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Magnetic ring structures are of particular interest, due to stable remanent configurations such as the vortex state, which have potential in future magnetic random access memory (MRAM) applications. Here we report results on the first experimental study of periodic ring arrays using off-specular polarized neutron reflectivity (PNR). The experiments were performed on a 7.5 mm x 5 mm NiFe array with an average ring diameter of 3.2/[micro]m, 0.7/[micro]m width, 25 nm thickness, and separation of 5.6/[micro]m. Using polarization analysis, a spin dependent diffraction of reflected and transmitted neutron beams were investigated as a function of external applied field. Index Terms--Magnetic domains, magnetization reversal, polarized neutron reflectivity.

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress. Meanwhile, heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and the SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect the Z-mediated virus budding and GP2-mediated fusion activity that is required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan-NMT inhibitor DDD85646 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlates with reduced Z budding activity and GP2-mediated fusion activity as well as with proteasome-mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic-fever-causing Junin (JUNV) and Lassa (LASV) mammarenaviruses. Our results support the exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses., Competing Interests: The authors declare no conflict of interest.

The eukaryotic chaperonin containing tailless complex polypeptide 1 ring complex (CCT, also known as TCP-1 Ring Complex, TRiC/CCT) participates in the folding of 5% to 10% of the cellular proteome and has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses, but the mechanisms by which the TRiC/CCT complex contributes to virus multiplication remain poorly understood. Here, we document that the nucleoprotein (NP) of the mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a substrate of the human TRiC/CCT complex, and that pharmacological inhibition of TRiC/CCT complex function, or RNAi-mediated knockdown of TRiC/CCT complex subunits, inhibited LCMV multiplication in human cells. We obtained evidence that the TRiC/CCT complex is required for the production of NP-containing virus-like particles (VLPs), and the activity of the virus ribonucleoprotein (vRNP) responsible for directing replication and transcription of the viral genome. Pharmacological inhibition of the TRIC/CCT complex also restricted multiplication of the live-attenuated vaccine candidates Candid#1 and ML29 of the hemorrhagic fever causing Junin (JUNV) and Lassa (LASV) mammarenaviruses, respectively. Our findings indicate that the TRiC/CCT complex is required for mammarenavirus multiplication and is an attractive candidate for the development of host directed antivirals against human-pathogenic mammarenaviruses. IMPORTANCE Host-directed antivirals have gained great interest as an antiviral strategy to counteract the rapid emergence of drug-resistant viruses. The chaperonin TRiC/CCT complex has been involved in the life cycle of several viruses, including dengue, Zika, and influenza viruses. Here, we have provided evidence that the chaperonin TRiC/CCT complex participates in mammarenavirus infection via its interaction with the viral NP. Importantly, pharmacological inhibition of TRiC/CCT function significantly inhibited multiplication of LCMV and the distantly related mammarenavirus JUNV in human cells. Our findings support that the TRiC/CCT complex is required for multiplication of mammarenaviruses and that the TRiC/CCT complex is an attractive host target for the development of antivirals against human-pathogenic mammarenaviruses.

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.

The mammarenavirus Lassa virus (LASV) causes a life-threatening acute febrile disease, Lassa fever (LF). To date, no US Food and Drug Administration (FDA)-licensed medical countermeasures against LASV are available. This underscores the need for the development of novel anti-LASV drugs. Here, we screen an FDA-approved drug library to identify novel anti-LASV drug candidates using an infectious-free cell line expressing a functional LASV ribonucleoprotein (vRNP), where levels of vRNP-directed reporter gene expression serve as a surrogate for vRNP activity. Our screen identified the pan-ErbB tyrosine kinase inhibitor afatinib as a potent inhibitor of LASV vRNP activity. Afatinib inhibited multiplication of lymphocytic choriomeningitis virus (LCMV) a mammarenavirus closely related to LASV. Cell-based assays revealed that afatinib inhibited multiple steps of the LASV and LCMV life cycles. Afatinib also inhibited multiplication of Junín virus vaccine strain Candid#1, indicating that afatinib can have antiviral activity against a broad range of human pathogenic mammarenaviruses., 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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Defective interfering particles (DIPs) are naturally occurring products during virus replication in infected cells. DIPs contain defective viral genomes (DVGs) and interfere with replication and propagation of their corresponding standard viral genomes by competing for viral and cellular resources, as well as promoting innate immune antiviral responses. Consequently, for many different viruses, including mammarenaviruses, DIPs play key roles in the outcome of infection. Due to their ability to broadly interfere with viral replication, DIPs are attractive tools for the development of a new generation of biologics to target genetically diverse and rapidly evolving viruses. Here, we provide evidence that in cells infected with the Lassa fever (LF) vaccine candidate ML29, a reassortant that carries the nucleoprotein (NP) and glycoprotein (GP) dominant antigens of the pathogenic Lassa virus (LASV) together with the L polymerase and Z matrix protein of the non-pathogenic genetically related Mopeia virus (MOPV), L-derived truncated RNA species are readily detected following infection at low multiplicity of infection (MOI) or in persistently-infected cells originally infected at high MOI. In the present study, we show that expression of green fluorescent protein (GFP) driven by a tri-segmented form of the mammarenavirus lymphocytic choriomeningitis virus (r3LCMV-GFP/GFP) was strongly inhibited in ML29-persistently infected cells, and that the magnitude of GFP suppression was dependent on the passage history of the ML29-persistently infected cells. In addition, we found that DIP-enriched ML29 was highly attenuated in immunocompetent CBA/J mice and in Hartley guinea pigs. Likewise, STAT-1 -/- mice, a validated small animal model for human LF associated hearing loss sequelae, infected with DIP-enriched ML29 did not exhibit any hearing abnormalities throughout the observation period (62 days).

