The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36 ؉ EPCs expanded and differentiated from CD34 ؉ HSCs and assessed the CD36 ؉ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36 ؉ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36 ؉ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36 ؉ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36 ؉ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100-and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

In an attempt to experimentally define the roles of viral proteins encoded by the B19 genome in the viral life cycle, we utilized the B19 infectious clone constructed in our previous study to create two groups of B19 mutant genomes: (i) null mutants, in which either a translational initiation codon for each of these viral genes was substituted by a translational termination codon or a termination codon was inserted into the open reading frame by a frameshift; and (ii) a deletion mutant, in which half of the hairpin sequence was deleted at both the 5 and the 3 termini. The impact of these mutations on viral infectivity, DNA replication, capsid protein production, and distribution was systematically examined. Null mutants of the NS and VP1 proteins or deletion of the terminal hairpin sequence completely abolished the viral infectivity, whereas blocking expression of the 7.5-kDa protein or the putative protein X had no effect on infectivity in vitro. Blocking expression of the proline-rich 11-kDa protein significantly reduced B19 viral infectivity, and protein studies suggested that the expression of the 11-kDa protein was critical for VP2 capsid production and trafficking in infected cells. These findings suggest a previously unrecognized role for the 11-kDa protein, and together the results enhance our understanding of the key features of the B19 viral genome and proteins.Parvovirus B19 is the only member of the Parvoviridae confirmed to cause disease in humans and is the type member of the Erythrovirus genus. B19 is highly erythrotropic, with infection of erythroid progenitor cells leading to cytotoxicity and interruption of erythrocyte production (27). The physiological conditions of the host and the extent of the immune antiviral response then contribute to the evolution and clinical manifestation of the infection (39). Infection causes fifth disease in children (1, 2), polyarthropathy syndromes in adults (23, 26), transient aplastic crisis in patients with underlying chronic hemolytic anemia (31,35), and chronic anemia due to persistent infection in immunocompromised patients (18,19). Infection during pregnancy can lead to hydrops fetalis with possible fetal loss (16) and/or congenital infection (6).In common with other parvoviruses, B19 has a small (22 nm), nonenveloped, icosahedral capsid, encapsidating a singlestranded DNA genome of 5,596 nucleotides (nt). The ends of the genome are long inverted terminal repeats (ITRs) of 383 nt, of which the distal 365 nt form an imperfect palindrome (9). Transcription of the B19 viral genome is controlled by the single promoter (p6) located at map unit 6, which regulates the synthesis of all nine viral transcripts (4, 29). The single nonspliced transcript encodes the nonstructural protein (NS) and, by a combination of different splicing events, the other eight transcripts encode the two capsid proteins (VP1 and VP2) and two smaller proteins of unknown function (7,29,38). In addition, a short open reading frame (ORF) putatively encoding protein X was found in the VP1 reg...

Merkel cell polyomavirus (MCPyV) is associated to Merkel cell carcinoma (MCC). We studied 113 MCC tumoral skin lesions originating from 97 patients. MCPyV detection was higher in fresh-frozen (FF) biopsies (94%) than in formalin-fixed paraffin-embedded biopsies (39-47%). Mean viral load in FF tumor was of 7.5 copies per cell with a very wide range (0.01-95.4). Nineteen complete sequences of LTAg were obtained, mainly from FF biopsies when the viral load was high. Seventeen showed stop codons, all localized downstream of the pRb protein binding domain. Sequence comparison and phylogenetic analysis showed that all sequences clustered in the large C clade of MCPyV strains. MCPyV integration was demonstrated in 19 out of 27 FF MCC DNA biopsies without evidence of specific host cellular genome integration site. In 13/19 cases, the viral junction was located within the second exon of the LTAg, after the pRB binding domain.

These results strongly suggest ongoing direct zoonotic acquisition of STLV-1 in humans through severe NHP bites during hunting activities.

Human parvovirus B19 (B19) has been, for decades, the only parvovirus known to be pathogenic in humans. Another pathogenic human parvovirus, human bocavirus (HBoV), was recently identified in respiratory samples from children with acute lower respiratory tract symptoms. Both B19 and HBoV are transmitted by the respiratory route. The vast majority of adults are IgG seropositive for HBoV, whereas the HBoV‐specific Th‐cell immunity has not much been studied. The aim of this study was to increase our knowledge on HBoV‐specific Th‐cell immunity by examining HBoV‐specific T‐cell proliferation, Interferon‐gamma (IFN‐γ), IL‐10 and IL‐13 responses in 36 asymptomatic adults. Recombinant HBoV VP2 virus‐like particles (VLP) were used as antigen. HBoV‐specific responses were compared with those elicited by B19 VP2 VLP. Proliferation, IFN‐γ and IL‐10 responses with HBoV and B19 antigens among B19‐seropositive subjects were statistically similar in magnitude, but the cytokine and proliferation responses were much more closely correlated in HBoV than in B19. Therefore, at the collective level, B19‐specific Th‐cell immunity appears to be more divergent than the HBoV‐specific one.

The variation in the amount of parvovirus B19 DNA and different classes of RNA in permissive and non-permissive infected cells was analysed by means of quantitative real-time PCR and RT-PCR assays. In the permissive bone marrow mononuclear cells, UT7/Epo and KU812Ep6 cells, viral DNA usually increased within 48 hpi, rarely exceeding 2 Logs with respect to input DNA. Viral RNA was always present within 2-6 hpi, its increase paralleled that of viral DNA up to 36-48 hpi, and all the different classes of viral RNA were constantly represented in stable relative amounts throughout the infection cycle. In the non-permissive TF-1 cells, viral DNA did not increase and only one most represented single class of viral RNA was detected. Our data do not support the current model for B19 virus replication and transcription, consisting in different early and late expression patterns, but suggest an alternative model, indicating that the B19 virus genome should be considered a single, two-state replicative and transcriptional unit.

Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

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