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S Vilcek

Publications and source records attributed to S Vilcek.

At least 37 records · Page 2Linked to original sources
PubMed1999

Genetic analysis of pestiviruses at the 3' end of the genome.

Specific PCR primers were selected for each pestivirus genotype which flanked the 3'-part of the NS5B gene and more than three quarters of the 3'-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3' untranslated region (3'-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type I (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3'-UTR region. Viruses of the BVDV lb group contained a characteristic deletion of 41 nucleotides. Compared to the 5'-UTR, the 3'-UTR was less conserved. The first 50-60 nucleotides were particularly variable, whilst the most conserved part was found at the 3' end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.

3' Untranslated Regions↗
PubMed1999

Phylogenetic comparison and molecular epidemiology of classical swine fever virus.

The genetic diversity of classical swine fever virus (CSFV) was studied by RT-PCR amplification and sequencing of a 409 bp fragment of the NS5B polymerase region. A total of 106 viruses isolated from 20 countries over a period of 52 years (1945-1997) were included in the phylogenetic study. The results showed that the viruses could be divided into two main groups. Group 1 consisted of Asian and South American isolates from the 1980s, as well as of old European and American isolates. Group 2 consisted mostly of recent European viruses from the 1980s and 1990s, and was further divided into three subgroups largely according to geographic origin and/or year of isolation. Five 1997 CSFV isolates from Germany, Netherlands and Italy clustered together indicating a common origin for these outbreaks, but two other 1997 isolations in different regions of Germany are likely due to different epidemiological events. The results show that the NSSB region of the genome gives a good resolution for phylogenetic studies of CSFV. Molecular epidemiology based on nucleotide sequence diversity is a useful tool for tracing virus spread and for developing disease control strategies.

Animals↗
PubMed1999

Genetic clustering of bovine viral diarrhoea viruses in cattle farms: genetic identification and analysis of viruses directly from cattle sera.

The herd-specific genetic clustering of bovine viral diarrhoea virus (BVDV) was studied by phylogenetic analysis of 42 sera collected between 1995-97 from persistently infected cattle on 16 farms in Sweden. The viruses were typed by sequencing a part of the 5' untranslated region of the genome, which had been amplified directly from serum by reverse transcription-polymerase chain reaction. All of the viruses were of BVDV I, either BVDV Ia (NADL-like) or BVDV Ib (Osloss-like) genotypes. No relationship was observed between the geographic region of origin and the character of clinical signs and the typing of the BVDV isolates. However, the phylogenetic analysis revealed a strict herd-specific genetic clustering of the virus. In 15 of the 16 herds, animals were infected with a single strain of BVDV characteristic for that herd. Direct nucleotide sequence analysis from serum can therefore be used as a tool for molecular epizootiology of BVDV infections.

Animals↗
PubMed1998

A novel nested reverse transcription PCR detects bovine viral diarrhoea virus in fluids from aborted bovine fetuses.

A nested reverse transcription-PCR (RT-PCR) was developed to detect pestivirus nucleic acid in fetal fluids and to study the number of bovine abortions associated with BVDV infection. Three techniques for the extraction of viral RNA from fetal fluids were compared; phenol:chloroform method, treatment with Catrimox-14 followed by guanidium isothiocyanate buffer and the Qiagen total RNA kit. The Qiagen kit was the most sensitive and reproducible and therefore adopted. After cDNA synthesis, initial amplification of a 288-base pair product using existing primers derived from the highly conserved 5'-untranslated region of the BVDV genome was achieved. Newly designed internal primers yielded a 171-base pair fragment which was visualised after electrophoresis on an ethidium bromide-stained gel. This assay detected 6.0 TCID50 of BVDV per 300 microl of artificially contaminated fetal fluid. One hundred fetal fluids were screened for the presence of BVDV RNA and the results compared with existing virus isolation methods. The BVDV antibody status of each fetus was determined. The nested RT-PCR detected BVDV RNA in eight of the hundred fetal fluids screened, whereas BVD virus was isolated from only one sample. The use of the nested RT-PCR will provide us with a more accurate picture of bovine embryonic infection due to BVDV.

Abortion, Veterinary↗
PubMed1998

Molecular characterization of the 3' noncoding region of classical swine fever virus vaccine strains.

