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The name arenavirus comes from the Latin arenosus (sandy). Ribosomes are packaged within arenavirus virions and these are responsible for the “sandy” appearance of the particles. Arenavirus virions are enveloped with helical nucleocapsids and are pleomorphic, ranging in size from 100 to 130 nm in diameter. Structural proteins include a nucleocapsid protein (N), a matrix-like protein (Z), and two glycoproteins derived from a single precursor. Arenaviruses are segmented RNA viruses that replicate using an overall strategy common to negative-strand RNA viruses. However, both genome segments (large (L) and small (S)) are ambisense in their coding strategy. Rodents are natural hosts of many arenaviruses though some can infect humans through contact with infected rodents or inhalation of infectious urine or feces.

Objectives This article is one of a series on acute, severe diseases of humans caused by emerging viruses for which there are no or limited licensed medical countermeasures. We approached this summary on South American Hemorrhagic Fevers from a clinical perspective, focusing on pathogenesis, clinical features, and diagnostics, with an emphasis on therapies and vaccines that have demonstrated potential for use in an emergency situation through their evaluation in nonhuman primates (NHPs) and/or in humans. Methods A standardized literature review was conducted on the clinical, pathological, vaccine, and treatment factors for South American Hemorrhagic Fevers as a group and for each individual virus/disease. Results We identified two treatments and one vaccine platform that have demonstrated potential benefit for treating or preventing infection in humans and four other potential treatments currently under investigation. Conclusion We provide succinct summaries of these countermeasures to give the busy clinician a head start in reviewing the literature if faced with a patient with a South American Hemorrhagic Fever. We also provide links to other authoritative sources of information.

We report a case of Argentine hemorrhagic fever diagnosed in a woman in Belgium who traveled from a disease-endemic area. Patient management included supportive care and combination therapy with ribavirin and favipiravir. Of 137 potential contacts, including friends, relatives, and healthcare and laboratory workers, none showed development of clinical symptoms of this disease., info:eu-repo/semantics/published

Bone marrow stromal cell antigen-2 (BST-2), also known as tetherin, is an interferon-inducible membrane-associated protein. It effectively targets enveloped viruses at the release step of progeny viruses from host cells, thereby restricting the further spread of viral infection. Junin virus (JUNV) is a member of Arenaviridae, which causes Argentine haemorrhagic fever that is associated with a high rate of mortality. In this study, we examined the effect of human BST-2 on the replication and propagation of JUNV. The production of JUNV Z-mediated virus-like particles (VLPs) was significantly inhibited by over-expression of BST-2. Electron microscopy analysis revealed that BST-2 functions by forming a physical link that directly retains VLPs on the cell surface. Infection using JUNV showed that infectious JUNV production was moderately inhibited by endogenous or exogenous BST-2. We also observed that JUNV infection triggers an intense interferon response, causing an upregulation of BST-2, in infected cells. However, the expression of cell surface BST-2 was reduced upon infection. Furthermore, the expression of JUNV nucleoprotein (NP) partially recovered VLP production from BST-2 restriction, suggesting that the NP functions as an antagonist against antiviral effect of BST-2. We further showed that JUNV NP also rescued the production of Ebola virus VP40-mediated VLP from BST-2 restriction as a broad spectrum BST-2 antagonist. To our knowledge, this is the first report showing that an arenavirus protein counteracts the antiviral function of BST-2., Journal of general virology, 101(6), pp.573-586; 2020

Despite tremendous advances in the development of anti-viral therapeutics, viral infections remain a chief culprit accounting for ongoing morbidity and mortality worldwide. Natural products, in particular animal venoms, embody a veritable cornucopia of exotic constituents, suggesting an immensurable source of anti-infective drugs. In this context, melittin, the principal constituent in the venom of the European honeybee Apis mellifera, has been demonstrated to exert anti-cancer, anti-inflammatory, anti-diabetic, anti-infective, and adjuvant properties. To our knowledge, there is no review appertaining to effects of melittin against viruses, prompting us to synopsize experimental investigations on its anti-viral activity throughout the past decades. Accumulating evidence indicates that melittin curbs infectivity of a diverse array of viruses including coxsackievirus, enterovirus, influenza A viruses, human immunodeficiency virus (HIV), herpes simplex virus (HSV), Junín virus (JV), respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), and tobacco mosaic virus (TMV). However, medication safety, different routes of administrations, and molecular mechanisms behind the anti-viral activity of melittin should be scrutinized in future studies.

Junin virus (JUNV) is a pathogen of biodefense importance due to its potential for aerosol transmission and mortality rates reaching 30%. Currently, there are no JUNV vaccines licensed by the United States Food and Drug Administration (FDA) for at-risk individuals. A vaccine based on recombinant vesicular stomatitis virus (rVSV) has been effectively used to prevent Ebola virus disease in humans. Here, we evaluated the protective efficacy of a rVSV expressing the JUNV glycoprotein (rVSVΔG-JUNVGP) in a guinea pig model of lethal JUNV disease. Two groups of guinea pigs, one prime and one prime-boost, were vaccinated with rVSVΔG-JUNVGP; six control animals remained unvaccinated. Survival for prime and prime-boost vaccinated animals was 100% while the challenge virus was uniformly lethal in all control animals. Animals in both vaccine groups developed robust, high avidity IgG antibody titers post-vaccination as well as detectable neutralizing antibodies while control animals failed to develop detectable antibody responses. This study demonstrates for the first time that rVSV expressing the JUNV GP fully protects guinea pigs from lethal JUNV challenge with a single injection vaccine.

