A Conserved Receptor-Binding Domain in the VP1u of Primate Erythroparvoviruses Determines the Marked Tropism for Erythroid Cells.

Parvovirus B19 (B19V) is a human pathogen with a marked tropism for erythroid progenitor cells (EPCs). The N-terminal of the VP1 unique region (VP1u) contains a receptor-binding domain (RBD), which mediates virus uptake through interaction with an as-yet-unknown receptor (VP1uR). Considering the central role of VP1uR in the virus tropism, we sought to investigate its expression profile in multiple cell types. To this end, we established a PP7 bacteriophage-VP1u bioconjugate, sharing the size and VP1u composition of native B19V capsids. The suitability of the PP7-VP1u construct as a specific and sensitive VP1uR expression marker was validated in competition assays with B19V and recombinant VP1u. VP1uR expression was exclusively detected in erythroid cells and cells reprogrammed towards the erythroid lineage. Sequence alignment and in silico protein structure prediction of the N-terminal of VP1u (N-VP1u) from B19V and other primate erythroparvoviruses (simian, rhesus, and pig-tailed) revealed a similar structure characterized by a fold of three or four α-helices. Functional studies with simian parvovirus confirmed the presence of a conserved RBD in the N-VP1u, mediating virus internalization into human erythroid cells. In summary, this study confirms the exclusive association of VP1uR expression with cells of the erythroid lineage. The presence of an analogous RBD in the VP1u from non-human primate erythroparvoviruses emphasizes their parallel evolutionary trait and zoonotic potential.

A Functional Minigenome of Parvovirus B19.

Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.

Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 µM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 µM) without prominent cytotoxicity (CC50 = 71.8 µM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 µM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 µM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

Virus-Host Interactions of Enteroviruses and Parvovirus B19 in Myocarditis.

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses.

Viral Infections and Systemic Lupus Erythematosus: New Players in an Old Story.

A causal link between viral infections and autoimmunity has been studied for a long time and the role of some viruses in the induction or exacerbation of systemic lupus erythematosus (SLE) in genetically predisposed patients has been proved. The strength of the association between different viral agents and SLE is variable. Epstein-Barr virus (EBV), parvovirus B19 (B19V), and human endogenous retroviruses (HERVs) are involved in SLE pathogenesis, whereas other viruses such as Cytomegalovirus (CMV) probably play a less prominent role. However, the mechanisms of viral-host interactions and the impact of viruses on disease course have yet to be elucidated. In addition to classical mechanisms of viral-triggered autoimmunity, such as molecular mimicry and epitope spreading, there has been a growing appreciation of the role of direct activation of innate response by viral nucleic acids and epigenetic modulation of interferon-related immune response. The latter is especially important for HERVs, which may represent the molecular link between environmental triggers and critical immune genes. Virus-specific proteins modulating interaction with the host immune system have been characterized especially for Epstein-Barr virus and explain immune evasion, persistent infection and self-reactive B-cell "immortalization". Knowledge has also been expanding on key viral proteins of B19-V and CMV and their possible association with specific phenotypes such as antiphospholipid syndrome. This progress may pave the way to new therapeutic perspectives, including the use of known or new antiviral drugs, postviral immune response modulation and innate immunity inhibition. We herein describe the state-of-the-art knowledge on the role of viral infections in SLE, with a focus on their mechanisms of action and potential therapeutic targets.

Human parvovirus B19: a review of clinical and epidemiological aspects in Brazil.

Since the first evidence of human parvovirus B19 (B19V) infection in late 80s, several studies have been conducted to clarify the spectrum of clinical diseases in Brazil. B19V infection is prevalent in the general population and has exhibited a cyclical pattern of occurrence every 4-5 years, with the predominance of genotype 1 over 3b. During epidemic periods the wide range of clinical conditions, such as ertythema infectiosum, arthropathy, transient aplastic crisis, nonimmune hydrops fetalis and B19V-hepatitis were diagnosed. However, many infections are likely asymptomatic or have a self-limiting clinical course and are not readly diagnosed. Besides, the similarity of the symptoms of ertythema infectiosum to other rash diseases and the broadly circulation of arboviruses makes differential diagnosis more difficult. In this article, we provide a historical comprehensive overview of the research on parvovirus B19 conducted in Brazil, with a focus on the clinical and epidemiological aspects of the infection.