The genomes of classical swine fever virus (CSFV) vaccine strains are poorly characterized, and the mechanisms for their attenuation remain unknown. The aim of the present study was to characterize the 3' noncoding region (3' NCR) of a number of attenuated vaccine strains of CSFV in order to examine changes in the viral genome after attenuation. The results showed that the 3' NCR:s of Porcivac, Rovac, Russian LK and original Chinese vaccine strain contain insertions very similar to that present in the published nucleotide sequence of the C-strain. Phylogenetic analysis showed that the vaccine strains Porcivac, Rovac and Russian LK vaccine were closely related to each other, but that these three strains showed a distant relationship with two tested variants of the Chinese vaccine strain (C-strain and original Chinese vaccine). The 3' NCR insertion is not likely to be a marker for attenuation of the virus, since many CSFV vaccine strains do not contain the insertion. The fact that the insertions occur in lapinized vaccine strains suggests that these genetic segments were incorporated during the adaptation of the virus to the rabbit host system.

Animals↗
PubMed1998

Genetic typing of pestiviruses from New Zealand.

AIM: To investigate the genetic type of 20 pestiviruses collected from New Zealand over the period 1967-97. METHODS: The pestiviruses were genetically typed by the sequencing of polymerase chain reaction (PCR) products. The primers selected were from the 5'-untranslated region (5'-UTR) of the pestivirus genome and consistently amplified a 288 bp fragment from all samples tested. RESULTS: Sequencing and phylogenetic analysis of PCR products revealed that all samples obtained from cattle represented bovine viral diarrhoea (BVDV) type I. Two sheep isolates were characterised as border disease virus (BDV). A pestivirus isolated from foetal calf serum of USA origin was typed as BVDV type II. CONCLUSIONS: The findings show that the evolution of pestiviruses in New Zealand has been similar to Europe and North America, indicating the occurrence of a conservative phylogenetic branch of BVDV type I in cattle and the presence of BDV in the sheep population.

Journal Article↗
PubMed1998

Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains.

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the previously established subgroups A and AB. A third pattern was produced by five Scandinavian strains and a fourth was observed from a single Dutch isolate. The genetic diversity of 27 strains of BRSV was investigated by comparative nucleotide sequence analysis of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were obtained from GenBank. The analysis revealed similarities of 88-100% among BRSV strains and 38-41% between BRSV and HRSV. A phylogenetic tree created for BRSV revealed two main branches, one of which divided into five further lineages, each representing a geographic cluster. A correlation was evident between the positions of some strains in the phylogenetic tree and their antigenic pattern. For HRSV strains, a genetic similarity of only 62% allowed the distinction of two antigenic subgroups, A and B, a pattern which was not seen for BRSV. This study showed that genetic analysis was an accurate method for discriminating BRSV strains and that these viruses should be regarded as a single genetic and antigenic group, within which variants can be distinguished.

Amino Acid Sequence↗
PubMed1997

Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species.

A pestivirus was transmitted by contact from a persistently infected (P.I.) bullock to pregnant sheep. This resulted in the birth of P.I. lambs, one of which in turn was able to transmit virus by contact to pregnant cattle. Two of these animals gave birth to P.I. calves, from one of which the virus was again transmitted by contact with pregnant sheep, leading to another generation of P.I. lambs. The expression of one or more epitopes on the E2 glycoprotein of the viruses isolated from this series of alternate cattle-sheep transmissions appeared to depend on the host species. Thus, several monoclonal antibodies which bound strongly to, and neutralised, viruses isolated from the bovine hosts, failed to bind or neutralise in the case of sheep isolates. The viral consensus sequences of the E2 gene as well as parts of the 5' untranslated region and of the Npro and capsid genes were compared between the different isolates. This revealed a high degree of genetic stability. However, a single codon change at amino acid position 9 of the E2 gene correlated with and was able to cause the loss of particular epitopes.

Animals↗
PubMed1997

Genetic heterogeneity of classical swine fever virus in Central Europe.

The aim of this work was to genetically characterize Central European isolates of classical swine fever virus (CSFV) and to evaluate the applicability of molecular analysis in the epizootiology of CSFV infections. Thirty four viruses, derived from Central European pigs or wild boar, were examined. All of these viruses were detected by each of three sets of oligonucleotide primers which had been designed for the specific RT-PCR amplification of different genomic regions. Comparative sequence analysis of the PCR products showed that they were of a genetic type common in Western Europe. Further discrimination of virus isolates was possible, into subgroups that largely coincided with their regions of origin in Poland, Slovakia, Hungary and Estonia. The discriminatory ability of the technique was improved by the analysis of a composite dataset consisting of all of the sequence data from all of the viruses. Using this approach we were able to distinguish between all of the viruses and to group them in a manner that precisely matched their geographical origins, apart from a single Estonian isolate which grouped with viruses from Eastern Poland.

Animals↗
PubMed1997

Organization and diversity of the 3'-noncoding region of classical swine fever virus genome.

