Biomedical subjects

A A de Vries

Publications and source records attributed to A A de Vries.

At least 19 recordsLinked to original sources

PubMed2001

Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors.

Gene therapy aims to complement or, ideally, correct defective genes. The broad clinical application of this emerging technology requires the development of safe high-capacity gene delivery vehicles that combine efficient transduction of dividing as well as quiescent cells with sustained transgene expression. Here we present a new hybrid vector system that unites favorable attributes of adenoassociated virus (AAV) and adenovirus (Ad) vectors in a single particle. This was achieved by inclusion of Ad packaging elements in different sized recombinant AAV genomes. In the presence of AAV replicative functions and a recombinant helper Ad, AAV/Ad hybrid particles were generated via encapsidation of AAV-dependent replicative intermediates into Ad capsids. In stringent in vitro models based on transduction of proliferating cells we show that AAV/Ad hybrid vectors are superior to Ad vectors in establishing prolonged transgene expression and can be used to deliver DNA fragments of at least 27 kb.

Adenoviruses, Human↗

PubMed2001

Recombinant equine arteritis virus as an expression vector.

Equine arteritis virus (EAV) is the prototypic member of the family Arteriviridae, which together with the Corona- and Toroviridae constitutes the order Nidovirales. A common trait of these positive-stranded RNA viruses is the 3'-coterminal nested set of six to eight leader-containing subgenomic mRNAs which are generated by a discontinuous transcription mechanism and from which the viral open reading frames downstream of the polymerase gene are expressed. In this study, we investigated whether the unique gene expression strategy of the Nidovirales could be utilized to convert them into viral expression vectors by introduction of an additional transcription unit into the EAV genome directing the synthesis of an extra subgenomic mRNA. To this end, an expression cassette consisting of the gene for a green fluorescent protein (GFP) flanked at its 3' end by EAV-specific transcription-regulating sequences was constructed. This genetic module was inserted into the recently obtained mutant infectious EAV cDNA clone pBRNX1.38-5/6 (A. A. F. de Vries, et al., 2000, Virology 270, 84-97) between the genes for the M and the G(L) proteins. Confocal fluorescence microscopy of BHK-21 cells electroporated with capped RNA transcripts derived from the resulting plasmid (pBRNX1.38-5/6-GFP) demonstrated that the GFP gene was expressed in the transfected cells, while the gradual spread of the infection through the cell monolayer showed that the recombinant virus was replication competent. The development of the cytopathic effect was, however, much slower than in cells that had received equivalent amounts of pBRNX1.38-5/6 RNA, indicating that the vector virus had a clear growth disadvantage compared to its direct precursor. Immunoprecipitation analyses of proteins from metabolically labeled BHK-21 cells infected with supernatant of the transfected cultures confirmed that the recombinant virus vector was viable and expressed viral genes as well as the GFP gene. Reverse transcription-PCR of the viral mRNAs extracted from cells infected with the vector virus revealed that it directed the synthesis of nine instead of eight different EAV RNAs. These findings were corroborated by hybridization analyses. Mapping of the leader-to-body junctions of the ninth mRNA indicated that the 3' part of the GFP gene contains cryptic transcription signals which gave rise to at least five different RNA species ranging in size from 1277 to 1439 nt [without oligo(A) tract]. Furthermore, translation of the unintended mRNA resulted in the production of an extended version of the EAV M protein. Serial passage of the recombinant virus vector led to its gradual replacement by viral mutants carrying deletions in the GFP gene. The reduction in viral fitness associated with the insertion of the expression cassette into the EAV genome apparently caused genetic instability of the recombinant virus.

Amino Acid Sequence↗

PubMed2000

Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material.

5' Untranslated Regions↗

PubMed2000

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E.

The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

Animals↗

PubMed1999

Identification of a novel structural protein of arteriviruses.

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny virus.

Amino Acid Sequence↗

PubMed1998

Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein.

The mouse hepatitis virus (MHV) membrane (M) protein contains only O-linked oligosaccharides. We have used this protein as a model to study the structural requirements for O-glycosylation. We show that MHV M is modified by the addition of a single oligosaccharide side chain at the cluster of 4 hydroxylamino acids present at its extreme amino terminus and identified Thr at position 5 as the functional acceptor site. The hydroxylamino acid cluster, which is quite conserved among O-glycosylated coronavirus M proteins, is not in itself sufficient for O-glycosylation. Downstream amino acids are required to introduce a functional O-glycosylation site into a foreign protein. In a mutagenic analysis O-glycosylation was found to be sensitive to some particular changes but no unique sequence motif for O-glycosylation could be identified. Expression of mutant M proteins in cells revealed that substitution of any 1 residue was tolerated, conceivably due to the occurrence of multiple UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situ glycosylation assay based on the co-expression of endoplasmic reticulum-retained forms of the GalNAc transferases with endoplasmic reticulum-resident MHV M mutants. The GalNAc transferases were found to have largely overlapping, but distinct substrate specificities. The requirement for a threonine as acceptor rather than a serine residue and the requirement for a proline residue three positions downstream of the acceptor site were found to be distinctive features.

