Concomitant human papillomavirus (HPV) vaccination and screening for elimination of HPV and cervical cancer.

HPV vaccination with concomitant HPV-based screening of young women has been proposed for faster cervical cancer elimination. We describe the baseline results of a population-based trial of this strategy to reduce the incidence of HPV. All 89,547 women born 1994-1999 and resident in the capital region of Sweden were personally invited to concomitant HPV vaccination and HPV screening with 26,125 women (29.2%) enrolled between 2021-05-03 and 2022-12-31. Baseline HPV genotyping of cervical samples from the study participants finds, compared to pre-vaccination prevalences, a strong decline of HPV16 and 18 in birth cohorts previously offered vaccination, some decline for cross-protected HPV types but no decline for HPV types not targeted by vaccines. Our dynamic transmission modelling predicts that the trial could reduce the incidence of high-risk HPV infections among the 1994-1998 cohorts by 62-64% in 3 years. Baseline results are prevalences of HPV infection, validated transmission model projections, and power estimates for evaluating HPV incidence reductions at follow-up (+/-0.1% with 99.9% confidence). In conclusion, concomitant HPV vaccination and HPV screening appears to be a realistic option for faster cervical cancer elimination. Clinicaltrials.gov identifier: NCT04910802; EudraCT number: 2020-001169-34.

Junín virus induces autophagy in human A549 cells.

Autophagy, a highly regulated degradative process that promotes cellular homeostasis, is increasingly recognised as a fundamental component of the cellular response against viral infection. In this study, we investigated the role of autophagy during Junín virus (JUNV) multiplication using human A549 cells. We found that JUNV infection induces an increment of the LC3-II/LC3-I ratio, an accumulation of punctate pattern in RFP-LC3-transfected cells and the colocalisation of viral nucleoprotein and LC3 protein, suggesting autophagosome formation. JUNV infection also induced the degradation of the autophagy receptor p62, suggesting that complete autophagic flux was triggered. In addition, we showed that inhibition of autophagy with bafilomycin A1 or 3-methyladenine significantly reduces viral multiplication. Moreover, viral yield was increased when autophagy was induced using rapamycin. Furthermore, JUNV infection induced the colocalisation of p62, ATG16, RAB5, RAB7A and LAMP1 with the autophagosomal LC3 protein. That suggests that phagosomes undergo the maturation process during viral infection. Finally, we demonstrated that siRNA experiments targeting essential autophagy genes (ATG5, ATG7 and Beclin 1) reduce viral protein synthesis and viral yield. Overall, our results indicate that JUNV activates host autophagy machinery enhancing its multiplication.

Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains.

The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.

Macrophages and galectin 3 play critical roles in CVB3-induced murine acute myocarditis and chronic fibrosis.

Macrophage influx and galectin 3 production have been suggested as major players driving acute inflammation and chronic fibrosis in many diseases. However, their involvement in the pathogenesis of viral myocarditis and subsequent cardiomyopathy are unknown. Our aim was to characterise the role of macrophages and galectin 3 on survival, clinical course, viral burden, acute pathology, and chronic fibrosis in coxsackievirus B3 (CVB3)-induced myocarditis. Our results showed that C3H/HeJ mice infected with CVB3 and depleted of macrophages by liposome-encapsulated clodronate treatment compared with infected untreated mice presented higher viral titres but reduced acute myocarditis and chronic fibrosis, compared with untreated infected mice. Increased galectin 3 transcriptional and translational expression levels correlated with CVB3 infection in macrophages and in non-depleted mice. Disruption of the galectin 3 gene did not affect viral titres but reduced acute myocarditis and chronic fibrosis compared with C57BL/6J wild-type mice. Similar results were observed after pharmacological inhibition of galectin 3 with N-acetyl-d-lactosamine in C3H/HeJ mice. Our results showed a critical role of macrophages and their galectin 3 in controlling acute viral-induced cardiac injury and the subsequent fibrosis. Moreover, the fact that pharmacological inhibition of galectin 3 induced similar results to macrophage depletion regarding the degree of acute cardiac inflammation and chronic fibrosis opens up the possibility of new pharmacological strategies for viral myocarditis.