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Ebola virus (EBOV) disease has killed thousands of West and Central Africans over the past several decades. Many who survive the acute disease later experience post-Ebola syndrome, a constellation of symptoms whose causative pathogenesis is unclear., Methods: We investigated EBOV-specific CD8+ and CD4+ T-cell responses in 37 Sierra Leonean EBOV disease survivors with (n = 19) or without (n = 18) sequelae of arthralgia and ocular symptoms. Peripheral blood mononuclear cells were infected with recombinant vesicular stomatitis virus encoding EBOV antigens. We also studied the presence of EBOV-specific immunoglobulin G, antinuclear antibodies, anti-cyclic citrullinated peptide antibodies, rheumatoid factor, complement levels, and cytokine levels in these 2 groups., Results: Survivors with sequelae had a significantly higher EBOV-specific CD8+ and CD4+ T-cell response. No differences in EBOV-specific immunoglobulin G, antinuclear antibody, or anti-cyclic citrullinated peptide antibody levels were found. Survivors with sequelae showed significantly higher rheumatoid factor levels., Conclusion: EBOV-specific CD8+ and CD4+ T-cell responses were significantly higher in Ebola survivors with post-Ebola syndrome. These findings suggest that pathogenesis may occur as an immune-mediated disease via virus-specific T-cell immune response or that persistent antigen exposure leads to increased and sustained T-cell responses., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects. Dihydroorotate dehydrogenase (DHODH) inhibitors, which block de novo pyrimidine biosynthesis, have antiviral activity against viruses from different families, including Arenaviridae , the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC 50 ] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8 + ) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity. IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine., (Copyright © 2020 Sakabe et al.)

Lassa virus infects hundreds of thousands of people each year across rural West Africa, resulting in a high number of cases of Lassa fever (LF), a febrile disease associated with high morbidity and significant mortality. The lack of approved treatments or interventions underscores the need for an effective vaccine. At least four viral lineages circulate in defined regions throughout West Africa with substantial interlineage nucleotide and amino acid diversity. An effective vaccine should be designed to elicit Lassa virus specific humoral and cell mediated immunity across all lineages. Most current vaccine candidates use only lineage IV antigens encoded by Lassa viruses circulating around Sierra Leone, Liberia, and Guinea but not Nigeria where lineages I-III are found. As previous infection is known to protect against disease from subsequent exposure, we sought to determine whether LF survivors from Nigeria and Sierra Leone harbor memory T cells that respond to lineage IV antigens. Our results indicate a high degree of cross-reactivity of CD8+ T cells from Nigerian LF survivors to lineage IV antigens. In addition, we identified regions within the Lassa virus glycoprotein complex and nucleoprotein that contributed to these responses while T cell epitopes were not widely conserved across our study group. These data are important for current efforts to design effective and efficient vaccine candidates that can elicit protective immunity across all Lassa virus lineages., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: RFG and LMB are co-founders of Zalgen Labs, LLC. The Viral Hemorrhagic Fever Consortium (vhfc.org) is a partnership of academic and industry scientists who are developing diagnostics, therapeutics and vaccines for LF and other severe diseases. Tulane University and various industry partners have filed United States and foreign patent applications on behalf of the consortium for several of these technologies. If commercial products are developed, consortium members may receive royalties or profits.

