RESUMEN

The 5' ends of all newly synthesized single-stranded (s1) DNA genomes of the autonomous parvovirus minute virus of mice are covalently linked to the major virally coded nonstructural protein NS-1, but later in infection this association is disrupted, giving rise to an abbreviated form of single-stranded DNA designated s2. Both s1 and s2 forms are encapsidated and migrate in velocity gradients as 110S particles, and, as such, both appear to be infectious. Most virions are released from A9 cells as s1 particles, but the NS-1 molecules are located on the outside of the virion where they are accessible to both antibodies and enzymes. These polypeptides are cleaved from the encapsidated DNA by nucleolytic or proteolytic digestion, which can occur either in the culture medium or upon subsequent entry into further host cells. Since the s1 to s2 cleavage can be minimized by blocking viral reentry, it is likely that most of the processing occurs after entry into the host cell. Incoming virus is rapidly converted to the s2 form when it is used to infect new host cells, but in vitro removal of the NS-1 molecules with proteases or nucleases fails to influence the infectivity of s1 particles under normal culture conditions. Limited proteolysis of s1 particles with trypsin demonstrates that NS-1 is linked to the DNA via its amino-terminal domain. Analysis of the 5' ends of s1 and s2 forms indicates that there are approximately 24 externally located nucleotides linking the NS-1 molecules to the 5.1-kilobase nuclease-resistant DNA core of the virion.

Asunto(s)

RESUMEN

Virions of minute virus of mice were purified by sedimentation in sucrose gradients and chromatography on DEAE-cellulose columns and shown to consist of single-stranded viral DNA and the viral capsid polypeptides VP-1 (83 kDa) and VP-2 (64.5 kDa). A 63-kDa polypeptide distinct from the viral capsid polypeptide VP-3 (61.4 kDa) was found in some virion preparations. Virions sedimented at 135 and 110 S. The genomic single strands associated with purified 135 and 110 S virions were covalently bound to a protein as judged by the anomalous electrophoretic mobility of the DNA in agarose gels at pH 12.5. The protein was removed from the DNA by Pronase but remained bound after heating at 98 degrees in the presence of 0.1% sodium dodecyl sulfate. Nuclease digestion of the purified DNA-protein complex released several polypeptides ranging in size from 58 to 65 kDa. Restriction enzyme analysis of the purified DNA protein complex following its conversion to a duplex RF DNA in vitro showed that the protein was attached to the 5' termini of the DNA.

Asunto(s)

Asunto(s)

RESUMEN

Minute virus of canines (MVC, canine parvovirus-1), originally isolated in 1970 from the feces of normal dogs, was compared with canine parvovirus type-2 (CPV-2). The two viruses, which differ in their host cell ranges and spectra of hemagglutination, also were found distinct in their antigenic and genomic properties. We demonstrated that the MVC replicates in dogs and is capable of producing pathologic changes that were most prominent in oronasally-exposed neonatal pups. Macroscopic and microscopic lesions were most prominent in the thymus and lymph nodes; minor changes were found in the duodenal crypts. The MVC strain used had been passaged 13 times in cell cultures and it may not represent the true virulence of naturally occurring virus.

Asunto(s)

RESUMEN

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.

Asunto(s)

Asunto(s)

RESUMEN

Canine parvovirus (CPV) strains were compared for haemagglutination (HA) activity and antigenicity and were divided into two types by HA activity. Strains Cp49 and 29-F showed temperature-dependent HA, like feline panleukopenia virus (FPLV) and mink enteritis virus (MEV), whereas strains Sp-80 and Y-1 showed temperature-independent HA with erythrocytes from eight species of animals. The results of a cross HA inhibition test using immunized rat sera suggested that of the two types of CPV those showing temperature-dependent HA were antigenically like FPLV and MEV whereas those showing temperature-independent HA were not. This antigenic difference between the two types was confirmed by a HA inhibition test with monoclonal antibodies. A chronological survey revealed that CPV isolates from earlier years have HA activity and antigenicity similar to those of FPLV and MEV, whereas current CPV isolates do not. There are some exceptional isolates from the transitional period which have similar antigenicity to FPLV and MEV but different HA activity. These results suggest that the haemagglutinin of CPV altered from one form resembling that of FPLV to a somewhat different structure during passage in dogs in nature.

