Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN α/β) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2−/− mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels.

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Coxsackieviruses B and have a critical role in the pathogenesis of virus-induced myocarditis. J Thromb Haemost 2015; 13: 271-82.See also Koupenova M, Freedman JE. Platelets: the unsung hero of the immune response. This issue, pp 268-70.Summary. Background: To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. Methods: Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times postinfection. Results: CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals.Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. Conclusions: Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.

To cite this article: Negrotto S, de Giusti CJ, Lapponi MJ, Etulain J, Rivadeneyra L, Pozner RG, Gomez RM, Schattner M. Expression and functionality of type I interferon receptor in the megakaryocytic lineage. J Thromb Haemost 2011; 9: 2477-85.Summary. Background: Type I interferons (IFN-I) negatively regulate megakaryo/thrombopoiesis. However, expression of the IFN-I receptor (IFNAR) in the megakaryocytic lineage is poorly characterized. Objectives: To study the expression and functionality of IFNAR in the megakaryocytic lineage. Methods and results: Although IFNAR mRNA was found in every cell type studied, its protein expression showed differences between them. According to flow cytometry and immunofluorescence, IFNAR1 was observed in Meg-01, Dami, CD34+ cells and megakaryocytes, but not in proplatelets or platelets. Immunoblotting assays showed that IFNAR1 and IFNAR2 were highly expressed in all cell types, except in platelets where it was barely detectable. Regarding IFNAR1, 130-and 90-kDa bands were detected in Meg-01 and Dami, whereas 130-and 60-kDa bands were found in CD34+ cells and megakaryocytes. Activation of megakaryocytic IFNAR by IFN-b induced pSTAT1/2 and upregulated the antiviral genes IRF7 and MXA. The latter response was completely suppressed by IFNAR blockade. In contrast, the low levels of IFNAR in platelets were not functional as pSTAT1/2, aggregation and Pselectin expression were not induced by IFN-I. In addition, megakaryocytes increased IFN-I transcript levels and produced IFN-b upon stimulation with PolyI:C, a synthetic dsRNA that mimics viral infection. Conclusions: Early progenitors and mature megakaryocytes, but not platelets, express functional IFNAR and synthetize/release IFN-b, revealing not only that megakaryo/thrombopoiesis regulation by IFN-I is associated with a specific interaction with its receptor, but also that megakaryocytes may play a role in the antiviral defense by being both IFN producers and responders.

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 10⁵-10⁶ plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.

To understand better how different genomic regions may confer pathogenicity for the coxsackievirus B (CVB), two intratypic CVB1 variants and a number of recombinant viruses were studied. Sequencing analysis showed 23 nucleotide changes between the parental non-pathogenic CVB1N and the pathogenic CVB1Nm. Mutations present in CVB1Nm were more conserved than those in CVB1N when compared to other CVB sequences. Inoculation in C3H/HeJ mice showed that the P1 region is critical for pathogenicity in murine pancreas and heart. The molecular determinants of disease for these organs partially overlap. Several P1 region amino acid differences appear to be located in the decay accelerating factor (DAF) footprint CVBs. CVB1N and CVB1Nm interacted with human CAR, but only CVB1N seemed to interact with human DAF, as determined using soluble receptors in a plaque reduction assay. However, the murine homologue Daf-1 did not interact with any virus assessed by haemagglutination. The results of this study suggest that an unknown receptor interaction with the virus play an important role in the pathogenicity of CVB1Nm. Further in vivo studies may clarify this issue.

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