Uptake of Gadolinium-Based Contrast Agents by Blood Cells During Contrast-Enhanced MRI Examination.

OBJECTIVES: Gadolinium-based contrast agents (GBCAs) are routinely used in magnetic resonance imaging (MRI) examinations. However, there is limited knowledge about the interaction with and distribution of the drug in human cells. This lack of knowledge is surprising, given that the first interaction of the drug occurs with blood cells. Moreover, recent studies reported gadolinium (Gd) deposition within organs, such as the brain. Hence, this study is aiming to determine the uptake of GBCA in blood cells of patients undergoing contrast-enhanced MRI (ce-MRI) examination. MATERIALS AND METHODS: Human blood was exposed to either gadoterate meglumine (Gd-DOTA) or Eu-DOTA in vitro or was collected from patients undergoing ce-MRI with Gd-DOTA. Uptake of contrast agents (CAs) by blood cells was quantified by Gd measurements using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) or, to confirm Gd-DOTA uptake, by a complementary method using Eu-DOTA by time-resolved fluorescence spectroscopy, respectively. RESULTS: Uptake of Gd-DOTA or Eu-DOTA into white blood cells (WBCs) ex vivo was detectable by SC-ICP-MS and time-resolved fluorescence spectroscopy. The intracellular concentrations were estimated to be in the range of 1-3 µM. However, no CA uptake into erythrocytes was detected with either method. In total, 42 patients between 30 and 84 years old (24 men, 18 women) were enrolled. White blood cells' uptake of Gd was measured by SC-ICP-MS. Isolated WBCs from patients who underwent ce-MRI examination showed substantial Gd uptake; however, the studied patient group showed an inhomogeneous distribution of Gd uptake. Measurements immediately after MRI examination indicated 21-444 attogram/WBC, corresponding to an intracellular Gd concentration in the range from 0.2 to 5.5 µM. CONCLUSIONS: This study confirms the ex vivo uptake of GBCA by WBCs and provides the first evidence that GBCA is indeed taken up by WBCs in vivo by patients undergoing ce-MRI examination. However, the observed Gd uptake in WBCs does not follow a log-normal distribution commonly observed in the fields of environmental studies, biology, and medicine. Whether cellular uptake of GBCA is linked to the observed deposition of Gd remains unclear. Therefore, studying the interaction between GBCA and human cells may clarify crucial questions about the effects of Gd on patients after MRI examinations.

Beyond Single-Cell Analysis of Metallodrugs by ICP-MS: Targeting Cellular Substructures.

Platinum compounds such as cisplatin (cisPt) embody the backbone of combination chemotherapy protocols against advanced lung cancer. However, their efficacy is primarily limited by inherent or acquired platinum resistance, the origin of which has not been fully elucidated yet, although of paramount interest. Using single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), this study quantifies cisPt in single cancer cells and for the first time in isolated nuclei. A comparison of cisPt uptake was performed between a wild type (wt) cancer cell line and related resistant sublines. In both, resistant cells, wt cells, and their nuclei, cisPt uptake was measured at different incubation times. A lower amount of cisPt was found in resistant cell lines and their nuclei compared to wt cells. Moreover, the abundance of internalized cisPt decreased with increasing resistance. Interestingly, concentrations of cisPt found within the nuclei were higher than compared to cellular concentrations. Here, we show, that SC-ICP-MS allows precise and accurate quantification of metallodrugs in both single cells and cell organelles such as nuclei. These findings pave the way for future applications investigating the potency and efficacy of novel metallodrugs developed for cancer treatment.

Conjugates Containing Two and Three Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Units as In Vitro Antiparasitic and Anticancer Agents.

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasmagondii tachyzoites constitutively expressing ß-galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T.gondii proliferation with IC50 values ranging from 90 to 539 nM, and seven derivatives displayed IC50 values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC50 values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC50 values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6-10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11-14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

Generation of Stable cisPt Resistant Lung Adenocarcinoma Cells.

