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PubMed1993

Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin.

A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.

Base Sequence↗
PubMed1994

Immunochemiluminescent Southern blot assay for polymerase chain reaction detection of human parvovirus B19 DNA.

Human parvovirus B19 is not only an acute self-limited infection causing erythema infectiosum, transient aplastic crisis, foetal hydrops and arthritis but can also be a chronic infection causing chronic anaemia and associated with chronic neuropathy and vasculitis. Serologic studies have proven to be the most sensitive way to detect acute infection in the immunologically normal patient while polymerase chain reaction (PCR) assays for B19 DNA are the most sensitive way to detect chronic infection. The ability to detect B19 in clinical specimens can be further increased with a second amplification step using nested primers. However, nested PCR is both time consuming and enhances the risk of false-positive results due to contaminating DNA. In this study, we developed a sensitive immunochemiluminescent Southern blot assay for detecting PCR amplified B19 DNA with a digoxigenin labelled primer. The sensitivity and specificity of this assay were comparable to nested PCR and at least 100-fold more sensitive than a single PCR amplification.

Blotting, Southern↗
PubMed1998

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma.

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10(3) copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100 microl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.

Animals↗
PubMed1993

Identification and characterization of a family of 11-kDa proteins encoded by the human parvovirus B19.

The human pathogenic parvovirus B19 directs the synthesis of two size classes of small abundant RNAs. It is shown that the smallest RNAs, of 500 and 600 nt, are translated into at least two 11-kDa proteins in B19-infected human leukemic bone marrow cells. A COS-7 cell expression system was used to demonstrate that the different forms of the protein result from translational initiation at multiple AUG codons in the same 94 aa ORF. The 11-kDa proteins were localized to the cytoplasm of transfected COS-7 cells using indirect immunofluorescence. However, their localization was at least partially nuclear in B19-infected cells. In COS-7 cells the expression of the major B19 structural and nonstructural proteins was not affected in the absence of the expression of the 11-kDa proteins.

Base Sequence↗
PubMed1993

NF kappa B upstream regulatory sequences of the HIV-1 LTR are involved in the inhibition of HIV-1 promoter activity by the NS proteins of autonomous parvoviruses H-1 and MVMp.

To investigate parvoviral interference with human immunodeficiency virus type 1 (HIV-1) in human cells that are normally susceptible to HIV-1 infection, nonstructural (NS) proteins of the parvoviruses H-1 virus and minute virus of mice were studied for their effect on the activity of the HIV-1 promoter in a variety of CD4+ cells. Transient cotransfection assays revealed a reduced HIV-1 promoter activity in the presence of parvoviral NS proteins. Stimulation of the HIV-1 promoter by phorbol 12-myristate 13-acetate (PMA) led to an increase in its sensitivity to NS-induced suppression. The inhibitory effect of NS polypeptides depended, at least in part, on the presence of the NF kappa B motifs of the HIV-1 long terminal repeat, suggesting an interaction of the parvoviral products with PMA-inducible cellular factors binding to these elements of the HIV-1 promoter.

Animals↗
PubMed1994

Chimeric parvovirus B19 capsids for the presentation of foreign epitopes.

Chimeric proteins consisting of the VP2 capsid protein of human parvovirus B19 and defined linear epitopes from human herpes simplex virus type 1 and mouse hepatitis virus A59 inserted at the N-terminus and at a predicted surface region were expressed by recombinant baculoviruses. The chimeric proteins expressed the inserted epitopes and assembled into empty capsids. Immunoelectron microscopy indicated that the epitopes inserted in the loop were exposed on the surface of the chimeric particles. The chimeric capsids were immunogenic in mice and antibodies specific for the inserted sequences were induced. In the case of MHV, antibodies were produced that recognized the epitope in the context of native virus. Mice immunized with the chimeric capsids were partially protected against a lethal challenge infection with either MHV or HSV.

Amino Acid Sequence↗
PubMed1994

Identification of a 40- to 42-kDa attachment polypeptide for canine parvovirus in A72 cells.

The attachment of canine parvovirus (CPV) to different cell lines was quantitated by a fluorescence-activated cell sorter assay. The viral attachment was observed to both permissive A72 and nonpermissive ST cells but not to nonpermissive MDBK cells. The binding of and infectivity for CPV to A72 cells was reduced upon prior treatment of cells with Vibrio cholerae neuraminidase or lectins, specific for sialic acid. Similarly, treatment of cells with any of several proteases reduced virus binding; however, phospholipase treatment had no effect indicating that one or more membrane glycoproteins were involved in virus binding. These proteins were characterized with a virus overlay protein blot assay. Virus bound to a protein with a molecular mass of 40 to 42 kDa in membranes prepared from A72 and ST cells and not from MDBK cells. The binding to this polypeptide was specific since increasing amounts of unlabeled virions competitively inhibited binding of radiolabeled virions in a dose-dependent manner. A polypeptide of similar molecular mass was immunoprecipitated from radiolabeled octyl glucoside (OG) extract of A72 cells using purified virions, virion-specific antiserum, and protein A. The binding to this polypeptide was decreased but not abolished upon prior treatment of the membrane with V. cholerae neuraminidase. CPV preferentially recognized a polypeptide of similar molecular size in the OG extract prepared from the biotinylated basolateral surface of polarized MDCK monolayer. Hence, we propose that the 40- to 42-kDa glycoprotein represents a specific attachment molecule for CPV in A72 cells.

