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P Becher

Publications and source records attributed to P Becher.

At least 19 recordsLinked to original sources
PubMed2005

[Thierry's spatula instrumental extraction: a study of foetal-maternal morbidity].

OBJECTIVE: Assisted delivery is necessary in many obstetrical conditions but is involved in maternal and foetal complications. The legal pressure and the commendable aim consisting in less neonatal morbidity and mortality have called forth a reflection about the type and the way of instrumental foetal extraction. In 1950, Thierry had already felt this problem and he invented spatula to replace obstetrical forceps. Although this instrument appears empirically little deleterious, literature about its evaluation is very poor. We studied this instrument in a retrospective 190 cases series. PATIENTS AND METHOD: Retrospective study of 190 Thierry's spatula extractions, over a seven-year period (January 1996 to December 2002), at the Centre Hospitalier General of Montbeliard. RESULTS: Out of a total of 8126 deliveries for the study period, the instrumental extraction rate was 5.3%, with 40.6% spatula extractions (190 cases). No failure of Thierry's spatula extraction was noted. DISCUSSION ET CONCLUSION: Our study concludes that spatula is efficient but does not usually permit anterior rotation of occipito-posterior presentation. Maternal and foetal morbidity is not frequent.

Birth Injuries↗
PubMed2004

Bovine viral diarrhea virus with deletions in the 5'-nontranslated region: reduction of replication in calves and induction of protective immunity.

Bovine viral diarrhea virus (BVDV) with deletions in the 5'-nontranslated region (5'-NTR) were tested for their suitability as live BVD vaccines. Firstly, the genetic stability of the mutants was established by culturing over 15 passages in bovine cells. Secondly, two deletion mutants and the parent strain CP7-5A were characterised with respect to in vivo replication competence, attenuation and induction of protective immunity against BVDV. Naïve calves (n = 5 per group) were inoculated with mutants d2-31 and d5-57 or CP7-5A and 5 weeks later, a challenge with the BVDV type 1 strain New York was performed. The mutants were found to be genetically and phenotypically stable. Moreover, the results indicate that the mutants were attenuated with regard to effects including pyrexia and drop in leucocyte counts. Infection with the mutants induced moderate to high titers of BVDV neutralizing antibodies and completely prevented viremia after challenge infection with a heterologous BVDV strain. Taken together, the 5'-NTR deletion mutants combine a good safety profile with good efficacy and are therefore well suited as candidate live vaccines.

5' Untranslated Regions↗
PubMed2004

Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus.

Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.

Amino Acid Sequence↗
PubMed2003

Genetic heterogeneity of bovine viral diarrhoea viruses from Spain.

Forty-four bovine viral diarrhoea virus (BVDV) isolates collected from the north of Spain between 1989 and 2000 were characterised at the molecular level. For 24 of these isolates the entire N(pro) gene sequence was determined. Phylogenetic analysis revealed that 21 isolates belong to subgroup BVDV-1b, while two BVDV-1c isolates and one BVDV-1h isolate were found. Furthermore, 20 additional virus isolates were analysed by differential reverse transcription-polymerase chain reaction (RT-PCR) and classified as BVDV-1. The results from our study demonstrate that BVDV-1b is the most prevalent subgroup present in bovine dairy herds of Spain, while there is no evidence for the presence of BVDV-2.

Amino Acid Sequence↗
PubMed2003

Parapoxvirus infection in harbor seals (Phoca vitulina) from the German North Sea.

