Pathology and Pathogenesis of Lassa Fever: Novel Immunohistochemical Findings in Fatal Cases and Clinico-pathologic Correlation.

BACKGROUND: Lassa fever is a zoonotic, acute viral illness first identified in Nigeria in 1969. An estimate shows that the "at risk" seronegative population (in Sierra Leone, Guinea, and Nigeria) may be as high as 59 million, with an annual incidence of all illnesses of 3 million, and fatalities up to 67 000, demonstrating the serious impact of the disease on the region and global health. METHODS: Histopathologic evaluation, immunohistochemical assay, and electron microscopic examination were performed on postmortem tissue samples from 12 confirmed Lassa fever cases. RESULTS: Lassa fever virus antigens and viral particles were observed in multiple organ systems and cells, including cells in the mononuclear phagocytic system and other specialized cells where it had not been described previously. CONCLUSIONS: The immunolocalization of Lassa fever virus antigens in fatal cases provides novel insightful information with clinical and pathogenetic implications. The extensive involvement of the mononuclear phagocytic system, including tissue macrophages and endothelial cells, suggests participation of inflammatory mediators from this lineage with the resulting vascular dilatation and increasing permeability. Other findings indicate the pathogenesis of Lassa fever is multifactorial and additional studies are needed.

Tetravalent Rabies-Vectored Filovirus and Lassa Fever Vaccine Induces Long-term Immunity in Nonhuman Primates.

BACKGROUND: The objective of this study is to evaluate the immunogenicity of adjuvanted monovalent rabies virus (RABV)-based vaccine candidates against Ebola virus (FILORAB1), Sudan virus (FILORAB2), Marburg virus (FILORAB3), Lassa virus (LASSARAB1), and combined trivalent vaccine candidate (FILORAB1-3) and tetravalent vaccine candidate (FILORAB1-3 and LASSARAB) in nonhuman primates. METHODS: Twenty-four Macaca fascicularis were randomly assigned into 6 groups of 4 animals. Each group was vaccinated with either a single adjuvanted vaccine, the trivalent vaccine, or the tetravalent vaccine at days 0 and 28. We followed the humoral immune responses for 1 year by antigen-specific enzyme-linked immunosorbent assays and RABV neutralization assays. RESULTS: High titers of filovirus and/or Lassa virus glycoprotein-specific immunoglobulin G were induced in the vaccinated animals. There were no significant differences between immune responses in animals vaccinated with single vaccines vs trivalent or tetravalent vaccines. In addition, all vaccine groups elicited strong rabies neutralizing antibody titers. The antigen-specific immune responses were detectable for 1 year in all groups. CONCLUSIONS: In summary, this study shows the longevity of the immune responses up to 365 days for a pentavalent vaccine-against Ebola virus, Sudan virus, Marburg virus, Lassa virus, and RABV-using a safe and effective vaccine platform.

Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses.

Antiviral therapies that impede virus entry are attractive because they act on the first phase of the infectious cycle. Drugs that target pathways common to multiple viruses are particularly desirable when laboratory-based viral identification may be challenging, e.g., in an outbreak setting. We are interested in identifying drugs that block both Ebola virus (EBOV) and Lassa virus (LASV), two unrelated but highly pathogenic hemorrhagic fever viruses that have caused outbreaks in similar regions in Africa and share features of virus entry: use of cell surface attachment factors, macropinocytosis, endosomal receptors, and low pH to trigger fusion in late endosomes. Toward this goal, we directly compared the potency of eight drugs known to block EBOV entry with their potency as inhibitors of LASV entry. Five drugs (amodiaquine, apilimod, arbidol, niclosamide, and zoniporide) showed roughly equivalent degrees of inhibition of LASV and EBOV glycoprotein (GP)-bearing pseudoviruses; three (clomiphene, sertraline, and toremifene) were more potent against EBOV. We then focused on arbidol, which is licensed abroad as an anti-influenza drug and exhibits activity against a diverse array of clinically relevant viruses. We found that arbidol inhibits infection by authentic LASV, inhibits LASV GP-mediated cell-cell fusion and virus-cell fusion, and, reminiscent of its activity on influenza virus hemagglutinin, stabilizes LASV GP to low-pH exposure. Our findings suggest that arbidol inhibits LASV fusion, which may partly involve blocking conformational changes in LASV GP. We discuss our findings in terms of the potential to develop a drug cocktail that could inhibit both LASV and EBOV.IMPORTANCE Lassa and Ebola viruses continue to cause severe outbreaks in humans, yet there are only limited therapeutic options to treat the deadly hemorrhagic fever diseases they cause. Because of overlapping geographic occurrences and similarities in mode of entry into cells, we seek a practical drug or drug cocktail that could be used to treat infections by both viruses. Toward this goal, we directly compared eight drugs, approved or in clinical testing, for the ability to block entry mediated by the glycoproteins of both viruses. We identified five drugs with approximately equal potencies against both. Among these, we investigated the modes of action of arbidol, a drug licensed abroad to treat influenza infections. We found, as shown for influenza virus, that arbidol blocks fusion mediated by the Lassa virus glycoprotein. Our findings encourage the development of a combination of approved drugs to treat both Lassa and Ebola virus diseases.

