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Biomedical subjects

P Tattersall

Publications and source records attributed to P Tattersall.

At least 55 records · Page 3Linked to original sources
PubMed1988

The terminal protein of minute virus of mice is an 83 kilodalton polypeptide linked to specific forms of double-stranded and single-stranded viral DNA.

A new assay (label transfer from DNA to protein) enabled the identification of a terminal protein (TP) in nucleoprotein complexes extracted from cells infected with the parvovirus, minute virus of mice, MVM. In SDS-PAGE, TP migrates as a major band at 83 kDa, with a minor 65 kDa component, each of which exactly co-migrates with the cellular forms of the virally coded polypeptide NS-1. In parallel, the analysis of nucleoproteins by SDS-agarose gel electrophoresis allowed us to observe that the major species of viral DNA molecules (mRF, dRF and ssDNA) are all present in the form of DNA-protein complexes. Three forms of mRF DNA were identified, two of which are protein-associated and one which appears to be protein-free.

Animals↗
PubMed1988

Fetal infection with human parvovirus B19.

The case of premature infant with nonimmune hydrops secondary to human parvovirus B19 infection is presented. The infection was not suspected clinically. The fetal autopsy showed intranuclear inclusions in erythroid cells that stained positively with a DNA hybridization probe for B19 parvovirus.

Edema↗
PubMed1988

The NS-1 polypeptide of minute virus of mice is covalently attached to the 5' termini of duplex replicative-form DNA and progeny single strands.

When A9 cells are infected with minute virus of mice, a small proportion of the virally coded NS-1 polypeptide becomes covalently attached to newly synthesized viral DNA. Antisera directed against NS-1 will specifically precipitate two forms of monomer duplex replicative-form DNA, multimeric duplex intermediates and progeny single strands, and restriction analysis of the duplex forms in these precipitates reveals that NS-1 is exclusively associated with extended-form conformers of the genomic termini. Pulse-labeled viral DNA, harvested at various times in a highly synchronized infection, can be almost quantitatively precipitated with any one of a series of antisera directed against different protein domains distributed throughout the NS-1 molecule but not with antibodies directed against other viral proteins. In each case the interaction with NS-1 can be shown to involve both termini of duplex DNA and single-strand forms, suggesting that in each case a full-length (83-kilodalton) copy of NS-1 is present. Precipitation of the replicating viral DNA with an antibody directed against a synthetic 16-amino-acid peptide containing the sequence at the extreme carboxy terminus of NS-1 can be quantitatively and specifically inhibited with the immunizing peptide in its unconjugated form, showing that the antibodies responsible for precipitating viral DNA are directed against the NS-1 sequence itself and not against a trace contaminant. Exonuclease digestion studies show that the association effectively blocks the 5' ends of the DNA molecules. Very little (less than 0.1%) of the newly synthesized [35S]methionine-labeled NS-1 made in highly synchronized cells during a 15-min pulse early in infection (6.25 to 6.5 h into the S phase) becomes associated with viral DNA immediately. However, pulse-chase experiments show that later in infection (10 to 13 h into the S phase), when viral DNA replication is reaching its peak, a few percent of the molecules in these preexisting pools of NS-1 do become covalently attached to the newly replicated DNA. Isolated viral DNA-protein complexes labeled with [35S]methionine in this way can be obtained by fractionation of the immunoprecipitated complexes on Sepharose CL4B in sodium dodecyl sulfate. Digestion of the purified complexes with nuclease releases an 83-kilodalton molecule which exactly comigrates with authentic NS-1 in sodium dodecyl sulfate-polyacrylamide gels.

Animals↗
PubMed1988

Evidence that developmentally regulated control of gene expression by a parvoviral allotropic determinant is particle mediated.

An infectious molecular clone of the immunosuppressive strain of the autonomous parvovirus minute virus of mice [MVM(i)] was constructed deriving left-hand terminal sequences from a rare encapsidated plus strand. Progeny virus was shown to package the same proportions of plus and minus strands as did authentic MVM(i) virions. Rescue of virus from this clone also resulted in the repair of a 21-base truncation at the junction between the right-hand end of the viral insert and the vector and generated the same heterogeneous 5' end as is present in standard MVM(i) DNA. Progeny virus rescued by transfection of this clone into mouse cell lines displayed the lymphotropic phenotype characteristic of the parental MVM(i) virus from which it was derived. However, analysis of viral RNA from transfected mouse fibroblasts revealed that the MVM(i) and MVM(p) genomic clones are transcribed at the same low level. Furthermore, transfected fibroblasts yielded similar numbers of infectious centers regardless of which MVM clone was introduced. These results contrast markedly with the different infectivities of MVM(i) and MVM(p) particles and with the observation that viral transcription in fibroblasts productively infected with MVM(p) virions is 100-fold greater than that seen in the restrictive MVM(i) particle-mediated infection. These results suggest that the developmentally regulated intracellular factors controlling host cell susceptibility at the level of viral transcription interact with a component of the incoming viral capsid, rather than with a sequence within the viral DNA.