Specific PCR primers were selected to amplify a 359 bp DNA fragment flanking the 3'-part of the polymerase gene and the 3'-noncoding (3'-NC) region of the genome of classical swine fever virus (CSFV). In RT-PCR the selected fragment was amplified from the genomes of 27 viral strains collected from Europe, America and Asia over a period of a half century as well as from three vaccine strains of CSFV. Eight PCR products were sequenced using an automatic sequencing device. Nucleotide sequence analysis was performed by computer programs DNASTAR and PHYLIP. The comparative studies revealed that the 3'-NC region contains a variable region of nucleotides which is located immediately after the stop codon TGA or TAA. Furthermore, a strongly conserved constant region was identified near to the extreme 3'-terminus. Two imperfect repeated sequences were found both in the variable and in the constant regions. In addition, the variable region was characterized by the occurrence of a 50 bp long poly AT track. Phylogenetic analysis with different mathematical approaches (MegAlign, Neighbor-Joining method, Maximum Likelihood, Parsimony) revealed that the studied CSFV strains were clustered into two main phylogenetic groups. Group I was comprised of the reference strain Brescia, together with old American and European field isolates and a Brazilian vaccine strain. Group II included the reference strain Alfort (Tübingen) and recent European field strains. The Congenital Tremor strain formed a distinct lineage which, although being highly divergent, was more closely related to group I than to group II. In conclusion, the present phylogenetic grouping yielded very similar results as previous studies base on comparison of the E2 (gp55) region. The agreement of the phylogenetic analysis in the two distinct regions confirms the reliability of the genetic grouping of CSFV strains into two main genogroups.

Animals↗
PubMed1997

Molecular characterization of ovine pestiviruses.

Forty-two ovine pestivirus isolates, collected over a period of 18 years, were compared by phylogenetic analysis. The viruses were mostly field isolates from Britain; two others originated from Sweden and two from New Zealand. RT-PCR products were obtained from two genomic regions, one within the 5'-noncoding (5'-NC) region, and the other encompassing parts of the p20 (Npro) and C coding regions. Direct sequencing of the 5'-NC PCR products, followed by computer-assisted phylogenetic analysis, divided the ovine pestiviruses into three main genotypes. The results demonstrated that sheep may naturally be infected not only with border disease virus (BDV), but also with bovine viral diarrhoea virus (BVDV) types I and II. The BDV isolates segregated into two principal subtypes represented by the Moredun strain from Scotland and the 137/4 strain from England. The BVDV-I group was composed of three clusters, two of them represented by BVDV reference strains NADL and Osloss, respectively, and the third by ovine isolates D1120/1 and D1432/P. The grouping of ovine pestiviruses, based on comparative nucleotide sequence analysis of the 5'-NC region, was confirmed by comparative analysis of the p20 (Npro) and C coding regions, performed both at the nucleotide and at the amino acid level. The presence of three genotypes in sheep, including BVDV-I and BVDV-II, indicates the inadequacy of the current hostspecies-based nomenclature and classification of pestiviruses.

Animals↗
PubMed1997

Genetic analysis of classical swine fever virus isolates from a small geographic area.

Twenty-eight field CSFV samples were isolated from outbreaks of CSF which have occurred in three small geographic areas in Slovakia in the period of December 1993-November 1994. All the organ homogenates were positive by virus isolation and RT-PCR assay using general pestivirus 324/326 primers selected from 5'-noncoding (5'-NC) genomic region. Specific discrimination of CSFV was confirmed by the cleavage of amplicons using Bgl1 and Ava 1. Four viral isolates (A, B, C, E) were selected for a comparative sequencing study. Sequencing of amplicons in the 5'-NC, gp55 (E2), p54 (NS2) and NS5B genomic regions revealed that A, B isolates (originated from SR geographic area) were the same but different from identical C (NR geographic area) and E (ER geographic area) isolates. Results of molecular-genetic study were very well supported with epizootological data. All CSFV isolates from Slovakia phylogenetically fell into the group of recent European isolates clearly separated from old European and American strains.

Animals↗
PubMed1997

Secondary structure of the 5'-noncoding region of border disease virus genome RNAs.

The computer predicted secondary structures from the 5'-NC genome region of four border disease virus (BDV) strains collected from sheep in England and Scotland over a period 1976-1986 were prepared. The FOLD program from GCG sequence analysis software package was used for the analysis of a 243 bp RNA fragment. Two typical shapes of secondary structures were observed which contained multiple imperfect stem-loop motifs. The shape of those structures exactly fit with the grouping of BDV strains to two phylogenetic groups. Secondary structures are typical only of BDV strains and they are different from the structures prepared for NADL (BVDV) and Alfort (CSFV) strains.