In the summer of 2000, proliferative lesions of the skin and oral mucosa were observed in 26 young harbor seals (Phoca vitulina) from a rehabilitation center in Schleswig-Holstein, Germany. Verrucose, roundish nodules, approximately 1-2 cm in diameter, were presented in the oral cavity, especially on the tongue. Some animals developed similarly sized spherical dermal elevations with ulceration on flippers, chest, neck, and perineum. Necropsy of one animal showed multifocal, verrucose nodules in the oral cavity and a mild tonsillitis. Histologically, the nodules were characterized by ballooning degeneration of the outer parts of the spiny layer and stratum granulosum, with large eosinophilic cytoplasmic inclusions and a perivascular to interstitial lymphohistiocytic infiltration accompanied by fibroblastic proliferation and neovascularization. Negative staining of mucosal tissue homogenates demonstrated parapoxvirus-like particles. The presence of parapoxvirus was confirmed by polymerase chain reaction, using primers specific for parapoxvirus of ungulates. By in situ hybridization, using a parapox-specific, digoxigenin-labeled DNA probe, abundant parapoxvirus DNA-positive epithelial cells were detected in the stratum granulosum and the outer parts of the spiny layer. There was no parapoxvirus-positive signal in the adjacent submucosa. Although DNA analysis revealed that the causative agent can clearly be distinct from terrestrial parapoxviruses, lesions resembled parapoxvirus infections in other terrestrial species, and the pattern of virus DNA distribution indicated a direct effect of the virus on keratinocytes. In contrast, changes in the corium may be considered an indirect response mediated by the virus or the immune system.

Animals↗
PubMed2002

Characterization of sealpox virus, a separate member of the parapoxviruses.

A disease outbreak characterized by lesions of the skin and mucosa of the oral cavity was recognized in harbor seals ( Phoca vitulina) from the German North Sea. Using electron microscopy typical parapoxvirus particles were observed. The presence of parapoxvirus was confirmed by PCR and nucleotide sequencing of part of the putative major envelope protein coding gene. Comparative sequence analysis revealed that the virus from seal is significantly different from the established parapoxvirus species Orf virus, Bovine papular stomatitis virus, Pseudocowpox virus, and Parapoxvirus of red deer in New Zealand. The results of our analysis provide evidence for inclusion of the seal parapoxvirus as member of a separate species within the genus Parapoxvirus.

Amino Acid Sequence↗
PubMed2001

Evidence for the presence of two novel pestivirus species.

The genus Pestivirus of the family Flaviviridae comprises four species, namely Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, Border disease virus (BDV), and Classical swine fever virus (CSFV). Comparative analyses of partial sequences have suggested that pestivirus isolates from giraffe (Giraffe-1) and reindeer (Reindeer-1) are distinct from the established species (Becher et al., Virology 262, 64--71, 1999). In this study, we report the complete genomic sequences of pestivirus strains Giraffe-1 and Reindeer-1. Comparative sequence analyses revealed considerable differences among Giraffe-1, Reindeer-1, and the currently recognized pestivirus species. Phylogenetic analysis of the complete coding sequences of these two strains, along with 13 other sequences representing the four established species, indicated that CSFV, BDV, and Reindeer-1 have bifurcated from one common branch and BVDV-1 and BVDV-2 from another. In the former branch BDV and the pestivirus from reindeer are more similar to each other than to CSFV. The giraffe pestivirus is equally distinct from both major branches. In addition, the antigenic relatedness of pestivirus isolates covering the observed major genetic groups was studied by cross-neutralization assays. A clustering procedure on the basis of antigenic differences indicated the presence of six major groups corresponding to the genetically defined groups. Taken together, the results of our analyses addressing both nucleotide sequence relatedness and serological relatedness argue for the inclusion of Giraffe-1 and Reindeer-1 as the first members of two separate novel species within the genus Pestivirus.

Amino Acid Sequence↗
PubMed2001

RNA recombination between persisting pestivirus and a vaccine strain: generation of cytopathogenic virus and induction of lethal disease.

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are >99% identical to the previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, and H.-J. Thiel, J. Virol. 72:8697-8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.

Animals↗
PubMed2000

Insertion of cellular NEDD8 coding sequences in a pestivirus.