Platinum compounds represent the backbone of combined chemotherapy protocols for advanced lung cancer. The mechanisms responsible for its frequent primary or acquired resistance to cisplatin (cisPt)-based chemotherapy remains enigmatic. The availability of two cell lines of the same origin, one resistant and the other sensitive, will facilitate research to reveal the mechanism of resistance formation. Lung adenocarcinoma cells, A240286S (A24), were cultivated in increasing cisPt concentrations over a prolonged time. After a significant increase in IC50 was measured, cultivation of the cells was continued in absence of cisPt and IC50s determined over a long period (>7 months). As a result, a cell line with lasting, high-level cisPt resistance, designated (D-)A24cisPt8.0, was obtained. The cells were cross-resistant to oxaliplatin and to pemetrexed at a low level. Previous publications have claimed that Leucine-rich repeat-containing protein 8 (LRRC8A and LRRC8D) of the volume-regulated anion channels (VRACs) affect cellular resistance to cisPt. Even though cisPt decreased LRRC8D expression levels, we showed by knockdown and overexpression experiments with LRRC8A and D that these proteins do not govern the observed cisPt resistance. The tumor cell sublines described here provide a powerful model to study the mechanisms of resistance to cisPt in lung cancer cells and beyond.

The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells.

Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells.

Cutthroat Trout Virus-Towards a Virus Model to Support Hepatitis E Research.

Cutthroat trout virus (CTV) is a non-pathogenic fish virus belonging to the Hepeviridae family, and it is distantly related to hepatitis E virus (HEV). Here, we report the development of an efficient cell culture system where CTV can consistently replicate to titers never observed before with a hepevirus. By using the rainbow trout gill (RTGill-W1) cell line, CTV reaches 1010 geq/mL intracellularly and 108 geq/mL extracellularly within 5-6 days in culture. We additionally established a qPCR system to investigate CTV infectivity, and developed a specific antibody directed against the viral capsid protein encoded by ORF2. With these methods, we were able to follow the progressive accumulation of viral RNA and the capsid protein, and their intracellular distribution during virus replication. Virus progeny purified through iodixanol density gradients indicated-that similar to HEV-CTV produced in cell culture is also lipid-associated. The lack of an efficient cell culture system has greatly impeded studies with HEV, a major human pathogen that causes hepatitis worldwide. Although several cell culture systems have recently been established, the replication efficiency of HEV is not robust enough to allow studies on different aspects of the virus replication cycle. Therefore, a surrogate virus that can replicate easily and efficiently in cultured cells would be helpful to boost research studies with hepeviruses. Due to its similarities, but also its key differences to HEV, CTV represents a promising tool to elucidate aspects of the replication cycle of Hepeviridae in general, and HEV in particular.

Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus.

UNLABELLED: Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. IMPORTANCE: In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle.

The Receptor-Binding Domain in the VP1u Region of Parvovirus B19.

Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system.

Specific Targeting of Proerythroblasts and Erythroleukemic Cells by the VP1u Region of Parvovirus B19.

Viruses are evolutionarily developed cell-entering nanomachines, which are frequently used as gene or drug delivery systems. Parvovirus B19 (B19V) shows a remarkably restricted tropism for erythropoietin-dependent erythroid differentiation stages, and thus this virus provides an opportunity to deliver cargo to these intermediate differentiated cells. Here we report the construction of a delivery system from B19V subunits that maintains the highly selective cell-entry of the native virus and offers versatile cargo transport. To obtain this specific carrier, we conjugated the cell-targeting VP1u region of B19V to NeutrAvidin as a loading platform for biotinylated cargos. The VP1u-NeutrAvidin conjugate delivered fluorophores, DNA, and toxic payloads specifically to erythroid cells around the proerythroblast differentiation stage, including erythroleukemic cells. The VP1u-NeutrAvidin represents a unique cell surface marker which exclusively detects intermediate erythroid differentiation stages. Furthermore, the cell-entering property of this viral-based targeting system offers opportunities for erythroid-specific drug delivery or gene therapy.

Parvovirus B19 uptake is a highly selective process controlled by VP1u, a novel determinant of viral tropism.

The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V.

Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19.

The lack of a permissive cell culture system hampers the study of human parvovirus B19 (B19V). UT7/Epo is one of the few established cell lines that can be infected with B19V but generates none or few infectious progeny. Recently, hypoxic conditions or the use of primary CD36+ erythroid progenitor cells (CD36+ EPCs) have been shown to improve the infection. These novel approaches were evaluated in infection and transfection experiments. Hypoxic conditions or the use of CD36+ EPCs resulted in a significant acceleration of the infection/transfection and a modest increase in the yield of capsid progeny. However, under all tested conditions, genome encapsidation was impaired seriously. Further analysis of the cell culture virus progeny revealed that differently to the wild-type virus, the VP1 unique region (VP1u) was exposed partially and was unable to become further externalized upon heat treatment. The fivefold axes pore, which is used for VP1u externalization and genome encapsidation, might be constricted by the atypical VP1u conformation explaining the packaging failure. Although CD36+ EPCs and hypoxia facilitate B19V infection, large quantities of infectious progeny cannot be generated due to a failure in genome encapsidation, which arises as a major limiting factor for the in vitro propagation of B19V.