Animals↗
PubMed1988

Mapping of the fibrotropic and lymphotropic host range determinants of the parvovirus minute virus of mice.

The fibrotropic and lymphotropic strains of minute virus of mice are each unable to grow lytically in the differentiated host cell type of the other strain. To map the viral sequence responsible for the target cell specificities of the two strains, we constructed chimeric viral genomes in vitro from infectious genomic clones. The phenotypes of viral progeny derived from the chimeric genomes were tested by transfecting the plasmids into fibroblast monolayers and assaying plaque formation and by testing stocks of the recombinant viruses for cytotoxicity in fibroblast and lymphocyte cultures. Both the fibrotropic and lymphotropic determinants mapped to the same 237-nucleotide sequence within the coding region of the virus structural gene. A second sequence, near the viral promoter at map unit 38, was also shown to affect viral growth in fibroblast host cells profoundly.

Animals↗
PubMed1988

Human parvovirus infection in pregnancy.

During an outbreak of fifth disease in the Hartford, Connecticut area in the winter of 1986, nine pregnant women were exposed to the parvovirus. Five of these nine had serologic evidence of previous exposure and immunity; all five had uneventful pregnancies. Three of the four women who exhibited serologic evidence of recent exposure to the parvovirus had hydropic fetuses who died, one of whom was anencephalic. Histologic and DNA hybridization studies suggest parvovirus infection as a cause for nonimmune hydrops and fetal death. A review of the literature suggests that of 37 women who were exposed and infected by human B19 parvovirus during pregnancy, 14 (38%) had adverse outcomes, including spontaneous abortions, intrauterine fetal death, and congenital anomalies. Women at high risk for exposure (school teachers) should have screening for immunoglobulin G parvovirus-specific antibodies; if these are absent, they should avoid close contact with schoolchildren. Pregnant women with evidence of recent infection should have a level II ultrasound. Because no vaccine is currently available, routine screening cannot be recommended.

Adolescent↗
PubMed1987

Human parvovirus B19 infection during pregnancy.

Human parvovirus B19 (B19) has been implicated as the cause of fifth disease and has been associated with fetal death. We identified pregnant women who were at risk of contracting B19 infection during an outbreak of fifth disease. The sera of 12 women classified at high risk of exposure and 19 classified at low risk were tested during prenatal care, at delivery, or at both times for IgG and IgM antibodies to B19. Four women at high risk but none at low risk were considered infected because they were IgM positive. One IgM-positive woman gave birth to a stillborn hydropic fetus whose tissues were positive for B19 DNA by a nucleic acid hybridization assay. The other three IgM-positive women gave birth to normal offspring, of whom one had IgM-positive cord serum. We conclude that B19 infection during pregnancy can lead to fetal infection with at least two associated outcomes--no adverse effect on the fetus or fetal death.

Adolescent↗
PubMed1986

The NS-1 polypeptide of the autonomous parvovirus MVM is a nuclear phosphoprotein.

Cells infected with the autonomous parvovirus MVM synthesize a major virally coded non-structural protein which accumulates in the nucleus and is phosphorylated to a significant extent. Peptide map analysis shows that this in vivo product has the same primary sequence as the NS-1 protein previously identified in the in vitro translation products of MVM encoded mRNA, and as such is the product of the largest messenger RNA species, a spliced 4.8 kb (R1) transcript. In vivo NS-1 exists in two predominant forms, a phosphorylated 84-85 kDa species and an unphosphorylated or poorly phosphorylated 83 kDa form. Both forms are preferentially extracted from the nucleus using an extraction procedure which enriches for replication complexes.

Cell Line↗
PubMed1986

The role of parvovirus B19 in aplastic crisis and erythema infectiosum (fifth disease).

In 1984, simultaneous outbreaks of aplastic crisis and erythema infectiosum occurred in northeastern Ohio. Sera were analyzed from 26 patients with aplastic crisis: 24 had IgM specific for parvovirus B19, five had B19-like particles by electron microscopy, and 13 had DNA from B19; no sera from 33 controls had evidence of recent infection with B19 (P less than .0001). DNA from B19 was also detected in specimens of throat gargle and urine from two patients with aplastic crisis. Sera from 36 of 51 children with erythema infectiosum had B19-specific IgM, compared with serum from one of 42 susceptible controls (P less than .0001). DNA from B19 was detected in sera from only two of 51 patients who had erythema infectiosum. The secondary attack rates among susceptible contacts decreased with age (overall total, 49.6%). Differential rates of asymptomatic infection were observed among black (68.8%) and white (20.0%) household members (P = .003). These were the first identified simultaneous outbreaks of aplastic crisis and erythema infectiosum. Their occurrence provided an opportunity to study the epidemiology and spectrum of B19 infection with geographically and temporally matched comparison groups; our results support the hypothesis that infection with parvovirus causes these two clinical entities.

Adolescent↗
PubMed1986

Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections.