The VP1u of Human Parvovirus B19: A Multifunctional Capsid Protein with Biotechnological Applications.

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.

Enhanced Cell-Based Detection of Parvovirus B19V Infectious Units According to Cell Cycle Status.

Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage. Two of the clones tested, B12 and E2, reached sensitivity levels higher than those of UT7/Epo-S1 and CD36+ erythroid progenitor cells. These findings highlight the importance of cell cycle status for sensitivity to B19V, and we propose a promising new straightforward cell-based method for quantifying B19V infectious units.

High prevalence of parvovirus B19 infection in patients with chronic kidney disease under hemodialysis: A multicenter study.

OBJECTIVES: Parvovirus B19 (B19V) infection is commonly acute and self-limited, but in chronic kidney disease (CKD) patients under dialysis treatment, this infection could increase susceptibility to acute and chronic anemia. The aim of this study was to evaluate the frequency and risk of B19V infection among Brazilian CKD patients under dialysis. METHODS: A study was conducted among 221 CKD patients and a control group of 142 blood donors. B19V infection was evaluated in serum samples by real-time PCR, and ELISA (anti-B19V IgM and IgG). RESULTS: B19V DNA was detected in 65% (145/221) of CKD patients, which was significantly higher (p < 0.001) than in the blood donors (6.3%). Simultaneous detection of B19V IgG and viremia was shown in 40.3% of CKD patients, which was indicative of persistent B19V infection. CKD patients showed an increased risk of developing B19V infection (OR = 28.1, CI = 13.5-58.5, p = 0.001). CONCLUSIONS: Despite an absence of clinical signs of B19V infection, these data highlight the importance of B19V infection in this high-risk population, since a persistent B19V infection could become clinically significant after renal transplant. Moreover, the persistent viremia should be considered as a potential risk, mainly because of the contamination of dialysis equipment.

No G-Quadruplex Structures in the DNA of Parvovirus B19: Experimental Evidence versus Bioinformatic Predictions.

Parvovirus B19 (B19V), an ssDNA virus in the family Parvoviridae, is a human pathogenic virus, responsible for a wide range of clinical manifestations, still in need of effective and specific antivirals. DNA structures, including G-quadruplex (G4), have been recognised as relevant functional features in viral genomes, and small-molecule ligands binding to these structures are promising antiviral compounds. Bioinformatic tools predict the presence of potential G4 forming sequences (PQSs) in the genome of B19V, raising interest as targets for antiviral strategies. Predictions locate PQSs in the genomic terminal regions, in proximity to replicative origins. The actual propensity of these PQSs to form G4 structures was investigated by circular dichroism spectroscopic analysis on synthetic oligonucleotides of corresponding sequences. No signature of G4 structures was detected, and the interaction with the G4 ligand BRACO-19 (N,N'-(9-{[4-(dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1-ylpropanamide) did not appear consistent with the stabilisation of G4 structures. Any potential role of PQSs in the viral lifecycle was then assessed in an in vitro infection model system, by evaluating any variation in replication or expression of B19V in the presence of the G4 ligands BRACO-19 and pyridostatin. Neither showed a significant inhibitory activity on B19V replication or expression. Experimental challenge did not support bioinformatic predictions. The terminal regions of B19V are characterised by relevant sequence and symmetry constraints, which are functional to viral replication. Our experiments suggest that these impose a stringent requirement prevailing over the propensity of forming actual G4 structures.

The Role of Human Parvovirus B19 in the Pediatric Patients with Pancytopenia?