Prion removal capacity of plasma protein manufacturing processes: a data collection from PPTA member companies.

BACKGROUND: The variant Creutzfeldt-Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large-scale manufacturing of human plasma-derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety. Collectively analyzing the results could reveal experimental reproducibility and detect trends and mechanisms driving prion removal. STUDY DESIGN AND METHODS: Plasma Protein Therapeutics Association member companies collected more than 200 prion removal studies on plasma protein manufacturing steps, including precipitation, adsorption, chromatography, and filtration, as well as combined steps. The studies used a range of model spiking agents and bench-scale process replicas. The results were grouped based on key manufacturing variables to identify factors impacting removal. The log reduction values of a group are presented for comparison. RESULTS: Overall prion removal capacities evaluated by independent groups were in good agreement. The removal capacity evaluated using biochemical assays was consistent with prion infectivity removal measured by animal bioassays. Similar reduction values were observed for a given step using various spiking agents, except highly purified prion protein in some circumstances. Comparison between combined and single-step studies revealed complementary or overlapping removal mechanisms. Steps with high removal capacities represent the conditions where the physiochemical differences between prions and therapeutic proteins are most significant. CONCLUSION: The results support the intrinsic ability of certain plasma protein manufacturing steps to remove prions in case of an unlikely contamination, providing a safeguard to products.

Characterization of the early steps of human parvovirus B19 infection.

The early steps of human parvovirus B19 (B19V) infection were investigated in UT7/Epo cells. B19V and its receptor globoside (Gb4Cer) associate with lipid rafts, predominantly of the noncaveolar type. Pharmacological disruption of the lipid rafts inhibited infection when the drug was added prior to virus attachment but not after virus uptake. B19V is internalized by clathrin-dependent endocytosis and spreads rapidly throughout the endocytic pathway, reaching the lysosomal compartment within minutes, where a substantial proportion is degraded. B19V did not permeabilize the endocytic vesicles, indicating a mechanism of endosomal escape without apparent membrane damage. Bafilomycin A(1) (BafA1) and NH(4)Cl, which raise endosomal pH, blocked the infection by preventing endosomal escape, resulting in a massive accumulation of capsids in the lysosomes. In contrast, in the presence of chloroquine (CQ), the transfer of incoming viruses from late endosomes to lysosomes was prevented; the viral DNA was not degraded; and the infection was boosted. In contrast to the findings for untreated or BafA1-treated cells, the viral DNA was progressively associated with the nucleus in CQ-treated cells, reaching a plateau by 3 h postinternalization, a time coinciding with the initiation of viral transcription. At this time, more than half of the total intracellular viral DNA was associated with the nucleus; however, the capsids remained extranuclear. Our studies provide the first insight into the early steps of B19V infection and reveal mechanisms involved in virus uptake, endocytic trafficking, and nuclear penetration.

Contribution to safety of immunoglobulin and albumin from virus partitioning and inactivation by cold ethanol fractionation: a data collection from Plasma Protein Therapeutics Association member companies.

BACKGROUND: Virus removal by partitioning into different fractions during cold ethanol fractionation has been described by several authors, demonstrating that cold ethanol fractionation can provide significant contribution to virus removal, even in those cases where virus removal is limited and must be supported by additional measures for virus inactivation during further processing. STUDY DESIGN AND METHODS: Plasma Protein Therapeutics Association (PPTA) member companies collected and evaluated 615 studies on virus removal by the steps of the cold ethanol fractionation process. The studies describe the precipitation and separation of Fraction (F)III or FI/III in the immunoglobulin fractionation process and precipitation and separation of FII/III, FI/II/III, and FIV/IV in the albumin fractionation process. RESULTS: The data indicate a significant contribution of cold ethanol fractionation to the overall clearance of a broad spectrum of viruses, at varied process variables such as pH, temperature, and alcohol concentration and demonstrate the robustness of virus removal by the cold ethanol fractionation process. CONCLUSIONS: The data presented here support the importance of the partitioning steps for virus safety for immunoglobulins and albumin. However, virus removal by cold ethanol fractionation alone cannot provide viral safety of human albumin and immunoglobulins and therefore must be completed by other virus inactivation and removal procedures.

