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The 2018 Nigerian Lassa fever season saw the largest ever recorded upsurge of cases, raising concerns over the emergence of a strain with increased transmission rate. To understand the molecular epidemiology of this upsurge, we performed, for the first time at the epicenter of an unfolding outbreak, metagenomic nanopore sequencing directly from patient samples, an approach dictated by the highly variable genome of the target pathogen. Genomic data and phylogenetic reconstructions were communicated immediately to Nigerian authorities and the World Health Organization to inform the public health response. Real-time analysis of 36 genomes and subsequent confirmation using all 120 samples sequenced in the country of origin revealed extensive diversity and phylogenetic intermingling with strains from previous years, suggesting independent zoonotic transmission events and thus allaying concerns of an emergent strain or extensive human-to-human transmission.

Dendritic cells connect innate and adaptive immune responses. This is a particularly important immune checkpoint in the case of emerging infections against which most of the population does not have preexisting antibody immunity. In this study, we sought to test whether antibody-based delivery of Ebola virus (EBOV) antigens to dendritic cells could be used as a vaccination strategy against Ebola virus disease. Our approach was to use antibodies targeting the endocytic receptor DEC-205 present in murine and human dendritic cells, to deliver the EBOV nucleoprotein or the model antigen ovalbumin (OVA). Our findings indicate that DEC-205 targeting stimulated antigen-specific T-cell responses in mice, which resulted in protection from EBOV or recombinant EBOV-OVA challenge. An added value of this strategy was the generation of resident memory T cells. We propose that dendritic cell targeting could be used to improve T-cell responses against filoviruses, a strategy that may complement current vaccination strategies., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2025. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Lassa virus (LASV) outbreaks in West Africa pose a significant public health threat. We investigated the infection phenotype and transmission (horizontal and vertical) of LASV strain Ba366 in its natural host, Mastomys natalensis. Here we analyze viral RNA levels in body fluids, virus titers in organs and antibody presence in blood. In adults and 2-week-old animals, LASV causes transient infections with subsequent seroconversion. However, mice younger than two weeks exhibit persistent infections lasting up to 16 months despite antibody presence. LASV can be detected in various body fluids, organs, and cell types, primarily in lung, kidney, and gonadal epithelial cells. Despite the systemic virus presence, no pathological alterations in organs are observed. Infected animals efficiently transmit the virus throughout their lives. Our findings underscore the crucial role of persistently infected individuals, particularly infected females and their progeny, in LASV dissemination within the host population., (© 2024. The Author(s).)

