Biomedical subjects

S Vydelingum

Publications and source records attributed to S Vydelingum.

16 recordsLinked to original sources

PubMed1998

Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens.

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n = 47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n = 96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.

African Swine Fever↗

PubMed1998

Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus.

The authors evaluated the ability of a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect classical swine fever virus (CSFV) in comparison with virus isolation and detection by an indirect immunoperoxidase assay (VI-IPA). To determine the specificity of the assay, samples from 60 spleens, 45 tonsils, ten submandibular lymph nodes, eight mesenteric lymph nodes and four kidneys, collected from pigs of various ages which had been slaughtered in abattoirs in Canada (a population free from CSFV), were tested. All the samples tested gave negative results by both VI-IPA and RT-PCR. A total of 20 samples were passaged in porcine kidney (PK) 15 cells and retested by both assays. All were found to be negative, giving a specificity of 100%. To determine the analytical sensitivity of the assay, a similar comparative study was conducted, using CSFV grown in tissue culture and tonsil tissues from a CSFV-infected pig. For both infected tissues and tissue culture fluids, RT-PCR was ten times more sensitive than VI-IPA. Amounts as small as 0.6 infectious units per 100 mg of tissue were detected by RT-PCR, compared to 6 infectious units by VI-IPA. Similarly, RT-PCR could detect as little as 0.1 infectious unit per ml in tissue culture fluids, compared to one infectious unit per ml by VI-IPA. To determine diagnostic sensitivity, three coded panels (two internal and one external), comprising 45 samples from 14 pigs, were tested. The diagnostic sensitivity of both RT-PCR and VI-IPA was found to be 100% for both internal panels. The results of the external panel, apart from two samples that were missed by both RT-PCR and VI-IPA, were found to be in total agreement. These two samples remained negative after amplification in PK15 cells. All the RT-PCR results were based on a single test whereas, for the VI-IPA results, positive results were obtained for five samples only after an amplification round in PK15 cells. Application of the RT-PCR assay for the diagnosis of CSFV would enable improved detection of the virus in a shorter time period.

Animals↗

PubMed1998

Long-term regulation of leptin expression is correlated with adipocyte number in obese rats.

OBJECTIVE: To investigate long-term regulation of leptin expression in adipose tissues of obese JCR:LA-corpulent rats, which have been shown to overexpress leptin. DESIGN: Manipulation of adipose tissue growth in obese rats by dietary restriction. INTERVENTIONS: Weanling female obese rats were maintained on 1 of 3 diets until 8 months old. One group was allowed to feed ad libitum, the second was pair-fed with lean rats, and the third had food intake restricted to maintain weights equal to those of age-matched lean rats. OUTCOME MEASURES: Body and fat pad weights, leptin messenger RNA (mRNA) levels, and size and number of adipocytes in retroperitoneal fat pads. RESULTS: Adipose tissue mass was increased 6-fold in the obese rats compared with the lean ones, despite equal body weight and intake restriction that was sufficient to impair growth. Although leptin mRNA level was down-regulated by intake restriction, it was still twice as elevated in the obese rats as in the lean ones, and was highly correlated with specific fat pad mass and adipocyte number, but not with size. CONCLUSIONS: These data suggest that leptin expression is correlated with adipocyte number within a fat pad, and that there is inappropriate hepatic de novo synthesis and storage of triacylglycerols in obese rats. A role for leptin in nutrient partitioning is proposed.

Adipocytes↗

PubMed1996

Evaluation of nucleic acid amplification methods for the detection of hog cholera virus.

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

African Swine Fever Virus↗

PubMed1995

Overexpression of the obese gene in the genetically obese JCR:LA-corpulent rat.

Expression of the obese (ob) gene in JCR:LA-cp rats was examined. A 360 bp fragment of the conserved region of the gene was obtained by RT-PCR using total RNA isolated from adipose tissues of Sprague-Dawley (SD), JCR:LA-cp obese and lean rats. The three gene fragments were sequenced and shown to be identical. They were over 90% identical to the mouse ob gene sequence. The amplified fragments encode for 120 amino acids and have a glutamine residue at position +49. The gene was shown to be expressed only in adipose tissues, both white and brown. A ten-fold increase in ob mRNA was detected in white adipose tissues of obese animals compared to the lean ones of the JCR-LA:cp strain of rat. Ob gene was expressed in adipocytes and preadipocytes from the obese rat whereas in the lean and SD rats, ob gene expression was found in adipocytes only. No ob mRNA was detected in preadipocytes from the lean or SD rats, indicating a differentiation or maturation-dependent expression in normal rats.