The mammarenavirus Lassa (LASV) is highly prevalent in West Africa where it infects several hundred thousand individuals annually resulting in a high number of Lassa fever (LF) cases, a febrile disease associated with high morbidity and significant mortality. Mounting evidence indicates that the worldwide-distributed prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. There are not Food and Drug Administration (FDA) licensed vaccines and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective and can cause significant side effects. Therefore, there is an unmet need for novel antiviral drugs to combat LASV. This task would be facilitated by the implementation of high throughput screens (HTS) to identify inhibitors of the activity of the virus ribonucleoprotein (vRNP) responsible for directing virus RNA genome replication and gene transcription. The use of live LASV for this purpose is jeopardized by the requirement of biosafety level 4 (BSL4) containment. We have developed a virus-free cell platform, where expression levels of reporter genes serve as accurate surrogates of vRNP activity, to develop cell-based assays compatible with HTS to identify inhibitors of LASV and LCMV mammarenavirus vRNP activities., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000⁻300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8 + T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013-2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8 + T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8 + T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8 + T cells to EBOV VP24, VP35, and VP40 also made CD8 + T cells to NP, but rarely to GP. We identified 34 CD8 + T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination., Competing Interests: The authors declare no conflict of interest.

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo., Competing Interests: The authors have declared that no competing interests exist.

Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines, and current antiarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus-host interactions suitable for targeting, as well as lead compounds to develop future antiarenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3-dihydrobenzo[ b][1,4] dioxin-6-yl)-4'-methoxy-[1,1'-biphenyl]-3-yl)thiophene-2-carboxamide] as having strong anti-lymphocytic choriomeningitis virus (LCMV) activity in cultured cells. KP-146 did not inhibit LCMV cell entry but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus-host interactions, as well as lead candidate structures to develop antiarenaviral drugs.

Unintended dural punctures with leakage of cerebrospinal fluid (CSF) are recognized as a frequent complication of spinal surgery. Although conservative or invasive options may be used to treat postoperative CSF leaks, the existing literature does not define either an algorithmic treatment approach or a universally accepted standard of care. We believe that a transforaminal epidural blood patch (EBP) can serve as a minimally invasive, cost-effective option to treat postsurgical CSF leaks that do not resolve with conservative management. We have performed an EBP via the transforaminal route to treat postsurgical CSF leaks in both the cervical and lumbar spine. The first case describes a patient who underwent an anterior cervical diskectomy and fusion with a complication of profuse CSF leakage. The application of a cervical transforaminal EBP at the levels of surgical repair was effective in stopping the dural leak. The second case involves a patient who experienced classic positional spinal headaches after a lumbar hemilaminectomy and diskectomy. After utilization of lumbar transforaminal EBPs, his symptoms revolved. This article presents the potential use of an EBP via the transforaminal route to treat postsurgical dural leaks in both the cervical and lumbar region., Level of Evidence: V., (Copyright © 2017 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.)

