PubMed Health⌕ Search

Biomedical subjects

S Vilcek

Publications and source records attributed to S Vilcek.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of herd-specific bovine viral diarrhoea virus isolates from infected cattle and sheep.

Thirteen pestiviruses isolated from ruminants on four different farms in Sweden were compared antigenically and genetically. On two farms, viruses were isolated from both cattle and sheep, a third farm contained only sheep and a fourth only cattle. Seven viruses were isolated from six different cattle and six viruses were isolated from five different sheep. Epitope conservation between the viruses was studied with a panel of 32 monoclonal antibodies, revealing that all of the isolates were BVDV-like. However, certain epitopes present in isolates from cattle were lost following virus transmission to sheep. In vitro amplification of the 5'-untranslated region of the 13 isolates by the polymerase chain reaction (PCR) and subsequent analyses of amplified products with restriction enzymes also indicated that all 13 isolates belong to the BVDV group of pestiviruses. A fragment of the E2 (gp53) gene of each virus was amplified by PCR and a comparison of the amplified sequence of 188 nucleotides separated the isolates into four groups each of which could be identified with a particular farm of origin. The 13 isolates were thus herd-specific rather than species-specific demonstrating that BVDV is readily transmitted between cattle and sheep.

Animals↗
PubMed1995

Detection of bovine herpesvirus 1 in clinical samples by the polymerase chain reaction.

A PCR assay with primers selected from the gl gene and flanking a 468 bp DNA fragment was tested on clinical samples. Of 27 samples (nasal swabs, lung, lymph nodes, tracheal mucosa) collected from 16 different outbreaks in Scotland, 18 were positive by PCR and 13 by virus isolation. Some samples of isolated DNA had to be diluted by a factor 50-100 to obtain a positive PCR result. PCR assay could detect the BHV-1 positive samples collected from different European regions, namely from Slovakia, Italy, England, Scotland as well as the IPV sample.

Animals↗
PubMed1994

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis.

A polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5' non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.

Animals↗
PubMed1994

Evaluation of a 'one tube' reverse transcription-polymerase chain reaction for the detection of ruminant pestiviruses.

A 'one tube' reverse transcription-polymerase chain reaction ('one tube' RT-PCR) using rTth DNA polymerase was compared with an existing RT-PCR using Taq DNA polymerase (Taq RT-PCR) to detect ruminant pestiviruses in infected cell cultures. The technically simpler and more convenient 'one tube' method was relatively insensitive detecting only 11 of the 34 samples tested, all of which were positive by Taq RT-PCR.

Animals↗
PubMed1994

Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay based on primers from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size was amplified using DNA from herpesviruses isolated from reindeer, red deer and goats. The PCR assay was able to detect virus in nasal swabs up to 14 days after experimental infection of cattle and there was a good correlation when PCR was compared with virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful.

Animals↗
PubMed1994

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.

Acute Disease↗
PubMed1994

[Development of PCR tests for the detection of bovine herpesvirus-1, bovine respiratory syncytial viruses and pestiviruses].

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.

DNA, Viral↗
PubMed1993

Detection of the bovine herpesvirus-1 (BHV-1) genome by PCR.

The amplification of the 468 bp fragment of the BHV-1 genome by PCR is described. The 22 bp oligomers from the BHV-1 gI gene were used as primers. For successful amplification the thermal denaturation (100 degrees C/8 min, ice) of the DNA sample was carried out prior to the cycling (95 degrees C for 1 min, 56 degrees C for 1 min, 73 degrees C for 1 min). The presence of glycerol markedly enhances the PCR.

Base Sequence↗
PubMed1993

The effect of the mode of sampling on BHV-1 detection in infected cattle by dot-blot hybridization.

