Search

Highly pathogenic Arenaviruses, like the Lassa Virus (LASV), pose a serious public health threat in affected countries. Research and development of vaccines and therapeutics are urgently needed but hampered by the necessity to handle these pathogens under biosafety level 4 conditions. These containment restrictions make large-scale screens of antiviral compounds difficult. Therefore, the Mopeia virus (MOPV), closely related to LASV, is often used as an apathogenic surrogate virus. We established for the first time trisegmented MOPVs (r3MOPV) with duplicated S segments, in which one of the viral genes was replaced by the reporter genes ZsGreen (ZsG) or Renilla Luciferase (Rluc), respectively. In vitro characterization of the two trisegmented viruses (r3MOPV ZsG/Rluc and r3MOPV Rluc/ZsG), showed comparable growth behavior to the wild type virus and the expression of the reporter genes correlated well with viral titer. We used the reporter viruses in a proof-of-principle in vitro study to evaluate the antiviral activity of two well characterized drugs. IC50 values obtained by Rluc measurement were similar to those obtained by virus titers. ZsG expression was also suitable to evaluate antiviral effects. The trisegmented MOPVs described here provide a versatile and valuable basis for rapid high throughput screening of broadly reactive antiviral compounds against arenaviruses under BSL-2 conditions. [ABSTRACT FROM AUTHOR]

Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells. [ABSTRACT FROM AUTHOR]

Natural hosts of most arenaviruses are rodents. The human-pathogenic Lassa virus and several non-pathogenic arenaviruses such as Morogoro virus (MORV) share the same host species, namely Mastomys natalensis (M. natalensis). In this study, we investigated the history of infection and virus transmission within the natural host population. To this end, we infected M. natalensis at different ages with MORV and measured the health status of the animals, virus load in blood and organs, the development of virus-specific antibodies, and the ability of the infected individuals to transmit the virus. To explore the impact of the lack of evolutionary virus–host adaptation, experiments were also conducted with Mobala virus (MOBV), which does not share M. natalensis as a natural host. Animals infected with MORV up to two weeks after birth developed persistent infection, seroconverted and were able to transmit the virus horizontally. Animals older than two weeks at the time of infection rapidly cleared the virus. In contrast, MOBV-infected neonates neither developed persistent infection nor were able to transmit the virus. In conclusion, we demonstrate that MORV is able to develop persistent infection in its natural host, but only after inoculation shortly after birth. A related arenavirus that is not evolutionarily adapted to M. natalensis is not able to establish persistent infection. Persistently infected animals appear to be important to maintain virus transmission within the host population. [ABSTRACT FROM AUTHOR]

Mastomys natalensis is the natural host of various arenaviruses, including the human-pathogenic Lassa virus. Homologous arenaviruses, defined here as those having M. natalensis as a natural host, can establish long-lasting infection in M. natalensis, while these animals rapidly clear arenaviruses having another rodent species as a natural host (heterologous viruses). Little is known about the mechanisms behind the underlying arenavirus–host barriers. The innate immune system, particularly the type I interferon (IFN) response, might play a role. In this study, we developed and validated RT-PCR assays to analyse the expression of M. natalensis interferon-stimulated genes (ISGs). We then used these assays to study if homologous and heterologous viruses induce different IFN responses in M. natalensis cells. Infection experiments were performed with the homologous Lassa and Morogoro viruses and the related but heterologous Mobala virus. Compared to the direct induction with IFN or Poly(I:C), arenaviruses generally induced a weak IFN response. However, the ISG-expression profiles of homologous and heterologous viruses were similar. Our data indicate that, at least in M. natalensis cells, the IFN system is not a major factor in the virus–host barrier for arenaviruses. Our system provides a valuable tool for future in vivo investigation of arenavirus host restrictions at the level of the innate immune response. [ABSTRACT FROM AUTHOR]

Highlights: [•] T-705 suppressed replication of Zaire EBOV by 4log units in cell culture. [•] Mice lacking the type I interferon receptor (IFNAR− / −) were used as in vivo model. [•] In this model, T-705 administration prevented a lethal outcome. [•] Treatment was effective even if initiated at day 6 post infection. [ABSTRACT FROM AUTHOR]

Background Malaria presents with unspecific clinical symptoms that frequently overlap with other infectious diseases and is also a risk factor for coinfections, such as non-Typhi Salmonella. Malaria rapid diagnostic tests are sensitive but unable to distinguish between an acute infection requiring treatment and asymptomatic malaria with a concomitant infection. We set out to test whether cytokine profiles could predict disease status and allow the differentiation between malaria and a bacterial bloodstream infection. Methods We created a classification model based on cytokine concentration levels of pediatric inpatients with either Plasmodium falciparum malaria or a bacterial bloodstream infection using the Luminex platform. Candidate markers were preselected using classification and regression trees, and the predictive strength was calculated through random forest modeling. Results Analyses revealed that a combination of 7–15 cytokines exhibited a median disease prediction accuracy of 88% (95th percentile interval, 73%–100%). Haptoglobin, soluble Fas-Ligand, and complement component C2 were the strongest single markers with median prediction accuracies of 82% (with 95th percentile intervals of 71%–94%, 62%–94%, and 62%–94%, respectively). Conclusions Cytokine profiles possess good median disease prediction accuracy and offer new possibilities for the development of innovative point-of-care tests to guide treatment decisions in malaria-endemic regions. [ABSTRACT FROM AUTHOR]