BACKGROUND: Parvoviruses are small DNA viruses causing erythema infectiosum, which is known as the fifth disease. The aim of this study was to investigate the presence of Parvovirus B19 DNA by Real-Time-PCR retrospectively in clinical samples of children diagnosed as acute leukemia and aplastic anemia when investigating the cause of pancytopenia and to investigate its relationship with the clinical manifestations. METHODS: The study samples were collected between March 2014 and March 2018 in Gazi University, Faculty of Medicine, Department of Pediatric Hematology. Sixty pediatric patients; 37 males and 23 females, were included in the study. Nucleic acid isolation was performed by using MagNA-Pure Compact Nucleic Acid Isolation Kit (Roche, Germany). Extracted DNA was studied with LightCycler® 2.0 using the Real-Time PCR method and LightCycler® Parvovirus B19 Quantification Kit (Roche, Germany), and the results were evaluated quantitatively. Parvovirus B19 DNA detection interval of the kit was 101 - 106 copies/mL. RESULTS: Sixty serum samples were investigated and 8.3% (5/60) Parvovirus B19 DNA positivity was determined. Of the five patients with Parvovirus B19 DNA positivity, three had acute lymphoblastic leukemia and two were diagnosed as aplastic anemia. Regarding viral load; 2/5, 1/5, 1/5, and 1/5 of the samples had a viral load of 102, 103, 104, and 105 copies/mL, respectively. Parvovirus B19 DNA positivity was detected in samples from March (2/5), April (2/5), and August (1/5). CONCLUSIONS: Patients with acute leukemia and aplastic anemia in childhood using immunosuppressive drugs, blood, and blood products during chemotherapy, encounter Parvovirus B19 infections in the follow-up period and are diagnosed by serological and molecular methods. As a result of the study, we suggest that the detection of Parvovirus B19 DNA by Real-Time PCR method in children being admitted with pancytopenia and diagnosed as acute leukemia and aplastic anemia is useful in the follow-up and treatment.

Advances in the Development of Antiviral Strategies against Parvovirus B19.

Parvovirus B19 (B19V) is a human pathogenic virus, responsible for an ample range of clinical manifestations. Infections are usually mild, self-limiting, and controlled by the development of a specific immune response, but in many cases clinical situations can be more complex and require therapy. Presently available treatments are only supportive, symptomatic, or unspecific, such as administration of intravenous immunoglobulins, and often of limited efficacy. The development of antiviral strategies against B19V should be considered of highest relevance for increasing the available options for more specific and effective therapeutic treatments. This field of research has been explored in recent years, registering some achievements as well as interesting future perspectives. In addition to immunoglobulins, some compounds have been shown to possess inhibitory activity against B19V. Hydroxyurea is an antiproliferative drug used in the treatment of sickle-cell disease that also possesses inhibitory activity against B19V. The nucleotide analogues Cidofovir and its lipid conjugate Brincidofovir are broad-range antivirals mostly active against dsDNA viruses, which showed an antiviral activity also against B19V. Newly synthesized coumarin derivatives offer possibilities for the development of molecules with antiviral activity. Identification of some flavonoid molecules, with direct inhibitory activity against the viral non-structural (NS) protein, indicates a possible line of development for direct antiviral agents. Continuing research in the field, leading to better knowledge of the viral lifecycle and a precise understanding of virus-cell interactions, will offer novel opportunities for developing more efficient, targeted antiviral agents, which can be translated into available therapeutic options.

Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection.

Globoside (Gb4) is considered the primary receptor of parvovirus B19 (B19V); however, its expression does not correlate well with the attachment and restricted tropism of the virus. The N terminus of VP1 (VP1u) of B19V interacts with an as-yet-unknown receptor required for virus internalization. In contrast to Gb4, the VP1u cognate receptor is expressed exclusively in cells that B19V can internalize. With the aim of clarifying the role of Gb4 as a B19V receptor, we knocked out the gene B3GalNT1 coding for the enzyme globoside synthase in UT7/Epo cells. Consequently, B3GalNT1 transcripts and Gb4 became undetectable in the knockout (KO) cells without affecting cell viability and proliferation. Unexpectedly, virus attachment, internalization, and nuclear targeting were not disturbed in the KO cells. However, NS1 transcription failed, and consequently, genome replication and capsid protein expression were abrogated. The block could be circumvented by transfection with a B19V infectious clone, indicating that Gb4 is not required after the generation of viral double-stranded DNA with resolved inverted terminal repeats. While in wild-type (WT) cells, occupation of the VP1u cognate receptor with recombinant VP1u disturbed virus binding and blocked the infection, antibodies against Gb4 had no significant effect. In a mixed population of WT and KO cells, B19V selectively infected WT cells. This study demonstrates that Gb4 does not have the expected receptor function, as it is dispensable for virus entry; however, it is essential for productive infection, explaining the resistance of the rare individuals lacking Gb4 to B19V infection.IMPORTANCE Globoside has long been considered the primary receptor of B19V. However, its expression does not correlate well with B19V binding and uptake and cannot explain the pathogenesis or the remarkable narrow tissue tropism of the virus. By using a knockout cell line, we demonstrate that globoside does not have the expected function as a cell surface receptor required for B19V entry, but it has an essential role at a postentry step for productive infection. This finding explains the natural resistance to infection associated with individuals lacking globoside, contributes to a better understanding of B19V restricted tropism, and offers novel strategies for the development of antiviral therapies.

