Biomedical subjects

N Osterrieder

Publications and source records attributed to N Osterrieder.

29 records · Page 2Linked to original sources

PubMed1997

Analysis of the contributions of the equine herpesvirus 1 glycoprotein gB homolog to virus entry and direct cell-to-cell spread.

Experiments to analyze the functions of the equine herpesvirus 1 (EHV-1) glycoprotein gB were performed. Cell lines which stably expressed either the full-length EHV-1 gB or only the extracellular portion of gB (amino acids 1 to 844) were constructed and were termed TCgBf and TCgB delta, respectively. Using the cell line TCgBf, a gB-negative viral mutant, L11delta gB, was generated by replacing a 2.1-kb BglII-NruI fragment in the EHV-1 strain RacL11 gB with the Escherichia coli LacZ gene. EHV-1 strain RacL11, the modified live vaccine strain RacH, and L11delta gB were used for functional studies. It was shown that: (i) EHV-1 gB is essential for virus growth in vitro since gB-negative L11delta gB exhibited titers of <10 PFU/ml when grown and titrated on noncomplementing cells. (ii) The cell line expressing truncated gB (TCgB delta) did not complement for the growth of L11delta gB, but the RacH virus grew to titers comparable to those of RacL11 in all cell lines tested. Since RacH had amino acids 944-980 of gB replaced by 7 missense amino acids as determined by nucleotide sequence analysis, the extreme carboxyterminus but not a domain between amino acid residues 845 and 943, probably the transmembrane domain, of EHV-1 gB is dispensable for virus growth in cultured cells. (iii) Single infected cells but no plaque formation were observed after infection of noncomplementing cells with L11delta gB, demonstrating the requirement of EHV-1 gB for direct cell-to-cell spread of infection. (iv) The attachment of gB-negative L11delta gB virions to target cells was similar to both phenotypically complemented L11delta gB and parent RacL11 virus. (v) L11delta gB viral titers could be enhanced by using the fusogen polyethylene glycol (PEG). The increase of L11delta gB titers by PEG treatment, however, was considerably lower compared to gB-negative pseudorabies virus, suggesting that EHV-1 gB might not be as stringently required for virus penetration as are its homologs in other Alphaherpesvirinae.

Amino Acid Sequence↗

PubMed1996

The equine herpesvirus 1 IR6 protein influences virus growth at elevated temperature and is a major determinant of virulence.

The diploid IR6 gene (ORF 67) of equine herpesvirus type 1 (EHV-1) is absent in the modified live EHV-1 vaccine strain RacH and is present in a mutated form in the avirulent EHV-1 strains RacM24 and RacM36, such that the IR6 protein fails to form the typical rod-like structures observed for wild-type EHV-1 RacL11. To assess the role of the IR6 protein in EHV-1 replication and virulence, two recombinant RacH viruses, HIR6-1 and HIR6-2, that harbor a single copy of the wild-type IR6 gene were engineered and characterized. It was shown that: (i) HIR6-1 or HIR6-2 virus encoded for an IR6 protein that was capable of forming the rod-like structures typical of cells infected with the wild-type virulent virus strain RacL11. (ii) Whereas the avirulent EHV-1 strains RacH and RacM36 are temperature-sensitive (in that virus replication at 40 degrees versus that at 37 degrees was reduced by as much as 7,500-fold), the HIR6-1 and HIR6-2 viruses, like RacL11 virus, were capable of significant replication at the elevated temperature. (iii) Electron microscopic analyses revealed that cells infected with the HIR6-1 or HIR6-2 virus, like those infected with virulent RacL11 virus, produced and released comparable numbers of virus particles at both 37 and 40 degrees. In cells infected with the RacH virus at 40 degrees, however, release of extracellular particles was inhibited by greater than 90% and relatively few of the particles were enveloped. (iv) Infections of BALB/cA mice revealed that both the HIR6-1 and HIR6-2 viruses, unlike the parent RacH virus, were as virulent as the wild-type RacL11 strain as judged by the criteria of body weight loss, development of clinical signs of EHV-1 infection, virus titers in the lung, and ability to cause viremia. These findings and those of our recent studies indicate that the IR6 protein is a major determinant of EHV-1 virulence and that the IR6 protein may play a role in virus maturation and/or egress.

