key: cord-0003702-59c9cpfe
authors: Brisse, Morgan E.; Ly, Hinh
title: Hemorrhagic Fever-Causing Arenaviruses: Lethal Pathogens and Potent Immune Suppressors
date: 2019-03-13
journal: Front Immunol
DOI: 10.3389/fimmu.2019.00372
sha: 3380d0cb8447aa4741878f8a3d300a3765ed9a40
doc_id: 3702
cord_uid: 59c9cpfe

Hemorrhagic fevers (HF) resulting from pathogenic arenaviral infections have traditionally been neglected as tropical diseases primarily affecting African and South American regions. There are currently no FDA-approved vaccines for arenaviruses, and treatments have been limited to supportive therapy and use of non-specific nucleoside analogs, such as Ribavirin. Outbreaks of arenaviral infections have been limited to certain geographic areas that are endemic but known cases of exportation of arenaviruses from endemic regions and socioeconomic challenges for local control of rodent reservoirs raise serious concerns about the potential for larger outbreaks in the future. This review synthesizes current knowledge about arenaviral evolution, ecology, transmission patterns, life cycle, modulation of host immunity, disease pathogenesis, as well as discusses recent development of preventative and therapeutic pursuits against this group of deadly viral pathogens.

Significant progress has been made in recent years to understand the role of modern means of travel in inadvertently exporting deadly arenaviruses from endemic regions, the basic biology of these viruses, their genomic evolution and modes of transmission and immune suppression, and the disease pathogenesis for which they are responsible. A detailed level of understanding of the viral life cycle, evolution, and interactions with the host's immune signaling pathways is necessary in order to design effective therapeutic and preventative measures against this group of deadly human pathogens.

The family Arenaviridae is divided into 3 genera based on their natural hosts-Mammarenaviridae, Reptarenaviridae, and Hartmaniviridae that include viruses infecting mammals, reptiles, and fish, respectively (1, 2) . Mammarenaviridae are further classified into the regions of their origins, such as the Old World (OW) viruses found in West Africa (3-9) and the New World (NW) viruses found in South America (5, (10) (11) (12) (13) (14) (15) (16) (17) , which are believed to have originated <23,000 and 41,000 years ago in those continents, respectively (18) (Figure 1) . Additionally, several NW arenaviral strains have been discovered in the United States, which are suspected to potentially cause human FIGURE 1 | Taxonomy and location of arenaviruses. The phylogenetic tree for OW and NW arenaviral strains and their geographic locations. Tree was generated from full-length genomic sequences for the L polymerase protein aligned by Clustalw analysis. Asterisks designate strains that cause natural human diseases, whereas hashtags designate strains that can cause laboratory-acquired diseases in animals.

disease (19, 20) . The OW and NW subgroups are polyphyletic and contain both human-pathogenic and non-pathogenic viral strains, with 10 strains in total known to cause human diseases (21). Unlike other hemorrhagic fever viruses, such as the Ebola virus (EBOV), arenaviral transmissions to humans have been found primarily as a result of human interactions with the rodents as the natural reservoirs of these viruses, as has been directly observed as recently as the 2017-2018 Lassa virus outbreak in Nigeria (9). However, human to human transmission may play a larger role in certain viral outbreaks, such as a 2014 outbreak where strains across larger geographical areas were found to cluster closely together (22). This transmission model presents an interesting challenge for determining the evolutionary history of arenaviruses. As with many zoonotic viruses that follow a co-speciation pattern to allow for infection of new hosts, arenaviruses have been previously thought to have originated in Asia along with the earliest rodents and later spread to Europe, Africa, and the Americas alongside the spread of the rodents (23, 24). However, arenaviral and rodent host phylogenetic trees almost never perfectly match (25) and some models result in rodent hosts that are randomly integrated into the arenaviral phylogeny (26). Additionally, only the Lymphocytic Choriomeningitis Virus (LCMV) has been found to circulate among European rodents (18, 27-30), but titers against LCMV have been found in human subjects worldwide (31, 32), suggesting that rodents and potentially other hosts for LCMV are more widespread than previously thought. Recent studies have revealed that LCMV can be isolated from ticks in the Ukraine (33) and in China (34) (though the low numbers of positively infected sample specimens and unknown capacity of LCMV to infect insect cells raise some doubts about ticks being a true reservoir rather than an intermittent viral carrier). Arenaviral evolution, therefore, may be more reflective of their adaptation to the available hosts based on geographic constraints (9, 18).