Animals↗
PubMed1995

Analysis of the cell and erythrocyte binding activities of the dimple and canyon regions of the canine parvovirus capsid.

Canine parvovirus (CPV) binds to a number of cell and erythrocyte receptors, some of which are involved in cell infection, while others are used for other viral functions. Little is known about the regions of the virus capsid which bind to the cell receptors. CPV binds sialic acid through a region within or adjacent to the dimple on the surface of the capsid (Barbis, D. P., Chang, S-F., and Parrish, C. R., 1992, Virology 191, 301-308). In order to map the sialic acid binding site in more detail and to examine other regions of the capsid for cell receptor binding, a variety of mutant capsids were analyzed which had changes in two depressions within the surface of the capsid--the "canyon" and "dimple." In most cases recombinant VP1 and VP2 proteins were stably expressed together in canine A72 cells from a plasmid expression vector. The purified empty capsids were tested for their ability to bind sialic acid and thereby hemagglutinate (HA) erythrocytes and for binding to permissive host cells. In addition, the ability of neutralizing monoclonal antibodies to block cell attachment was also examined. Mutations of amino acids on a wall of the dimple eliminated or severely decreased HA. Changing various residues within the canyon had no effect on binding to either sialic acids or other receptors on feline lymphoblastoid cells, suggesting that the canyon is not the site of cell receptor attachment. Neutralizing monoclonal antibodies against both major antigenic determinants had variable effects on cell binding, but no consistent inhibition of binding was observed by antibodies directed against either of those two major antigenic determinants of the capsid.

Amino Acid Sequence↗
PubMed1996

Structural analysis of a mutation in canine parvovirus which controls antigenicity and host range.

A single mutation in canine parvovirus (CPV) of VP2 residue 300 from alanine to aspartic acid causes a loss of canine host range and alters the antigenic properties of the virus. The three-dimensional structure of this mutant has been solved to 3.25 A resolution. Crystals of full particles were triclinic, with cell dimensions of a = 267.6, b = 268.5, c = 274.3 A. alpha = 61.9, beta = 62.6, and gamma = 60.2 degrees. The native structure of CPV was used as an initial model. Phases were improved by real-space electron density averaging. In spite of the relative low percentage of observed reflections (32.5% of the data between 15.0 and 3.25 A resolution), the presence of 60-fold noncrystallographic redundancy allowed the averaging procedure to converge smoothly. The mutant aspartic acid at residue 300 forms a salt bridge with Arg81 in an icosahedrally threefold-related subunit, inducing local changes within the antigenic site B on the CPV surface. In addition, the loop between residues 359 and 374 adopts a conformation similar to that displayed by feline panleukopenia virus. The ability of the Ala300-->Asp mutant to evade antibody binding can be associated with the change of charge distribution and structure in the antigenic binding site. The variation in host range behavior may be due to the increased stability as a result of formation of the salt bridge between adjacent subunits.

Animals↗
PubMed1998

Differences in the evolutionary pattern of feline panleukopenia virus and canine parvovirus.

Canine parvovirus (CPV) suddenly appeared in the late 1970s after which it showed continuous antigenic changes. Virological and molecular genetic analyses mainly focused on feline panleukopenia virus (FPLV) were conducted in this study because FPLV is the suspected ancestor of CPV; the way in which FPLV evolves may help to explain the emergence of CPV. Analysis of escape mutants against FPLV-specific monoclonal antibody showed that viruses possessing CPV-like properties were not easily detected in FPLV virus stocks. Phylogenetic analysis revealed that the nonstructural protein 1 (NS1) and capsid protein 2 (VP2) genes of FPLV changed with time. A similar tendency, however, was not observed in the FPLV VP2 proteins. In contrast, the topology of the phylogenetic tree of VP2 proteins of CPV basically concurred with that of the VP2 genes. Analysis of the ratio of nonsynonymous and synonymous substitutions revealed that synonymous substitutions exceeded nonsynonymous substitutions in both the NS1 and VP2 genes of FPLV, even when the analysis focused on specific regions in the VP2 gene that are known to be located on the capsid surface. Comparison of the CPV VP2 genes revealed that nonsynonymous substitution was found to dominate over synonymous substitution in one specific region in the VP2 gene. These results suggested that FPLV has changed mainly by random genetic drift. In contrast, after the appearance of CPV, changes in the CPV VP2 gene appear to be partly selected by certain positive selection forces. CPV and FPLV are known to be closely related viruses genetically and biologically, but the evolutionary mechanisms of the two viruses appeared to be different.