Background: The standard of care for Lassa fever is the use of ribavirin with supportive therapy. There is little information on the course of viremia and its relationship with clinical outcomes in patients treated with ribavirin., Methods: We conducted a retrospective analysis of virologic and clinical parameters of 152 reverse transcription polymerase chain reaction-confirmed Lassa fever cases admitted and treated with ribavirin therapy. We describe the Lassa virus RNA kinetics in blood in relation to the clinical course of the patients., Results: The overall mortality was 9%. The median duration (interquartile range [IQR]) of illness before admission was 8 (5-12) days. Median (IQR) Ct values on admission ( t 0 ) were lower among patients who died (21 [20-27]) than in those who survived (34 [30-37]; P < .01). The receiver operating characteristics curve of the association between outcome and Ct value at t 0 had a high classification performance, with an AUC of 0.92 (95% CI, 0.86-0.98). The median time to viral clearance (IQR) was 10 (5-15) days. The viral load decreased steadily with the duration of treatment, and all survivors achieved viral clearance within 25 days of hospitalization., Conclusions: Our study demonstrates that the Ct value on admission has prognostic value and Lassa fever patients treated with ribavirin typically clear the virus within 3-4 weeks of hospitalization. This kinetics has implications for the design of clinical case management and future clinical trial protocols., Competing Interests: Potential conflicts of interest. The authors: no reported conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Electroencephalography (EEG) has been used for almost a century in well-equipped medical centers to facilitate the diagnosis of epilepsy and other brain disorders. Lassa fever (LF) and other emerging viral infections (EVI) are known to cause neurological complications, including meningitis, seizures, and encephalopathy, though to date it remains unclear whether these are secondary to metabolic disturbances caused by the disease or by direct involvement of the central nervous system (CNS). To better characterize how Lassa virus (LASV) affects the CNS, we established an EEG diagnostic unit in the LF isolation ward at Irrua Specialist Teaching Hospital in Edo State, Nigeria. Here, we report on the specific difficulties to successful implementation of EEG in this highly challenging setting. Technical artefacts due to electrical interferences and interrupted power supply, artefacts deriving from a partly improvised EEG setup within a high consequence pathogen isolation ward, and environmental factors, such as heat in the endemic West African setting are among the main difficulties encountered when setting up this diagnostic facility. It takes experienced neurophysiologists to distinguish such artefacts from actual EEG abnormalities as many of them are not commonly encountered to this extent in well-equipped EEG laboratories and can easily be confused with pathologies. The EEG recording process is further complicated by biosafety considerations and the necessity of wearing extensive personal protective equipment. Nevertheless, with the help of experienced neurophysiologists, it is possible to correctly set up the facility and interpret recordings. Taking the above into consideration, EEG is valuable in identifying CNS involvement in emerging infections, particularly regarding assessment of encephalitis, differential diagnosis of impaired consciousness and treatment adjustment in patients with symptomatic seizures. Although highly challenging under these circumstances, EEG can be an important, noninvasive diagnostic tool for neurological complications in EVI where other more advanced imaging modalities are not available., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mueller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lipid droplets (LDs) are organelles involved in lipid storage, maintenance of energy homeostasis, protein sequestration, signaling events and inter-organelle interactions. Recently, LDs have been shown to favor the replication of members from different viral families, such as the Flaviviridae and Coronaviridae. In this work, we show that LDs are essential organelles for members of the Arenaviridae family. A virus-driven reduction of LD number was observed in cultures infected with Junín mammarenavirus (JUNV), caused in part by action of the viral nucleoprotein. Notably, we identified a new pool of nucleoprotein and viral RNA that localizes in the vicinity of LDs, suggesting that LDs play a role during the viral replication cycle. Regarding the mechanism behind LD exhaustion, we found evidence that lipophagy is involved in LD degradation with the resulting fatty acids being substrates of fatty acid β-oxidation, which fuels viral multiplication. This work highlights the importance of LDs during the replication cycle of JUNV, contributing to the knowledge of the metabolic changes these mammarenaviruses cause in their hosts., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Previous studies have described that Ebola virus (EBOV) infection of human monocyte-derived dendritic cells (moDCs) inhibits dendritic cell (DC) maturation, resulting in poor T-cell activation. However, it is unknown how other DC subsets distinct from moDCs respond to EBOV infection., Methods: To better understand how DCs initiate T-cell activation during EBOV infection, we assessed the response of conventional mouse DCs (cDCs) to EBOV infection utilizing a recombinant EBOV expressing the model antigen ovalbumin., Results: In contrast to moDCs, mouse cDC2s and cDC1s were poorly infected with EBOV but were highly activated. DCs were able to prime CD8 T cells via cross-presentation of antigens obtained from cell debris of EBOV-infected cells. EBOV infection further enhanced DC cross-presentation., Conclusions: Our findings indicate that EBOV infection of cDCs results in activation rather than inhibition, leading to high levels of T-cell activation. With that we propose a mechanistic explanation for the excess T-cell activation observed in human Ebola virus disease., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The Third International Conference on Crimean-Congo Hemorrhagic Fever (CCHF) was held in Thessaloniki, Greece, September 19-21, 2023, bringing together a diverse group of international partners, including public health professionals, clinicians, ecologists, epidemiologists, immunologists, and virologists. The conference was attended by 118 participants representing 24 countries and the World Health Organization (WHO). Meeting sessions covered the epidemiology of CCHF in humans; Crimean-Congo hemorrhagic fever virus (CCHFV) in ticks; wild and domestic animal hosts; molecular virology; pathogenesis and animal models; immune response related to therapeutics; and CCHF prevention in humans. The concluding session focused on recent WHO recommendations regarding disease prevention, control strategies, and innovations against CCHFV outbreaks. This meeting report summarizes lectures by the invited speakers and highlights advances in the field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice ( Mastomys natalensis ), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae , family Orthoherpesviridae ), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

Background: Managing Lassa fever (LF) patients is challenging because of the complexity of this life-threatening infectious disease, the necessary isolation measures, and the limited resources in countries where it is endemic. Point-of-care ultrasonography (POCUS) is a promising low-cost imaging technique that may help in guiding the management of patients., Methods: We conducted this observational study at the Irrua Specialist Teaching Hospital in Nigeria. We developed a POCUS protocol, trained local physicians who applied the protocol to LF patients and recorded and interpreted the clips. These were then independently re-evaluated by an external expert, and associations with clinical, laboratory and virological data were analyzed., Findings: We developed the POCUS protocol based on existing literature and expert opinion and trained two clinicians, who then used POCUS to examine 46 patients. We observed at least one pathological finding in 29 (63%) patients. Ascites was found in 14 (30%), pericardial effusion in 10 (22%), pleural effusion in 5 (11%), and polyserositis in 7 (15%) patients, respectively. Eight patients (17%) showed hyperechoic kidneys. Seven patients succumbed to the disease while 39 patients survived, resulting in a fatality rate of 15%. Pleural effusions and hyper-echoic kidneys were associated with increased mortality., Interpretation: In acute LF, a newly established POCUS protocol readily identified a high prevalence of clinically relevant pathological findings. The assessment by POCUS required minimal resources and training; the detected pathologies such as pleural effusions and kidney injury may help to guide the clinical management of the most at-risk LF patients., Competing Interests: Declaration of Competing Interest All authors report no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Lassa virus (LASV) can cause severe acute systemic infection in humans. No approved antiviral drugs or vaccines are currently available. Antibody-based therapeutics are considered a promising treatment strategy in the management of LASV disease., Methods: We used chimeric Ifnar -/- C57BL/6 (Ifnar -/- Bl6 ) mice, a lethal LASV mouse model, to evaluate the protective efficacy of polyclonal antibodies purified from sera of rabbits hyperimmunized with virus-like particles displaying native-like LASV glycoprotein GP spikes., Results: Polyclonal anti-LASV GP antibodies provided 100% protection against lethal LASV infection in a pre- and post-exposure treatment setting and prevented LASV disease. Treatment also significantly lowered viremia level and virus load in organs. When treatment was initiated at the onset of symptoms, the hyperimmune antibodies provided partial protection and increased the survival rate by 80%., Conclusions: Our findings support the consideration of animal-derived hyperimmune antibodies targeting GP as an effective treatment option for highly pathogenic LASV.