Adolescent↗
PubMed1986

DNA sequence of the lymphotropic variant of minute virus of mice, MVM(i), and comparison with the DNA sequence of the fibrotropic prototype strain.

The sequence of molecular clones of the genome of MVM(i), a lymphotropic variant of minute virus of mice, was determined and compared with that of MVM(p), the fibrotropic prototype strain. At the nucleotide level there are 163 base changes: 129 transitions and 34 transversions. Most nucleotide changes are silent, with only 27 amino acids changes predicted, of which 22 are conservative. Notable differences between the MVM(i) and MVM(p) genomes which may account for the cell specificities of these viruses occur within the 3' nontranslated regions. The differences discussed include the absence of a 65-base-pair direct in MVM(i), the presence of only two polyadenylation sites in MVM(i) compared with four in MVM(p), and sequences that bear a resemblance to enhancer sequences. Also included in this paper is an important correction to the MVM(p) sequence (C.R. Astell, M. Thomson, M. Merchlinsky, and D. C. Ward, Nucleic Acids Res. 11:999-1018, 1983).

Base Sequence↗
PubMed1986

Organization of nonstructural genes of the autonomous parvovirus minute virus of mice.

Regions of open reading frame (ORF) from the genome of the autonomous parvovirus minute virus of mice (MVM) were cloned into a procaryotic expression vector, and bacterial fusion proteins containing MVM-specific amino acid sequences were isolated. Antibodies raised against these proteins were used to immunoprecipitate viral proteins synthesized in vitro in a rabbit reticulocyte lysate translation system programmed with mRNA isolated from cells infected with MVM and a number of different parvoviruses. These studies demonstrated that: the 83-kilodalton nonstructural protein NS-1 and the 25-kilodalton nonstructural protein NS-2 have a common amino-terminal sequence which is encoded by the single ORF located between nucleotides 225 and 534 in the viral genome; the ORF located between nucleotides 1110 and 1638 is only expressed in the NS-1 protein; and the sequence encoded in a small alternative ORF between nucleotides 2075 and 2291 is expressed exclusively in NS-2. These data confirm that NS-1 is the product of the 4.8-kilobase R1 viral transcript and demonstrate that NS-2 is synthesized from the 3.3-kilobase R2 transcript which arises from the left-hand promoter at map unit 4 on the viral genome. Antibodies against the MVM fusion proteins also cross-reacted with similar proteins encoded by the viruses H-1 and LuIII, but although antibodies against the carboxy-terminal half of NS-1 cross-reacted with a similar protein in CPV, we were unable to demonstrate an NS-2 protein encoded by this virus.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis.

The nucleotide sequence of an almost-full-length clone of human parvovirus B19 was determined. Whereas the extreme left and right ends of this genomic clone are incomplete, the sequence clearly indicates that the two ends of viral DNA are related by inverted terminal repeats similar to those of the Dependovirus genus. The coding regions are complete in the cloned DNA, and the two large open reading frames which span almost the entire genome are restricted to one strand, as has been found for all other parvoviruses characterized to date. From the DNA sequence we conclude that the organization of the B19 transcription units is similar although not identical to those of other parvoviruses. In particular, we predict that the B19 genome may utilize a fourth promoter to transcribe mRNA encoding the major structural polypeptide, VP2. Analysis of the putative polypeptides confirms that B19 is only distantly related to the other parvoviruses but reveals that there is a small region in the gene probably encoding the major nonstructural protein of B19, which is closely conserved between all of the parvovirus genomes for which sequence information is currently available.

Amino Acid Sequence↗
PubMed1986

Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments.

Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights of 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Restriction endonuclease fragments of this cloned B19 genome were treated with BAL 31 and shotgun cloned into the open reading frame expression vector pJS413. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

Adolescent↗
PubMed1986

Human parvovirus B19-induced epidemic acute red cell aplasia in patients with hereditary hemolytic anemia.

From March to August 1984, 26 patients with hereditary hemolytic anemia in northeastern Ohio developed acute, profound red cell aplasia. The patients included 14 males and 12 females 2 to 23 years old, with sickle cell anemia (20 cases), hemoglobin SC-disease (4 cases), sickle-beta-thalassemia (1 case), or hereditary spherocytosis (1 case). All had an acute onset of severe reticulocytopenia and anemia and prodromal symptoms of illness including fever, abdominal symptoms, headache, and arthralgias. Twenty-two received transfusions. Reticulocytosis occurred spontaneously within 2 to 14 days of presentation. In five acute-phase sera, 10(8) to 10(12) viral particles/mL were detected by electron microscopy. Human parvovirus B19 DNA was demonstrated in high concentration by hybridization in the same five acute-phase sera and in low concentration in sera of eight additional patients. The five highly viremic sera inhibited erythroid colony formation in vitro. B19-specific IgM was detected in sera of 24/26 patients, and B19-specific IgG in 21 of 22 patients tested. Our results indicate that human parvovirus B19 was the etiologic agent in this large epidemic of life-threatening acute red cell aplasia in patients with hereditary hemolytic anemia.

Adolescent↗