Amino Acid Substitution↗
PubMed1998

Assaying for structural variation in the parvovirus capsid and its role in infection.

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.

Animals↗
PubMed2000

U94, the human herpesvirus 6 homolog of the parvovirus nonstructural gene, is highly conserved among isolates and is expressed at low mRNA levels as a spliced transcript.

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B, respectively) encode homologs (U94) of the parvovirus nonstructural gene, ns1 or rep. Here we describe the HHV-6B homolog and analyze its genetic heterogeneity and transcription. U94 nucleotide and amino acid sequences differ by approximately 3.5% and 2.5%, respectively, between HHV-6A and HHV-6B. Among a collection of 17 clinically and geographically disparate HHV-6 isolates, intravariant nucleotide and amino acid sequence divergence was less than 0.6% and 0.2%, respectively; all 13 HHV-6B isolates had identical amino acid sequences. The U94 transcript is spliced to remove a 2.6-kb intron and is expressed at very low levels relative to other HHV-6B genes, reaching approximately 10 copies per cell 3 days after infection. The mRNA has several small AUG-initiated open reading frames upstream of the U94 open reading frame, a hallmark of proteins expressed at low levels. Consistent with this, the U94-encoded protein was immunologically undetectable in HHV-6B-infected cells. The high degree of sequence conservation suggests that the gene function is nearly intolerant of sequence variation. The low abundance of U94 transcripts and the presence of encoded inefficient translation initiation suggest that the U94 protein may be required only in small amounts during infection.

Base Sequence↗
PubMed1999

Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses.

Amino Acid Sequence↗
PubMed2000

Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization.

We cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid. After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity. Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG. Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting. The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted. Mutagenesis of predicted contact residues in three heavy-chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2. The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type. Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells.

Amino Acid Sequence↗
PubMed2000

Different patterns of restriction to B19 parvovirus replication in human blast cell lines.

B19 parvovirus can replicate in erythroid progenitor cells and in a small number of human blast cell lines. To better understand and analyze the B19 virus replicative cycle, we performed and compared the infection of bone marrow cells and of different blast cell lines with erythroblastoid and megakaryoblastoid phenotypic characteristics (UT-7, TF-1, M-07, and B1647). Following in vitro infection, B19-specific nucleic acids were characterized with regard to the genome-replicative intermediates, the transcription pattern, and the localization of virus-specific nucleic acids inside infected cells. While all cell lines tested proved to be susceptible to B19 virus infection, two different patterns of restriction to replication of B19 virus were observed. In the first restriction pattern, observed in UT-7 cells, the single-stranded viral DNA was converted to double-stranded replicative intermediates, identical to those found in bone marrow cells, and a full set of viral transcripts were observed. However, replication and transcription were restricted to a small subset of cells, and production of capsid proteins was not detected. In the second restriction pattern, observed in TF-1, M-07, and B1647 cells, the single-stranded viral DNA was not converted to double-stranded replicative intermediates.

Bone Marrow Cells↗
PubMed2001

Canine parvovirus capsid assembly and differences in mammalian and insect cells.

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Animals↗
PubMed2001

The small 11-kDa protein from B19 parvovirus binds growth factor receptor-binding protein 2 in vitro in a Src homology 3 domain/ligand-dependent manner.

The small 11-kDa proteins of B19 parvovirus contain three proline-rich regions which conform to consensus Src homology 3 (SH3) ligand sequences present in signaling molecules within the cell. We have shown that the B19 11-kDa proteins specifically interact with the growth factor receptor-binding protein 2 (Grb2) in vitro. Mutation of prolines within one of the three SH3 ligand-like sequences decreases the binding of B19 11-kDa proteins to Grb2, suggesting that the proline-rich region is involved in the B19 11-kDa/Grb2 interaction. Therefore, the B19 11-kDa proteins may function to alter Grb2-mediated signaling by disrupting SH3 domain/ligand interactions. These results implicate the 11-kDa proteins in B19 pathogenesis through perturbation of normal cellular signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria.

E3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants. This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus. Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs. A second construct, containing a different promoter, showed delayed growth and genome instability which, based on the size difference between these inserts, suggests a maximum packaging size of 106 to 109% wild-type genome size for CAV-1.

Adenovirus E3 Proteins↗