Animals↗

PubMed1996

The equine herpesvirus 1 IR6 protein is nonessential for virus growth in vitro and modified by serial virus passage in cell culture.

The IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated. Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies. In the 256th passage (RacH), both copies of the IR6 gene were absent. As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide. It is shown that (i) the IR6 protein is nonessential for virus growth in vitro. (ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed. (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells. In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles. (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions. (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization. (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein. In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures. (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures.

Amino Acid Sequence↗

PubMed1996

Alterations in the equine herpesvirus type-1 (EHV-1) strain RacH during attenuation.

The equine herpesvirus type-1 modified live-vaccine strain RacH (256th passage on porcine embryonic kidney cells) was investigated by restriction-enzyme analysis and compared to representative plaque isolates of the 12th passage (RacL11, RacL22) and 185th passage (RacM24, RacM36). The restriction patterns of all Rac plaque isolates differed compared with reference strain Ab4. The left UL terminus was shortened by 0.1 kbp and a missing BamHI site led to the fusion of the f and t fragments. In some Rac derivatives, losses of restriction sites without deletions were observed: 1. One BamHI site located in the ribosyl reductase gene was missing in RacH, RacM24, RacM36, and RacL22; and 2. An SalI site mapping to the gp14 (gB) gene was absent in RacM24, RacM36 and RacH. An identical deletion of 0.85 kbp in size was found in both copies of the inverted repeat (IR) regions of RacH. The deletion was present only in the terminal IR of the medium-passage derivative RacM36. By contrast, in the genomes of the apathogenic RacM24, as well as the pathogenic plaque isolates RacL11 and RacL22, no deletions in the IRs were detectable. Nucleotide-sequence and Northern-blot analyses revealed that the deletions led to the elimination of one or both copies of the gene 67 (IR6) open-reading frame in RacM36 and RacH and affected the gene 68 (EUS1) in RacH.

Animals↗

PubMed1996

The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions.

Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.

Base Sequence↗

PubMed1995

Protection against EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gp14 (gB).

Four formulations of the equine herpesvirus type 1 (EHV-1) glycoprotein gp 14 (gB), were tested for their ability to protect mice against intranasal (inas) EHV-1 challenge infection. The preparations tested included (i) a truncated gp14 produced in Escherichia coli or (ii) a truncated gp14 expressed in insect cells by a recombinant baculovirus, (iii) truncated gp14 coexpressed with human immunodeficiency virus type 1 (HIV-1) gag virus-like particles (VLP) in insect cells, and (iv) a gp14-DNA vaccine under the control of the cytomegalovirus immediate early promoter. All antigen preparations and the DNA vaccine elicited a humoral and delayed-type hypersensitivity (DTH) immune response to EHV-1 after intramuscular (im) immunization. After inas immunization, only the VLP-gp14 preparation produced both a good humoral and a prominent DTH immune response; gp14 expressed by insect cells elicited high titers of EHV-1-specific antibodies, whereas gp14 produced in E. coli and the DNA vaccine elicited only low antibody titers. Glycoprotein gp14 expressed by bacteria, however, induced a strong DTH reaction after inas application. Mice were completely protected against EHV-1 challenge infection after both the im and the inas immunization with the VLP-gp14 preparation. Protection was less efficient after immunization with the E. coli and insect cell gp14s as well as after DNA vaccination. Although the transmembrane domain of EHV-1 gp14 was deleted, recombinant gp14 could be demonstrated in insect cell membranes at late times postinfection and aggregated with the VLPs. It is suggested that the transmembrane domain is not required for gp14-association with membranes in that system.

Administration, Intranasal↗

PubMed1994

Identification of binding sites for neutralizing monoclonal antibodies on the 14-kDa fusion protein of orthopox viruses.

A 14-kDa gene-specific probe of vaccinia virus Western Reserve (WR) hybridized to homologous sequences in the genomes of the orthopox virus species cowpox, camelpox, mousepox, and monkeypox virus. The corresponding genes were mapped and sequenced. Homologies of more than 95% were found when compared to the 14-kDa gene of vaccinia virus WR. However, point mutations which led to alterations in the amino acid sequences were mainly located between residues 26 and 40. By use of synthetic peptides, this part of the 14-kDa fusion protein could be identified as the binding site for four different neutralizing monoclonal antibodies.