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose serious public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. We have documented that a recombinant LCMV containing the glycoprotein (GPC) gene of LASV within the backbone of the immunosuppressive clone 13 (Cl-13) variant of the Armstrong strain of LCMV (rCl-13/LASV-GPC) exhibited Cl-13-like growth properties in cultured cells, but in contrast to Cl-13, rCl-13/LASV-GPC was unable to establish persistence in immunocompetent adult mice, which prevented its use for some in vivo experiments. Recently, V459K and K461G mutations within the GP2 cytoplasmic domain (CD) of rCl-13/LASV-GPC were shown to increase rCl-13/LASV-GPC infectivity in mice. Here, we generated rCl-13(GPC/VGKS) by introducing the corresponding revertant mutations K465V and G467K within GP2 of rCl-13 and we show that rCl-13(GPC/VGKS) was unable to persist in mice. K465V and G467K mutations did not affect GPC processing, virus RNA replication, or gene expression. In addition, rCl-13(GPC/VGKS) grew to high titers in cultured cell lines and in immunodeficient mice. Further analysis revealed that rCl-13(GPC/VGKS) infected fewer splenic plasmacytoid dendritic cells than rCl-13, yet the two viruses induced similar type I interferon responses in mice. Our findings have identified novel viral determinants of Cl-13 persistence and also revealed that virus GPC-host interactions yet to be elucidated critically contribute to Cl-13 persistence., Importance: The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), provides investigators with a superb experimental model system to investigate virus-host interactions. The Armstrong strain (ARM) of LCMV causes an acute infection, whereas its derivative, clone 13 (Cl-13), causes a persistent infection. Mutations F260L and K1079Q within GP1 and L polymerase, respectively, have been shown to play critical roles in Cl-13's ability to persist in mice. However, there is an overall lack of knowledge about other viral determinants required for Cl-13's persistence. Here, we report that mutations K465V and G467K within the cytoplasmic domain of Cl-13 GP2 resulted in a virus, rCl-13(GPC/VGKS), that failed to persist in mice despite exhibiting Cl-13 wild-type-like fitness in cultured cells and immunocompromised mice. This finding has uncovered novel viral determinants of viral persistence, and a detailed characterization of rCl-13(GPC/VGKS) can provide novel insights into the mechanisms underlying persistent viral infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Hemorrhagic fever arenaviruses (HFAs) pose important public health problems in regions where they are endemic. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. We have recently shown that the noncoding intergenic region (IGR) present in each arenavirus genome segment, the S and L segments (S-IGR and L-IGR, respectively), plays important roles in the control of virus protein expression and that this knowledge could be harnessed for the development of live-attenuated vaccine strains to combat HFAs. In this study, we further investigated the sequence plasticity of the arenavirus IGR. We demonstrate that recombinants of the prototypic arenavirus lymphocytic choriomeningitis virus (rLCMVs), whose S-IGRs were replaced by the S-IGR of Lassa virus (LASV) or an entirely nonviral S-IGR-like sequence (Ssyn), are viable, indicating that the function of S-IGR tolerates a high degree of sequence plasticity. In addition, rLCMVs whose L-IGRs were replaced by Ssyn or S-IGRs of the very distantly related reptarenavirus Golden Gate virus (GGV) were viable and severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. Our findings indicate that replacement of L-IGR by a nonviral Ssyn could serve as a universal molecular determinant of arenavirus attenuation., Importance: Hemorrhagic fever arenaviruses (HFAs) cause high rates of morbidity and mortality and pose important public health problems in regions where they are endemic. Implementation of live-attenuated vaccines (LAVs) will represent a major step to combat HFAs. Here we document that the arenavirus noncoding intergenic region (IGR) has a high degree of plasticity compatible with virus viability. This observation led us to generate recombinant LCMVs containing nonviral synthetic IGRs. These rLCMVs were severely attenuated in vivo but able to elicit protective immunity against a lethal challenge with wild-type LCMV. These nonviral synthetic IGRs can be used as universal molecular determinants of arenavirus attenuation for the rapid development of safe and effective, as well as stable, LAVs to combat HFA., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Hemorrhagic fever arenaviruses (HFA) pose important public health problems in regions where they are endemic. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa, causing a large number of Lassa fever cases associated with high morbidity and mortality. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. The Mopeia virus (MOPV)/LASV reassortant (ML29) is a LASV candidate live-attenuated vaccine (LAV) that has shown promising results in animal models. Nevertheless, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. Development of LAVs based on well-defined molecular mechanisms of attenuation will represent a major step in combatting HFA. We used the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to develop a general molecular strategy for arenavirus attenuation. Our approach involved replacement of the noncoding intergenic region (IGR) of the L genome segment with the IGR of the S genome segment to generate a recombinant LCMV, rLCMV(IGR/S-S), that was highly attenuated in vivo but induced protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV(IGR/S-S) was associated with a stable reorganization of the control of viral gene expression. This strategy can facilitate the rapid development of LAVs with the antigenic composition of the parental HFA and a mechanism of attenuation that minimizes concerns about increased virulence that could be caused by genetic changes in the LAV., Importance: Hemorrhagic fever arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in the regions where they are endemic. Implementation of live-attenuated vaccines (LAV) will represent a major step in combatting HFA. Here we have used the prototypic arenavirus LCMV to document a general molecular strategy for arenavirus attenuation that can facilitate the rapid development of safe and effective, as well as stable, LAV to combat HFA., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406., Importance: Hemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the two trans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5' and 3' termini of the viral genome., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Modern drug discovery efforts rely, to a large extent, on lead compounds from two classes of small organic molecules; namely, natural products (i.e., secondary metabolites) and designed compounds (i.e., synthetic molecules). In this article, we demonstrate how these two domains of lead compounds can be merged through total synthesis and molecular design of analogs patterned after the targeted natural products, whose promising biological properties provide the motivation. Specifically, the present study targeted the naturally occurring biyouyanagins A and B and their analogs through modular chemical synthesis and led to the discovery of small organic molecules possessing anti-HIV and anti-arenavirus properties.