Ebola virus infection causes severe hemorrhagic fever, and its mortality rates varied from 25 to 90% in the previous outbreaks. The highly infectious and lethal nature of this virus highlights the need for reliable and sensitive diagnostic methods to distinguish it from other diseases present with similar clinical symptoms. Based on multiplex polymerase chain reaction (PCR) and oligonucleotide microarray technology, a cost-effective, multipathogen and high-throughput method was developed for simultaneous detection of Ebola virus and other pathogens associated with hemorrhagic fever, including Marburg virus, Lassa fever virus, Junin virus, Machupo virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, malaria parasite, hantavirus, severe fever with thrombocytopenia syndrome virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus, and influenza B virus. This assay had an excellent specificity for target pathogens, without overlap signal between the probes. The limit of detection was approximately 103 pathogen copies/μl. A total of 60 positive nucleic acid samples for different pathogens were detected, a concordance of 100% was observed between microarray assay and real-time PCR analysis. Consequently, the described oligonucleotide microarray may be specific and sensitive assay for diagnosis and surveillance of infections caused by Ebola virus and other species of hemorrhagic fever pathogens.

Live-attenuated virus vaccines are highly effective in preventing viral disease but carry intrinsic risks of residual virulence and reversion to pathogenicity. The classically derived Candid#1 virus protects seasonal field workers in Argentina against zoonotic infection by Junin virus (JUNV) but is not approved in the United States, in part due to the potential for reversion at the attenuating locus, a phenylalanine-to-isoleucine substitution at position 427 in the GP2 subunit of the GPC envelope glycoprotein. Previously, we demonstrated facile reversion of recombinant Candid#1 (rCan) in cell culture and identified an epistatic interaction between the attenuating I427 and a secondary K33S mutation in the stable signal peptide (SSP) subunit of GPC that imposes an evolutionary barrier to reversion. The magnitude of this genetic barrier is manifest in our repeated failures to rescue the hypothetical revertant virus. In this study, we show that K33S rCan is safe and attenuated in guinea pigs and capable of eliciting potent virus-neutralizing antibodies. Immunized animals are fully protected against lethal challenge with virulent JUNV. In addition, we employed a more permissive model of infection in neonatal mice to investigate genetic reversion. RNA sequence analysis of the recovered virus identified revertant viruses in pups inoculated with the parental rCan virus and none in mice receiving K33S rCan (P

Arenaviral infections often result lethal hemorrhagic fevers, affecting primarily in African and South American regions. To date, there is no FDA-approved licensed vaccine against arenaviruses and treatments have been limited to supportive therapy. Hence, the study was employed to design a highly immunogenic cross-reactive vaccine against Arenaviridae family using reverse vaccinology approach. The whole proteome of Lassa virus (LASV), Lymphocytic Choriomeningitis virus (LCMV), Lujo virus and Guanarito virus were retrieved and assessed to determine the most antigenic viral proteins. Both T-cell and B-cell epitopes were predicted and screened based on transmembrane topology, antigenicity, allergenicity, toxicity and molecular docking analysis. The final constructs were designed using different adjuvants, top epitopes, PADRE sequence and respective linkers and were assessed for the efficacy, safety, stability and molecular cloning purposes. The proposed epitopes were highly conserved (84%–100%) and showed greater cumulative population coverage. Moreover, T cell epitope GWPYIGSRS was conserved in Junin virus (Argentine mammarenavirus) and Sabia virus (Brazilian mammarenavirus), while B cell epitope NLLYKICLSG was conserved in Machupo virus (Bolivian mammarenavirus) and Sabia virus, indicating the possibility of final vaccine construct to confer a broad range immunity in the host. Docking analysis of the refined vaccine with different MHC molecules and human immune receptors were biologically significant. The vaccine-receptor (V1-TLR3) complex showed minimal deformability at molecular level and was compatible for cloning into pET28a(+) vector of E. coli strain K12. The study could be helpful in developing vaccine to combat arenaviral infections in the future. However, further in vitro and in vivo trials using model animals are highly recommended for the experimental validation of our findings., Arenavirus; Hemorrhagic fevers; Nucleoprotein; Recombinant vaccine; Molecular docking

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins–a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion–the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes., Author summary Pathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa GPC-mediated fusion was augmented by the human LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

Correction: Volume65, Issue6 Article Numbere00653- 21 DOI10.1128/AAC.00653-21 Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus (JUNV], lymphocytic choriomeningitis virus (LCMV), and Pichinde virus (PICA). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.

Junin virus (JUNV) is a New World arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammarenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV)., Author summary The continual development of safe, effective vaccines against emerging diseases is one of the greatest challenges facing the scientific community. The New World group of mammarenaviruses contains multiple human pathogens, each capable of causing severe hemorrhagic disease. Among these, only Junin virus has a distributed vaccine. By utilizing this vaccine, we are able to determine vaccine development strategies for related New World viruses that represent an emerging threat. Here we demonstrate that manipulation of the viral Z protein is able to produce an incompatibility that ultimately attenuates the virus. This provides yet another tool for future vaccine development to further global public health.

KEY FINDINGS There is currently no evidence to support the use of virgin coconut oil in the adjunctive treatment of COVID-19. • Virgin coconut oil is naturally extracted from fresh coconut kernel and is rich in medium chain triglycerides, with lauric acid as the predominant fatty acid.• Virgin coconut oil is currently explored as an adjunctive treatment for patients with COVID-19 due to its antiviral and immunomodulatory properties.• In vitro studies show that lauric acid or its derivative exert inhibitory activities against viruses with similar structure to coronavirus (enveloped ssRNA virus) such as Junin virus, vesicular stomatitis virus, human immunodeficiency virus type 1 (HIV-1), and Semliki Forest virus.• Animal studies demonstrate antiviral activity of monolaurin, the pharmacologically active metabolite of lauric acid, on avian influenza virus and Simean immunodeficiency virus, which are both enveloped ssRNA viruses.• Clinical trials among patients with HIV report that virgin coconut oil can increase CD4+ T lymphocyte counts and reduce viral load.• In vitro and animal studies demonstrate anti-inflammatory properties of virgin coconut oil.• At present, there are no studies that investigate the effectiveness of virgin coconut oil in the adjunctive treatment of COVID-19 infection.• There is currently one ongoing clinical trial in the Philippines evaluating the use of virgin coconut oil in the adjunctive treatment of COVID-19.• Nausea, vomiting, mild diarrhea, and abdominal pain have been reported, but no serious adverse events have been identified with the use of virgin coconut oil.• To date, there are no guidelines that mention virgin coconut oil as an option for the adjunctive treatment of COVID-19.