The BHV-1 genome in nasal swabs and washings was detected by dot-blot hybridization using the 32P-pUR-1 probe (1.8 kb EcoRI-HindIII random fragment of BHV-1 DNA ligated into the pUC-9 plasmid) as early as on day 1 after the experimental infection of cattle. In dependence on the sampling method, differences were observed in the maximum of hybridization signals. During nasal swab analyses maximum amounts of BHV-1 differed in the individual samples (day 1-3). Hybridization signals obtained at the analysis of BHV-1 DNA nasal washings did not vary but showed a continuous maximum on day 2 after infection. Nasal washings proved to be more advantageous for detection of the BHV-1 genome by the hybridization technique.

Animals↗
PubMed1993

Detection of bovine herpesvirus 1 with various types of DNA probes.

The method of dot-blot hybridization on nitrocellulose filters by various types of DNA probes (ds recombinant plasmids, ss recombinant M13 phages and a 42bp synthetic oligonucleotide) was used for BHV-1 detection. The highest sensitivity was achieved with the 32P-pUR1 probe (1.8 kb random EcoRI-HindIII fragment inserted into pUC9) which detected the BHV-1 genome in 5 x 10(3) infected MDBK cells. Using the pUR1 probe, no cross hybridization was observed with other herpesviruses: BHV-2, 3, 4, and Aujeszky's disease virus. The 32P-pUR1 probe detected BHV-1 in nasal swabs of calves as early as on day 1 after experimental infection. The maximum intensity of BHV-1 detection occurred on day 1-3. The 32P-pUR1 probe also detected BHV-1 in field samples of nasal swabs from cows and calves.

Animals↗
PubMed1993

[Detection of bovine herpesvirus-1 using the dot-blot hybridization method].

With the development of molecular biology in the 1980s methods of microorganism detection start to innovate. One of the main advantages of the molecular-genetic methods, namely hybridization of nucleic acids and PCR methods, is the detection of genome of microorganism without the need for cellular cultivation. To detect BHV-1 (etiological agens IBR-IPV) the dot-blot hybridization method on nitrocellulose filters was used together with different types of DNA probes (two-fiber recombinant plasmids, one-fiber recombinant phages M 13 and 40 bp synthetic oligonucleotide). Genome DNA BHV-1 was isolated from samples (virions, infested cells, nasal smears and secretions by phenol extraction). The highest sensitivity of detection was achieved with 32P-pUR-1 probe (1.8kb random EcoRI-Hind III fragment ligated into plasmid pUC 9) which detected genome BHV-1 in 5 x 10(3) infested MDBK cells. This probe did not respond with herpetic viruses BHV-2, BHV-3, BHV-4 and the virus of Aujeszky's disease. The quality of pUR-1 probe was further tested for IBR diagnostics in animals experimentally infested with the virus BHV-1 (intranasal infection). BHV-1 could be detected in nasal smears and secretions in experimentally infested calves as early as on the first day following infection, while the agens amount reached its peak on the days 2-3 and on the days 6-7 the occurrence of virus fell markedly. When digoxigenin-pUR-1, i.e. non-radioactively marked probe, the virus presence was confirmed only on the days 2-3, in the time of the highest occurrence of infection agens. To detect the virus through the dot-blot hybridization nasal secretions were confirmed as better compared with nasal smears. The technology of virus isolation on cell cultures confirmed also the occurrence of agens as soon as on the first day from infection, with maximum on the days 2-5, but much more reliably it detected the virus on the days observed from the day 3 and their peak was obtained on the day 6 from infection. Experiments, comparing classical methods of IBR diagnostics (detection of specific antibodies, the method of isolation on cellular cultures) with the dot-blot hybridization using the samples obtained from farms with natural occurrence of IBM, are under progress.

Animals↗
PubMed1993

[In vitro amplification of genome fragments of the mucosal disease virus (BVD-MD) using the PCR method].