Background: There is limited epidemiological evidence on Lassa fever in pregnant women with acute gaps on prevalence, infection incidence, and risk factors. Such evidence would facilitate the design of therapeutic and vaccine trials and the design of control programs. Our study sought to address some of these gaps by estimating the seroprevalence and seroconversion risk of Lassa fever in pregnant women., Methodology/principal Findings: We conducted a prospective hospital-based cohort between February and December 2019 in Edo State, Southern Nigeria, enrolling pregnant women at antenatal clinic and following them up at delivery. Samples were evaluated for IgG antibodies against Lassa virus. The study demonstrates a seroprevalence of Lassa IgG antibodies of 49.6% and a seroconversion risk of 20.8%. Seropositivity was strongly correlated with rodent exposure around homes with an attributable risk proportion of 35%. Seroreversion was also seen with a seroreversion risk of 13.4%., Conclusions/significance: Our study suggests that 50% of pregnant women were at risk of Lassa infection and that 35.0% of infections might be preventable by avoiding rodent exposure and conditions which facilitate infestation and the risk of human-rodent contact. While the evidence on rodent exposure is subjective and further studies are needed to provide a better understanding of the avenues of human-rodent interaction; public health measures to decrease the risk of rodent infestation and the risk of spill over events may be beneficial. With an estimated seroconversion risk of 20.8%, our study suggests an appreciable risk of contracting Lassa fever during pregnancy and while most of these seroconversions may not be new infections, given the high risk of adverse outcomes in pregnancy, it supports the need for preventative and therapeutic options against Lassa fever in pregnancy. The occurrence of seroreversion in our study suggests that the prevalence obtained in this, and other cohorts may be an underestimate of the actual proportion of women of childbearing age who present at pregnancy with prior LASV exposure. Additionally, the occurrence of both seroconversion and seroreversion in this cohort suggests that these parameters would need to be considered for the development of Lassa vaccine efficacy, effectiveness, and utility models., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kayem et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Soh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Evidence from previous studies suggest that Lassa fever, a viral haemorrhagic fever endemic to West Africa has high case fatalities, particularly in pregnancy. While there have been remarkable innovations in vaccine development, with some Lassa vaccines undergoing early clinical trials. An understanding of Lassa antibody kinetics and immune responses will support vaccine design and development. However, there is currently no evidence on the antibody kinetics of Lassa (LASV) in pregnancy. Our study sought to estimate the efficiency of transplacental transfer of LASV IgG antibodies from the mother to the child., Methodology/principal Findings: The study made use of data from a prospective hospital-based cohort of pregnant women enrolled at the antenatal clinic and followed up at delivery between February and December 2019. Blood samples from mother-child pairs were evaluated for antibodies against Lassa virus. The study demonstrates a transplacental transfer of LASV IgG of 75.3% [60.0-94.0%], with a significant positive correlation between maternal and cord concentrations and a good level of agreement. The study also suggests that transfer may be more variable in women with 'de novo' antibodies compared to those with pre-existing antibodies., Conclusions/significance: The study shows that maternal antibody levels play an important role in determining transfer efficiency of Lassa antibodies to the new-born; and while the evidence is preliminary, the study also suggests that transfer efficiency may be less stable in acute or recent infection, as such timing of vaccination before pregnancy, that is in women of childbearing age may be more appropriate for protection of both pregnant women and their neonates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kayem et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Lassa fever is endemic in large parts of West Africa. The recommended antiviral treatment is ribavirin. Two treatment regimens are currently endorsed in Nigeria: the "McCormick regimen" based on a study published in 1986 and the "Irrua regimen" constituting a simplified schedule developed at the Irrua Specialist Teaching Hospital, Nigeria. Evidence for the safety and efficacy of ribavirin in Lassa fever patients is poor and pharmacokinetic data for both regimens are lacking., Methods: Polymerase chain reaction-confirmed Lassa fever patients with mild to moderate disease severity were invited to participate in this prospective, observational pharmacokinetic study. Pharmacokinetics of ribavirin, clinical, virologic, and clinical laboratory parameters were assessed., Results: Using a population pharmacokinetic approach, plasma concentrations of ribavirin were best described by a 3-compartment model. Drug exposure was remarkably consistent between participants. Overall, drug clearance was 28.5% lower in female compared with male participants. Median (5th-95th percentile) time above half maximal inhibitory concentration (IC50) was 37.3% (16.9%-73.1%), 16.7% (8.2%-58.5%), and 9.6% (4.9%-38.4%) on days 1, 7, and 8, respectively. Clinical laboratory parameters indicated reduction of cell damage and development of hemolytic anemia in the course of the treatment period., Conclusions: This observational study characterizes the pharmacokinetics of ribavirin in the treatment of Lassa fever indicating consistent exposure across patients. Whereas only a short time interval of concentrations above the IC50 implies rather low antiviral efficacy in vivo, the prominent reduction of cell damage markers might point to indirect-potentially anti-inflammatory-effects of ribavirin. The role of ribavirin in the treatment of Lassa fever requires further scrutiny., Competing Interests: Potential conflicts of interest. S. G. reports support for attending meetings and/or travel from Keystone Symposia. E. O. E. and S. O. report other financial or nonfinancial interests in CEPI—Lassa Epidemiological Programme as a study team member at ISTH, Nigeria. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Lassa fever is a viral hemorrhagic fever treated with supportive care and the broad-spectrum antiviral drug ribavirin. The pathophysiology, especially the role of hyperinflammation, of this disease is unknown. We report successful remission of complicated Lassa fever in 2 patients in Nigeria who received the antiinflammatory agent dexamethasone and standard ribavirin.