The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes, and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease, but it has been shown to protect against Argentine hemorrhagic fever-like disease in marmosets infected with the New World mammarenavirus Junín virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant TCRV (rTCRV). We successfully rescued a wild-type (WT) rTCRV (a trisegmented form of TCRV expressing two reporter genes [r3TCRV]) and a bisegmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39-nucleotide (nt) deletion (Δ39) in the noncoding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, an rTCRV containing this 39-nt deletion in the L-IGR (rTCRV/Δ39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or vaccine vectors. IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide protection against Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially other viral infections. Here, we describe for the first time the feasibility of generating recombinant TCRV (rTCRV) using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

Several arenaviruses are highly pathogenic to humans, causing hemorrhagic fever. Discovery of anti-arenavirus drug candidates is urgently needed, although the molecular basis of the host- and organ-specific pathogenicity remains to be fully elucidated. The arenavirus Z protein facilitates production of virus-like particles (VLPs), providing an established method to assess virus budding. In this study, we examined the efficiency of VLP production by solely expressing Z protein of several different arenaviruses. In addition, we analyzed the role of the late (L)-domain of the arenavirus Z protein, which is essential for the interaction with ESCRT proteins, in VLP production among different cell lines. VLP assay was performed using Z proteins of Junin virus (JUNV), Machupo virus (MACV), Tacaribe virus (TCRV), Latino virus (LATV), Pichinde virus (PICV), and Lassa virus (LASV) in six different cell lines: HEK293T, Huh-7, A549, Vero76, BHK-21, and NIH3T3 cells. JUNV, MACV, and LASV Z proteins efficiently produced VLPs in all tested cell lines, while the efficiencies of VLP production by the other arenavirus Z proteins were cell type-dependent. The contribution of the L-domain(s) within Z protein to VLP production also highly depended on the cell type. These results suggested that each arenavirus has its own particle-production mechanism, which is different among the cell types., Frontiers in Microbiology, 11, art.no.562814; 2020

Argentine hemorrhagic fever (AHF) is a serious endemic disease in Argentina, produced by Junín virus, whose host is the Sigmodontinae rodent Calomys musculinus. Within the endemic area, human incidence and proportion of infected rodents remains high for 5-10 years after the first appearance of the disease (epidemic [E] zone) and then gradually declines to sporadic cases (historic [H] zone). We tested the hypothesis that host populations within the E zone are large and well connected by gene flow, facilitating the transmission and maintenance of the virus, whereas those in the H and nonendemic (NE) zones are small and isolated, with the opposite effect. We estimated parameters affected by levels of gene flow and population size in 14 populations of C. musculinus: population effective size (Ne), genetic variability, and mean relatedness. Our hypothesis was not supported: the lowest levels of variability and of Ne and the highest genetic relatedness among individuals were found in the H zone. Populations from the NE zone displayed opposite results, whereas those in the E zone showed intermediate values. If we consider that populations are first NE, then E, and finally H, a correlative decrease in Ne was observed. Chronically infected females have a low reproductive success. We propose that this would lower Ne because each cohort would originate from a fraction of females of the previous generation, and affect other factors such as proportion of individuals that develop acute infection, probability of viral transmission, and evolution of virulence, which would explain, at least partly, the changing incidence of AHF. Fil: Chiappero, Marina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Piacenza, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Ciencias Naturales; Argentina Fil: Provensal, María Cecilia. Universidad Nacional de Río Cuarto; Argentina Fil: Calderón, Gladys Ethel. Instituto Nacional de Enfermedades Virales Humanas Dr. Julio I. Maiztegui; Argentina Fil: Gardenal, Cristina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Polop, Jaime Jose. Universidad Nacional de Río Cuarto; Argentina

Presently, the Arenavirus genus (Arenaviridae family) includes 26 individual species of viruses. It is divided into two main groups – Old World arenaviruses and New World arenaviruses. The New World arenaviruses comprise four clades: А, B, C, D; pathogenic for humans New World arenaviruses are attributed to clade B. Lassa, Lujo, Machupo, Junin, Guanarito and Sabia viruses are the agents of extremely hazardous hemorrhagic fevers (Lassa hemorrhagic fever, Lujo hemorrhagic fever, Bolivian hemorrhagic fever, Argentinean hemorrhagic fever, Venezuelan hemorrhagic fever, Brazilian hemorrhagic fever, accordingly). These arenaviruses pose a potential threat to national public health due to the possibility of their accidental importation into the territory of the Russian Federation. The vaccination of risk group is the most effective and money-saving means of protection against epidemic spread. Objective of this review is to analyze the specific preparations for arenaviral hemorrhagic fever prevention that are currently under development. Production of live vaccines based on attenuated strains of the agents, the DNA vaccines, vector recombinant vaccines and vaccines on the basis of RNA-replicons is viewed as the main trends in the area. Тhe most important results in the development of effective prophylactic means against arenaviral hemorrhagic fevers are discussed in this paper.

The aim of this study was to investigate the effect of A771726, the active metabolite of leflunomide, (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad against the infection with Junín virus (JUNV), agent of Argentine hemorrhagic fever (AHF). The treatment with non-cytotoxic concentrations of A771726 of Vero and A549 cells infected with JUNV inhibited virus replication in a dose-dependent manner, as determined by virus yield reduction assay. The antiviral effectiveness of A771726 was not importantly affected by the multiplicity of infection and the virus strain. Moreover, the combination of A771726 and ribavirin had a significantly more potent antiviral activity than each single drug treatment. Mechanistic studies showed that the main action of A771726 is exerted before 6 h of JUNV infection. Accordingly, inhibition of viral RNA synthesis was detected in treated infected cells by real time RT-PCR. The exogenous addition of uridine or orotic acid produced a partial reversal of the inhibitory effect of A771726 on infective virus production whereas a total reversion was detected on JUNV RNA synthesis, probably by restoration of the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and the intracellular pyrimidine pools. In conclusion, these results suggest that the antiviral target would be viral RNA synthesis through pyrimidine depletion, but any other effect of the compound on JUNV infection cannot be excluded. This study opens the possibility of the therapeutic application of a wide spectrum host-targeted compound alone or in combination with ribavirin to combat AHF as well as other human pathogenic arenaviruses. Fil: Sepúlveda, Claudia Soledad. Universidad de Buenos Aires; Argentina Fil: Garcia, Cybele. Universidad de Buenos Aires; Argentina Fil: Damonte, Elsa Beatriz. Universidad de Buenos Aires; Argentina

