanced by T-lymphocyte-derived interleukin-2.

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5+ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-gamma) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.

Animals↗
PubMed1997

Modulation of bovine leukemia virus-associated spontaneous lymphocyte proliferation by monoclonal antibodies to lymphocyte surface molecules.

Both human T lymphotropic virus (HTLV) and bovine leukemia virus (BLV) infections are characterized by in vitro proliferation of peripheral blood lymphocytes in the absence of exogenous antigens or mitogens. Differential expression of lymphocyte surface molecules in HTLV and BLV infection suggests that lymphocyte dysregulation may involve signaling through surface molecules involved in immune regulation. We examined the expression of adhesion and major histocompatibility (MHC) molecules on circulating lymphocytes from BLV-infected cows with persistent lymphocytosis and the ability of monoclonal antibodies to these molecules to modulate spontaneous lymphocyte proliferation. The integrin molecule, CD11c, and both MHC class I and MHC class II molecules were upregulated on B and T lymphocytes from PL cows. Anti-CD11c antibody was stimulatory to lymphocyte proliferation regardless of BLV status and had a greater stimulatory effect on spontaneously proliferating lymphocytes from persistently lymphocytotic cows than on normal bovine lymphocytes. Antibodies to bovine class I and class II inhibited spontaneous lymphocyte proliferation. Results suggest that lymphocyte dysregulation in BLV-induced persistent lymphocytosis involves upregulation of and signaling through lymphocyte surface molecules which are involved in immune activation of lymphocytes.

Animals↗
PubMed1997

Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1.

Sonic hedgehog (Shh) is a putative morphogen secreted by the floor plate and notochord, which specifies the fate of multiple cell types in the ventral aspect of the vertebrate nervous system. Since in Drosophila the actions of Hh have been shown to be transduced by Cubitus interruptus (Ci), a zinc finger transcription factor, we examined whether a vertebrate homolog of this protein can mediate the functions of Shh in the vertebrate nervous system. Here, we demonstrate that expression of Gli-1, one of three vertebrate homologs of Ci, can be induced by Shh in the neural tube. Further, ectopic expression of Gli-1 in the dorsal midbrain and hindbrain of transgenic mice mimics the effects of ectopically expressed Shh-N, leading to the activation of ventral neural tube markers such as Ptc, HNF-3beta, and Shh; to the suppression of dorsal markers such as Pax-3 and AL-1; and to the formation of ectopic dorsal clusters of dopaminergic and serotonergic neurons. These findings demonstrate that GLI-1 can reproduce the cell patterning actions of Shh in the developing nervous system and provide support for the hypothesis that it is a mediator of the Shh signal in vertebrates.

Animals↗