Parvovirus variation for disease: a difference with RNA viruses?

The Parvoviridae, a family of viruses with single-stranded DNA genomes widely spread from invertebrates to mammal and human hosts, display a remarkable evolutionary capacity uncommon in DNA genomes. Parvovirus populations show high genetic heterogeneity and large population sizes resembling the quasispecies found in RNA viruses. These viruses multiply in proliferating cells, causing acute, persistent or latent infections relying in the immunocompetence and developmental stage of the hosts. Some parvovirus populations in natural settings, such as carnivore autonomous parvoviruses or primate adeno associated virus, show a high degree of genetic heterogeneity. However, other parvoviruses such as the pathogenic B19 human erythrovirus or the porcine parvovirus, show little genetic variation, indicating different virus-host relationships. The Parvoviridae evolutionary potential in mammal infections has been modeled in the experimental system formed by the immunodeficient scid mouse infected by the minute virus of mice (MVM) under distinct immune and adaptive pressures. The sequence of viral genomes (close to 10(5) nucleotides) in emerging MVM pathogenic populations present in the organs of 26 mice showed consensus sequences not representing the complex distribution of viral clones and a high genetic heterogeneity (average mutation frequency 8.3 x 10(-4) substitutions/nt accumulated over 2-3 months). Specific amino acid changes, selected at a rate up to 1% in the capsid and in the NS2 nonstructural protein, endowed these viruses with new tropism and increased fitness. Further molecular analysis supported the notion that, in addition to immune pressures, the affinity of molecular interactions with cellular targets, as the Crml nuclear export receptor or the primary capsid receptor, as well as the adaptation to tissues enriched in proliferating cells, are major selective factors in the rapid parvovirus evolutionary dynamics.

Genome replication and postencapsidation functions mapping to the nonstructural gene restrict the host range of a murine parvovirus in human cells.

The infection outcome of the Parvoviridae largely relies on poorly characterized intracellular factors modulated by proliferation, differentiation, and transformation of host cells. We have studied the interactions displayed by the highly homologous p and i strains of the murine parvovirus minute virus of mice (MVM), with a series of transformed cells of rat (C6) and human (U373, U87, SW1088, SK-N-SH) nervous system origin, seeking for molecular mechanisms governing parvovirus host range. The MVMp infection of C6 and U373 cells was cytotoxic and productive, whereas the other nervous cells behaved essentially as resistant to this virus. In contrast, MVMi did not complete its life cycle in any of the human nervous cells, though it efficiently killed the astrocytic tumor cells by two types of nonproductive infections: (i) normal synthesis of all viral macromolecules with a late defect in infectious virion maturation and release to the medium in U373; and (ii) high levels of accumulation of the full set of viral messenger RNAs and of both nonstructural (NS-1) and structural (VP-1 and VP-2) proteins, under a very low viral DNA amplification, in U87 and SW1088 cells. Further analyses showed that U87 was permissive for nuclear transport of MVMi proteins, leading to efficient assembly of empty viral capsids with a normal phosphorylation and VP1-to-VP2 ratio. The DNA amplification blockade in U87 occurred after conversion of the incoming MVMi genome to the monomeric replicative form, and it operated independently of the delivery pathway used by the viral particle, since it could not be overcome by transfection with cloned infectious viral DNA. Significantly, a chimeric MVMi virus harboring the coding region of the nonstructural (NS) gene replaced with that of MVMp showed a similar pattern of restriction in U87 cells as the parental MVMi virus, and it attained in U373 cultures an infectious titer above 100-fold higher under equal levels of DNA amplification and genome encapsidation. The results suggest that the activity of complexes formed by the NS polypeptides and recruited cellular factors restrict parvovirus DNA amplification in a cell type-dependent manner and that NS functions may in addition determine MVM host range acting at postencapsidation steps of viral maturation. These data are relevant for understanding the increased multiplication of autonomous parvovirus in some transformed cells and the transduction efficacy of nonreplicative parvoviral vectors, as well as a general remark on the mechanisms by which NS genes may regulate viral tropism and pathogenesis.