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9μM) and MPA (0.8-2.5μM). 6-Azauridine toxicity was 6.1-17.5μM with NR, VTG, and WST-1, compared to 48-92μM with ALB-A and ALB-F (P

Several clade B New World arenaviruses (NWAs) can cause severe and often fatal hemorrhagic fever, for which preventive and therapeutic measures are severely limited. These NWAs use human transferrin receptor 1 (hTfR1) as a host cell receptor for virus entry. The most prevalent of the pathogenic NWAs is Junín virus (JUNV), the etiological agent of Argentine hemorrhagic fever. Small animal models of JUNV infection are limited because most laboratory rodent species are refractory to disease. Only guinea pigs are known to develop disease following JUNV infection, but the underlying mechanisms are not well characterized. In the present study, we demonstrate marked susceptibility of Hartley guinea pigs to uniformly lethal disease when challenged with as few as 4 plaque-forming units of the Romero strain of JUNV. In vitro, we show that infection of primary guinea pig macrophages results in greater JUNV replication compared to infection of hamster or mouse macrophages. We provide evidence that the guinea pig TfR1 (gpTfR1) is the principal receptor for JUNV, while hamster and mouse orthologs fail to support viral entry/infection of pseudotyped murine leukemia viruses expressing pathogenic NWA glycoproteins or JUNV. Together, our results indicate that gpTfR1 serves as the primary receptor for pathogenic NWAs, enhancing viral infection in guinea pigs. IMPORTANCE JUNV is one of five known NWAs that cause viral hemorrhagic fever in humans. Countermeasures against JUNV infection are limited to immunization with the Candid#1 vaccine and immune plasma, which are available only in Argentina. The gold standard small animal model for JUNV infection is the guinea pig. Here, we demonstrate high sensitivity of this species to severe JUNV infection and identify gpTfR1 as the primary receptor. Use of hTfR1 for host cell entry is a feature shared by pathogenic NWAs. Our results show that expression of gpTfR1 or hTfR1 comparably enhances JUNV virus entry/infectivity. Our findings shed light on JUNV infection in guinea pigs as a model for human disease and suggest that similar pathophysiological mechanisms related to iron sequestration during infection and regulation of TfR1 expression may be shared between humans and guinea pigs. A better understanding of the underlying disease process will guide development of new therapeutic interventions.

Arenaviral infections often resulting in lethal Hemorrhagic Fevers (HF) affect primarily African and South American regions. To date, there is no FDA-approved licensed vaccine against arenaviruses and treatments have been limited to supportive therapies. Hence, the study was employed to design a highly immunogenic heterosubtypic vaccine candidate against Arenaviridae family. The whole proteomes of Lassa virus (LASV), Lymphocytic Chorio Meningitis Virus (LCMV), Lujo virus and Guanarito virus were retrieved from NCBI database and assessed to determine the most antigenic viral proteins. Only the conserved sequences were used for T cell and B cell epitope prediction to ensure protective response against a wide range of viral strains. For each virus, nucleoproteins were identified as most antigenic which generated a plethora of antigenic epitopes. The proposed epitopes were highly conserved (up to 100%) and showed high cumulative population coverage. Moreover, results revealed that among the top epitopes, T cell epitope GWPYIGSRS were conserved in Argentine mammarenavirus (Junin virus) and Brazilian mammarenavirus (Sabia virus), while B cell epitope NLLYKICLSG were conserved in Bolivian mammarenavirus (Machupo virus) and Brazilian mammarenavirus (Sabia virus), indicating the possibility of final vaccine constructs to confer broad range immunity in the host. A total 3 constructs were designed by the combination of top epitopes from each protein along with suitable adjuvant and linkers. Different physicochemical properties revealed the superiority of construct V1 in terms of safety and efficacy. Docking analysis of the refined vaccine structure with different MHC molecules and human immune receptors were also biologically significant. The vaccine receptor complex (V1-TLR3) showed minimal deformability at molecular level. Moreover, construct V1 was compatible for insertion into pET28a(+) vector and heterologous cloning in E. coli srain K12. However, the results were based on different sequence analysis and various immune databases. Further wet lab based studies using model animals are highly recommended for the experimental validation of the designed vaccine candidates.

The arenaviruses Lassa virus (LASV), Junín virus (JUNV), and Machupo virus (MACV) can cause severe and fatal diseases in humans. Although these pathogens are closely related, the host immune responses to these virus infections differ remarkably, with direct implications for viral pathogenesis. LASV infection is immunosuppressive, with a very low-level interferon response. In contrast, JUNV and MACV infections stimulate a robust interferon (IFN) response in a retinoic acid-inducible gene I (RIG-I)-dependent manner and readily activate protein kinase R (PKR), a known host double-stranded RNA (dsRNA) sensor. In response to infection with RNA viruses, host nonself RNA sensors recognize virus-derived dsRNA as danger signals and initiate innate immune responses. Arenavirus nucleoproteins (NPs) contain a highly conserved exoribonuclease (ExoN) motif, through which LASV NP has been shown to degrade virus-derived immunostimulatory dsRNA in biochemical assays. In this study, we for the first time present evidence that LASV restricts dsRNA accumulation during infection. Although JUNV and MACV NPs also have the ExoN motif, dsRNA readily accumulated in infected cells and often colocalized with dsRNA sensors. Moreover, LASV coinfection diminished the accumulation of dsRNA and the IFN response in JUNV-infected cells. The disruption of LASV NP ExoN with a mutation led to dsRNA accumulation and impaired LASV replication in minigenome systems. Importantly, both LASV NP and RNA polymerase L protein were required to diminish the accumulation of dsRNA and the IFN response in JUNV infection. For the first time, we discovered a collaboration between LASV NP ExoN and L protein in limiting dsRNA accumulation. Our new findings provide mechanistic insights into the differential host innate immune responses to highly pathogenic arenavirus infections. IMPORTANCE Arenavirus NPs contain a highly conserved DEDDh ExoN motif, through which LASV NP degrades virus-derived, immunostimulatory dsRNA in biochemical assays to eliminate the danger signal and inhibit the innate immune response. Nevertheless, the function of NP ExoN in arenavirus infection remains to be defined. In this study, we discovered that LASV potently restricts dsRNA accumulation during infection and minigenome replication. In contrast, although the NPs of JUNV and MACV also harbor the ExoN motif, dsRNA readily formed during JUNV and MACV infections, accompanied by IFN and PKR responses. Interestingly, LASV NP alone was not sufficient to limit dsRNA accumulation. Instead, both LASV NP and L protein were required to restrict immunostimulatory dsRNA accumulation. Our findings provide novel and important insights into the mechanism for the distinct innate immune response to these highly pathogenic arenaviruses and open new directions for future studies.