Animals↗
PubMed1996

Genetic identification of pestivirus strain Frijters as a border disease virus from pigs.

A pig pestivirus isolate, the Frijters strain, was characterized by using reverse transcription-PCR (RT-PCR), followed by RFLP and direct sequencing of the amplicons. Restriction endonuclease enzyme AvaI and Bg/I digestion of 5' NC genomic region PCR products suggested that Frijters strain belongs to the border disease viruses (BDV). This finding was confirmed by nucleotide sequencing of the amplified part of the 5-NC genomic region, which revealed 94% and 95% nucleotide similarity between the Frijters strain and the BDV prototype strains Moredun cp and Moredun ncp, respectively. On the other hand, 55-77% nucleotide identity was observed between the Frijters strain and prototypes of BVDV and CSFV. The amino acid similarity in the Npro (p20) region was 89% between Frijters and the Moredun strains and 70-77% between Frijters and the BVDV of CSFV strains. It was concluded that the Frijters virus is a member of the BDV group of the Pestivirus genus. Considering that this virus circulates in the large swine populations of Europe, the spread of certain pestiviruses to more than one animal species should not be considered sporadic. Since the presence of BDV in swine herds might be a confusing factor during classical swine fever eradication programmes, highly specific methods are required for reliable identification of these isolates. RT-PCR, followed by RFLP and sequencing of the amplicons proved to be a reliable approach for the rapid detection and identification of pestiviruses.

Animals↗
PubMed1996

Genetic variability of classical swine fever virus.

The genetic variability of classical swine fever virus was studied by comparative nucleotide sequence analysis of 76 virus isolates, collected during a half century from three continents. Parts of the E2 (gp55) and the polymerase gene coding regions of the viral genome were amplified by RT-PCR and DNA fragments of 254 and 207 bp, respectively, were sequenced. The comparative sequence analysis of the E2 region revealed two main phylogenetic groups of CSFV, indicating that the virus apparently evolved from two ancestor nodes. Group I (represented by Brescia strain) consisted of old and recent American and Asian viruses, as well as old English isolates from the 1950s. This group was subdivided into three subgroups, termed I.A-I.C. Group II (represented by Alfort strain) consisted of relatively recent isolates from Europe, together with strain Osaka, which was isolated in Japan from a pig of European origin. Based on genetic distances the group was divided into subgroups II.A and II.B. Malaysian isolates were branched into both groups, indicating multiple origins for contemporaneous outbreaks in that country. All ten vaccine strains tested were branched in group I, implying a common ancestor. The Japanese Kanagawa strain, isolated in 1974, and the British Congenital Tremor strain from 1964 were the most distinct variants of CSFV in our collection. The comparison of the nucleotide sequences of the polymerase coding region of 32 European strains distinguished subgroups II.A and II.B which were similar to the corresponding subgroups of the E2 phylogenetic tree. Thus, the results revealed that the E2 region and the polymerase coding regions seem to be appropriate for the grouping of CSFV isolates from all over the world, distinguishing two major groups of the virus. The reliability of these regions for phylogenetic analysis is indicated by the similarity of the results obtained from the two separate parts of the CSFV genome.

Base Sequence↗
PubMed1996

The distinction of serologically related ruminant alphaherpesviruses by the polymerase chain reaction (PCR) and restriction endonuclease analysis.

The amplification and analysis of a 468bp fragment from the gB gene of the serologically related ruminant alphaherpesviruses bovine herpesvirus-1.1 and 1.2 (BHV-1.1 and BHV-1.2), caprine herpesvirus-1 (CapHV-1), cervine herpesvirus-1 (CerHV-1) and rangiferine herpesvirus-1 (RanHV-1) by PCR and restriction endonuclease analysis is described. As primers, 22bp oligomers selected from the BHV-1 gB gene sequences were used for the amplification of the DNA from the five viruses. The amplification product from each virus was analysed by the restriction endonuclease enzymes BglI, HinfI, SmaI and AvaI. The specific amplification obtained demonstrate the existence of the gB gene sequences for each of the five alphaherpesviruses. However, sequences from some of the fragments were found to be different from those predicted from the gB gene following restriction endonuclease analysis. All five amplification products generated the same number of fragments after digestion with HinfI except for two additional bands evident in CapHV-1. The CerHV-1 and RanHV-1 fragments contained slightly different BglI restriction sites from those of the other three. While BHV-1.1, BHV-1.2, CapHV-1 and CerHV-1 contained SmaI and AvaI restriction sites, the RanHV-1 amplification product lacked both SmaI and AvaI restriction sites.

Animals↗