DNA in-vitro amplification when a PCR (polymerase chain reaction) method is used (Saiki et al., 1985) provides for a simple technique of marked amplification of a selected DNA fragment. The length of a DNA amplified fragment is determined by two synthetic primers which spontaneously (at an appropriate temperature) hybridize with the opposite ends of antiparallelly oriented strains of denatured DNA. The enzyme Taq polymerase completes the synthetisation of new DNA strains from the primers. Repetition of these cycles (denaturing, primer bonds, DNA synthesis) enhances the DNA amplification of a defined strain length to such an extent that is possible to prove this process by e.g. electrophoretic analysis. For the purposes of a proof of BVD-MD genome in cattle the fragment 315 bp was chosen from the virus-coding gene gp 80. The primers P1-5'-GTAGGTAGAGTGAAACCCGG-3' and P2-5'-CGGGACCTGGACTTCATAGC-3' (Hertig et al., 1991) determined the length of the amplified fragment. Virus RNA was isolated from the infectious BVD-MD-containing medium (Ph strain) using a phenol-chloroform (1:1) mixture, and before amplification it was transcribed to cDNA in the P2 presence by the effect of AMV reverse transcriptase. cDNA without isolation from the transcription reaction mixture was directly used for PCR. DNA was denatured at 94 degrees C for 10 minutes before the outset of amplification. These reaction conditions are suitable for the PCR method: P1 and P2 primer concentrations per 100 microliters reaction solution-1 microM, dNTP-100 microM, 2-4 U Taq polymerase, 25-35 amplification cycles with the temperature regime: 94 degrees C/l min, 56 degrees C/l min, 73 degrees C/l min, and prolonged incubation 73 degrees C/7 min in the last cycle. Proof of the amplified product 315 bp DNA-electrophoretic analysis of 1.5 to 2% agarose gel in TAE buffer and ethidium bromide staining of gel are suitable. The introduced PCR method gives opportunities for innovations of BVD-MD diagnosis in cattle on the basis of virus genome proof without any cell cultivation need.

DNA, Viral↗
PubMed1992

[Isolation and control of the functional quality of mRNA of bovine leukocyte interferon].

For the construction of the cDNA library total cellular RNA was separated from bovine leucocytes induced with NDV (inductor of interferons) for five hours. Guanidinisothiocyanate and ultracentrifugation in the gradient of CsCl were used for the separation of the total RNA (Chirgwin et al., 1979). Bands 18S and 28S were detected in the samples of RNA by electrophoresis in an MOPS buffer (Fig. 1). mRNA was separated from the mixture of RNA via affinity chromatography on oligo(dT)-cellulose (Aviv and Leder, 1972). The function quality of mRNA BoIFN-alpha was verified by microinjection into the oocytes of Xenopus laevis using a microinjector of our own construction (Fig. 2). The microinjector was calibrated by injection of a radioactive 51Cr solution (Fig. 3). It was found out on the basis of CPE inhibition measurements that the injected oocytes synthesized 320-640 U/ml BoIFN-alpha (16-32 U/50 microliters; Tab. I).

Animals↗
PubMed1986

Hydrolysis of adenylyl(2'-5')adenosine in chick embryo sera.

Similar to adult chick sera in chick embryo sera the enzyme activity hydrolyzing the core of the dimer of A-5A - adenylyl(2' -5')adenosine is present. The enzyme activity possesses the same properties in both sera. Heating of the serum to 60 degrees C for 30 minutes does not affect the enzyme activity. A marked decrease in activity occurs after heating of the serum to above 65 degrees C. Hydrolysis of A2' p5' A in the serum is more efficient under alkaline conditions (pH 9-10) than at physiological pH 7.2-7.4.

Adenosine Monophosphate↗
PubMed1984

Stability of (2'-5')oligoriboadenylates in various sera.

Avian and mammalian sera were found to contain an enzyme activity degrading 2-5A oligonucleotides. The most extensive degradation of the A2' p5' A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56 degrees C for 30 min. The 5-mU3' p5' A has also been degraded in chicken serum.

Adenine Nucleotides↗