Amino Acid Sequence↗

PubMed1994

Characterization of the gene encoding the A-type inclusion body protein of mousepox virus.

The gene of the mousepox virus strain MP-1 coding for the protein of A-type inclusions was identified and sequenced. The gene maps in the HindIII-A fragment at the same position as the cowpox-virus Brighton 160 kD gene (1) and the closely related 94 kD LS gene of vaccinia virus strain Western Reserve (2,3). The encoded protein consists of 1049 amino acids with a predicted molecular weight of 122.5 kD, which corresponds well to the apparent molecular weight of 130 kD estimated after polyacrylamide gel electrophoresis and Western blotting. The protein is a characteristic feature of mousepox virus and could be demonstrated for five independently isolated strains. The MP-1 ATI protein displays a similarity of 82.3% to the cowpox virus Brighton 160 kD protein, the first 340 amino acids being almost identical. However, two major deletions of 55 and 92 amino acids were observed from amino acid positions 671 to 732 and 794 to 885, respectively. Furthermore, the C-terminal residues (position 998-1049 of the MP-1 sequence) did not match at satisfactory levels with cowpox nor with vaccinia virus sequences. According to Western blot and protein alignment data as well as hydropathy predictions, the 130 and 160 kD proteins closely resemble each other, indicating similar structure and function.

Amino Acid Sequence↗

PubMed1994

A touchdown PCR for the differentiation of equine herpesvirus type 1 (EHV-1) field strains from the modified live vaccine strain RacH.

More than 50 reference strains and field isolates of equine herpesvirus type 1 (EHV-1) were examined by a touchdown PCR. Primers for specific amplification of EHV-1 DNA were chosen from the terminal and internal repeat regions of the EHV-1 genome where the high-passaged live vaccine strain RacH displays symmetric 850 bp deletions. The positive strand and one negative strand primer were designed to encompass the deletions present in RacH, and the second negative strand primer was designed to hybridize within these deletions. Discrimination between field isolates and the vaccine strain was achieved by the generation of amplification products of different size: In all EHV-1 reference strains and field isolates, a 495 bp DNA fragment was amplified specifically, whereas a 310 bp fragment was amplified when DNA of the vaccine strain RacH was used as a template. PCR amplification was only obtained in the presence of 8-10% dimethylsulfoxide and when the primer annealing temperatures were decreased stepwise from 72 degrees C to 60 degrees C. Under these conditions as little as 100 fg template DNA, corresponding to about 100 genome equivalents, could be detected. The PCR assay allows fast and sensitive discrimination of the modified live vaccine strain RacH from field strains of EHV-1 since it is applicable to viral DNA extracted from organ samples and paraffin-embedded tissues. It may thus be helpful for examining the potential involvement of the RacH live vaccine strain in abortions of vaccinated mares.

Base Sequence↗

PubMed1994

Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses.

The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gp14, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant E. coli expressed a fusion protein of M(r) 135K which was composed of the truncated gp14 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a 115K to 118K glycoprotein which was cleaved intracellularly into two subunits of M(r) 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M(r) 120K to 122K. The recombinant proteins produced in E. coli and in insect cells elicited EHV-1-specific antibodies in goats as demonstrated by Western blot analysis. The gp14 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gp14 expressed by E. coli failed to elicit neutralizing antibodies. The results suggest that post-translational modification of the EHV-1 gp14 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.

Animals↗

PubMed1993

Differentiation of species of the genus Orthopoxvirus in a dot blot assay using digoxigenin-labeled DNA-probes.

A dot blot assay using five different digoxigenin-labeled probes was established for specific detection and differentiation of four species belonging to the genus Orthopoxvirus. As little as 20 pg orthopoxvirus DNA can be detected, corresponding to approximately 8 x 10(4) DNA molecules. A total of 37 orthopoxvirus strains and isolates of different origin were investigated and could be assigned to the species vaccinia, cowpox, camelpox, and mousepox virus. These findings agree with their identification by biological means. Additionally, a simple procedure to isolate orthopoxvirus DNA directly from scab material was developed. This allows a fast classification without isolating the virus.

Animals↗