Background: Malaria presents with unspecific clinical symptoms that frequently overlap with other infectious diseases and is also a risk factor for coinfections, such as non-Typhi Salmonella. Malaria rapid diagnostic tests are sensitive but unable to distinguish between an acute infection requiring treatment and asymptomatic malaria with a concomitant infection. We set out to test whether cytokine profiles could predict disease status and allow the differentiation between malaria and a bacterial bloodstream infection.Methods: We created a classification model based on cytokine concentration levels of pediatric inpatients with either Plasmodium falciparum malaria or a bacterial bloodstream infection using the Luminex platform. Candidate markers were preselected using classification and regression trees, and the predictive strength was calculated through random forest modeling.Results: Analyses revealed that a combination of 7-15 cytokines exhibited a median disease prediction accuracy of 88% (95th percentile interval, 73%-100%). Haptoglobin, soluble Fas-Ligand, and complement component C2 were the strongest single markers with median prediction accuracies of 82% (with 95th percentile intervals of 71%-94%, 62%-94%, and 62%-94%, respectively).Conclusions: Cytokine profiles possess good median disease prediction accuracy and offer new possibilities for the development of innovative point-of-care tests to guide treatment decisions in malaria-endemic regions. [ABSTRACT FROM AUTHOR]

Background: The pathophysiology of Ebola virus disease (EVD) is still poorly understood. This study aimed at identifying soluble biomarkers that inform on disease mechanisms.Methods: Fifty-four soluble mediators of the immune, coagulation, and endothelial system were measured in baseline and follow-up samples from hospitalized patients with EVD, using Luminex technology. Cross-sectional expression levels and changes over time were correlated with outcome.Results: Levels of circulating proinflammatory cytokines and chemokines, as well as markers of endothelial dysfunction and coagulopathy, were elevated on admission to hospital in patients who died from EVD as compared to survivors. These markers further increased in patients who died and/or decreased over time in survivors. In contrast, markers of gut integrity and T-cell response were higher in survivors and increased until discharge.Conclusions: Inflammatory response, endothelial integrity, gastric tissue protection, and T cell immunity play a role in EVD pathophysiology. [ABSTRACT FROM AUTHOR]

Lassa fever (LASF) is a highly severe viral syndrome endemic to West African countries. Despite the annual high morbidity and mortality caused by LASF, very little is known about the pathophysiology of the disease. Basic research on LASF has been precluded due to the lack of relevant small animal models that reproduce the human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV) and, to date, only immunodeficient mice, or mice expressing human HLA, have shown some degree of susceptibility to experimental infection. Here, transplantation of wild-type bone marrow cells into irradiated type I interferon receptor knockout mice (IFNAR-/-) was used to generate chimeric mice that reproduced important features of severe LASF in humans. This included high lethality, liver damage, vascular leakage and systemic virus dissemination. In addition, this model indicated that T cell-mediated immunopathology was an important component of LASF pathogenesis that was directly correlated with vascular leakage. Our strategy allows easy generation of a suitable small animal model to test new vaccines and antivirals and to dissect the basic components of LASF pathophysiology. [ABSTRACT FROM AUTHOR]

We studied the therapeutic potential of favipiravir (T-705) for Lassa fever, both alone and in combination with ribavirin. Favipiravir suppressed Lassa virus replication in cell culture by 5 log10 units. In a novel lethal mouse model, it lowered the viremia level and the virus load in organs and normalized levels of cell-damage markers. Treatment with 300 mg/kg per day, commenced 4 days after infection, when the viremia level had reached 4 log10 virus particles/mL, rescued 100% of Lassa virus-infected mice. We found a synergistic interaction between favipiravir and ribavirin in vitro and an increased survival rate and extended survival time when combining suboptimal doses in vivo. [ABSTRACT FROM AUTHOR]

Background: Mice lacking the type I interferon receptor (IFNAR−/− mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR−/− mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus. Methodology/Principal Findings: CCHF virus-infected IFNAR−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6–2.8 µg/ml; IC90 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects. Conclusions/Significance: Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin. [ABSTRACT FROM AUTHOR]

Several of the human-pathogenic arenaviruses cause hemorrhagic fever and have to be handled under biosafety level 4 conditions, including Lassa virus. Rapid and safe inactivation of specimens containing these viruses is fundamental to enable downstream processing for diagnostics or research under lower biosafety conditions. We established a protocol to test the efficacy of inactivation methods using the low-pathogenic Morogoro arenavirus as surrogate for the related highly pathogenic viruses. As the validation of chemical inactivation methods in cell culture systems is difficult due to cell toxicity of commonly used chemicals, we employed filter devices to remove the chemical and concentrate the virus after inactivation and before inoculation into cell culture. Viral replication in the cells was monitored over 4 weeks by using indirect immunofluorescence and immunofocus assay. The performance of the protocol was verified using published inactivation methods including chemicals and heat. Ten additional methods to inactivate virus in infected cells or cell culture supernatant were validated and shown to reduce virus titers to undetectable levels. In summary, we provide a robust protocol for the validation of chemical and physical inactivation of arenaviruses in cell culture, which can be readily adapted to different inactivation methods and specimen matrices. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b+ DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103+ DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome. [ABSTRACT FROM AUTHOR]

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA. [ABSTRACT FROM AUTHOR]