Ebola virus disease (EVD) is a complex infectious disease characterized by high inflammation, multiorgan failure, the dysregulation of innate and adaptive immune responses, and coagulation abnormalities. Evidence accumulated over the last 2 decades indicates that, during fatal EVD, the infection of antigen-presenting cells (APC) and the dysregulation of T cell immunity preclude a successful transition between innate and adaptive immunity, which constitutes a key disease checkpoint. In order to better understand the contribution of the APC-T cell crosstalk to EVD pathophysiology, we have developed avatar mice transplanted with human, donor-specific APCs and T cells. Here, we show that the transplantation of T cells and APCs from Ebola virus (EBOV)-naive individuals into avatar mice results in severe disease and death and that this phenotype is dependent on T cell receptor (TCR)-major histocompatibility complex (MCH) recognition. Conversely, avatar mice were rescued from death induced by EBOV infection after the transplantation of both T cells and plasma from EVD survivors. These results strongly suggest that protection from EBOV reinfection requires both cellular and humoral immune memory responses. IMPORTANCE The crosstalk between dendritic cells and T cells marks the transition between innate and adaptive immune responses, and it constitutes an important checkpoint in EVD. In this study, we present a mouse avatar model in which T cell and dendritic cell interactions from a specific donor can be studied during EVD. Our findings indicate that T cell receptor-major histocompatibility complex-mediated T cell-dendritic cell interactions are associated with disease severity, which mimics the main features of severe EVD in these mice. Resistance to an EBOV challenge in the model was achieved via the transplantation of both survivor T cells and plasma.

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.

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. IC 50 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.

Sensitive and specific serological tests are mandatory for epidemiological studies evaluating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence as well as coronavirus disease 2019 (COVID-19) morbidity and mortality rates. The accuracy of results is challenged by antibody waning after convalescence and by cross-reactivity induced by previous infections with other pathogens. By employing a patented platform technology based on capturing antigen-antibody complexes with a solid-phase-bound Fcγ receptor (FcγR) and truncated nucleocapsid protein as the antigen, two SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISAs), featuring different serum and antigen dilutions, were developed. Validation was performed using a serum panel comprising 213 longitudinal samples from 35 COVID-19 patients and a negative-control panel consisting of 790 pre-COVID-19 samples from different regions of the world. While both assays show similar diagnostic sensitivities in the early convalescent phase, ELISA 2 (featuring a higher serum concentration) enables SARS-CoV-2 IgG antibody detection for a significantly longer time postinfection (≥15 months). Correspondingly, analytical sensitivity referenced to indirect immunofluorescence testing (IIFT) is significantly higher for ELISA 2 in samples with a titer of ≤1:640; for high-titer samples, a prozone effect is observed for ELISA 2. The specificities of both ELISAs were excellent not only for pre-COVID-19 serum samples from Europe, Asia, and South America but also for several challenging African sample panels. The SARS-CoV-2 IgG FcγR ELISAs, methodically combining antigen-antibody binding in solution and isotype-specific detection of immune complexes, are valuable tools for seroprevalence studies requiring the (long-term) detection of anti-SARS-CoV-2 IgG antibodies in populations with a challenging immunological background and/or in which spike-protein-based vaccine programs have been rolled out.

The aim of this study was to evaluate the use of a capture enzyme-linked immunosorbent assay (ELISA) for the detection of LASV-reactive IgG antibodies in Mastomys rodents. The assay was used for laboratory-bred Mastomys rodents, as well as for animals caught in the wild in various regions of West Africa. The ELISA reached an accuracy of 97.1% in samples of known exposure, and a comparison to an immunofluorescence assay (IFA) revealed a very strong agreement between the ELISA and IFA results (Cohen's kappa of 0.81). The agreement is valid in Nigeria, and in Guinea and Sierra Leone where the lineages II and IV are circulating, respectively. Altogether, these results indicate that this capture ELISA is suitable for LASV IgG serostatus determination in Mastomys rodents as an alternative to IFA. This assay will be a strong, accurate, and semi-quantitative alternative for rodent seroprevalence studies that does not depend on biosafety level 4 infrastructures, providing great benefits for ecology and epidemiology studies of Lassa fever, a disease listed on the Research and Development Blueprint of the WHO.