Lymphocytic choriomeningitis virus (LCVM) nucleoprotein (NP) counteracts the host type I interferon (IFN) response by inhibiting activation of the IFN regulatory factor 3 (IRF3). In this study, we have mapped the regions and specific amino acid residues within NP involved in its anti-IFN activity. We identified a region spanning residues 382 to 386 as playing a critical role in the IFN-counteracting activity of NP. Alanine substitutions at several positions within this region resulted in NP mutants that lacked the IFN-counteracting activity but retained their functions in virus RNA synthesis and assembly of infectious particles. We used reverse genetics to rescue a recombinant LCMV strain carrying mutation D382A in its NP [rLCMV/NP*(D382A)]. Compared to wild-type (WT) LCMV, rLCMV/NP*(D382A) exhibited a higher level of attenuation in IFN-competent than IFN-deficient cells. In addition, A549 cells infected with rLCMV/NP*(D382A), but not with WT LCMV, produced IFN and failed to rescue replication of the IFN-sensitive Newcastle disease virus.

We have documented that the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus is an antagonist of the type I interferon response. In this study we tested the ability of NPs encoded by representative arenavirus species from both Old World and New World antigenic groups to inhibit production of interferon. We found that, with the exception of Tacaribe virus (TCRV), all NPs tested inhibited activation of beta interferon and interferon regulatory factor 3 (IRF-3)-dependent promoters, as well as the nuclear translocation of IRF-3. Consistent with this observation, TCRV-infected cells also failed to inhibit interferon production.

Borna disease virus (BDV) is an enveloped virus. Its non-segmented, negative-stranded RNA genome has the coding capability for six main polypeptides and has an organization characteristic of members of the order Mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae. Here, the establishment of a reverse genetics system for BDV is described. Intracellular synthesis of a BDV RNA analogue or minigenome (MG) from a plasmid was driven by RNA polymerase I. Co-transfection with plasmids expressing the BDV polymerase (L), nucleoprotein (N) and phosphoprotein (P) under the control of RNA polymerase II allowed for BDV MG replication and expression. This process depended on a delicate N:P ratio, whereas the L:P ratio was less critical. Two isoforms of N, Np40 and Np38, are present in BDV-infected cells but only Np40 was strictly required for virus polymerase activity. BDV p10 polypeptide encoded by the P gene exhibited a strong inhibitory effect on BDV MG expression.

Borna disease virus (BDV) has a non-segmented, negative-strand (NNS) RNA genome. In contrast to all other known NNS RNA animal viruses, BDV replication and transcription occur in the nucleus of infected cells. Moreover, BDV uses RNA splicing for the regulation of its genome expression. Two introns (I and II), both present in two viral primary transcripts of 2.5 and 7.2 kb, have been reported in BDV. Here, evidence is provided of a new BDV intron, intron III, generated by alternative 3' splice-site choice. Intron III-spliced mRNAs were detected at early times post-infection and found to be present in cells from different types and species. Intron III-spliced mRNAs have coding capability for two new viral proteins with predicted molecular masses of 8.4 and 165 (p165) kDa. p165 is a deleted form of the BDV L polymerase, containing three RGD motifs and a signal peptide signal that could target it into the secretory pathway. These findings underscore the proteomic complexity exhibited by BDV.

We have investigated the entry pathway of Borna disease virus (BDV). Virus entry was assessed by detecting early viral replication and transcription. Lysosomotropic agents (ammonium chloride, chloroquine, and amantadine), as well as energy depletion, prevented BDV infection, indicating that BDV enters host cells by endocytosis and requires an acidic intracellular compartment to allow membrane fusion and initiate infection. Consistent with this hypothesis, we observed that BDV-infected cells form extensive syncytia upon low-pH treatment. Entry of enveloped viruses into animal cells usually requires the membrane-fusing activity of viral surface glycoproteins (GPs). BDV GP is expressed as two products of 84 and 43 kDa (GP-84 and GP-43, respectively). We show here that only GP-43 is present at the surface of BDV-infected cells and therefore is likely the viral polypeptide responsible for triggering fusion events. We also present evidence that GP-43, which corresponds to the C terminus of GP-84, is generated by cleavage of GP-84 by the cellular protease furin. Hence, we propose that BDV GP-84 is involved in attachment to the cell surface receptor whereas its furin-cleaved product, GP-43, is involved in pH-dependent fusion after internalization of the virion by endocytosis.