Local host adaption of arenaviruses is further evidenced by recent insights into diversity of arenaviruses in reptilian and marine hosts. The full genomic sequences have been determined for three reptarenaviruses isolated from boa constrictors and annulated tree boas: the CAS virus (35), Golden Gate virus (35) and University of Helsinki virus (36), necessitating the addition of the genus Reptarenaviridae into the Arenaviridae family in 2015 (1) . These three reptarenaviruses have been found to be causative agents of Inclusion Body Disease (IBD), a fatal condition in snakes characterized by neurological abnormalities (37, 38), large eosinophilic aggregations inside infected cells (37-40) consisting of a 68 kDa protein (39) thought to be reptarenaviral nucleoproteins (NPs) (36, 41) and the primary source of lethality being secondary infections (38-40). Preliminary evidence has indicated that reptarenaviruses might have specifically adapted to boa constrictors, with viral replication being optimal at the reptilian 30 • C and attenuated when grown at the mammalian 37 • C condition (41), and infected boa constrictors having decreased rates of IBD and increased chances of becoming asymptomatic viral carriers than other snake species despite a high viral load (37, 38, 42) . Like rodent reservoirs, reptarenaviruses have also been found to transmit vertically (43). Whereas, the genome of mammarenaviruses are bi-segmented with a small (S) segment and a large (L) segment, those of the reptarenaviruses are potentially more complicated, with some infected snakes carrying up to 4 different S segments and 11 different L segments per individual (44). Further adding to arenaviral genome diversity is the recent discovery of two new arenaviruses infecting ray-finned fish (2) with trisegmented genomes, making them a potential intermediate between arenaviruses and their close "cousin" virus, the trisegmented Bunyaviridae family found widely in arthropods (2, 45 ) that can also cause severe and lethal hemorrhagic infections in humans.

The focus of this article is on the OW and NW mammarenaviruses. Phylogenetically, changes in the viral polymerase of the mammarenaviruses may be more associated with older adaptive events dating to the speciation of NW arenaviruses (18), while more current adaptation, at least with the OW Lassa virus (LASV), appears to more prominently associate with the viral surface glycoproteins (46). This observation is recapitulated when comparing the genetic similarity of the four arenaviral proteins (Figure 2) . The arenaviral polymerases have less homology between OW and NW viruses as well as among OW and NW strains compared to the other 3 arenaviral proteins, suggesting an earlier genetic divergence. A recent report further supports this observation by noting that a reassorted virus containing the S genomic segment that encodes the viral glycoprotein (GP) and nucleoprotein (NP) from the known virulent LASV Josiah and the L genomic segment that encodes the viral polymerase (L) and matrix protein (Z) from the known less virulent 2015/Liberia LASV strain retained the disease pathogenicity in guinea pigs (Figure 3 ). In contrast, another reassorted virus carrying the viral GP and NP from the 2015 Liberian strain was severely attenuated (47). Taken together, this suggests that earlier adaption to a geographically available host required fundamental changes to the internal viral proteins, such as the L polymerase, while more recent evolution is driven by optimizing viral entry efficiency and immune evasion mediated by viral GP and NP, respectively. The concept of viral proteins mediating host immune evasion will be discussed further below.

Mammarenaviruses enter their hosts by inhalation of airborne viral particles or by eating and/or drinking virus contaminated food or water, respectively. The social structures of arenavirus endemic regions present a particular challenge for viral containment with this transmission model. Homes are often open-air spaces and contain small spaces for rats to cohabitate with humans when built from locally available materials. As a result, rodent reservoirs move easily from home to home (48) and previous surveys have found significant numbers of homes containing rodent reservoirs and instances of contact with rodent or rodent waste products, particularly at night when the activity of rodents is at its highest level (49). Rice is often the stable crop in endemic areas (particularly in western Africa), which is typically grown in fields or low-lying swamps that encourage rodent habitation (50). The challenges presented by the educational and communication infrastructure in endemic regions also prevent optimal disease control, with a recent survey finding that 76% of residents in a Nigerian urban town had inadequate knowledge of Lassa fever and 51% had poor control practices (51). Practices such as hunting rodents for food and for use in sacrificial ceremonies have also been documented to increase risks for infection (50). Future measures of disease prevention will not only need to focus on enforcing individual habits of rodent control, but also on encouraging larger political and incentive policies conducive to foster sensible habits (52, 53) and infrastructural building, as diagnostic laboratory capable of carrying out viral genomic PCR to detect arenaviral infections is often not available in endemic regions (54-56). Computational modeling of regions at risk of arenaviral pandemics will also continue to be necessary as predictive/preventive measures (53, 57-60).