Asunto(s)

RESUMEN

Two nonstructural proteins of bovine parvovirus (BPV) with apparent molecular sizes of 75,000 and 83,000 daltons have been detected. The proteins were immunoprecipitated from lung cells infected with various isolates of BPV and from in vitro translations of infected cell mRNA. These proteins were expressed as nuclear phosphoproteins and were synthesized early in infection, before the peak of capsid protein synthesis. Early in infection, the 75-kilodalton-size species could be resolved into two bands of equal intensity, but later in infection, the lower-molecular-size form predominated. Antibodies directed against bacterial fusion proteins encoding amino acid sequences from a highly conserved region of the NS-1 polypeptides of two other parvoviruses, minute virus of mice and the human virus B19, gave specific nuclear fluorescence with BPV-infected cells, although the antibodies failed to immunoprecipitate any viral proteins. The noncapsid proteins appear to be homologous to the previously characterized NS-1 proteins of other autonomous parvoviruses.

Asunto(s)

RESUMEN

The major capsid and noncapsid proteins of the pathogenic parvovirus B19, propagated in vitro, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation, and immunoblot of the erythroid fraction of infected human bone marrow cell cultures. There were two capsid proteins of 58 kilodaltons (kDa; the major species) and 84 kDa (the minor species). Newly synthesized capsid viral proteins were present in the supernatants of infected cultures. The major noncapsid protein of 77 kDa was localized to the nucleus.

Asunto(s)

RESUMEN

The immunoblot analysis of canine parvovirus (CPV) polypeptides with 47 rat monoclonal antibodies (RH) has revealed four different types of reaction. Many of these antibodies do not recognize the electroblotted proteins. However, among these monoclonals that do react, 12 recognize all three viral polypeptides (VP67, VP70 and VP85), 3 recognize preferentially the VP85 and one is exclusively directed against VP70. Thus, three antigenic regions are proposed which correspond to domains of the CPV polypeptides. In addition, these results together with evidence for the biological activities of the monoclonals suggest that the C and N terminals of VP85 are exposed at the surface of the viral particle.

Asunto(s)

RESUMEN

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.

Asunto(s)

Asunto(s)

RESUMEN

Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA.

Asunto(s)

RESUMEN

We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.

Asunto(s)

Asunto(s)

RESUMEN

Isolates of ADV replicate to rather high quantities in lungs from neonatally infected mink kits. The virus was analysed for polypeptide composition, and for the first time high molecular weight polypeptides have been observed in in vivo produced ADVs. These polypeptides are analogous to those of in vitro produced ADVs. The molecular weights of the structural polypeptides of the low virulence Pullman ADV and the highly virulent DK and Utah I isolates of ADV were found to be 88kD and 78kD and in vivo produced ADV-G polypeptides were found to be 85kD and 75kD, the same molecular weights as those described for in vitro produced ADV-G. Presence of the ADV coded, non-structural polypeptide with the molecular weight of 71kD (p71) was also demonstrated in the lung tissue from mink kits.

Asunto(s)

RESUMEN

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

Asunto(s)

RESUMEN

In the spring of 1980, an epidemic of an illness that resembled erythema infectiosum occurred in Manitoba, Canada. We initiated prospective epidemiologic, clinical, and microbiologic studies of this illness among elementary-school children and their families. Initial microbiologic studies failed to identify the cause of the exanthem. After a similar illness associated with serologic evidence of human parvovirus infection occurred in London, stored specimens of 12 patients with exanthem were investigated for parvovirus infection. Eleven patients had parvovirus-specific IgM antibody, as did two family contacts and a teacher with nonexanthematous illnesses, and two asymptomatic family members. None of 28 children with measles or rubella had serologic evidence of recent parvovirus infection. Human parvovirus was detected by DNA hybridization and immune electron microscopy in the serum of one patient who later had a rash and in one unaffected family contact. Parvovirus DNA was also detected in the pharyngeal specimen of the teacher who was ill but did not have a rash. We conclude that human parvovirus infection can be asymptomatic or cause a variety of clinical manifestations, including nonexanthematous illness and an illness resembling erythema infectiosum.

Asunto(s)

RESUMEN

Half of the genomic DNA of the human parvovirus (B19) was cloned in the plasmid pBR322. The cloned DNA was used as a molecular probe for the detection of parvovirus in serum by means of a dot hybridization test. In an assay of 26 samples, the dot hybridization test was found to be of comparable sensitivity and to be as rapid as radioimmunoassay for viral antigen detection; it is potentially useful as a diagnostic test.

Asunto(s)

Asunto(s)