Fatal Case of Lassa Fever, Bangolo District, Côte d'Ivoire, 2015.

Lassa fever has not been reported in Côte d'Ivoire. We performed a retrospective analysis of human serum samples collected in Côte d'Ivoire in the dry seasons (January-April) during 2015-2018. We identified a fatal human case of Lassa fever in the Bangolo District of western Côte d'Ivoire during 2015.

Structures of arenaviral nucleoproteins with triphosphate dsRNA reveal a unique mechanism of immune suppression.

A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3'-5' exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5' triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

Development of a new drug candidate for the inhibition of Lassa virus glycoprotein and nucleoprotein by modification of evodiamine as promising therapeutic agents.

The Lassa virus (LASV), an RNA virus prevalent in West and Central Africa, causes severe hemorrhagic fever with a high fatality rate. However, no FDA-approved treatments or vaccines exist. Two crucial proteins, LASV glycoprotein and nucleoprotein, play vital roles in pathogenesis and are potential therapeutic targets. As effective treatments for many emerging infections remain elusive, cutting-edge drug development approaches are essential, such as identifying molecular targets, screening lead molecules, and repurposing existing drugs. Bioinformatics and computational biology expedite drug discovery pipelines, using data science to identify targets, predict structures, and model interactions. These techniques also facilitate screening leads with optimal drug-like properties, reducing time, cost, and complexities associated with traditional drug development. Researchers have employed advanced computational drug design methods such as molecular docking, pharmacokinetics, drug-likeness, and molecular dynamics simulation to investigate evodiamine derivatives as potential LASV inhibitors. The results revealed remarkable binding affinities, with many outperforming standard compounds. Additionally, molecular active simulation data suggest stability when bound to target receptors. These promising findings indicate that evodiamine derivatives may offer superior pharmacokinetics and drug-likeness properties, serving as a valuable resource for professionals developing synthetic drugs to combat the Lassa virus.

Lassa Fever Virus Binds Matriglycan-A Polymer of Alternating Xylose and Glucuronate-On α-Dystroglycan.

Lassa fever virus (LASV) can cause life-threatening hemorrhagic fevers for which there are currently no vaccines or targeted treatments. The late Prof. Stefan Kunz, along with others, showed that the high-affinity host receptor for LASV, and other Old World and clade-C New World mammarenaviruses, is matriglycan-a linear repeating disaccharide of alternating xylose and glucuronic acid that is polymerized uniquely on α-dystroglycan by like-acetylglucosaminyltransferase-1 (LARGE1). Although α-dystroglycan is ubiquitously expressed, LASV preferentially infects vascular endothelia and professional phagocytic cells, which suggests that viral entry requires additional cell-specific factors. In this review, we highlight the work of Stefan Kunz detailing the molecular mechanism of LASV binding and discuss the requirements of receptors, such as tyrosine kinases, for internalization through apoptotic mimicry.

Detection of Antibody and Antigen for Lassa Virus Nucleoprotein in Monkeys from Southern Nigeria.