Several modeling studies have indicated that several African regions in close proximity to current endemic regions are at serious risk for arenavirus spread (53, 57, 58), and new reservoirs for human pathogenic arenaviruses are continuing to be found, adding to the potential for interregional spread (61). However, the biggest factor in restricting arenaviral pandemics appears to be a strong inter-species host transmission barrier (62) . Current models suggest about 10% of rodents in endemic areas are seropositive for local arenaviruses, and that rodents clear the virus in a time period significantly shorter than their life span to produce antibodies (63, 64) with some preliminary evidence suggesting that anti-arenaviral antibodies may have a small correlation with decreased survival and increased rodent capture (65) .

There is increasing concern about the potential for arenaviruses to spread across regions and initiate worldwide pandemics. LASV remains the only documented arenavirus to be imported by travelers who have visited endemic regions for a variety of reasons (Supplemental Table 1 ). While most documented cases occurred prior to 2000 (66) , more recent cases of Lassa fever importation have been reported in the United States [Pennsylvania (67) , Minnesota (68) , and Georgia (69)], Ghana (70), Sweden (71) , and Germany (72, 73) . A common theme among these cases is that, while no secondary infections as of yet have been documented, an intermittent period of remission between initial treatment and reemergence of symptoms presents a potential risk for protracted disease and transmission (74) . A salient fact that arenaviruses also have the potential to be used as biological weapons (75, 76) is of particular concern for endemic regions that are engaged in constant political and military conflicts (77) . Human-to-human transmissions, although are rare occurrences, have been documented through the use of contaminated medical instruments in standard and specialized FIGURE 2 | The L RdRp polymerase exhibits the least conservation among the arenaviral proteins. Full-length protein coding sequences from arenaviral strains were aligned by Clustlw analysis, and the matrix for pair-wise score similarity (# of shared amino acid residues/alignment length*100) was converted into a heatmap by the pheatmap module for R. The GenBank accession ID's used for alignments in Frontiers in Immunology | www.frontiersin.org medical procedures, such as organ transplantations (78) . As a whole, the threat of arenaviral spread and exportation from endemic regions should be of concern for public health considerations despite natural barriers that currently help curb major outbreaks.

Arenaviruses are enveloped, ambisense single-stranded RNA viruses. Their structure consists of a membrane envelope containing the surface glycoprotein (GP) subunits surrounding a capsid, which consists of the Z matrix protein ( Figure 4A ). Inside the capsid are the L RNA-dependent RNA polymerase (L RdRp) and the viral bi-segmented genome encapsulated by the nucleoprotein (NP). Cell entry is mediated by GP, which is first expressed as the glycoprotein precursor complex (GPC). The host subtilase SK1-S1P proteolytically cleaves GPC into its three final subunits (79) (80) (81) : GP1, GP2, and SSP (Stable Signal Peptide), which form heterotrimers on the cell membrane surface (82) (83) (84) . GP1 forms spikes protruding from the viral envelope and is responsible for interacting with entry receptors, while GP2 is a class 1 fusion transmembrane protein by virtue of its 6 helix domain (85, 86) . SSP (87) is an unusually small, hydrophobic and long-lived (88) signaling protein implicated in viral fusion (89, 90) , transport of the GPC complex through the SK1-S1P containing Golgi (91) and as a GPC folding chaperone (92, 93) . While recombinant arenaviruses expressing a SSP from a different strain are viable, recombinant viruses expressing a non-arenaviral signal peptide are not (94) , indicating the specific adaptation of SSP for arenaviral proteins. This observation is strengthened by work from our laboratory, which shows that certain SSP residues attenuate viral growth independently of cell entry efficiency, illustrating the multiple roles of SSP in the viral life cycle that remain to be fully elucidated (95) . GP1 and GP2 also contain N-glycosylation residues at multiple sites (96) , while SSP is myristoylated (89) . While the purpose of these post-translational modifications has yet to be described in detail, GP1/2 glycosylation has been linked to viral protein transport (96) and SSP myristoylation has been found to be vital for directing membrane anchor and viral fusion (89, 90) .