Argentine hemorrhagic fever is a potentially lethal disease that is caused by Junin virus (JUNV). There are currently around 5 million individuals at risk of infection within regions of endemicity in Argentina. The live attenuated vaccine strain Candid #1 (Can) is approved for use in regions of endemicity and has substantially decreased the number of annual Argentine hemorrhagic fever (AHF) cases. The glycoprotein (GPC) gene is primarily responsible for attenuation of the Can strain, and we have shown that the absence of an N-linked glycosylation motif in the subunit G1 of the glycoprotein complex of Can, which is otherwise present in the wild-type pathogenic JUNV, causes GPC retention in the endoplasmic reticulum (ER). Here, we show that Can GPC aggregates in the ER of infected cells, forming incorrect cross-chain disulfide bonds, which results in impaired GPC processing into G1 and G2. The GPC fails to cleave into its G1 and G2 subunits and is targeted for degradation within lysosomes. Cells infected with the wild-type Romero (Rom) strain do not produce aggregates that are observed in Can infection, and the stress on the ER remains minimal. While the mutation of the N-linked glycosylation motif (T168A) is primarily responsible for the formation of aggregates, other mutations within G1 that occurred earlier in the passage history of the Can strain also contribute to aggregation of the GPC within the ER. IMPORTANCE The development of vaccines and therapeutics to combat viral hemorrhagic fevers remains a top priority within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. The Can strain, derived from the pathogenic XJ strain of JUNV, has been demonstrated to be both safe and protective against AHF. While the vaccine strain is approved for use in regions of endemicity within Argentina, the mechanisms of Can attenuation have not been elucidated. A better understanding of the viral genetic determinants of attenuation will improve our understanding of the mechanisms contributing to disease pathogenesis and provide critical information for the rational design of live attenuated vaccine candidates for other viral hemorrhagic fevers.

The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF). Previous studies of human macrophage infection by the Old-World arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus replicates and activates human macrophages, the pathogenic Lassa virus replicates but fails to activate human macrophages. Less is known in regard to the impact of New World arenavirus infection on the human macrophage immune response. Macrophage activation is critical for controlling infections but could also be usurped favoring immune evasion. Therefore, it is crucial to understand how the JUNV infection modulates macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the JUNV virus in human macrophage cultures. The results showed that both JUNV strains similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively regulates the immune response, was down-regulated by C#1 strain and up-regulated by P strain infection. Similarly, the target genes of MERTK activation, the cytokine suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P strain infection. Together, this differential activation/polarization pattern of macrophages elicited by P strain suggests a more evasive immune response and may have important implications in the pathogenesis of AHF and underpinning the development of new potential therapeutic strategies, Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular, Centro de Investigación y Desarrollo en Fermentaciones Industriales

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

Junín virus (JUNV), a member of the family Arenaviridae, is the etiological agent of Argentine hemorrhagic fever (AHF), a potentially deadly endemic-epidemic disease affecting the population of the most fertile farming land of Argentina. Autophagy is a degradative process with a crucial antiviral role; however, several viruses subvert the pathway to their benefit. We determined the role of autophagy in JUNV-infected cells by analyzing LC3, a cytoplasmic protein (LC3-I) that becomes vesicle membrane associated (LC3-II) upon induction of autophagy. Cells overexpressing enhanced green fluorescent protein (EGFP)-LC3 and infected with JUNV showed an increased number of LC3 punctate structures, similar to those obtained after starvation or bafilomycin A1 treatment, which leads to autophagosome induction or accumulation, respectively. We also monitored the conversion of LC3-I to LC3-II, observing LC3-II levels in JUNV-infected cells similar to those observed in starved cells. Additionally, we kinetically studied the number of LC3 dots after JUNV infection and found that the virus activated the pathway as early as 2 h postinfection (p.i.), whereas the UV-inactivated virus did not induce the pathway. Cells subjected to starvation or pretreated with rapamycin, a pharmacological autophagy inductor, enhanced virus yield. Also, we assayed the replication capacity of JUNV in Atg5 knockout or Beclin 1 knockdown cells (both critical components of the autophagic pathway) and found a significant decrease in JUNV replication. Taken together, our results constitute the first study indicating that JUNV infection induces an autophagic response, which is functionally required by the virus for efficient propagation. IMPORTANCE Mammalian arenaviruses are zoonotic viruses that cause asymptomatic and persistent infections in their rodent hosts but may produce severe and lethal hemorrhagic fevers in humans. Currently, there are neither effective therapeutic options nor effective vaccines for viral hemorrhagic fevers caused by human-pathogenic arenaviruses, except the vaccine Candid no. 1 against Argentine hemorrhagic fever (AHF), licensed for human use in areas of endemicity in Argentina. Since arenaviruses remain a severe threat to global public health, more in-depth knowledge of their replication mechanisms would improve our ability to fight these viruses. Autophagy is a lysosomal degradative pathway involved in maintaining cellular homeostasis, representing powerful anti-infective machinery. We show, for the first time for a member of the family Arenaviridae, a proviral role of autophagy in JUNV infection, providing new knowledge in the field of host-virus interaction. Therefore, modulation of virus-induced autophagy could be used as a strategy to block arenavirus infections. Fil: Roldan, Julieta Suyay. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Candurra, Nélida Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina Fil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Delgui, Laura Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

