Biomedical subjects

M Orlich

Publications and source records attributed to M Orlich.

At least 19 recordsLinked to original sources

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↗

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↗

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

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

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↗

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↗

PubMed1995

Trypsin-resistant protease activation mutants of an influenza virus.

New classes of mutants of influenza virus A/seal/Mass/1/80 are described in which the haemagglutinins (HA) have lost their protease cleavability by trypsin, but can be activated by elastase, chymotrypsin or thermolysin in different cell types. The same proteases that were required for activation of infectivity of the mutants also activated haemolysis and cell-fusing properties. The protease activation (pa)-mutants were non-pathogenic for chickens, but induced a protective immune response against a highly pathogenic challenge virus. The failure of the mutants to be activated by trypsin, but instead to be activated by the other proteases employed, was related to amino acid exchanges around the HA cleavage site. The cleavability of the chymotrypsin and elastase pa-mutants is most likely determined by replacement of Arg-1 by neutral amino acids such as Ile, Thr, Met or Leu, depending on the substrate specificity of the activating proteases. Cleavage activation of the thermolysin pa-mutants, on the other hand, became possible by insertion of a single Leu residue at position 4 of the HA2 polypeptide, which compensates for the loss of the Gly residue at the N terminus of the fusion peptide due to thermolysin cleavage. The correction of the mutations in revertants confirmed the conclusions drawn from sequence analyses of the pa-mutants.

Amino Acid Sequence↗

PubMed1994

Nonhomologous recombination between the hemagglutinin gene and the nucleoprotein gene of an influenza virus.

Mutants of the influenza virus A/seal/Mass/1/80 (H7N7) are described which contain an insertion of 60 nucleotides in the hemagglutinin (HA) gene, derived most probably by recombination between the HA gene and the nucleoprotein gene of the same virus. The nonhomologous RNA recombination resulted in an enhanced hemagglutinin cleavability associated with broadening of the host cell spectrum, increased hemolytic activity, and increased pathogenicity for chickens.

Amino Acid Sequence↗

PubMed1994

Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.

Amino Acid Sequence↗

PubMed1993

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes.

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides GM3 (II3Neu5Ac-LacCer), neolacto-series gangliosides (IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer) containing C24:1, and to some extent C22:0; and C16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac alpha 2-3Gal and/or Neu5Ac alpha 2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions.

Carbohydrate Sequence↗

PubMed1992

Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R.

The hemagglutinin of influenza virus A/FPV/Rostock/34 (H7) was altered at its multibasic cleavage site by site-directed mutagenesis and assayed for proteolytic activation after expression in CV-1 cells. The results indicated that the cellular protease responsible for activation recognizes the tetrapeptide motif R-X-K/R-R that must be presented in the correct sequence position. Studies on plaque variants of influenza virus A/fowl/Victoria/75 (H7N7) showed that alteration of the consensus sequence resulted in a loss of pathogenicity for chickens.

Amino Acid Sequence↗

PubMed1990

Structural variation occurring in the hemagglutinin of influenza virus A/turkey/Oregon/71 during adaptation to different cell types.

The influenza virus A/turkey/Oregon/71 (H7N3) has been adapted to grow in MDCK or chicken embryo cells (CEC) in the absence of trypsin. Changes occurred in the biological properties of the virus variants selected, depending on the cell type used for adaptation. They coincided with enhanced hemagglutinin (HA) activation by intracellular proteolytic cleavage. In the case of MDCK cell selected variants growth, plaque formation, and HA cleavability were restricted to this cell type, whereas the CEC-derived variants displayed altered activities in a broad range of host cells. Unlike the wild-type virus and its MDCK cell-derived variants, CEC variants had acquired pathogenic properties for chickens. By nucleotide sequence analysis of the HA genes of the MDCK cell variants several point mutations were found, which were localized predominantly at the distal, globular part of the HA molecule. The mechanism by which these point mutations increased HA cleavability has not been defined. In the CEC-derived variants besides point mutations, an insertion of 54 nucleotides adjacent to the cleavage site was observed, which corresponds in its sequence to a region in the 28 S ribosomal RNA. This insertion is probably responsible for the altered cleavability of the CEC variants' HA, leading to increased growth potential and pathogenicity.

Adaptation, Physiological↗

PubMed1990

Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes.

Influenza virus A/seal/Mass/1/80 (H7N7) was adapted to grow in MDCK cells and chicken embryo cells (CEC) in the absence of exogenous protease. The biological properties of the virus variants obtained coincided with intracellular activation of the hemagglutinin (HA) by posttranslational proteolytic cleavage and depended on the cell type used for adaptation. MDCK cell-adapted variants contained point mutations in regions of the HA more distant from the cleavage site. It is proposed that these mutations are probably responsible, through an unknown mechanism, for enhanced cleavability of HA in MDCK cells. Such virus variants were apathogenic in chickens. CEC-adapted variants, on the other hand, contained an insertion of basic amino acids at the HA cleavage site, in addition to scattered point mutations. The insertions converted the cleavage sites in the variant virus HAs so that they came to resemble the cleavage site found in highly pathogenic avian influenza viruses. CEC variants with such cleavage site modifications were highly pathogenic for chickens. The lethal outcome of the infection in chickens demonstrated for the first time that an influenza virus derived from a mammalian species can be modified during adaptation to a new cell type to such an extent that the resulting virus variant becomes pathogenic for an avian species.

Amino Acid Sequence↗