Adipocytes↗

PubMed1995

Replication of measles virus in human monocytes and T cells.

Replication of measles virus (MV) in populations of peripheral blood mononuclear cells enriched for T cells and monocytes was studied using a temperature-sensitive mutant, MV ts38, and the parent counterpart, MV Lec. Stimulation of the cells was required for a full cycle of virus replication in both cell types. More infectious virus was released after stimulation from MV-infected populations enriched for T cells, T cell-enriched than from monocyte-enriched populations. However, similar amounts of viral mRNA, genomic RNA, and viral proteins of the expected size were found in both cell populations. The results indicate that MV-specific macromolecular synthesis is similar in both T cells and monocytes, but the assembly and (or) release of infectious virus is greatly reduced in monocytes as compared with T cells.

Cell Division↗

PubMed1994

Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1).

The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identified 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topoisomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV-encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.

African Swine Fever Virus↗

PubMed1993

African swine fever virus genome content and variability.

A 55 kilobase pair (kb) region from the right end of the virulent African swine fever virus isolate, Malawi LIL20/1, has been sequenced. The 68 major open reading frames (ORFs) encoded are generally closely spaced and read from both DNA strands across the complete sequence. Comparison of the amino acid sequences of predicted ORFs with sequence databases identified 15 ORFs which encode proteins that are similar to proteins of known function. Two ORFs are homologous to copies of multigene family 360 (MGF360) and one ORF is homologous to copies of multigene family 110 (MGF110). Both of these multigene families have been described previously.

African Swine Fever Virus↗

PubMed1993

Duplicated genes within the variable right end of the genome of a pathogenic isolate of African swine fever virus.

The right variable region of the genome of a pathogenic strain of African swine fever virus (ASFV), Malawi LIL20/1, has been sequenced and 15 open reading frames (ORFs) identified by computer analysis. Eight of these ORFs were found to be similar to previously described ASFV ORFs and three of these belong to two previously described multiple gene families (MGF), 360 and 110. Four of the remaining five ORFs belong to a novel MGF, designated MGF 100, and the last ORF encodes a protein that is similar to the virus structural protein, p22. Copies of MGF 110 and the gene coding for p22 have previously been characterized only at the left end of the ASFV genome. The organization of these genes suggests evolution by duplications, deletions and sequence transposition from one end of the genome to the other. Sequence comparisons of members of MGF 360 suggest that the Malawi LIL20/1 genome has undergone separate DNA rearrangements compared to the Ba71V genome. Lastly, one ORF was found to be similar to the myeloid differentiation primary response protein, MyD116 and to the herpes simplex virus neurovirulence-associated factor ICP34.5.

African Swine Fever Virus↗

PubMed1993

Three African swine fever virus genes encoding proteins with homology to putative helicases of vaccinia virus.

Sequence analysis of the SalI g, h, i and j restriction fragments of the African swine fever virus (ASFV) genome from the virulent isolate Malawi (LIL20/1) identified three open reading frames (ORFs) encoding predicted proteins of 125.0K (g10L), 80.4K (j10L) and 58.0K (j11L) which showed homology to members of the DNA and RNA helicase superfamily. ORFj10L was related to protein 4 of the Kluyveromyces lactis killer plasmid pKG12 and to two putative helicases, D6R and D11L, of vaccinia virus. ORF g10L was most closely related to ASFVj10L and to vaccinia virus D11L. ORFj11L was homologous to A18R, a third putative helicase of vaccinia virus. The possible functions of these genes in the replication of ASFV are discussed and the evolutionary implications are considered.

African Swine Fever Virus↗

PubMed1993

African swine fever virus encodes a serine protein kinase which is packaged into virions.

Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions.

African Swine Fever Virus↗

PubMed1992

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.