The globoside receptor triggers structural changes in the B19 virus capsid that facilitate virus internalization.

Globoside (Gb4Cer), Ku80 autoantigen, and α5ß1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.

Chloroquine and its derivatives exacerbate B19V-associated anemia by promoting viral replication.

BACKGROUND: An unexpectedly high seroprevalence and pathogenic potential of human parvovirus B19 (B19V) have been observed in certain malaria-endemic countries in parallel with local use of chloroquine (CQ) as first-line treatment for malaria. The aims of this study were to assess the effect of CQ and other common antimalarial drugs on B19V infection in vitro and the possible epidemiological consequences for children from Papua New Guinea (PNG). METHODOLOGY/PRINCIPAL FINDINGS: Viral RNA, DNA and proteins were analyzed in different cell types following infection with B19V in the presence of a range of antimalarial drugs. Relationships between B19V infection status, prior 4-aminoquinoline use and anemia were assessed in 200 PNG children <10 years of age participating in a case-control study of severe infections. In CQ-treated cells, the synthesis of viral RNA, DNA and proteins was significantly higher and occurred earlier than in control cells. CQ facilitates B19V infection by minimizing intracellular degradation of incoming particles. Only amodiaquine amongst other antimalarial drugs had a similar effect. B19V IgM seropositivity was more frequent in 111 children with severe anemia (hemoglobin <50 g/L) than in 89 healthy controls (15.3% vs 3.4%; P = 0.008). In children who were either B19V IgM or PCR positive, 4-aminoquinoline use was associated with a significantly lower admission hemoglobin concentration. CONCLUSIONS/SIGNIFICANCE: Our data strongly suggest that 4-aminoquinoline drugs and their metabolites exacerbate B19V-associated anemia by promoting B19V replication. Consideration should be given for choosing a non-4-aminoquinoline drug to partner artemisinin compounds in combination antimalarial therapy.

Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

BACKGROUND: Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. STUDY DESIGN AND METHODS: The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). RESULTS: Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. CONCLUSION: The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.

Interaction of parvovirus B19 with human erythrocytes alters virus structure and cell membrane integrity.

The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA(2) motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA(2) motif as well as a proportional increase of the PLA(2) activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA(2) motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA(2) activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA(2) constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells.

Molecular mechanism underlying B19 virus inactivation and comparison to other parvoviruses.

BACKGROUND: B19 virus (B19V) is a human pathogen frequently present in blood specimens. Transmission of the virus occurs mainly via the respiratory route, but it has also been shown to occur through the administration of contaminated plasma-derived products. Parvoviridae are highly resistant to physicochemical treatments; however, B19V is more vulnerable than the rest of parvoviruses. The molecular mechanism governing the inactivation of B19V and the reason for its higher vulnerability remain unknown. STUDY DESIGN AND METHODS: After inactivation of B19V by wet heat and low pH, the integrity of the viral capsid was examined by immunoprecipitation with two monoclonal antibodies directed to the N-terminal of VP1 and to a conformational epitope in VP2. The accessibility of the viral DNA was quantitatively analyzed by a hybridization-extension assay and by nuclease treatment. RESULTS: The integrity of the viral particles was maintained during the inactivation procedure; however, the capsids became totally depleted of viral DNA. The DNA-depleted capsids, although not infectious, were able to attach to target cells. Comparison studies with other members of the Parvoviridae family revealed a remarkable instability of B19V DNA in its encapsidated state. CONCLUSION: Inactivation of B19V by heat or low pH is not mediated by capsid disintegration but by the conversion of the infectious virions into DNA-depleted capsids. The high instability of the viral DNA in its encapsidated state is an exclusive feature of B19V, which explains its lower resistance to inactivation treatments.

Pathogen inactivation and removal procedures used in the production of intravenous immunoglobulins.

Patients with immunodeficiencies or some types of autoimmune diseases rely on a safe therapy with intravenous immunoglobulins (IVIGs) manufactured from human plasma, the only available source for this therapeutic. Since plasma is predisposed to contamination by a variety of blood-borne pathogens, ascertaining and ensuring the pathogen safety of plasma-derived therapeutics is a priority among manufacturers. State-of-the-art manufacturing processes provide a high safety standard by incorporating virus elimination procedures into the manufacturing process. Based on their mechanism these procedures are grouped into three classes: partitioning, inactivation, and virusfiltration.