Phosphorylation status of the parvovirus minute virus of mice particle: mapping and biological relevance of the major phosphorylation sites.

The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the (32)P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.

Implanted BDNF-producing fibroblasts prevent neurotoxin-induced serotonergic denervation in the rat striatum.

Degeneration of serotonergic fibers in the rat striatum was produced by local administration of the serotonergic neurotoxin 5, 7-dihydroxytryptamine (5,7-DHT) or the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), which is also toxic to serotonergic neurons. One week before neurotoxin administration, fibroblasts engineered to express the human BDNF gene were grafted into the mesencephalon, dorsal to the substantia nigra. Rats implanted with fibroblasts expressing the LacZ gene were used as controls, as well as sham-operated animals (not injected with any neurotoxin). After a survival period of 1 week, the serotonergic innervation of the striatum was assessed by measuring serotonin (5-HT) content and by immunohistochemical detection of 5-HT positive fibers. BDNF-producing cells prevented the striatal 5-HT loss induced by local administration of either 5,7-DHT or MPP(+), as well as the striatal dopamine (DA) loss induced by the latter neurotoxin. Grafting of fibroblasts carrying the BDNF or the Lac-Z gene did not modify striatal 5-HT or DA content in sham-operated animals. In 5, 7-DHT-lesioned rats, implanted or not with control Lac-Z fibroblasts, a striking reduction in the density of 5-HT immunoreactive fibers was observed. By contrast, the density of 5-HT fibers was similar in rats implanted with BDNF-producing fibroblasts as compared to sham-operated controls. The protective effect of BDNF on the damage to serotonergic terminals induced by the two neurotoxins suggests the interest of this neurotrophin in the treatment of behavioral disorders associated to neurodegenerative diseases.

A beta-stranded motif drives capsid protein oligomers of the parvovirus minute virus of mice into the nucleus for viral assembly.

The determinants of nuclear import in the VP-1 and VP-2 capsid proteins of the parvovirus minute virus of mice strain i (MVMi) synthesized in human fibroblasts were sought by genetic analysis in an infectious plasmid. Immunofluorescence of transfected cells revealed that the two proteins were involved in cooperative cytoplasmic interactions for nuclear cotransport. However, while VP-1 translocated regardless of extension of deletions and did not form capsid epitopes by itself, VP-2 seemed to require cytoplasmic folding and the overall conformation for nuclear transport. The sequence (528)KGKLTMRAKLR(538) was found necessary for nuclear uptake of VP-2, even though it was not sufficient to confer a nuclear localization capacity on a heterologous protein. In the icosahaedral MVMi capsid, this sequence forms the carboxy end of the amphipathic beta-strand I (betaI), and all its basic residues are contiguously positioned at the face that in the unassembled subunit would be exposed to solvent. Mutations in singly expressed VP-2 that either decrease the net basic charge of the exposed face (K530N-R534T), perturb the hydrophobicity of the opposite face (L531E), or distort the betaI conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by betaI mutants indicated that the basic residues clustered at one face of betaI drive VP oligomers into the nucleus preceding and uncoupled to assembly and that the nuclear environment is required for MVMi capsid formation in the infected cell. The degree of VP-1/VP-2 transport cooperativity suggests that VP trimers are the morphogenetic intermediates translocating through the nuclear pore. The results support a model in which nuclear transport signaling preserves the VP-1/VP-2 stoichiometry necessary for efficient intranuclear assembly and in which the beta-stranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis.

Biochemical and physical characterization of parvovirus minute virus of mice virus-like particles.

The VP-2 major capsid protein of the prototype strain of the parvovirus minute virus of mice (MVMp) was expressed, using a baculovirus vector, in Sf9 insect cells. Immunogold electron microscopy of infected Sf9 cells showed VP-2 localized in the nucleus and cytoplasm as is observed in mammalian cells during natural infections. The VP-2 subunits self-assembled into empty parvovirus-like particles (VLPs), which appeared morphologically similar to and immunogenically indistinguishable from native empty MVMp particles, which also contain the minor capsid protein, VP1. Incubations under different pH and temperature conditions showed that the MVMp VLPs and native empty MVMp capsids share comparable stability. Once heated the particles can be similarly and specifically cleaved by trypsin at the VP-2 N-terminal domain. This process mimics the further maturation of the "rat-like" parvovirus virions, following viral DNA encapsidation, indicating that biologically relevant features of the MVMp capsid are maintained in the VLPs. Crystals have been obtained for the MVMp VLPs which were isomorphous to those used for the high-resolution structure determination of virions and native empty particles of the immunosuppressive strain of MVM (MVMi). The VLP crystals diffracted X rays to beyond 3-A resolution and are in space group C2 (a = 448.7, b = 416.6, c = 306.1 A, and beta = 95.9 degrees ). This is the first report of crystals from parvoviral particles produced in a heterologous system diffracting X rays to high resolution, indicating that VP-2 of some parvovirus capsids can self-assemble into ordered T = 1 icosahedral capsids in the absence of other viral and host cell functions.

Severe leukopenia and dysregulated erythropoiesis in SCID mice persistently infected with the parvovirus minute virus of mice.

Parvovirus minute virus of mice strain i (MVMi) infects committed granulocyte-macrophage CFU and erythroid burst-forming unit (CFU-GM and BFU-E, respectively) and pluripotent (CFU-S) mouse hematopoietic progenitors in vitro. To study the effects of MVMi infection on mouse hemopoiesis in the absence of a specific immune response, adult SCID mice were inoculated by the natural intranasal route of infection and monitored for hematopoietic and viral multiplication parameters. Infected animals developed a very severe viral-dose-dependent leukopenia by 30 days postinfection (d.p.i.) that led to death within 100 days, even though the number of circulating platelets and erythrocytes remained unaltered throughout the disease. In the bone marrow of every lethally inoculated mouse, a deep suppression of CFU-GM and BFU-E clonogenic progenitors occurring during the 20- to 35-d.p.i. interval corresponded with the maximal MVMi production, as determined by the accumulation of virus DNA replicative intermediates and the yield of infectious virus. Viral productive infection was limited to a small subset of primitive cells expressing the major replicative viral antigen (NS-1 protein), the numbers of which declined with the disease. However, the infection induced a sharp and lasting unbalance of the marrow hemopoiesis, denoted by a marked depletion of granulomacrophagic cells (GR-1(+) and MAC-1(+)) concomitant with a twofold absolute increase in erythroid cells (TER-119(+)). A stimulated definitive erythropoiesis in the infected mice was further evidenced by a 12-fold increase per femur of recognizable proerythroblasts, a quantitative apoptosis confined to uninfected TER-119(+) cells, as well as by a 4-fold elevation in the number of circulating reticulocytes. Therefore, MVMi targets and suppresses primitive hemopoietic progenitors leading to a very severe leukopenia, but compensatory mechanisms are mounted specifically by the erythroid lineage that maintain an effective erythropoiesis. The results show that infection of SCID mice with the parvovirus MVMi causes a novel dysregulation of murine hemopoiesis in vivo.

Relevance of myeloablative conditioning in the engraftment of limiting numbers of normal and genetically marked lympho-hematopoietic stem cells.

We have studied the relevance of using myeloablative conditioning in the engraftment of limiting numbers of normal and retrovirally transduced bone marrow (BM) cells. The administration of high doses (10-12 Gy) of split-dose irradiation (4 h or 24 h intervals) to mice transplanted with small grafts (10(4)-10(6) cells) of normal BM not only minimized the endogenous reconstitution of recipients but also increased, with respect to single irradiation protocols, the survival rate of the animals in the long-term. The efficiency of these myeloablative regimens in BM transplantation protocols involving the use of transduced grafts was tested in mice transfused with 5 x 10(4)-2 x 10(6) genetically marked BM cells. Ninety percent of recipients survived in the long-term and, in most cases, predominant engraftment of the transduced population was apparent for up to 11 months post-transplantation. This was confirmed in hematopoietic samples corresponding to the CFU-S, the preCFU-S and the long-term repopulating cells of primary recipients. It was of significance, however, that reductions in the engraftment of the genetically marked cells were not associated with the engraftment of exogenous untransduced cells, but rather with rises in the extent of endogenous repopulation, revealing the difficulties of preventing the repopulation of residual endogenous stem cells when limiting numbers of transduced cells are transplanted. Our results emphasize the relevance of using efficient myeloablative conditioning regimens in those cases in which predominant and sustained engraftment of limiting numbers of transduced repopulating cells is required.

Parvovirus minute virus of mice strain i multiplication and pathogenesis in the newborn mouse brain are restricted to proliferative areas and to migratory cerebellar young neurons.

Newborn BALB/c mice intranasally inoculated at birth with a lethal dose of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) developed motor disabilities and intention tremors with a high incidence by the day 6 postinfection (dpi). These neurological syndromes paralleled the synthesis of virus intermediate DNA replicative forms and yield of infectious particles in the brain, with kinetics that peaked by this time. The preferred virus replicative sites in the brain were established early in the infection (2 dpi) and at the onset of clinical symptoms (6 dpi) and were compared with major regions of cellular proliferative activity found after intraperitoneal injection of bromodeoxyuridine 24 h before encephalons were subjected to immunohistochemistry detection. At 2 dpi, viral capsid antigen was located in the laterodorsal thalamic and the pontine nuclei but not in the extensive proliferative regions of the mouse brain at this postnatal day. At 6 dpi, however, the neurotropism of the MVMi was highlighted by its ability to target the subventricular zone of the ventricles, the subependymal zone of the olfactory bulb, and the dentate gyrus of the hippocampus, which are the three main germinal centers of the cerebrum in mouse postbirth neurogenesis. Unexpectedly, in the cerebellum, the MVMi capsid antigen was confined exclusively to cells that have undergone mitosis and have migrated to the internal granular layer (IGL) and not to the proliferative external granular layer (EGL), which was stained with antiproliferative cell nuclear antigen antibody and is the main target in other parvovirus infections. This result implies temporal or differentiation coupling between MVMi cycle and neuroblast morphogenesis, since proliferative granules of the EGL should primarily be infected but must migrate in a virus carrier state into the IGL in order to express the capsid proteins. During migration, many cells undergo destruction, accounting for the marked hypocellularity specifically found in the IGL and the irregular alignment of Purkinje cell bodies, both consistent histopathological hallmarks of animals developing cerebellar symptoms. We conclude that MVMi impairs postmitotic neuronal migration occurring in the first postnatal week, when, through the natural respiratory route of infection, the virus titer peaks in the encephalon. The results illustrate the intimate connection between MVMi neuropathogenesis and mouse brain morphogenetic stage, underscoring the potential of parvoviruses as markers of host developmental programs.

Myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i).

The in vivo myelosuppressive capacity of strain i of the parovirus minute virus of mice (MVMi) was investigated in newborn BALB/c mice inoculated with a lethal intranasal dose. MVMi infection reached maximum levels of DNA synthesis and infectious titers in lymphohemopoietic organs at 4 to 6 days postinoculation and was restricted by an early neutralizing humoral immune response. After viral control (by 10 days postinoculation), a significant decrease in femoral and splenic cellularity, as well as in granulocyte-macrophage colony-forming unit and erythroid burst-forming unit hemopoietic progenitors, was observed in most inoculated animals. This delayed myeloid depression, although it may be not a major cause of the lethality of the infection, implies indirect pathogenic mechanisms induced by MVMi infection in a susceptible host.

Transcriptional inhibition of the parvovirus minute virus of mice by constitutive expression of an antisense RNA targeted against the NS-1 transactivator protein.

We have assessed a genetic resistance approach based on antisense RNA to interfere with the prototype Minute Virus of Mice (MVMp), an autonomous parvovirus. MVMp is a cytolytic virus when infecting the permissive A9 mouse fibroblast cell line, and its gene expression is largely regulated at the level of transcription initiation by the nonstructural transactivator NS-1 protein, a multifunctional polypeptide also involved in viral DNA replication and cytotoxicity. An NS-1 specific antisense RNA constitutively expressed in transfected A9 clones increased several fold the proliferative viability of the cells upon high multiplicity virus infection, and cultures infected at low multiplicity reached confluence overcoming virus progression. All clones shared a common phenotype of resistance characterized by a lowered synthesis of viral DNA replicative intermediates and genomic forms, a significant reduction in the accumulation of the three viral messengers in the cytoplasmic and nuclear compartments, and a specific inhibition in viral protein synthesis. These results indicate that the constitutive antisense RNA mediates an overall repression of viral macromolecular synthesis by preventing the onset of NS-1 functions. Therefore, cytocidal parvoviruses may be hampered by engineered antisense RNA targeted against early regulators of virus growth.

Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells.

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5-FU treatment were incubated under IL-3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU-GM and CFU-S12 progenitor pools was observed (130- and 15-fold, respectively), with no apparent impairment in long-term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU-GM susceptibility to the infection by supernatants containing a Moloney-derived ecotropic retroviral vector carrying the neor gene. The designed protocol thus involved the infection of freshly harvested 5-FU-treated bone marrow, followed by expansion under IL-3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU-GM and 50 CFU-S12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neor gene and expressing resistance to G418 5 months after the transplantation indicates that long-term lympho-haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self-renewal and differentiation properties. This gene-transfer methodology may improve the development of gene therapy protocols where the purging of non-transduced precursors would guarantee a lasting and uniform expression of exogenous genes.

Comparison of electrophysiologic effects of quinidine and amiodarone in sustained ventricular tachyarrhythmias associated with coronary artery disease.

Fifteen patients with spontaneous ventricular tachyarrhythmias underwent electrophysiologic studies at baseline and during therapy with quinidine and amiodarone. In 9, ventricular tachycardia (VT) with a similar QRS morphology was induced with quinidine, amiodarone and under the control state. Both quinidine and amiodarone significantly increased QRS duration and the VT cycle length. Amiodarone increased the VT cycle length more than quinidine (85 vs 121 ms, p < 0.05). Amiodarone increased the percent QRS duration (during sinus rhythm, ventricular pacing and VT) significantly less than percent VT cycle length, whereas quinidine did so only at slow rates (at faster rates the percent increase in QRS duration is not different from the percent increase in VT cycle length). The percent increase in QRS duration produced by quinidine correlated significantly with the percent increase in VT cycle length (the best correlation was observed during pacing, r = 0.78). In contrast, no such significant correlations were obtained for amiodarone. Thus, amiodarone prolongs VT cycle length more than quinidine (at the doses used). The effects of quinidine on conduction in tissue mostly unrelated to tachycardia origin predict effects in the tachycardia cycle length. In contrast, the effects of amiodarone on the latter are more intense but not predicted by those on tissue unrelated to the tachycardia origin.

Treatment of ventricular arrhythmias in patients with hypertrophic cardiomyopathy.

In patients with hypertrophic cardiomyopathy, asymptomatic episodes of non-sustained ventricular tachycardia are frequent and of prognostic significance for sudden cardiac death in adults, particularly in those whose consciousness is impaired. Low-dose amiodarone, found to be beneficial in mildly symptomatic non-sustained ventricular tachycardia, but detrimental in a subgroup with prominent non-arrhythmic symptoms, could be an alternative to an electrophysiological approach in patients with syncope but preserved functional capacity. In cardiac arrest survivors, an electrophysiological approach seems preferable to empiric amiodarone, together with implantation of a defibrillator, at least in those in whom inducible arrhythmias cannot be suppressed.

Protein species of the parvovirus minute virus of mice strain MVMp: involvement of phosphorylated VP-2 subtypes in viral morphogenesis.

The pattern of induced protein species of the prototype strain of the parvovirus minute virus of mice was determined in permissive A9 mouse fibroblast cells by high-resolution two-dimensional gel electrophoresis. Identities of the viral proteins in the gels were assigned by probing two-dimensional blots with antisera raised against either purified capsids (recognizing VP-1 and VP-2) or specific coding regions of the nonstructural proteins (NS-1 and NS-2) expressed as beta-galactosidase fusion products in bacteria. All viral proteins showed posttranslational modifications, phosphate being a common substituent. The NS-1 protein migrated as a basic polypeptide in the pI range of 7.4 to 7.8 with multiple stages of modification and as a likely minor but hyperphosphorylated component in the neutral region of the gel. The NS-2 isoforms were resolved at a pI value close to 5.5 as three groups of unevenly phosphorylated polypeptides, each composed of at least two protein species. Both VP-1 and VP-2 structural polypeptides were induced as heterogeneous phosphoproteins. The major VP-2 protein could be resolved in the form of a consistent pattern of three abundant (a to c), two intermediate (d and e), and one meager (f) neutral isoelectric focusing species or subtypes. This posttranslational modification precedes and is uncoupled from viral assembly, and all of the VP-2 subtypes are packaged into empty capsids at the induced stoichiometry. However, intracellular full virions harbored additional phosphorylated subtypes (g to l) and a subtle rearrangement in the whole VP-2 composition, while mature virions purified from lysed cultures lacked these subtypes, coordinately with the emergence of six neutral VP-3 subtypes. Thus, the virion coat undergoes a chemical transition entailed by genome encapsidation, in which phosphates seem to play a major role, triggering the preferential proteolytic cleavage of the more acidic VP-2 subtypes to VP-3. Parvoviruses, with small coding capacity, may regulate some morphogenetic steps, such as assembly, genome encapsidation, and maturation, by posttranslational modifications of their structural proteins.

In vitro myelosuppressive effects of the parvovirus minute virus of mice (MVMi) on hematopoietic stem and committed progenitor cells.

The interaction of two strains of the parvovirus minute virus of mice (MVM) with the mouse hematopoietic system has been studied. The immunosuppressive strain MVMi, but not the prototype virus MVMp, inhibited hematopoiesis in vitro, as judged by colony-forming assays of the erythroid burst-forming unit and granulocyte-monocyte colony-forming unit (CFU-GM) progenitors. Interestingly, primitive hematopoietic cells of the stem compartment (CFU-S12d), were equally susceptible to the MVMi cytotoxic infection, unravelling an unprecedented feature of virus-hematopoiesis interactions. The replication of both strains of MVM virus was evaluated in primary myeloid cells of long-term bone marrow cultures. A high viral DNA synthesis and maturation was observed in MVMi-infected myeloid cells, but it was undetectable in MVMp infections; moreover, the expression of the cytotoxic nonstructural NS-1 protein, a more reliable parameter of cell permissiveness to MVM infection, was only detected in MVMi-infected cells. Correspondingly, MVMi was propagated to high titers of infectious virus and it mediated an acute myelosuppression in these cultures. We conclude that MVMi has a wider tropism than was previously suspected and it is proposed that cytotoxic infection of hematopoietic stem cells, besides that of committed progenitors, may provide an additional basis to understand the pathogenesis of certain animal and human bone marrow failures of viral etiology.

Cytotoxic infection of hematopoietic stem and committed progenitor cells by the parvovirus minute virus of mice. Propagation of an acute myelosuppression in culture.

We have investigated the ability of two strains of the parvovirus minute virus of mice to impair mouse hematopoiesis in vitro. We found that the BFU-E and CFU-GM committed progenitors, CFU-Mix pluripotent progenitor, as well as the CFU-S12d, one of the most primitive hematopoietic precursors of the stem cell compartment detectable by colony technique, were similarly inhibited in their proliferative capacity by the immunosuppressive strain MVMi, but not by the prototype virus MVMp. The inhibitory effect correlated with the input of purified MVMi and was reversed by neutralizing MVM antiserum, showing that cytotoxic mechanisms underlying infectious MVMi replication and not operating in MVMp-infected cells were responsible for the reproductive death of hematopoietic precursors. In agreement with this, myeloid nonadherent cells of long-term bone marrow cultures were selectively permissive for MVMi but not for MVMp replication, as judged by viral DNA synthesis, the expression of the nonstructural cytotoxic NS-1 protein, and virus propagation in these cells. Altogether, the suppressive effects mediated by the MVMi cytotoxic infection define a wide lympho-myelotropism not previously reported for this virus. The MVM-mouse model highlights the role that unsuspected virus-hematopoietic compartment interactions may play in bone marrow failures of immunocompromised animal or human hosts.

Multigene families in African swine fever virus: family 110.

The genome of African swine fever virus was screened for the existence of repetitive sequences by hybridization between different cloned restriction fragments covering the viral DNA. Several sets of repeated sequences were detected in fragments located close to the DNA ends. One of these groups of repetitions involved fragments located at both ends of the genome. The remaining groups involved fragments that were located exclusively at the left end. The sequence of a 3.2-kilobase segment spanning from 7.5 to 11 kilobases from the left DNA end, which showed a complex pattern of cross-hybridizations, was determined. Two short and three long blocks of direct repeated sequences were found in this DNA region, which accounted for the hybridization results. The repeated sequences formed a family of five homologous genes with an average length of 116 codons (multigene family 110), one of which had a dimeric structure. Transcripts of the five members of the family were detected both in RNA synthesized in vitro by purified African swine fever virions and in RNA isolated at early times after infection. Comparison of the predicted protein sequences revealed a striking conservation of a cysteine-rich domain in the central part of the proteins. In addition, a highly hydrophobic NH2-terminal sequence present in all the proteins suggests that these proteins are processed through the endoplasmic reticulum.

Multigene families in African swine fever virus: family 360.

A group of cross-hybridizing DNA segments contained within the restriction fragments RK', RL, RJ, and RD' of African swine fever virus DNA were mapped and sequenced. Analysis of these sequences revealed the presence of a family of homologous open reading frames in regions close to the DNA ends. The whole family is composed of six open reading frames with an average length of 360 coding triplets (multigene family 360), four of which are located in the left part of the genome and two of which are in the right terminal EcoRI fragment. In close proximity to the right terminal inverted repeat, we found an additional small open reading frame which was homologous to the 5'-terminal portion of the other open reading frames, suggesting that most of that open reading frame has been deleted. These repeated sequences account for the previously described inverted internal repetitions (J.M. Sogo, J.M. Almendral, A. Talavera, and E. Viñuela, Virology 133:271-275, 1984). Most of the genes of multigene family 360 are transcribed in African swine fever virus-infected cells. A comparison of the predicted protein sequences of family 360 indicated that several residues are conserved, suggesting that an overall structure is maintained for every member of the family. The transcription direction of each open reading frame, as well as the evolutionary relationships among the genes, suggests that the family originated by gene duplication and translocation of sequences between the DNA ends.

Procainamide-induced slowing of ventricular tachycardia with insights from analysis of resetting response patterns.

To investigate the mechanism of slowing of the rate of ventricular tachycardias (VTs) by procainamide, resetting response patterns were characterized in 24 VTs in 22 patients. All patients had coronary artery disease and inducible sustained VT during procainamide therapy. Only tachycardias with the same surface QRS morphology before and after procainamide were studied: all were slowed by procainamide. The mean cycle length was 292 +/- 61 ms before and 374 +/- 61 ms after procainamide (p less than 0.05). The mean effective refractory period, measured at the right ventricle, was 241 +/- 21 ms before and 261 +/- 24 ms after procainamide (p less than 0.05). During procainamide therapy, single and double extrastimuli were delivered during VT and resetting response patterns identified. Patterns were characterized as flat, increasing or flat plus increasing. Resetting was seen in 17 (71%) of these VTs and resetting response patterns were identified in 16 (94%) of these. The resetting response pattern was flat in 7, flat plus increasing in 5 and increasing in 4. The finding of some flat portion at the end of resetting response patterns in 12 VTs after procainamide indicates that the reentrant impulse conducts through fully recovered tissue within the circuit. It suggests that procainamide slowed these VTs by slowing conduction velocity in fully recovered tissue due to sodium channel blockade and not by prolongation of action potentials and refractory periods.