African Swine Fever Virus↗

PubMed1989

Infection of human peripheral blood mononuclear cells with a temperature-sensitive mutant of measles virus.

A stable temperature-sensitive mutant of measles virus (MV ts38) was used to study the mechanism of virus-mediated immune suppression of peripheral blood mononuclear cells in vitro. Both unstimulated and phytohemagglutinin-stimulated cultures released infectious virus at 32 degrees C, whereas no virus was released at 37 degrees C, although both viral RNA and viral proteins were synthesized. However, the response of the lymphoid cells to phytohemagglutinin, concanavalin A, and herpes simplex virus antigen was decreased in the presence of MV ts38 at 37 degrees C. The viability of infected cells was not diminished, therefore excluding cell death as a reason for immunosuppression. Interleukin 2 did not play a role in the inhibitory effect of MV ts38. Antibodies to alpha interferon partially reversed the inhibitory effect of the virus infection on lymphocyte mitogenesis, thus implying that alpha interferon plays a role in the immunosuppression. Depletion experiments indicated that adherent cells play a greater role in the measles virus-induced immunosuppression than nonadherent cells. However, monocyte maturation to macrophages had no effect on the degree of immunosuppression.

Cell Differentiation↗

PubMed1985

The role of interleukins in viral inhibition of lymphocyte mitogenesis.

Several different types of retroviruses have been shown to inhibit the lectin-driven blastogenesis of co-incubated mouse spleen cells. This abrogation of responsiveness is infection-independent, and can be obtained using live as well as UV-inactivated virus particles. Levels of both interleukin-1 (IL-1) and T-cell growth factor (TCGF) activities are greatly reduced in cultures of virus co-incubated cells. However, the inclusion of indomethacin in the culture medium, together with virus, enables responsiveness to Concanavalin A to occur at moderately efficient levels. The addition of exogenous IL-1 activity to virus co-incubated cells is also partially restorative to Con A-driven lymphocyte responsiveness, while at the same time restoring levels of detectable TCGF activity to near-control levels. Finally, the inclusion of exogenous TCGF activity also leads to efficient lymphocyte mitogenesis but does not result in increased levels of IL-1.

Animals↗

PubMed1984

Reversible interference with TCGF activity by virus particles.

Several types of virus particles including retroviruses and herpes viruses can impede the ability of human peripheral blood lymphocytes to respond to mitogenic stimuli. This is true even in the case of viruses that have been inactivated by u.v. light, indicating that the mechanisms involved are infection independent, at least in part. The observed inhibition could be shown to correlate with a reduced level of production of TCGF activity in the virus co-incubated cultures. In addition, these same viruses are apparently able to complex directly with TCGF activity and to render it biologically inactive. This was shown by allowing known quantities of virus to interact with TCGF activity for different periods of time, followed by centrifugation of any virus-TCGF complexes. This interference is reversible, however, and the dissociation of these virus-TCGF complexes by mild detergent, followed by viral centrifugation, yields TCGF activity in the supernatant.

Cells, Cultured↗

PubMed1983

Viral inhibition of lymphocyte mitogenesis: interference with the synthesis of functionally active T cell growth factor (TCGF) activity and reversal of inhibition by the addition of same.

We have investigated the mechanisms whereby co-incubation of several types of virus particles with human lymphoid cells in the presence of T cell lectins leads to inhibition of the proliferative response that otherwise ensues. The data indicate that, in the absence of infection, such inhibition can be reversed by the addition to cultures of relatively high concentrations of fluids rich in T cell growth factor (TCGF) activity. The ability of these fluids to achieve such reversal of inhibition is both concentration- and time-dependent. Addition of the factor to virus co-incubated cells more than 26 hr after culture initiation does not restore responsiveness. We have also shown that virus co-incubated cultures are deficient with respect to their ability to synthesize detectable levels of TCGF activity in the presence of phytohemagglutinin. In contrast, the use of relatively dilute virus preparations (less than 10 particles per cell) permits partial responsiveness to lectin as well as the synthesis of moderate levels of TCGF. These finding suggest that viral inhibition of lymphocyte mitogenesis is mediated directly or indirectly by interference with the synthesis of functionally active TCGF activity.

Animals↗