Arenavirus is a genetic term for viruses belonging to the family Arenaviridae and is presented from lymphocytic choriomeningitis virus (LCMV), which shows almost no pathogenicity to humans, to Lassa virus, Junin virus, Machupo virus, Chapare virus, Lujo virus, Sabia virus, and Guanarito virus, which shows high pathogenicity to humans. These viruses except for LCMV are risk group 4 pathogens specified by World Health Organization. Based on this designation, it is designated as Class I pathogens in Japan. Although there have been no reports excluding one imported case of the Lassa fever patient, it is not surprising whenever imported cases occur in our country. Considering the disease severity and mortality rate, it is an urgent matter to develop vaccines and therapeutic drugs in endemic areas, and maintenances of these are also important in countries other than endemic areas. However, basic research on highly pathogenic arenavirus infections and development of therapeutic drugs are not easily progressed, because handling in highly safe research facilities is indispensable. In this article, we will outline the current knowledge from the recent basic research on arenavirus to the development situation of antivirals against arenaviruses.

Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.

Objective There is no specific antiviral treatment available for coronavirus disease 2019 (COVID-19). Among the possible natural constituents is carrageenan, a polymer derived from marine algae that possesses a variety of antiviral properties. The purpose of this review was to summarize the evidence supporting carrageenan subtypes' antiviral activity against the emerging severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19. Methods PubMed/MEDLINE and Google Scholar searches were conducted for publications using the terms ‘carrageenan', ‘iota carrageenan', ‘kappa carrageenan', lambda-carrageenan', 'coronavirus', ‘common cold', 'rhinovirus', and ‘SARS-CoV-2′ search was also done in grey literature to increase our understanding. A search for the word "carrageenan" was also carried out. Most of the publications were discussed in narrative. Results Carrageenan has been shown to have potent antiviral activity against both coronaviruses (coronavirus NL63, SARS-CoV-2) and non-coronaviruses such as dengue virus, herpes simplex virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, sindbis virus, human immunodeficiency virus, influenza virus, human papillomavirus, rabies virus, junin virus, tacaribe virus, African swine fever, bovine herpes virus, suid herpes virus, and rhinovirus. No in vivo study has been conducted using carrageenan as an anti-SARS-CoV-2 agent. The majority of the in vivo research was done on influenza, a respiratory virus that causes common cold together with coronavirus. Thus, various clinical trials were conducted to determine the transferability of these in vitro data to clinical effectiveness against SARS-CoV-2. When combined with oral ivermectin, nasally administered iota-carrageenan improved outcome in COVID-19 patients. It is still being tested in clinics for single-dose administration. Conclusion Though the carrageenan exhibited potent antiviral activity against SARS-CoV-2 and was used to treat COVID-19 under emergency protocol in conjunction with oral medications such as ivermectin, there is no solid evidence from clinical trials to support its efficacy. Thus, clinical trials are required to assess its efficacy for COVID-19 treatment prior to broad application.

Mammarenaviruses, including lethal pathogens such as Lassa virus and Junín virus, can cause severe hemorrhagic fever in humans. Entry is a key step for virus infection, which starts with binding of the envelope glycoprotein (GP) to receptors on target cells and subsequent fusion of the virus with target cell membranes. The GP precursor is synthesized as a polypeptide, and maturation occurs by two cleavage events, yielding a tripartite GP complex (GPC) formed by a stable signal peptide (SSP), GP1 and GP2. The unique retained SSP interacts with GP2 and plays essential roles in virion maturation and infectivity. GP1 is responsible for binding to the cell receptor, and GP2 is a class I fusion protein. The native structure of the tripartite GPC is unknown. GPC is critical for the receptor binding, membrane fusion and neutralization antibody recognition. Elucidating the molecular mechanisms underlining the structure–function relationship of the three subunits is the key for understanding their function and can facilitate novel avenues for combating virus infections. This review summarizes the basic aspects and recent research of the structure–function relationship of the three subunits. We discuss the structural basis of the receptor-binding domain in GP1, the interaction between SSP and GP2 and its role in virion maturation and membrane fusion, as well as the mechanism by which glycosylation stabilizes the GPC structure and facilitates immune evasion. Understanding the molecular mechanisms involved in these aspects will contribute to the development of novel vaccines and treatment strategies against mammarenaviruses infection. [Image: see text]

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

Herein we describe the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles from carbohydrates with D-ribo and D-xylo configuration. The antiviral activity of these compounds was tested against Junin virus (the etiological agent of Argentine hemorrhagic fever). The p-chlorophenyl derivatives showed antiviral activity in a range of micromolar concentration.

The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to clade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cell’s innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2α. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR. IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV.