Junín virus infection of human hematopoietic progenitors impairs in vitro proplatelet formation and platelet release via a bystander effect involving type I IFN signaling.

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 alpha/beta) 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.

Characterization of monoclonal antibodies to Junin virus nucleocapsid protein and application to the diagnosis of hemorrhagic fever caused by South American arenaviruses.

Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare virus are members of New World arenavirus clade B and are the etiological agents of viral hemorrhagic fevers that occur in South America. In this study, we produced three monoclonal antibodies (MAbs) to the recombinant nucleocapsid protein of JUNV, designated C6-9, C11-12, and E4-2. The specificity of these MAbs was examined by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, and an epitope-mapping method. Using these MAbs, we developed antigen (Ag) capture ELISA systems. We showed that by using MAb C6-9, JUNV Ag was specifically detected. On the other hand, by using MAb C11-12 or E-4-2, the Ags of all human pathogenic South American arenaviruses were detected. The combined use of these Ag capture ELISA systems in the present study may be useful for the diagnosis of acute-phase viral hemorrhagic fever due to infection by a South American arenavirus.

Argentine hemorrhagic fever diagnostic test based on recombinant Junín virus N protein.

Junín arenavirus is the etiologic agent of Argentine hemorrhagic fever. Due to its morbidity and high mortality rate in untreated cases, this endemic disease is of mandatory report in Argentina. Secure and accurate diagnostic methods are needed for the epidemiological surveillance of the disease. Current assays rely on antigens prepared from lysates of virus infected mammalian cells. The bio-safety issue related to the manipulation of large quantities of virus restricts such antigen production to laboratories with the appropriate containment facilities. In this report, we describe the development of an enzyme linked immunosorbent assay for the etiologic confirmation of the disease, based on recombinant antigens expressed in insect cells. Eight different variables of the assay were optimized with the Taguchi approach for experimental design (L18 design, seven three-level factors and one two-level factor). The area under the receiver operating characteristics (ROC) curve was 0.966, showing the high accuracy of the test discriminating positive from negative samples. Taking into account the biosafety benefits, the high yields of antigen in cell culture, and the general performance of the assay, it is expected that it will be a useful alternative to the current ELISA for the detection of antibodies in sera from convalescent patients.

Astrocyte response to Junín virus infection.

In a previous study of experimental murine encephalitis induced by Junín virus (JV), an arenavirus, we showed increased expression of iNOS by unidentified cells, concomitant with the astrocyte reaction. The specific inhibition of iNOS was associated with greater mortality but lower astrocytosis, suggesting that the protective role of nitric oxide (NO) synthesized by iNOS was related to enhanced astrocyte activation, representing a beneficial cellular response to virus-induced central nervous system damage. In the present work, cultured astrocytes were used to study whether JV infection could trigger iNOS expression and assess its eventual relationship with viral replication, glial fibrilary acidic protein (GFAP) expression levels and the presence of apoptosis. We found that JV infection of astrocytes did not induce apoptosis but produced both increased iNOS synthesis, detected by immunocytochemistry and fluorescence activated cell sorting (FACS) analysis, and increased NO, which was indirectly measured by nitrite/nitrate levels. These changes occurred early relative to the increases in GFAP expression, as detected by immunocytochemistry, FACS analysis and RT-PCR. The fact that iNOS inhibition abolished enhanced GFAP expression in infected monolayers suggests that NO was directly involved. In addition, iNOS inhibition enhanced virus replication. Together with data from confocal microscopy, these results suggest that JV induces iNOS expression in infected astrocytes and that the resulting NO has an important role both in reducing viral replication and in enhancing subsequent astrocyte activation.