Clinical Case of Parvovirus B19 Infection in Pregnant Woman with Β-Thalassemia in Bulgaria.

A pregnant 30-year-old female in the 34th gestational week was admitted at University "Maichin Dom" Hospital prior to childbirth. The patient is diagnosed with ß-thalassemia. During laboratory screening hemoglobin of 98 g/L was established. Blood smear shows mild microcytic hypochromic anemia: RBC 5.15 x 1012/L, HGB 98 g/L, MCV 65.8 fL, MCH 19.4 pg, MCHC 295 g/L. Serum iron concentration is 12.9 µmol/L and ferritin 17.5 µg/L. For the delivery process cesium was considered. Two days after procedure a rash presented on face, hands and breasts. Although the mother was positive for parvovirus B19 infection, the baby was negative. This was confirmed by se-rological and molecular investigations. We discovered only the mother's B19V IgG antibodies in the newborn. In connection to the main disease, namely ß-thalassemia, acute virus infection could cause aplastic crisis. After consultation with a hematologist, serum hepcidin concentration (an iron homeostasis regulator) was quantified: 19.4 µg/L. ELISA test was used to prove B19V IgM antibodies in the mother. PCR analysis shows the presence of B19V DNA. During infection, inflammatory cytokines increase hepcidin secretion, leading to iron deposition into cells.

Parvovirus B19 Uncoating Occurs in the Cytoplasm without Capsid Disassembly and It Is Facilitated by Depletion of Capsid-Associated Divalent Cations.

Human parvovirus B19 (B19V) traffics to the cell nucleus where it delivers the genome for replication. The intracellular compartment where uncoating takes place, the required capsid structural rearrangements and the cellular factors involved remain unknown. We explored conditions that trigger uncoating in vitro and found that prolonged exposure of capsids to chelating agents or to buffers with chelating properties induced a structural rearrangement at 4 °C resulting in capsids with lower density. These lighter particles remained intact but were unstable and short exposure to 37 °C or to a freeze-thaw cycle was sufficient to trigger DNA externalization without capsid disassembly. The rearrangement was not observed in the absence of chelating activity or in the presence of MgCl2 or CaCl2, suggesting that depletion of capsid-associated divalent cations facilitates uncoating. The presence of assembled capsids with externalized DNA was also detected during B19V entry in UT7/Epo cells. Following endosomal escape and prior to nuclear entry, a significant proportion of the incoming capsids rearranged and externalized the viral genome without capsid disassembly. The incoming capsids with accessible genomes accumulated in the nuclear fraction, a process that was prevented when endosomal escape or dynein function was disrupted. In their uncoated conformation, capsids immunoprecipitated from cytoplasmic or from nuclear fractions supported in vitro complementary-strand synthesis at 37 °C. This study reveals an uncoating strategy of B19V based on a limited capsid rearrangement prior to nuclear entry, a process that can be mimicked in vitro by depletion of divalent cations.

Telbivudine Reduces Parvovirus B19-Induced Apoptosis in Circulating Angiogenic Cells.