For the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP 821, a duplication of the genomic region encoding part of NS2, NS3, NS4A, and part of NS4B together with a nonviral insertion was detected. Further analyses including molecular cloning and sequencing of the putative cellular recombination partner showed that the insertion in CP 821 originated from a bovine mRNA encoding the cellular protein NEDD8, which is 58% identical to ubiquitin. To our knowledge the genome of CP 821 represents the first viral RNA with a NEDD8 coding insertion. Remarkably, the insertion site differs from that described for insertions of ubiquitin. The NEDD8 sequence allows an additional cleavage of the viral polyprotein, whereby an NS3 with an unusual N-terminus is generated. Furthermore, the CP 821-specific genomic alterations were introduced into an infectious noncytopathogenic (noncp) BVDV cDNA clone. After transfection of bovine cells with the respective RNA, a cp virus was recovered. This showed that the NEDD8 coding insertion together with the duplicated viral sequences represents the genetic basis for cytopathogenicity of CP 821. In addition to the recovered cp virus, noncp BVDV rapidly evolved after transfection. This is the first time that a change from the cp to the noncp phenotype was demonstrated in the course of replication in tissue culture cells.

Amino Acid Sequence↗
PubMed2000

The genetic basis for cytopathogenicity of pestiviruses.

Two biotypes of pestiviruses, cytopathogenic (cp) and noncp viruses, can be distinguished by their effects on tissue culture cells. Identification of cp bovine viral diarrhea virus (BVDV) has been frequently reported since antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. In contrast to the BVDV system, only few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Serological analyses and sequence comparison studies showed that cp pestiviruses arise from noncp viruses by mutation. Elaborate studies during the last 10 years revealed that in most cases RNA recombination is responsible for the generation of the cp viruses. Recent results showed a second way for the development of a cp pestivirus which is based on the introduction of a set of point mutations within the NS2 gene.

Animals↗
PubMed2000

Mutations in the 5' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics.

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5' NTR mutants correlated with reduced amounts of accumulated viral RNAs.

5' Untranslated Regions↗
PubMed1999

Genetic diversity of pestiviruses: identification of novel groups and implications for classification.

The complete Npro coding sequences were determined for 16 pestiviruses isolated from cattle, pig, and several wild ruminant species including reindeer, bison, deer, and bongo. Phylogenetic analysis enabled the segregation of pestiviruses into the established species bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, border disease virus (BDV), and classical swine fever virus (CSFV). For BVDV-1 five distinct subgroups were identified, while BVDV-2, BDV, and CSFV were each subdivided into two subgroups. The virus isolates from bongo and deer as well as one porcine virus isolate belong to BVDV-1. Interestingly, the isolates from reindeer and bison are distinct from the established pestivirus species. The Npro sequences from these two viruses are more similar to BDV than to the other pestivirus species. Calculation of the pairwise evolutionary distances allowed a clear separation of the categories species, subgroup, and isolate only when the reindeer/bison viruses were considered as members of an additional pestivirus species. Furthermore, the entire E2 coding sequences of a representative set of virus isolates covering all recognized species and subgroups were studied. Segregation of pestiviruses based on the E2 region was identical with that obtained with the N(pro) sequences.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of an iridovirus from Hermann's tortoises (Testudo hermanni).

A virus was isolated from tissues of 2 diseased Hermann's tortoises (Testudo hermanni) and preliminarily characterized as an iridovirus. This conclusion was based on the presence of inclusion bodies in the cytoplasm of infected cells, sensitivity to chloroform, inhibition of virus replication by 5-iodo-2'-desoxyuridine and the size and icosahedral morphology of viral particles. The virus was able to replicate in several reptilian, avian and mammalian cell lines at 28 degrees C, but not at 37 degrees C. Restriction enzyme analysis showed resistance of the ral DNA to digestion with HpaII due to methylation of the internal cytosine at CCGG sequences. Part of the genomic region encoding the major capsid protein was amplified by PCR and subjected to sequence analysis. Comparative analysis of the obtained nucleotide sequence revealed that the isolate is closely related to frog virus 3, the type species of the genus Ranavirus.