There are two predominant subgroups in the Arenaviridae family of viruses, the Old World and the New World viruses, that use distinct cellular receptors for entry. While New World viruses typically elicit good neutralizing antibody responses, the Old World viruses generally evade such responses. Antibody-based immune responses are directed against the glycoprotein spike complexes that decorate the viruses. A thick coat of glycans reduces the accessibility of antibodies to the surface of spike complexes from Old World viruses, but other mechanisms may further hamper the development of efficient humoral responses. Specifically, it was suggested that the GP1 receptor-binding module of the Old World Lassa virus might help with evasion of the humoral response. Here we investigated the immunogenicity of the GP1 domain from Lassa virus and compared it to that of the GP1 domain from the New World Junín virus. We found striking differences in the ability of antibodies that were developed against these immunogens to target the same GP1 receptor-binding domains in the context of the native spike complexes. Whereas GP1 from Junín virus elicited productive neutralizing responses, GP1 from Lassa virus elicited only nonproductive responses. These differences can be rationalized by the conformational changes that GP1 from Lassa virus but not GP1 from Junín virus undergoes after dissociating from the trimeric spike complex. Hence, shedding of GP1 in the case of Lassa virus can indeed serve as a mechanism to subvert the humoral immune response. Moreover, the realization that a recombinant protein may be used to elicit a productive response against the New World Junín virus may suggest a novel and safe way to design future vaccines. IMPORTANCE Some viruses that belong to the Arenaviridae family, like Lassa and Junín viruses, are notorious human pathogens, which may lead to fatal outcomes when they infect people. It is thus important to develop means to combat these viruses. For developing effective vaccines, it is vital to understand the basic mechanisms that these viruses utilize in order to evade or overcome host immune responses. It was previously noted that the GP1 receptor-binding domain from Lassa virus is shed and accumulates in the serum of infected individuals. This raised the possibility that Lassa virus GP1 may function as an immunological decoy. Here we demonstrate that mice develop nonproductive immune responses against GP1 from Lassa virus, which is in contrast to the effective neutralizing responses that GP1 from Junín virus elicits. Thus, GP1 from Lassa virus is indeed an immunological decoy and GP1 from Junín virus may serve as a constituent of a future vaccine.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species. Fil: Leske, Anne. Friedrich Loeffler Institut; Alemania Fil: Waßmann, Irke. Friedrich Loeffler Institut; Alemania Fil: Schnepel, Kevin. Friedrich Loeffler Institut; Alemania Fil: Shifflett, Kyle. National Institutes of Health; Estados Unidos Fil: Holzerland, Julia. Friedrich Loeffler Institut; Alemania Fil: Bostedt, Linus. Friedrich Loeffler Institut; Alemania Fil: Bohn, Patrick. Friedrich Loeffler Institut; Alemania Fil: Mettenleiter, Thomas C.. Friedrich Loeffler Institut; Alemania Fil: Briggiler, Ana Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Brignone, Julia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Enria, Delia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina Fil: Cordo, Sandra Myriam. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hoenen, Thomas. Friedrich Loeffler Institut; Alemania. National Institutes of Health; Estados Unidos Fil: Groseth, Allison. Friedrich Loeffler Institut; Alemania. National Institutes of Health; Estados Unidos

Resumen La fiebre hemorrágica argentina (FHA) es una enfermedad zoonótica endémica en una amplia zona de la pampa húmeda de Argentina. El agente etiológico es el virus Junin que es mantenido en la naturaleza por el roedor Calomys musculinus y transmitido, principalmente, al humano a través de aerosoles generados de las secreciones y excreciones. Objetivos: Caracterizar la composición y diversidad de los ensambles de pequeños roedores, determinar la abundancia del hospedador C. musculinus y la prevalencia del virus de la FHA en las zonas epidémica, histórica y no endémica de dicha enfermedad en Argentina. Métodos: Para el muestreo de roedores en cada una de las zonas se demarcaron un área central y dos periféricas para 18 localidades de la región central de Argentina (incluyendo las provincias de Córdoba, Buenos Aires y Santa Fe) muestreadas en dos años. Se comparó la abundancia de C. musculinus entre zonas y entre las áreas dentro de cada zona y áreas cercanas entre zonas, utilizando modelos de análisis de varianza anidados. Resultados. Dentro de cada zona, el ensamble de roedores mostró diferencia espacial en la composición específica, diversidad y abundancia de C. musculinus. La zona epidémica registró mayor número de especies y mayor abundancia del hospedador. En zona histórica se capturó el menor número de especies (de roedores) y Akodon azarae fue la más abundante. En zona no endémica la composición del ensamble y la abundancia de C. musculinus variaron entre los dos años. Sólo se detectó infección por virus Junin en C. musculinus correspondientes a la zona epidémica con una prevalencia de 2,7 y 1,1% para los años 2007 y 2008, respectivamente. Conclusión: En este sistema, la abundancia del hospedador estaría afectando la dinámica espacial de este virus, más que la diversidad del ensamble o la presencia de A. azarae.

The family Arenaviridae is divided into three genera: Mammarenavirus, Reptarenavirus, and Hartmanivirus. The Mammarenaviruses contain viruses responsible for causing human hemorrhagic fever diseases including New World viruses Junin, Machupo, Guanarito, Sabia, and Chapare virus and Old World viruses Lassa, and Lujo virus. These two groups of arenaviruses share the same genome organization composed of two ambisense RNA segments. These segments contain four open reading frames that encode for four proteins: the nucleoprotein, glycoprotein precursor, L protein, and Z. Despite their genome similarities, these groups exhibit marked differences in their replication life cycles. This includes differences in attachment, entry, and immune evasion. By understanding the intricacy of replication in each of these viral species we can work to develop counter measures against human diseases. This includes the development of vaccines and antivirals for these emerging viral threats. Currently only the vaccine against Junin virus, Candid#1, is in use as well as Ribavirin for treatment of Lassa Fever. In addition, small molecule inhibitors can be developed to target various aspects of the virus life cycle. In these ways an understanding of the arenavirus replication cycle can be used to alleviate the mortality and morbidity of these infections worldwide.

Background: Thiazole is a good nucleus owing to its various pharmaceutical applications. Thiazole containing compounds (thiazoles) have shown various biological activities like antioxidant, analgesic, antibacterial, anticancer, antiallergic, antihypertensive, antiinflammatory, antimalarial, antifungal and antipsychotic. The scaffold is present in more than 18 FDA approved drugs and also in more than 70 experimental drugs. Only a few reviews are available in the literature despite its great medicinal importance. During the course of time, this scaffold has been studied extensively for its antiviral activities and provided compounds with activity in the nM range. However, no focused review is available on the compilation of antiviral activities shown by this scaffold. Objective: In the present review, we have made an effort to compile antiviral literature of thiazoles reported from the year 2011 to till date. Methods: We searched the SciFinder database (excluding patent literature) with keywords like “antiviral”, “anti-HIV” and “virus”. Further filters were applied for the year of publication and keywords thiazole, reviews etc. to find relevant literature reported on the antiviral activities of thiazoles. Results: Nearly, 50 research articles were selected to compile and review the antiviral literature of thiazoles reported from the year 2011 to till date. Compounds 8, 25, 40, 62, 72, 73, 91, 112, 113, 131, 137, 175, 198, 200, 201 and 213 were reported in the literature with potent antiviral activity against CVB, SARS, RSV, HCV, HRV, VZV, TMV, FMDV, DENV, YFV, influenza virus, Junin virus, HIV-1, HSV, VV and EBV, respectively. Conclusion: There is further scope for the synthesis and evaluation of novel thiazole compounds by taking the most active compounds as lead structures. In conclusion, this review provides an overview of antiviral activities of thiazole compounds reported from the year 2011 to till date.