Lassa fever is a deadly viral haemorrhagic fever caused by Lassa Virus (LASV). Rodents, especially, Mystomys natalensis, are the known reservoirs of LASV and humans are the defined hosts. Monkeys share many illnesses with humans and experimental LASV infections in monkeys are fatal but natural LASV infection of monkeys has not been reported. Serum samples obtained between August 2015 and December 2017 from 62 monkeys belonging to six species in Southern Nigeria were tested for LASV as part of an ongoing surveillance of monkeys in the region for zoonotic pathogens. Commercially available Recombinant LASV (ReLASV) Pan-Lassa enzyme-linked immunosorbent assay (ELISA) test kits (Zalgen Labs, Germantown, MD, USA) were used to detect antibodies (IgG and IgM) and antigen specific for LASV nucleoprotein in the sera. Lassa-fever-specific IgG and IgM, and antigen specific for LASV nucleoprotein were detected in 5/62, 0/62, and 1/62 samples, respectively. The presence of LASV-specific antibodies in the sera suggests natural exposure to the virus, while the presence of LASV antigen may mean that monkeys are carriers of the virus. There is a need to broaden Lassa fever surveillance to include nonhuman primates (NHPs) for their probable role in the epidemiology of the disease.HIGHLIGHTS.• Rodents are the natural reservoirs of Lassa fever virus (LASV) and humans are the defined hosts.• Experimental LASV infections in non-human primates (NHP) are fatal but natural infection of NHP with the virus have not been reported.• We detected antigen and antibody specific for LASV in free-living Monkeys from southern Nigeria which implies that monkeys in the region are naturally exposed to LASV and are probable carriers of the virus.

Diagnostics for Lassa fever virus: a genetically diverse pathogen found in low-resource settings.

Lassa fever virus (LASV) causes acute viral haemorrhagic fever with symptoms similar to those seen with Ebola virus infections. LASV is endemic to West Africa and is transmitted through contact with excretions of infected Mastomys natalensis rodents and other rodent species. Due to a high fatality rate, lack of treatment options and difficulties with prevention and control, LASV is one of the high-priority pathogens included in the WHO R&D Blueprint. The WHO LASV vaccine strategy relies on availability of effective diagnostic tests. Current diagnostics for LASV include in-house and commercial (primarily research-only) laboratory-based serological and nucleic acid amplification tests. There are two commercially available (for research use only) rapid diagnostic tests (RDTs), and a number of multiplex panels for differential detection of LASV infection from other endemic diseases with similar symptoms have been evaluated. However, a number of diagnostic gaps remain. Lineage detection is a challenge due to the genomic diversity of LASV, as pan-lineage sensitivity for both molecular and immunological detection is necessary for surveillance and outbreak response. While pan-lineage ELISA and RDTs are commercially available (for research use only), validation and external quality assessment (EQA) is needed to confirm detection sensitivity for all known or relevant strains. Variable sensitivity of LASV PCR tests also highlights the need for improved validation and EQA. Given that LASV outbreaks typically occur in low-resource settings, more options for point-of-care testing would be valuable. These requirements should be taken into account in target product profiles for improved LASV diagnostics.

GILT restricts the cellular entry mediated by the envelope glycoproteins of SARS-CoV, Ebola virus and Lassa fever virus.

Interferons (IFNs) control viral infections by inducing expression of IFN-stimulated genes (ISGs) that restrict distinct steps of viral replication. We report herein that gamma-interferon-inducible lysosomal thiol reductase (GILT), a lysosome-associated ISG, restricts the infectious entry of selected enveloped RNA viruses. Specifically, we demonstrated that GILT was constitutively expressed in lung epithelial cells and fibroblasts and its expression could be further induced by type II interferon. While overexpression of GILT inhibited the entry mediated by envelope glycoproteins of SARS coronavirus (SARS-CoV), Ebola virus (EBOV) and Lassa fever virus (LASV), depletion of GILT enhanced the entry mediated by these viral envelope glycoproteins. Furthermore, mutations that impaired the thiol reductase activity or disrupted the N-linked glycosylation, a posttranslational modification essential for its lysosomal localization, largely compromised GILT restriction of viral entry. We also found that the induction of GILT expression reduced the level and activity of cathepsin L, which is required for the entry of these RNA viruses in lysosomes. Our data indicate that GILT is a novel antiviral ISG that specifically inhibits the entry of selected enveloped RNA viruses in lysosomes via disruption of cathepsin L metabolism and function and may play a role in immune control and pathogenesis of these viruses.

Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.

Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.

Construction of pseudovirus reference standard containing five kinds of hemorrhagic fever virus / 军事医学

Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.