Background: There is anecdotal evidence for Lassa virus persistence in body fluids. We aimed to investigate various body fluids after recovery from acute Lassa fever, describe the dynamics of Lassa virus RNA load in seminal fluid, and assess the infectivity of seminal fluid., Methods: In this prospective, longitudinal, cohort study we collected plasma, urine, saliva, lacrimal fluid, vaginal fluid, and seminal fluid from Lassa fever survivors from Irrua Specialist Teaching Hospital in Edo State, Nigeria. Inclusion criteria for participants were RT-PCR-confirmed Lassa fever diagnosis and age 18 years or older. Samples were taken at discharge from hospital (month 0) and at months 0·5, 1, 3, 6, 9, 12, 18, and 24 after discharge. The primary objective of this study was to quantitatively describe virus persistence and clearance and assess the infectivity of seminal fluid. Lassa virus RNA was detected using real-time RT-PCR. Infectivity was tested in cell culture and immunosuppressed mice. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time., Findings: Between Jan 31, 2018, and Dec 11, 2019, 165 participants were enrolled in the study, of whom 159 were eligible for analysis (49 women and 110 men). Low amounts of Lassa virus RNA were detected at month 0 in plasma (49 [45%] of 110 participants), urine (37 [34%]), saliva (five [5%]), lacrimal fluid (ten [9%]), and vaginal fluid (seven [21%] of 33 female participants). Virus RNA was cleared from these body fluids by month 3. However, 35 (80%) of 44 male participants had viral RNA in seminal fluid at month 0 with a median cycle threshold of 26·5. Lassa virus RNA remained detectable up to month 12 in seminal fluid. Biostatistical modelling estimated a clearance rate of 1·19 log 10 viral RNA copies per month and predicted that 50% of male survivors remain Lassa virus RNA-positive in seminal fluid for 83 days after hospital discharge and 10% remain positive in seminal fluid for 193 days after discharge. Viral RNA persistence in seminal fluid for 3 months or more was associated with higher viraemia (p=0·006), more severe disease (p=0·0075), and longer hospitalisation during the acute phase of Lassa fever (p=0·0014). Infectious virus was isolated from 48 (52%) of 93 virus RNA-positive seminal fluid samples collected between month 0 and 12., Interpretation: Lassa virus RNA is shed in various body fluids after recovery from acute disease. The persistence of infectious virus in seminal fluid implies a risk of sexual transmission of Lassa fever., Funding: German Federal Ministry of Health, German Research Foundation, Leibniz Association., Competing Interests: Declaration of interests SG is member of the Scientific Advisory Group to advise WHO on the implementation of the WHO R&D Blueprint for action to prevent epidemics. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Specific serological tests are mandatory for reliable SARS-CoV-2 diagnostics and seroprevalence studies. Here, we assess the specificities of four commercially available SARS-CoV-2 IgG ELISAs in serum/plasma panels originating from Africa, South America, and Europe., Methods: 882 serum/plasma samples collected from symptom-free donors before the COVID-19 pandemic in three African countries (Ghana, Madagascar, Nigeria), Colombia, and Germany were analysed with three nucleocapsid-based ELISAs (Euroimmun Anti-SARS-CoV-2-NCP IgG, EDI™ Novel Coronavirus COVID-19 IgG, Mikrogen recomWell SARS-CoV-2 IgG), one spike/S1-based ELISA (Euroimmun Anti-SARS-CoV-2 IgG), and in-house common cold CoV ELISAs., Results: High specificity was confirmed for all SARS-CoV-2 IgG ELISAs for Madagascan (93.4-99.4%), Colombian (97.8-100.0%), and German (95.9-100.0%) samples. In contrast, specificity was much lower for the Ghanaian and Nigerian serum panels (Ghana: NCP-based assays 77.7-89.7%, spike/S1-based assay 94.3%; Nigeria: NCP-based assays 39.3-82.7%, spike/S1-based assay 90.7%). 15 of 600 African sera were concordantly classified as positive in both the NCP-based and the spike/S1-based Euroimmun ELISA, but did not inhibit spike/ACE2 binding in a surrogate virus neutralisation test. IgG antibodies elicited by previous infections with common cold CoVs were found in all sample panels, including those from Madagascar, Colombia, and Germany and thus do not inevitably hamper assay specificity. Nevertheless, high levels of IgG antibodies interacting with OC43 NCP were found in all 15 SARS-CoV-2 NCP/spike/S1 ELISA positive sera., Conclusions: Depending on the chosen antigen and assay protocol, SARS-CoV-2 IgG ELISA specificity may be significantly reduced in certain populations probably due to interference of immune responses to endemic pathogens like other viruses or parasites., (© 2021 The Authors Tropical Medicine & International Health Published by John Wiley & Sons Ltd.)