Acetylgalactosamine↗

PubMed1998

Identification of the leader-body junctions for the viral subgenomic mRNAs and organization of the simian hemorrhagic fever virus genome: evidence for gene duplication during arterivirus evolution.

Simian hemorrhagic fever virus (SHFV) was recently reclassified and assigned to the new virus family Arteriviridae. During replication, arteriviruses produce a 3' coterminal, nested set of subgenomic mRNAs (sgRNAs). These sgRNAs arise by discontinuous transcription, and each contains a 5' leader sequence which is joined to the body of the mRNA through a conserved junction sequence. Only the 5'-most open reading frame (ORF) is believed to be transcribed from each sgRNA. The SHFV genome encodes nine ORFs that are presumed to be expressed from sgRNAs. However, reverse transcription-PCR analysis with leader- and ORF-specific primers identified only eight sgRNA species. The consensus sequence 5'-UCNUUAACC-3' was identified as the junction motif. Our data suggest that sgRNA 2 may be bicistronic, expressing both ORF 2a and ORF 2b. SHFV encodes three more ORFs on its genome than the other arteriviruses. Comparative sequence analysis suggested that SHFV ORFs 2a, 2b, and 3 are related to ORFs 2 through 4 of the other arteriviruses. Evidence which suggests that SHFV ORFs 4 through 6 are related to ORFs 2a through 3 and may have resulted from a recombination event during virus evolution is presented.

Amino Acid Sequence↗

PubMed1997

Equine arteritis virus.

Equine arteritis virus (EAV) is a small, enveloped, positive-stranded RNA virus, in the family Arteriviridae , W.H.ich can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Marzz and geldings eliminate the virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed virus continuously in the semen, and can transmit the virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a virus neutralization (VN) test. If positive at a titer of >/= 1:4, the stallion should be tested for persistent infection by virus isolation from the sperm-rich fraction of the ejaculate, or by test mating Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination, the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of equine viral arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen.

Journal Article↗

PubMed1997

[Equine arteritis virus: clinical symptoms and prevention].

Sero-epidemiological surveys have revealed that equine arteritis virus (EAV) is prevalent in most European countries. The virus causes sporadic cases of respiratory disease and abortion in horses, the incidence of which has increased in recent years. Mares and geldings eliminate virus after acute infection, but 30% to 60% of stallions become persistently infected. In these animals, EAV is maintained within the reproductive tract and is shed continuously in the semen. Persistent infection with EAV in stallions has no negative consequences for fertility but mares inseminated with virus-contaminated semen can have an acute infection. These mares shed large amounts of virus in respiratory secretions and urine, leading to lateral spread of the virus to other susceptible horses. Acute infection at later stages of gestation can lead to abortion. Effective control of the spread of EAV infection depends on the identification of virus-shedding stallions. Persistently infected stallions should not be used for breeding or should be bred only to seropositive mares. Mares bred to shedding stallions should be isolated from other animals for a period of 3 weeks following insemination to prevent the lateral spread of EAV.

Abortion, Veterinary↗

PubMed1996

Equine arteritis virus: a review of clinical features and management aspects.

Abortion, Veterinary↗

PubMed1995

Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus.

A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1%, respectively, compared with EAV neutralization test and the recombinant antigen did not crossreact in ELISA with equine sera directed against other common equine respiratory viruses. Three post-EAV infection equine sera raised against different EAV isolates reacted strongly in the ELISA, as did two equine sera raised against EAV vaccines, indicating that the viral epitope was conserved between the viruses tested. Following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen ELISA preceded the development of a virus-neutralizing response. The study demonstrates the potential application of rGL 55-98 as a diagnostic antigen.

Animals↗

PubMed1995

Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL.

Complementary DNAs encoding ORFs 2 to 7 equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-specific synthetic peptide (residues 75 through 97) induced EAV-neutralizing antibody in vaccinated horses. The defined antigenic region of GL is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

Animals↗

PubMed1995

Comparison of equine arteritis virus isolates using neutralizing monoclonal antibodies and identification of sequence changes in GL associated with neutralization resistance.

Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize non-identical epitopes. Susceptibility to virus neutralization could not be used to distinguish viruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutralization-sensitive virus isolates revealed amino acid sequence changes at positions 99 and 100 which correlate with the NR phenotype. Additional unique changes in the amino acid sequence of MAb NR viruses at positions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the field isolates DL8 and DL11 possess two sites, both of which are used. Most of the non-conservative amino acid sequence changes were located within the second half of the N-terminal hydrophilic domain. Sequence changes within the first half of the N-terminal ectodomain, the predicted transmembrane domain and the C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.