LASV and LCMV were first discovered to utilize αdystroglycan as their cellular entry receptor (97) , which was later confirmed to be the case for all OW and clade C NW arenaviruses (98) (Figure 4B ). Glycosylation of α-dystroglycan by the glycosyltransferase LARGE is necessary for recognition by arenaviruses (99) (100) (101) , which mimics the interaction between α-dystroglycan and its natural ligand laminin (102) . Regulation of α-dystroglycan appears to be a driving genetic force in arenaviral endemic regions, as wide-scale genomic studies have found evidence of positive selection of polymorphisms of LARGE and other α-dystroglycan regulatory proteins in Nigerian populations (103) (104) (105) . The C-terminal GP1 subunit directly interacts with α-dystroglycan (97, (106) (107) (108) , while the C-terminal GP2 subunit has been found to be critical for stabilizing the αdystroglycan-GP complex (109, 110) . Residue 260 on GP1 was the first residue found to be important for interaction with αdystroglycan (98, (111) (112) (113) . X-ray crystallography analysis later identified the loop 1 structure, and specifically residues 153, 155, and 190 of GP1, to be responsible for directly interacting with α-dystroglycan (107). Residues 260 and 136 were also found to be critical for cell entry, but these residues were both located up to 25 Angstroms away from loop 1. Given that full-length GP is required for cellular entry, it is theorized that these extra residues may facilitate cell entry by complex and unknown mechanisms involving the full length protein (107) . Supporting this idea, residues at the trimeric interface of the GP were recently found to also be critical for cell entry (108) , highlighting the importance of the full GP for efficient cell entry. The endosomal cellular protein LAMP1 has been identified as a critical host factor for mediating a "pH-switch" mechanism of virus-cellular membranes fusion and endocytosis of the virion particle, which can partly explain previous observations that chicken cell lines are among the few not able to be infected by LASV as they lack LAMP1 expression (106, 114) . Conserved histidine residues on the GP1 subunit among OW arenaviruses have been found to mediate recognition by LAMP1 (108) , and structural analysis has found a shift in GP1 conformation following its interaction with LAMP1, possibly serving as an immune cloaking mechanism (115) . Another interesting mechanism has also been proposed for LAMP1-mediated LASV endocytosis, where LAMP1 raises the pH threshold for acidic endocytosis, thereby increasing virus yield by preventing deactivation of the viruses by the acidic environment inside the endosomes (116). Additional host factors and possible alternative entry receptors have also been identified for entry of arenaviruses that might involve the sodium hydrogen exchangers (117) and the phosphatidylserine receptors Axl (118) and TIM-1 (119), which need further analyses (120) . A recent study has also found that the cellular NRP2 factor and CD63 appear to serve as substitutes for α-dystroglycan and LAMP1 to mediate entry of the Lujo virus (LUJV) (121), implicating the conservation of similar mechanisms in other viruses as well as the importance of conserving these mechanisms during viral evolution.

NW arenaviruses use different cellular entry receptors depending on their clade. Clade B NW arenaviruses that are pathogenic in humans have been found to use human transferrin receptor 1 (hTrf1) (122, 123) while non-pathogenic clade B viruses use hTrf1 orthologs (124, 125) . A number of other viral receptors and host factors have also been found to allow Junin virus (JUNV) entry, with hTrf1 being the major candidate cellular receptor (123) , and DC-SIGN/L-SIGN (126), voltage-gated calcium channels (127) , and proteins involved in the clathrinmediated pathways such as the dyn2/Eps15 endocytic pathway (128) being possible co-factors. TRIM2 has recently been identified in a siRNA screen as an inhibitor of NW arenaviral cell entry by a yet undetermined mechanism (129) . The receptors for the other clades of NW arenaviruses are still unknown, though it has been shown that the tentative clade D viruses do not use hTrf1 (130) . Viral particles enter cells by clathrin-mediated pathways for α-dystroglycan-independent NW arenaviruses (131) , and a clathrin-independent mechanism that involves PI3K-mediated formation of multivesicular bodies during late endocytosis and the ESCRT sorting pathway for α-dystroglycan-dependent viruses (132) (Figure 4B ). GP1 disassociates from the viral particles upon exposure to acidic pH, exposing GP2 residues prior to viral fusion (133, 134) . Viral fusion then ensues, of which the hydrophobic regions in GP2 (109, 135) as well as the cytosolic tail (110) have been found to play a critical role.