Background: Lassa fever is endemic in several west African countries. Case-fatality rates ranging from 21% to 69% have been reported. The pathophysiology of the disease in humans and determinants of mortality remain poorly understood. We aimed to determine host protein biomarkers capable of determining disease outcome., Methods: In this observational study, we analysed left-over blood samples from patients who tested positive for Lassa fever at Irrua Specialist Teaching Hospital, Nigeria, between January, 2014, and April, 2017. We measured viral load, concentrations of clinical chemistry parameters, and levels of 62 circulating proteins involved in inflammation, immune response, and haemostasis. Patients with a known outcome (survival or death) and at least 200 μL of good-quality diagnostic sample were included in logistic regression modelling to assess the correlation of parameters with Lassa fever outcome. Individuals who gave consent could further be enrolled into a longitudinal analysis to assess the association of parameters with Lassa fever outcome over time. Participants were divided into two datasets for the statistical analysis: a primary dataset (samples taken between Jan 1, 2014, and April 1, 2016), and a secondary dataset (samples taken between April 1, 2016, and April 1, 2017). Biomarkers were ranked by area under the receiver operating characteristic curve (AUC) from highest (most predictive) to lowest (least predictive)., Findings: Of 554 patients who tested positive for Lassa fever during the study period, 201 (131 in the primary dataset and 70 in the secondary dataset) were included in the biomarker analysis, of whom 74 (49 in the primary dataset and 25 in the secondary dataset) had died and 127 (82 in the primary dataset and 45 in the secondary dataset) had survived. Cycle threshold values (indicating viral load) and levels of 18 host proteins at the time of admission to hospital were significantly correlated with fatal outcome. The best predictors of outcome in both datasets were plasminogen activator inhibitor-1 (PAI-1; AUC 0·878 in the primary dataset and 0·876 in the secondary dataset), soluble thrombomodulin (TM; 0·839 in the primary dataset and 0·875 in the secondary dataset), and soluble tumour necrosis factor receptor superfamily member 1A (TNF-R1; 0·807 in the primary dataset and 0·851 in the secondary dataset), all of which had higher prediction accuracy than viral load (0·774 in the primary dataset and 0·837 in the secondary dataset). Longitudinal analysis (150 patients, of whom 36 died) showed that of the biomarkers that were predictive at admission, PAI-1 levels consistently decreased to normal levels in survivors but not in those who died., Interpretation: The identification of PAI-1 and soluble TM as markers of fatal Lassa fever at admission, and of PAI-1 as a marker of fatal Lassa fever over time, suggests that dysregulated coagulation and fibrinolysis and endothelial damage have roles in the pathophysiology of Lassa fever, providing a mechanistic explanation for the association of Lassa fever with oedema and bleeding. These novel markers might aid in clinical risk stratification and disease monitoring., Funding: German Research Foundation, Leibniz Association, and US National Institutes of Health., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

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.

Background: SARS-CoV-2 molecular diagnostics is facing material shortages and long turnaround times due to exponential increase of testing demand., Objective: We evaluated the analytic performance and handling of four rapid Antigen Point of Care Tests (AgPOCTs) I-IV (Distributors: (I) Roche, (II) Abbott, (III) MEDsan and (IV) Siemens)., Methods: 100 RT-PCR negative and 84 RT-PCR positive oropharyngeal swabs were prospectively collected and used to determine performance and accuracy of these AgPOCTs. Handling was evaluated by 10 healthcare workers/users through a questionnaire., Results: The median duration from symptom onset to sampling was 6 days (IQR 2-12 days). The overall respective sensitivity were 49.4 % (CI95 %: 38.9-59.9), 44.6 % (CI95 %: 34.3-55.3), 45.8 % (CI95 %: 35.5-56.5) and 54.9 % (CI95 %: 43.4-65.9) for tests I, II, III and IV, respectively. In the high viral load subgroup (containing >10 6 copies of SARS-CoV-2 /swab, n = 26), AgPOCTs reached sensitivities of 92.3 % or more (range 92.3 %-100 %). Specificity was 100 % for tests I, II (CI95 %: 96.3-100 for both tests) and IV (CI95 %: 96.3-100) and 97 % (CI95 %: 91.5-98.9) for test III. Regarding handling, test I obtained the overall highest scores, while test II was considered to have the most convenient components. Of note, users considered all assays, with the exception of test I, to pose a significant risk for contamination by drips or spills., Discussion: Besides some differences in sensitivity and handling, all four AgPOCTs showed acceptable performance in high viral load samples. However, due to the significantly lower sensitivity compared to RT-qPCR, a careful consideration of pro and cons of AgPOCT has to be taken into account before clinical implementation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters in Hamburg, Germany., Methods: Metagenomic RNA-sequencing and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg., Results: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed us to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters., Conclusions: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection clusters can provide further resolution on transmission events indistinguishable at a consensus sequence level., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We report two outbreaks of Lassa fever that occurred in Benin in 2014 and 2016 with 20 confirmed cases and 50% (10/20) mortality. Benin was not previously considered to be an endemic country for Lassa fever, resulting in a delay to diagnose the disease and its human transmission. Molecular investigations showed the viral genomes to be similar to that of the Togo strain, which is genetically very different from other known strains and confirms the existence of a new lineage. Endemic circulation of Lassa virus in a new territory and the genetic diversity thus confirm that this virus represents a growing threat for West African people. Given the divergence of the Benin strain from the prototypic Josiah Sierra Leone strain frequently used to generate vaccine candidates, the efficacy of vaccine candidates should also be demonstrated with this strain.

Background: The ongoing SARS-CoV-2 pandemic presents a unique challenge to diagnostic laboratories. There are preliminary studies correlating qRT-PCR results from different materials to clinical outcomes, yet, comparability is limited due to the plethora of different assays used for diagnostics. In this study we evaluate clinical performance and linear range for the SARS-CoV-2 IVD (cobas6800/8800 system, a fully automated sample-to-result platform) in different clinically relevant matrix materials outside official specifications., Methods: Assay performance was assessed in human plasma, BAL/BL and transport medium following chemical inactivation. For analytical evaluation, respective matrix materials were spiked with SARS-CoV-2 RNA in ten-fold dilution series. The efficacy of chemical inactivation by guanidine hydrochloride solution was confirmed in cell culture infectivity experiments. For correlation, a total of 289 predetermined clinical samples including respiratory swabs, plasma and lower respiratory tract specimens were subjected to the SARS-CoV-2 IVD test and results were compared., Results: The SARS-CoV-2 IVD showed excellent linearity over four to six log steps depending on matrix material. Chemical inactivation resulted in a reduction in plaque forming units of at least 3.5 log steps, while having no significant impact on assay performance. Inter-run consistency from three different testing sites demonstrated excellent comparability of RT-PCR results (maximum deviation was 1.53 CT). Clinical evaluation for respiratory swabs showed very good agreement with the comparator assay (Positive agreement 95.7 %, negative agreement 98.9 %)., Conclusion: The SARS-CoV-2 IVD test for the cobas6800/8800 systems offers excellent linear range and inter-run consistency for quantification of SARS-CoV-2 RNA in different matrices outside official specifications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T-cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here, we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF, we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific and antigenically unrelated T cells. However, severe and fatal LF cases were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of nonspecific T cells with homing capacity to inflamed tissues are associated with severe LF. IMPORTANCE Lassa fever may cause severe disease in humans, in particular in areas of endemicity like Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here, we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus and poor Lassa virus-specific effector T-cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflamed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T-cell targeting as a potential postexposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models, such as nonhuman primates., (Copyright © 2020 Port et al.)

