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P Becher

Publications and source records attributed to P Becher.

27 records · Page 2Linked to original sources
PubMed1997

Phylogenetic analysis of pestiviruses from domestic and wild ruminants.

Infections with pestiviruses occur in cattle, sheep, pigs and also in numerous other ungulate species. In the present study, pestiviruses from goat, buffalo, deer and giraffe were analysed at the molecular level; unusual strains from cattle and pigs were also included. A phylogenetic analysis of the respective pestiviruses was undertaken on the basis of a fragment from the 5' noncoding region as well as the gene encoding autoprotease Npro. Statistical analyses of the respective phylogenetic trees-based on the 5' NCR revealed low confidence levels for most of the branches, while the structure of the tree based on the Npro gene was supported by high bootstrap values. Accordingly, the isolates from goat, buffalo and deer can be grouped together with bovine viral diarrhoea virus (pestivirus type 1); within this genotype three subgroups and one disparate virus have been identified. One isolate from pig and one from cattle belong to the group of 'true' border disease virus (pestivirus type 3), which can be further subdivided into two major subgroups. Interestingly, the giraffe isolate does not belong to one of the four established pestivirus genotypes. The phylogenetic analysis strongly suggests that genotype 1 pestiviruses occur world-wide in many ruminant species. Furthermore, phylogenetic trees based on the Npro gene nucleotide sequences show that the respective sequences do not segregate into discrete lineages based on host-species origin.

Amino Acid Sequence↗
PubMed1996

Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.

Amino Acid Sequence↗
PubMed1996

Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome.

Two border disease virus (BDV) pairs each consisting of cytopathogenic (cp) and non-cp viruses have been analyzed at the molecular level. Within the NS2-3 (p125) encoding region of both cp viruses, insertions of cellular sequences were identified which were absent in the corresponding non-cp isolates. A comparative sequence analysis revealed that within each pair the cp and non-cp viruses are almost identical. This strongly suggests that the cp BDV isolates developed from the non-cp viruses by RNA recombination between the viral genome and cellular sequences. Nonstructural protein NS3 (p80) was demonstrated after infection with both cp BDV strains. In addition, fusion proteins composed of cellular and viral sequences were identified. In contrast, expression of NS3 and the fusion proteins was not found after infection with the respective non-cp counterparts.

Amino Acid Sequence↗
PubMed1996

Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7.

The pestivirus genome encodes a single polyprotein which is subject to co- and posttranslational processing by cellular and viral proteases. The map positions of all virus-encoded proteins are known with the exception of a hypothetical peptide (p?) which interlinks the glycoprotein E2 and the nonstructural protein NS2-3 approximately between amino acid positions 1060 and 1130. Expression studies with recombinant vaccinia viruses bearing a set of C-terminally truncated E2-p?-NS2-encoding sequences derived from a bovine viral diarrhea virus (BVDV) strain led to the identification of a minor fraction of E2 which had an increased molecular mass due to a C-terminal extension. This larger form of E2 (E2p7) was specifically recognized by an antiserum raised against the amino acid sequence from 1065 to 1125. In addition, the antibodies revealed a BVDV-encoded 7-kDa protein (p7) in infected cells. By radiosequencing it was determined that Val-1067 was the N-terminal amino acid of in vitro-synthesized p7. Analyses of BVDV and classical swine fever virus virions suggest that neither p7 nor E2p7 is a major structural constituent.

Amino Acid Sequence↗
PubMed1995

Further characterization of border disease virus isolates: evidence for the presence of more than three species within the genus pestivirus.

The molecular analysis of three ovine pestivirus strains revealed the existence of two distinct groups of sheep-derived pestiviruses, namely "true" border disease virus strains (BDV) and bovine viral diarrhea virus (BVDV)-like strains. As an extension of these studies RT-PCR and nucleotide sequencing of the autoprotease (Npro) and nucleocapsid protein (C) encoding regions of additional serologically defined ovine pestivirus strains were performed. A comparison of Npro and C revealed that three ovine isolates belong to the group of true BDV while two were clearly different from these as well as from BVDV and CSFV. The amino acid identity between the latter two ovine strains is 85% for Npro and 92% for C and thus similar to values found within the three pestivirus species defined so far. In order to allow comparison with additional pestiviruses the nucleotide sequences of a part of the 5' noncoding region were determined for the ovine pestivirus strains. The comparative analysis showed that recently described BVDV strains associated with acute lethal infection were very similar to two ovine isolates. The latter represent a fourth group within the genus pestivirus different from the so far defined pestivirus species BVDV, true BDV, and CSFV. The data lead to the question whether the current nomenclature of pestivirus species according to the host origin and the induced diseases is still appropriate.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of border disease virus, a pestivirus from sheep.

Three serologically different pestivirus strains isolated from sheep were selected for molecular analysis. cDNA and deduced amino acid sequences of the genomic regions encoding glycoproteins E1 and E2 were obtained from the three strains. A comparison with amino acid sequences of bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) revealed that one of the three ovine pestivirus strains can be grouped together with BVDV. The other two strains, however, were clearly different from both BVDV and CSFV. Surprisingly, the amino acid sequences from these two viruses were more similar to CSFV than to BVDV. The identity between so-called "true" BDV strains at the amino acid level is about 95% for E1 and 86% for E2 and thus similar to homologies found between CSFV strains. For one "true" BDV strain the genomic region encompassing the nonstructural protein p125 was also cloned and sequenced. The respective comparative analysis led to results which are similar to the ones obtained for the two structural glycoproteins. Taken together the data demonstrate that "true" border disease virus strains represent a separate group within the genus pestivirus.

Amino Acid Sequence↗
PubMed1988

Isoelectric focusing of basic proteins: the problem of oxidation of cysteines.

Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314-325). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e.g. more than 4 h at greater than or equal to 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2-mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2-ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.

Adult↗
PubMed1988

A new sample applicator for isoelectric focusing in horizontal polyacrylamide gels.

A new sample applicator for horizontal flat gels is described. The applicator is practically safe against contamination from adjacent samples and can be used for all types of electrophoretic separations including a concentration step for either the sample (i.e. disc electrophoresis) or the separated zones (i.e. isoelectric focusing). The applicator is a piece of flat glass with 26 or 51 parallel 2 mm wide grooves, drilled at distances of 9 or 4.5 mm. Samples, maximally 25 or 50, are applied to the areas between the grooves. By inverting the applicator, the samples are brought into close vicinity to the gel surface and the pendant droplets expand by capillary attraction into the slits between the glass and gel with resultant even distribution across the lanes of 2.5 or 7 mm width. The applicator can be used for separations with and without protection of the electrophoretic setup by paraffin oil and allows for fast multiple handling of samples by means of appropriate syringes and microtiter plates.

Genetic Variation↗