Replication of the viral genome occurs in the cytosol following its release from the endosome. The viral NP and L proteins are the minimum known components required for viral genome replication and transcription (136) (137) (138) , and the viral Z protein is an inhibitor of the viral polymerase's transcriptional initiation function (137) (138) (139) (140) .

The viral genomic RNA structural elements play important roles in the regulatory mechanisms of viral genome replication, transcription, and gene expression. The 5 ′ and 3 ′ untranslated regions (UTRs) of arenaviral genomes contain complementary sequences that are predicted to form panhandle structures and are required for effective viral RNA replication and transcription (141, 142) . Specifically, 19 nucleotides located at the terminal 3 ′ UTR of the LCMV genome were found to serve as a minimally required promoter element and that the sequences located at the complementary 5 ′ UTR to form the panhandle structure appear to be required for efficient genome synthesis (142) . Foscaldi and colleagues have reported that the 5 ′ UTR of the Tacaribe virus (TCRV) appears to contain translational stimulatory signal, whereas some sequences within the 3 ′ UTR can down-regulate viral gene expression (142, 143) . The intergenic regions (IGRs) fold into hairpin secondary structures that are thought to help terminate transcription (12, 144, 145) and to protect the unique non-polyadenylated viral mRNA transcripts from degradation by the cellular exoribonucleases (145) . These sequences appear to have a large tolerance for sequence variations that can be exploited for the development of attenuated vaccine candidates (21, 146, 147) . It is noteworthy that during viral genome replication, genomic primers slip backwards from their initial binding site, resulting in a non-templated 5 ′ ppGpp residue (148) that is thought to act as a viral RNA decoy by competitively inhibiting the viral RNA sensing by the cellular innate-immune machinery (149) . The molecular mechanisms of viral RNA sensing and innate immune evasion strategies by arenaviruses as well as on efforts to develop vaccines will be discussed in detail below.

The localization of arenaviral RNAs has also been found to be important for replication control, with both sense and anti-sense RNAs associating with cytosolic compartments containing viral NP and host factors involved in RNA metabolism (150) . In this regard, recent advances in antibody-mediated dsRNA visualization (151) as well as specific probing for genomic and anti-genomic arenaviral RNA (152) show much promise for elucidating the dynamics of arenaviral RNA during the infectious life cycle. Preliminary evidence suggests that the timing of viral RNA replication is under regimented control mechanisms, as NP and L genes being transcribed and translated first and directly from the negative-sense viral genome, whereas GPC and Z are expressed several hours later after the viral genomic RNA segments encoding these genes have been replicated and then transcribed (152, 153) ( Figure 4C ). Additionally, virally infected cells appear to amplify viral genes in cyclical waves of expression and viral clearance, the molecular mechanism of which is unclear (152) . Future studies in this area will include method development and optimization in order to image RNA species at lower copy numbers and to expand the RNA repertoire available for targeted imaging.

After the viral RNAs are replicated, transcribed, and translated, virion assembly ensues and is mediated by both viral and host cellular proteins. The C-terminal domains of the viral Z matrix protein is a central player for viral assembly, budding, and release from the infected cells. Z-L interactions (140, 154) , Z-NP interactions (155) , and Z-GP interactions (156, 157) have been shown to ensure co-localization of viral proteins for assembly. Z then interacts with the cellular Tsg-101 ESCRT pathway proteins to allow viral budding process to begin (158, 159) , which is aided by a myristoylation residue at the N-terminus of Z to allow for interaction with the cellular membrane (160, 161) . Z proteins has also been found to be capable of self-budding and the production of the viral like particles (VLPs) in the absence of the viral RNA or other proteins indicates that Z is both necessary and sufficient for budding (162) (163) (164) (165) . A recent report suggests that the late domains located in the C-terminal part of the Z protein may also be required for the release of the so-called defective interfering (DI) virion particles from the infected cells but not necessarily for the release of infectious virions, a process that may be regulated by phosphorylation of certain residues in the viral Z protein (163) . DI particles are produced by many viruses, including arenaviruses, during infection and are similar to infectious virion particles in appearance and protein contents but they cannot produce productive infection (166) as their genomes contain large and deleterious deletions (167) . Such deletions have been observed in the 3'UTR regions of the LCMV genome and potentially elsewhere in the viral genome, but their specific functions in inhibiting viral replication have yet to be studied in detail (168) . Arenaviruses have been found to produce high levels of DI particles in cell culture (169) and in animal infection models (170) , and DI particles have been theorized to contribute to viral persistence (166, 171, 172) .