Lassa mammarenavirus (LASV) is a rodent-borne arenavirus endemic to several West African countries. It is the causative agent of human Lassa fever, an acute viral hemorrhagic fever disease. To date, no therapeutics or vaccines against LASV have obtained regulatory approval. Polyclonal neutralizing antibodies derived from hyperimmunized animals may offer a useful strategy for prophylactic and therapeutic intervention to combat human LASV infections. The LASV envelope surface glycoprotein complex (GP) is the major target for neutralizing antibodies, and it is the main viral antigen used for the design of an LASV vaccine. Here, we assessed the immunogenic potential of mammalian cell-derived virus-like particles (VLPs) expressing GP from the prototypic LASV strain Josiah in a native-like conformation as the sole viral antigen. We demonstrate that an adjuvanted prime-boost immunization regimen with GP-derived VLPs elicited neutralizing antibody responses in rabbits, suggesting that effective antigenic epitopes of GP were displayed. Notably, these antibodies exhibited broad reactivity across five genetic lineages of LASV. VLP-based immunization strategies may represent a powerful approach for generating polyclonal sera containing cross-reactive neutralizing antibodies against LASV., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany., (Copyright © 2020 Pfefferle et al.)

Lassa virus (LASV) is the causative agent of Lassa fever (LF), an often-fatal hemorrhagic disease. LF is endemic in Nigeria, Sierra Leone and other West African countries. Diagnosis of LASV infection is challenged by the genetic diversity of the virus, which is greatest in Nigeria. The ReLASV Pan-Lassa Antigen Rapid Test (Pan-Lassa RDT) is a point-of-care, in vitro diagnostic test that utilizes a mixture of polyclonal antibodies raised against recombinant nucleoproteins of representative strains from the three most prevalent LASV lineages (II, III and IV). We compared the performance of the Pan-LASV RDT to available quantitative PCR (qPCR) assays during the 2018 LF outbreak in Nigeria. For patients with acute LF (RDT positive, IgG/IgM negative) during initial screening, RDT performance was 83.3% sensitivity and 92.8% specificity when compared to composite results of two qPCR assays. 100% of samples that gave Ct values below 22 on both qPCR assays were positive on the Pan-Lassa RDT. There were significantly elevated case fatality rates and elevated liver transaminase levels in subjects whose samples were RDT positive compared to RDT negative.

In 2014, our research network was involved in the evaluation of favipiravir, an anti-influenza polymerase inhibitor, against Ebola virus. In this review, we discuss how mathematical modeling was used, first to propose a relevant dosing regimen in humans, and then to optimize its antiviral efficacy in a nonhuman primate (NHP) model. The data collected in NHPs were finally used to develop a model of Ebola pathogenesis integrating the interactions among the virus, the innate and adaptive immune response, and the action of favipiravir. We conclude the review of this work by discussing how these results are of relevance for future human studies in the context of Ebola virus, but also for other emerging viral diseases for which no therapeutics are available., (© 2020 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Introduction: Lassa fever (LF) is a severe and often fatal systemic disease in humans and affects a large number of countries in West Africa. Treatment options are limited to supportive care and the broad-spectrum antiviral agent ribavirin. However, evidence for ribavirin efficacy in patients with LF is poor and pharmacokinetic (PK) data are not available.Irrua Specialist Teaching Hospital (ISTH) developed an intravenous ribavirin regimen different to the WHO recommendation. Apart from a lower total daily dose the drug is usually administered once per day which reduces the exposure of personnel to patients with LF. The aim of this study is to characterise the PK of the Irrua ribavirin regimen., Methods and Analysis: This prospective, observational clinical study will assess PK properties of the Irrua ribavirin regimen on routinely ribavirin-treated patients with LF at ISTH, a referral hospital serving 19 local governmental areas in a LF endemic zone in Nigeria. Participants will be adults with PCR-confirmed LF. The primary objective is to describe classical PK parameters for ribavirin (maximum plasma drug concentration, time to maximum plasma drug concentration, area under the plasma drug concentration vs time curve, half-life time T1/2, volume of distribution). Blood samples will be collected at 0.5, 1, 3, 5, 8, 12 and 24 hours after doses on day 1, day 4 and day 10 of ribavirin treatment. Ribavirin plasma concentrations will be determined using liquid chromatography coupled to tandem mass spectrometry., Ethics and Dissemination: The study will be conducted in compliance with the protocol, the Declaration of Helsinki, Good Clinical Practice (GCP) and the Nigerian National Code for Health Research Ethics. The protocol has received approval by the Health Research Ethics Committee of ISTH. Results will be made available to LF survivors, their caregivers, the funders, LF research society and other researchers., Registration Details: ISRCTN11104750., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Rocaglates, a class of natural compounds isolated from plants of the genus Aglaia, are potent inhibitors of translation initiation. They are proposed to form stacking interactions with polypurine sequences in the 5'-untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex. Since virus replication relies on the host translation machinery, it is not surprising that the rocaglate Silvestrol has broad-spectrum antiviral activity. Unfortunately, synthesis of Silvestrol is sophisticated and time-consuming, thus hampering the prospects for further antiviral drug development. Here, we present the less complex structured synthetic rocaglate CR-31-B (-) as a novel compound with potent broad-spectrum antiviral activity in primary cells and in an ex vivo bronchial epithelial cell system. CR-31-B (-) inhibited the replication of corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses and, to a lesser extent, hepatitis E virus (HEV) at non-cytotoxic low nanomolar concentrations. Since HEV has a polypurine-free 5'-UTR that folds into a stable hairpin structure, we hypothesized that RNA clamping by Silvestrol and its derivatives may also occur in a polypurine-independent but structure-dependent manner. Interestingly, the HEV 5'-UTR conferred sensitivity towards Silvestrol but not to CR-31-B (-). However, if an exposed polypurine stretch was introduced into the HEV 5'-UTR, CR-31-B (-) became an active inhibitor comparable to Silvestrol. Moreover, thermodynamic destabilization of the HEV 5'-UTR led to reduced translational inhibition by Silvestrol, suggesting differences between rocaglates in their mode of action, most probably by engaging Silvestrol's additional dioxane moiety., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country. IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines., (Copyright © 2019 Ehichioya et al.)