Amino Acid Sequence↗

PubMed1995

The small envelope glycoprotein (GS) of equine arteritis virus folds into three distinct monomers and a disulfide-linked dimer.

The small membrane glycoprotein (GS) of equine arteritis virus (EAV) is a minor virion component but is abundantly expressed in EAV-infected cells. In this study, we have analyzed its membrane topology, folding, oligomerization, and intracellular transport. We show that GS is a class I integral membrane protein with one functional N-glycosylation site. Gel electrophoresis under nonreducing conditions revealed that GS occurs in EAV-infected cells in four monomeric conformations and as disulfide-linked homodimers. The slowest-migrating monomeric form corresponded to the fully reduced GS protein; the three faster-migrating monomeric species are probably generated by the formation of alternative intrachain disulfide bonds between the three luminal cysteines in the molecule. The GS monomers were selectively retained in the endoplasmic reticulum, as judged by their permanent susceptibility to endoglycosidase H, whereas the GS dimers were specifically incorporated into virus particles and became endoglycosidase H resistant and sialylated during passage through the Golgi apparatus.

Amino Acid Sequence↗

PubMed1995

The two major envelope proteins of equine arteritis virus associate into disulfide-linked heterodimers.

In a coimmunoprecipitation assay with monospecific antisera, the two major envelope proteins GL and M of equine arteritis virus were found to occur in heteromeric complexes in virions and infected cells. While the GL protein associated with M rapidly and efficiently, newly synthesized M protein was incorporated into complexes at a slower rate, which implies that it interacts with GL molecules synthesized earlier. Analysis under nonreducing conditions revealed that the GL/M complexes consist of disulfide-linked heterodimeric structures. Pulse-chase experiments showed that virtually all GL monomers ended up in heterodimers, whereas a fraction of the M protein persisted as monomers. The M protein also formed covalently linked homodimers, but only the heterodimers were incorporated into virus particles.

Amino Acid Sequence↗

PubMed1994

Monoclonal antibodies to equine arteritis virus proteins identify the GL protein as a target for virus neutralization.

Monoclonal antibodies (MAbs) to equine arteritis virus (EAV) proteins were produced and characterized. The protein specificities of eight MAbs were determined definitively by immunoprecipitation of EAV proteins expressed from vaccinia virus recombinants (VVRs). Included were two new VVRs produced for this study, expressing the M and the GL proteins, respectively. Three MAbs were determined to be N-specific and five MAbs recognized the GL protein. One GL-specific MAb, 17F5, of the IgA class, efficiently neutralized EAV infectivity. In competitive binding assays (CBAs), the N-specific MAbs defined a single antigenic domain on this protein. Four GL-specific MAbs, including MAb 17F5, demonstrated strong reciprocal competition in binding to the GL protein but differed in their virus-neutralizing ability. Thus the antigenic domain defined by these MAbs is probably composed of overlapping or closely adjacent epitopes. The fifth GL-specific MAb, a non-neutralizing antibody, may define an epitope adjacent to this antigenic domain as reciprocal CBAs demonstrated lower competition.

Animals↗

PubMed1992

Structural proteins of equine arteritis virus.

We have recently shown that the genome of equine arteritis virus (EAV) contains seven open reading frames (ORFs). We now present data on the structural proteins of EAV and the assignment of their respective genes. Virions are composed of a 14-kDa nucleocapsid protein (N) and three membrane proteins designated M, GS, and GL. M is an unglycosylated protein of 16 kDa, and GS and GL are N-glycosylated proteins of 25 and 30 to 42 kDa, respectively. The broad size distribution of GL results from heterogeneous N-acetyllactosamine addition since it is susceptible to digestion by endo-beta-galactosidase. Using monospecific antisera as well as an antivirion serum, and by expression of individual ORFs, the genes for the structural proteins were identified: ORF 7 codes for N, ORF 6 for M, ORF 5 for GL, and ORF 2 for GS. With the exception of GS, the proteins are about equally abundant in EAV virions, being present at a molar ratio of 3 (N):2 (M):3 (GL). The GS protein, which is expressed at a level similar to that of M in infected cells, is strikingly underrepresented in virus particles (1 to 2%). Our data justify a distinct taxonomic position for EAV, together with lactate dehydrogenase-elevating virus and simian hemorrhagic fever virus; although coronavirus- and toroviruslike in features of transcription and translation, the virion architecture of EAV is fundamentally different.

Amino Acid Sequence↗

PubMed1991

Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.

Amino Acid Sequence↗