Aims: Human parvovirus B19 (B19V) infection directly induces apoptosis and modulates CXCR4 expression of infected marrow-derived circulating angiogenic cells (CACs). This leads to dysfunctional endogenous vascular repair. Treatment for B19V-associated disease is restricted to symptomatic treatment. Telbivudine, a thymidine analogue, established in antiviral treatment for chronic hepatitis B, modulates pathways that might influence induction of apoptosis. Therefore, we tested the hypothesis of whether telbivudine influences B19V-induced apoptosis of CAC. Methods and Results: Pretreatment of two CAC-lines, early outgrowth endothelial progenitor cells (eo-EPC) and endothelial colony-forming cells (ECFC) with telbivudine before in vitro infection with B19V significantly reduced active caspase-3 protein expression (-39% and -40%, both p < 0.005). Expression of Baculoviral Inhibitor of apoptosis Repeat-Containing protein 3 (BIRC3) was significantly downregulated by in vitro B19V infection in ECFC measured by qRT-PCR. BIRC3 downregulation was abrogated with telbivudine pretreatment (p < 0.001). This was confirmed by single gene PCR (p = 0.017) and Western blot analysis. In contrast, the missing effect of B19V on angiogenic gene expression postulates a post-transcriptional modulation of CXCR4. Conclusions: We for the first time show a treatment approach to reduce B19V-induced apoptosis. Telbivudine reverses B19V-induced dysregulation of BIRC3, thus, intervening in the apoptosis pathway and protecting susceptible cells from cell death. This approach could lead to an effective B19V treatment to reduce B19V-related disease.

Synthesis of 3-(Imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one Derivatives and Study of Their Antiviral Activity against Parvovirus B19.

Parvovirus B19 (B19V) is a human pathogenic virus associated with a wide range of clinical conditions. Currently, there are no recognized antiviral drugs for B19V treatment; therefore, efforts in the search for compounds inhibiting B19V replication are now being pursued. Coumarins (chromen-2-ones) are considered a privileged structure for designing novel orally bioavailable and non-peptidic antiviral agents. To further contribute to the development of new drugs against B19V, our research was focused on the synthesis, characterization and evaluation of antiviral activity of some new 3-(imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one derivatives. The effects of the synthesized compounds on cell viability and viral replication were investigated by employing two relevant cellular systems, the myeloblastoid cell line UT7/EpoS1 and primary erythroid progenitor cells (EPCs). Some of the tested compounds showed inhibitory activity both on cell viability and on viral replication, depending on the cellular system. These results suggest that the mechanism involved in biological activity is sensitive to small structural changes and that it is possible to direct the activity of the 3-(imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one core.

Combined Laboratory Approach to Detection of Parvovirus B19 and Coxiella Burnetii in Patients with Fever of Unknown Origin.

BACKGROUND: Fever of unknown origin (FUO) is one of the greatest challenges for clinicians and patients. There are more than 200 etiological agents of FUO, among these the most common is the role of infection, neoplasms, and diseases of connective tissue. The aim of the present study is to investigate the role of the infectious agents parvovirus B19 (B19V) and Coxiella burnetii (C. burnetii) in the development of fever of unknown origin by a set of immunoenzymatic and molecular methods. METHODS: The present study included a total of 70 adult patients diagnosed with FUO and hospitalized in Bulgarian Hospitals. A control group of 26 healthy people were also included. Serological (indirect enzyme immunoassay test for detection of B19V and C. burnetii Ph. II specific IgM/IgG) and molecular (extraction and detection of infectious nucleic acids) methods were used. RESULTS: From all patients with FUO, a positive result for B19V-IgM was obtained in 18/70 (25.71%, 95% CI: 15.47 - 35.95) and the highest percentage was found in age groups 0 - 9 and 10 - 19 years. Protective B19V immunity and past viral infection was reported in 41/70 (58.57%, 95% CI: 47.03 - 70.11), and this percentage corresponded with the control group 16/26 (61.54%, 95% CI: 42.84 - 80.24). Anti-C. burnetii Ph. II-IgM was demonstrated in 13/70 (18.57%, 95% CI: 9.46 - 27.68). A relatively high percentage of affected patients were ≤ 40 years. Anti-C. burnetii Ph. II-IgG was detected in 24/70 (34.29%, 95% CI: 23.17 - 45.41). The control group has a 100% negative result for acute B19V and C. burnetii infection. A positive B19V-DNA result was obtained in 12/70 (17.14%, 95% CI: 8.31 - 25.97) patients. In 11/12 (91.67%) it was in combination with positive B19V-IgM marker. Of the total 70 sera tested, a positive PCR results for C. burnetii-DNA were obtained in 11 (15.71%, 95% CI: 7.18 - 24.24). According to clinical manifestation and concomitant symptoms, a high percentage of B19V and C. burnetii positives were associated with FUO and fever, headache, chills, and rash. CONCLUSIONS: It is of particular importance for a correct diagnosis of FUO to use a combined laboratory approach to prove acute or persistent infection and to test for a set of etiological agents.