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.

Differences in T-cell phenotype, particularly the expression of markers of T-cell homeostasis, have been observed in fatal and nonfatal Ebola virus disease (EVD). However, the relationship between these markers with T-cell function and virus clearance during EVD is poorly understood. To gain biological insight into the role of T cells during EVD, combined transcriptomics and T-cell receptor sequencing was used to profile blood samples from fatal and nonfatal EVD patients from the recent West African EVD epidemic. Fatal EVD was characterized by strong T-cell activation and increased abundance of T-cell inhibitory molecules. However, the early T-cell response was oligoclonal and did not result in viral clearance. In contrast, survivors mounted highly diverse T-cell responses, maintained low levels of T-cell inhibitors, and cleared Ebola virus. Our findings highlight the importance of T-cell immunity in surviving EVD and strengthen the foundation for further research on targeting of the dendritic cell-T cell interface for postexposure immunotherapy.

Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays. IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective vaccine or therapy is urgently needed. Here, we describe efforts to produce cross-reactive monoclonal antibodies that bind to both New and Old World arenaviruses. All of our MAbs seem to be nonneutralizing and nonprotective and target subunit 2 of the glycoprotein. Due to the lack of reagents such as recombinant glycoproteins and antibodies for rapid detection assays, our MAbs could be beneficial as analytic and diagnostic tools., (Copyright © 2018 Amanat et al.)

Ribavirin is a broad spectrum antiviral which inhibits Lassa virus (LASV) replication in vitro but exhibits a minor effect on viremia in vivo. However, ribavirin significantly improves the disease outcome when administered in combination with sub-optimal doses of favipiravir, a strong antiviral drug. The mechanisms explaining these conflicting findings have not been determined, so far. Here, we used an interdisciplinary approach combining mathematical models and experimental data in LASV-infected mice that were treated with ribavirin alone or in combination with the drug favipiravir to explore different putative mechanisms of action for ribavirin. We test four different hypotheses that have been previously suggested for ribavirin's mode of action: (i) acting as a mutagen, thereby limiting the infectivity of new virions; (ii) reducing viremia by impairing viral production; (iii) modulating cell damage, i.e., by reducing inflammation, and (iv) enhancing antiviral immunity. Our analysis indicates that enhancement of antiviral immunity, as well as effects on viral production or transmission are unlikely to be ribavirin's main mechanism mediating its antiviral effectiveness against LASV infection. Instead, the modeled viral kinetics suggest that the main mode of action of ribavirin is to protect infected cells from dying, possibly reducing the inflammatory response.

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 -/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics., (Copyright © 2017 American Society for Microbiology.)

Background: Human Ebola infection is characterized by a paralysis of the immune system. A signature of αβ T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014-2015 occurred in West Africa, and to assess their association with the clinical outcome., Methodology/principal Findings: Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome., Conclusions/significances: Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells.

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.

Background: By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fluid, including clearance parameters., Methods: In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fluid at follow-up every 3-6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time., Findings: We enrolled 26 participants and tested 130 seminal fluid specimens; median follow up was 197 days (IQR 187-209 days) after enrolment, which corresponded to 255 days (228-287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73-181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fluid of -0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fluid at 115 days (90% prediction interval 72-160) and 294 days (212-399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in affected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice., Interpretation: Time to clearance of Ebola virus RNA from seminal fluid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks., Funding: This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking., (Copyright © 2017 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY license. Published by Elsevier Ltd.. All rights reserved.)