An important step in the initiation of the innate immune response to virus infection is the recognition of non-self, viral RNA, including double-stranded RNA (dsRNA), by cytoplasmic pattern recognition receptors (PRRs). For many positive-sense RNA viruses and DNA viruses, the production of viral dsRNA, and the interaction of viral dsRNA and PRRs are well characterized. However, for negative-sense RNA viruses, viral dsRNA was thought to be produced at low to undetectable levels and PRR recognition of viral dsRNA is still largely unclear. In the case of arenaviruses, the nucleocaspid protein (NP) has been identified to contain an exoribonuclease activity that preferentially degrades dsRNA in biochemical studies. Nevertheless, pathogenic New World (NW) arenavirus infections readily induce an interferon (IFN) response in a RIG-I dependent manner, and also activate the dsRNA-dependent Protein Kinase R (PKR). To better understand the innate immune response to pathogenic arenavirus infection, we used a newly identified dsRNA-specific antibody that efficiently detects viral dsRNA in negative-sense RNA virus infected cells. dsRNA was detected in NW arenavirus infected cells colocalizing with virus NP in immunofluorescence assay. Importantly, the dsRNA signals also colocalized with cytoplasmic PRRs, namely, PKR, RIG-I and MDA-5, as well as with the phosphorylated, activated form of PKR in infected cells. Our data clearly demonstrate the PRR recognition of dsRNA and their activation in NW arenavirus infected cells. These findings provide new insights into the interaction between NW arenaviruses and the host innate immune response.

Several Old World and New World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antivirals, or specific treatments are available, except for Junín virus. However, protection of nonhuman primates against Lassa fever virus (LASV) is possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified by using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in NP of MOPV, we found that this activity was essential for multiplication in antigen-presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPV ExoN6b ) that is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to those of MOPV, and (iii) still promotes a strong type I interferon (IFN) response. MOPV ExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV or Lujo, Machupo, Guanarito, Chapare, or Sabia virus in order to broaden specific antigenicity while preserving the hyperattenuated characteristics of the parental strain. Our MOPV-based vaccine candidate for LASV, MOPEVAC LASV , was used in a one-shot immunization assay in nonhuman primates and fully protected them from a lethal challenge with LASV. Thus, our hyperattenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses. IMPORTANCE Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus and early care for treatment and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyperattenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected nonhuman primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic, and efficacious in nonhuman primates.

Background Machupo virus (MACV) is a member of the Mammarenavirus genus, Arenaviridae family and is the etiologic agent of Bolivian hemorrhagic fever, which causes small outbreaks or sporadic cases. Several other arenaviruses in South America Junín virus (JUNV) in Argentina, Guanarito in Venezuela, Sabiá in Brazil and Chapare in Bolivia, also are responsible for human hemorrhagic fevers. Among these arenaviruses, JUNV caused thousands of human cases until 1991, when the live attenuated Candid #1 vaccine, was used. Other than Candid #1 vaccine, few other therapeutic or prophylactic treatments exist. Therefore, new strategies for production of safe countermeasures with broad spectrum activity are needed. Findings We tested a tri-segmented MACV, a potential vaccine candidate with several mutations, (r3MACV). In cell culture, r3MACV showed a 2-log reduction in infectious virus particle production and the MACV inhibition of INF-1β was removed from the construct and produced by infected cells. Furthermore, in an animal experiment, r3MACV was able to protect 50% of guinea pigs from a simultaneous lethal JUNV challenge. Protected animals didn’t display clinical symptoms nor were virus particles found in peripheral blood (day 14) or in organs (day 28 post-inoculation). The r3MACV provided a higher protection than the Candid #1 vaccine. Conclusions The r3MACV provides a potential countermeasure against two South America arenaviruses responsible of human hemorrhagic fever. Electronic supplementary material The online version of this article (10.1186/s12985-018-1009-4) contains supplementary material, which is available to authorized users.

We have previously shown that the infection of cell cultures with the arenaviruses Junín (JUNV), Tacaribe (TCRV), and Pichindé promotes the phosphorylation of mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1 and 2 (ERK1/2) and that this activation is required for the achievement of a productive infection. Here we examined the contribution of ERK1/2 in early steps of JUNV and TCRV multiplication. JUNV adsorption, internalization, and uncoating were not affected by treatment of cultured cells with U0126, an inhibitor of the ERK1/2 signaling pathway. In contrast, U0126 caused a marked reduction in viral protein expression and RNA synthesis, while JUNV RNA synthesis was significantly augmented in the presence of an activator of the ERK1/2 pathway. Moreover, U0126 impaired the expression of a reporter gene in a TCRV-based replicon system, confirming the ability of the compound to hinder arenavirus macromolecular synthesis. By using a cell-based assay, we determined that the inhibitor did not affect the translation of a synthetic TCRV-like mRNA. No changes in the phosphorylation pattern of the translation factor eIF2α were found in U0126-treated cells. Our results indicate that U0126 impairs viral RNA synthesis, thereby leading to a subsequent reduction in viral protein expression. Thus, we conclude that ERK1/2 signaling activation is required for an efficient arenavirus RNA synthesis. Fil: Brunetti, Jesús Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina Fil: Foscaldi, Sabrina Andrea. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quintana, Verónica Mara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Scolaro, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina Fil: Lopez, Nora Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; Argentina Fil: Castilla, Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina