Assessing public health risk in the London polonium-210 incident, 2006.

OBJECTIVES: Mr Alexander Litvinenko died in a London hospital on 23 November 2006, allegedly from poisoning with the radionuclide polonium-210 ((210)Po). Associated circumstances required an integrated response to investigate the potential risk of internal contamination for individuals exposed to contaminated environments. STUDY DESIGN: Descriptive study. METHODS: Contaminated locations presenting a potential risk to health were identified through environmental assessment by radiation protection specialists. Individuals connected with these locations were identified and assessed for internal contamination with (210)Po. RESULTS: In total, 1029 UK residents were identified, associated with the 11 most contaminated locations. Of these, 974 were personally interviewed and 787 were offered urine tests for (210)Po excretion. Overall, 139 individuals (18%) showed evidence of probable internal contamination with (210)Po arising from the incident, but only 53 (7%) had assessed radiation doses of 1mSv or more. The highest assessed radiation dose was approximately 100mSv. CONCLUSIONS: Although internal contamination with (210)Po was relatively frequent and was most extensive among individuals associated with locations judged a priori to pose the greatest risk, a high degree of assurance could be given to UK and international communities that the level of health risk from exposure to the radionuclide in this incident was low.

Characterization and molecular cloning of a human parvovirus genome.

The genome of the small human virus serologically associated with erythrocyte aplasia and erythema infectiosum (fifth disease) is shown to be a linear, nonpermuted, single-stranded DNA molecule with self-priming hairpin termini, properties which are characteristic of the genomes of the family Parvoviridae. This human parvovirus chromosome was molecularly cloned into bacterial plasmid vectors and the cloned DNA was used to explore its relatedness to other mammalian parvovirus serotypes by DNA:DNA hybridization. It is not related to the human adeno-associated viruses but does show a distant evolutionary relationship to genomes of the helper-independent parvoviruses of rodents. This strongly suggests that it is an autonomous parvovirus, and as such is the first example of a member of this group of common animal pathogens to cause disease in man.

Two new members of the emerging KDWK family of combinatorial transcription modulators bind as a heterodimer to flexibly spaced PuCGPy half-sites.

Initially recognized as a HeLa factor essential for parvovirus DNA replication, parvovirus initiation factor (PIF) is a site-specific DNA-binding complex consisting of p96 and p79 subunits. We have cloned and sequenced the human cDNAs encoding each subunit and characterized their products expressed from recombinant baculoviruses. The p96 and p79 polypeptides have 40% amino acid identity, focused particularly within a 94-residue region containing the sequence KDWK. This motif, first described for the Drosophila homeobox activator DEAF-1, identifies an emerging group of metazoan transcriptional modulators. During viral replication, PIF critically regulates the viral nickase, but in the host cell it probably modulates transcription, since each subunit is active in promoter activation assays and the complex binds to previously described regulatory elements in the tyrosine aminotransferase and transferrin receptor promoters. Within its recognition site, PIF binds coordinately to two copies of the tetranucleotide PuCGPy, which, remarkably, can be spaced from 1 to 15 nucleotides apart, a novel flexibility that we suggest may be characteristic of the KDWK family. Such tetranucleotides are common in promoter regions, particularly in activating transcription factor/cyclic AMP response element-binding protein (ATF/CREB) and E-box motifs, suggesting that PIF may modulate the transcription of many genes.

Functional implications of the structure of the murine parvovirus, minute virus of mice.

BACKGROUND: Minute virus of mice (MVM) is a single-stranded (ss) DNA-containing, murine parvovirus with a capsid built up of 60 icosahedrally related polypeptide chains, each of which consists of the C-terminal region common to two structural proteins, VP1 and VP2. In infectious virions, most VP2 molecules are cleaved to VP3 by the removal of about 20 amino acids from the N terminus. Of the 587 amino acids in VP2, approximately half are identical to those in the analogous capsid protein of the antigenically distinct canine parvovirus (CPV), the crystal structure of which has previously been determined. The three-dimensional structure determination of MVMi (the immunosuppressive strain of MVM) was previously reported to 3.5 A resolution. RESULTS: We report here an analysis of the MVMi virus structure and provide insights into tissue tropism, antigenicity and DNA packaging. Amino acids determining MVM tissue tropism were found to cluster on, or near, the viral surface. A conserved, glycine-rich, N-terminal peptide was seen to run through a cylindrical channel along each fivefold axis and may have implications for antigenicity. Density within the virion was interpreted as 29 ssDNA nucleotides per icosahedral asymmetric unit, and accounts for over one-third of the viral genome. CONCLUSIONS: The presence of the glycine-rich sequence in the fivefold channels of MVMi provides a possible mechanism to explain how the unique N-terminal region of VP1 becomes externalized in infectious parvovirions. Residues that determine tropism may form an attachment recognition site for a secondary host-cell factor that modulates tissue specificity. The ordering of nucleotides in a similar region of the interior surface in the CPV and MVMi capsids suggests the existence of a genomic DNA-recognition site within the parvoviral capsid.

A consensus DNA recognition motif for two KDWK transcription factors identifies flexible-length, CpG-methylation sensitive cognate binding sites in the majority of human promoters.