Amino Acid Sequence↗
PubMed1999

Nonhomologous RNA recombination in bovine viral diarrhea virus: molecular characterization of a variety of subgenomic RNAs isolated during an outbreak of fatal mucosal disease.

Four bovine viral diarrhea virus type 2 (BVDV-2) pairs consisting of cytopathogenic (cp) and noncp BVDV-2 were isolated during an outbreak of mucosal disease. Comparative sequence analysis showed that the four noncp BVDV-2 isolates were almost identical. For the cp BVDV-2 isolates, viral subgenomic RNAs were shown by Northern blot to have a length of about 8 kb, which is about 4.3 kb shorter than the genome of noncp BVDV. Cytopathogenicity and the expression of NS3 were both strictly correlated to the presence of viral subgenomic RNAs. By reverse transcription-PCR, Southern blot analysis, and nucleotide sequencing, a set of 11 unique subgenomes was identified with up to 5 different subgenomes isolated from one animal. To our knowledge, this is the first report on isolation of a set of pestiviral subgenomes from individual animals. Common features of the BVDV-2 subgenomic RNAs include (i) deletion of most of the genomic region encoding the structural proteins, as well as the nonstructural proteins p7 and NS2, and (ii) insertion of cellular (poly)ubiquitin coding sequences. Three subgenomes also comprised 15 to 75 nucleotides derived from the 5' part of the NS2 gene. Comparisons of the obtained nucleotide sequences revealed that the different BVDV-2 subgenomes evolved from the respective noncp BVDV-2 by RNA recombination. The presence of short regions of sequence similarity at several crossing-over sites suggests that base pairing between the nascent RNA strand and the acceptor RNA template facilitates template switching of the BVDV RNA-dependent RNA polymerase.

Animals↗
PubMed1998

Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus.

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.

Amino Acid Sequence↗
PubMed1998

Complete genomic sequence of border disease virus, a pestivirus from sheep.

The genus Pestivirus of the family Flaviviridae comprises three established species, namely, bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus from sheep (BDV). In this study, we report the first complete nucleotide sequence of BDV, that of strain X818. The genome is 12,333 nucleotides long and contains one long open reading frame encoding 3, 895 amino acids. The 5' noncoding region (NCR) of BDV X818 consists of 372 nucleotides and is thus similar in length to the 5' NCR reported for other pestiviruses. The 3' NCR of X818 is 273 nucleotides long and thereby at least 32 nucleotides longer than the 3' NCR of pestiviruses analyzed thus far. Within the 3' NCR of BDV X818, the sequence motif TATTTATTTA was identified at four locations. The same repeat was found at two or three locations within the 3' NCR of different CSFV isolates but was absent in the 3' NCR of BVDV. Analysis of five additional BDV strains showed that the 3' NCR sequences are highly conserved within this species. Comparison of the deduced amino acid sequence of X818 with the ones of other pestiviruses allowed the prediction of polyprotein cleavage sites which were conserved with regard to the structural proteins. It has been reported for two BVDV strains that cleavage at the nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B is mediated by the NS3 serine protease and for each site a conserved leucine was found at the P1 position followed by either serine or alanine at P1' (N. Tautz, K. Elbers, D. Stoll, G. Meyers, and H.-J. Thiel, J. Virol. 71:5415-5422, 1997; J. Xu, E. Mendez, P. R. Caron, C. Lin, M. A. Murcko, M. S. Collett, and C. M. Rice, J. Virol. 71:5312-5322). Interestingly, P1' of the predicted NS5A/5B cleavage site of BDV is represented by an asparagine residue. Transient expression studies demonstrated that this unusual NS5A/5B processing site is efficiently cleaved by the NS3 serine protease of BDV.

Amino Acid Sequence↗