Parvovirus initiation factor (PIF), identified in HeLa cells as a host factor essential for parvoviral DNA replication, is a ubiquitous heterodimeric cellular transcription factor. This protein complex was simultaneously identified as glucocorticoid modulatory element binding protein (GMEB) by its ability to bind to the glucocorticoid modulating element (GME) upstream of the tyrosine transaminase promoter. Here, we show that the two PIF/GMEB subunits form site-specific DNA-binding heterodimers when co-expressed from recombinant baculoviruses and homodimers when expressed separately. Degenerate oligonucleotide selection experiments, combined with analysis of dissociation rates, established that the three complexes bind to flexibly spaced tetranucleotide half-sites that conform to the consensus ACGPy N(1-9) PuCGPy, with an optimum of N=6. Binding of all three complexes is extremely sensitive to methylation of the cytosine residues in the invariant CpG half-site core, suggesting a means by which PIF/GMEB binding could be regulated in vivo. Because CpG dinucleotides are suppressed in eukaryotic genomes, such binding sites would be expected to be very rare. However, analysis of 100 human promoters showed that over half of them contained at least one site conforming to the consensus, a significant deviation from the expected random distribution. In many of these, the binding site is within 100 nucleotides of the transcriptional start site, indicating that PIF/GMEB may be involved in regulation of these genes. Oligonucleotides corresponding to five of these sequences, chosen to represent the range of half-site separations identified by the consensus, were tested for PIF/GMEB binding by mobility shift assay. All five probes bound the heterodimer efficiently and, in each case, binding was completely abrogated by 5-methylation of the C residues in the CpGs of the putative half-sites.

cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.

The replication of viral genomes and the production of recombinant viral vectors from infectious molecular clones of parvoviruses MVMp and H1 were greatly improved by the introduction of a consensus NS-1 nick site at the junction between the left-hand viral terminus and the plasmid DNA. Progressive deletions of up to 1600 bp in the region encoding the structural genes as well as insertions of foreign DNA in replacement of those sequences did not appreciably affect the replication ability of the recombinant H1 virus genomes. In contrast, the incorporation of these genomes into recombinant particles appeared to depend on in cis-provided structural gene sequences. Indeed, the production of H1 viral vectors by cotransfection of recombinant clones and helper plasmids providing the structural proteins (VPs) in trans, drastically decreased when more than 800 bp was removed from the VP transcription unit. Furthermore, titers of viral vectors, in which most of the VP-coding region was replaced by an equivalent-length sequence consisting of reporter cDNA and stuffer DNA, were reduced more than 50 times in comparison with recombinant vectors in which stuffer DNA was not substituted for the residual VP sequence. In addition, viral vector production was restricted by the overall size of the genome, with a mere 6% increase in DNA length leading to an approximately 10 times lower encapsidation yield. Under conditions fulfilling the above-mentioned requirements for efficient packaging, titers of virus vectors from improved recombinant molecular DNA clones amounted to 5 x 10(7) infectious units per milliliter of crude extract. These titers should allow the assessment of the therapeutic effect of recombinant parvoviruses expressing small transgenes in laboratory animals.

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.

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.

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.

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.

ICRP Publication 125: Radiological Protection in Security Screening.

The use of technologies to provide security screening for individuals and objects has been increasing rapidly, in keeping with the significant increase in security concerns worldwide. Within the spectrum of technologies, the use of ionizing radiation to provide backscatter and transmission screening capabilities has also increased. The Commission has previously made a number of statements related to the general topic of deliberate exposures of individuals in non-medical settings. This report provides advice on how the radiological protection principles recommended by the Commission should be applied within the context of security screening. More specifically, the principles of justification, optimisation of protection, and dose limitation for planned exposure situations are directly applicable to the use of ionising radiation in security screening. In addition, several specific topics are considered in this report, including the situation in which individuals may be exposed because they are concealed ('stowaways') in a cargo container or conveyance that may be subject to screening. The Commission continues to recommend that careful justification of screening should be considered before decisions are made to employ the technology. If a decision is made that its use is justified, the framework for protection as a planned exposure situation should be employed, including optimization of protection with the use of dose constraints and the appropriate provisions for authorisation and inspection.

Replication of the parvovirus MVM. I. Dependence of virus multiplication and plaque formation on cell growth.

A plaque assay has been developed for the minute virus of mice. The infectious unit is a single particle. Plaque size is determined by the extent of cell division in the infected monolayer. Infection of quiescent and serum-stimulated cells suggests that virus multiplication depends on host function(s) that are not normally expressed in resting cells.

Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice.

Viral and cellular factors responsible for parvovirus target cell specificity have been examined for two serologically indistinguishable strains of the minute virus of mice which infect mouse cells of dissimilar differentiated phenotype. Both the prototype strain and the immunosuppressive strain grow in and form plaques on monolayers of simian virus 40-transformed human fibroblasts, a finding that has allowed the comparison of several aspects of their virus-host cell interactions. Although closely related by antigenic and genomic criteria, both the prototype strain and the immunosuppressive strain are restricted for lytic growth in each other's murine host cell, that is, in T cells and fibroblasts, respectively. The host range of each virus variant appears to be specified by a genetic determinant that is stably inherited in the absence of selection. In the restrictive virus-host interaction lytic growth is limited to a small or, in some cases, undetectable subset of the host cell population, and the majority of the infected cells remain viable, continuing to grow at the normal rate without expressing viral antigens. The susceptible host cell phenotype is dominant in T lymphocyte x fibroblast fusion hybrids, implying that different cell types express different developmentally regulated virus helper functions that can only be exploited by the virus variant that carries the appropriate strain-specific determinant.

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.

A genome-linked copy of the NS-1 polypeptide is located on the outside of infectious parvovirus particles.

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.

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.

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.