Mammarenaviral disease profiles tend to be heterogenous (173) (174) (175) (176) . Lassa fever, the most severe arenaviral disease, is estimated to cause up to 300,000 cases and 5,000 deaths per year in endemic regions of West Africa (5, 173) . A recent outbreak occurred in Nigeria has resulted in 431 laboratory confirmed cases that include 37 health care workers and with a 25% overall fatality rate (177) (178) (179) . Previous outbreaks have recorded lower mortality rate in hospitalized patients (180) . However, the fatality rate can be as high as 87% among infected women during the third trimester of pregnancy (181) , and maternalfetal transmission of arenaviruses has been demonstrated in rodent models (64) as well as in one recent reported case in humans (182) . Many other complications arise from severe arenaviral infections including liver (183) (184) (185) (186) and vascular (185, 187, 188) damage, both of which have been recapitulated in a guinea pig arenaviral animal model (189) (190) (191) as well as in immunocompromised mice (192, 193) and a novel hamster NW arenavirus model (194) . While liver damage tends to be highly prominent in infections by OW arenaviruses with platelet factors and heme breakdown products as significant biomarkers of LASV disease, hemorrhaging, and vascular damage occur more frequently in NW arenavirus infections (195) (Figure 5) . Sensorineural hearing loss (SNHL) also occurs in 29% of the survivors of Lassa fever (196) (197) (198) (199) which may be attributed to a pathogenic inflammatory response in the auditory nervous system (200, 201) . Other neurological complications have also been reported for arenaviral infection (202, 203) .

Monocytic cells such as alveolar macrophages and dendritic cells are the earliest target for arenaviral cell entry. However, these cells are not activated upon viral entry, as evidenced by the lack of increase in the levels of activating markers, such as CD80, CD86, CD40, CD54, and HLAs as well as cytokines TNFα, IL1β, IL6, and IL12 (204, 205) . For this reason, monocytic cells are thought to act as a viral "reservoir" early in infection, where the virus can easily enter, and virus later spreads when these cells enter draining lymph nodes (206, 207) . The failure of these cells to become activated is consistent with the observation of the generalized immunodepression in severely and fatally infected individuals (208, 209) . Virus replication has also been directly observed in a number of cell types, including respiratory epithelial cells following infection in human (210) and animal models (211) .

Studies on human and rodent survivors of mammarenaviral infection have all indicated that proper functioning of the immune responses (innate and T-cell mediated immunity) are critical to minimizing viral growth rates, presentation of symptoms, and mortality rates (209, 212) . The innate immune response is a compilation of non-specific defense mechanisms against foreign antigens that is critical for early detection and Frontiers in Immunology | www.frontiersin.org inhibition of pathogen growth before the adaptive immune response has time to produce proper cell-mediated immunity, such as the development of antibodies and cytotoxic Tlymphocyte responses (CTL) against the invading pathogen and/or the pathogen-infected cells (212) . Innate immunity is enacted within a few hours of host recognition of a pathogenassociated molecular pattern (PAMP). The most common viral PAMPs recognized by immune cells are unique molecular features of the viral genome, such as 5 ′ triphosphorylated RNA and double stranded RNA (Figure 6 ). Pattern recognition receptor proteins (PRRs), such as the RIG-I-like receptors (RLRs) RIG-I and MDA5, are activated by PAMPs and result in cytokine expression and the activation and recruitment of innate immune cells such as macrophages, neutrophils, and dendritic cells (213, 214) .

RIG-I and MDA5, upon binding to PAMP dsRNAs in the cytosol during the process of viral replication, change their conformations from a closed to an open and activated state (215) . Activated RLRs (RIG-I and MDA5) initiate several complex molecular cascades, including activation of the mitochondrial anti-viral signaling protein (MAVS), which eventually results in the translocation of transcription factors such as interferonregulatory factors (IRF) IRF3 and IRF7 as well as nuclear factor kappa B (NF-kB) into the nucleus of the cells to activate the expression of the type 1 interferons (IFN1) that include IFNα and IFNβ, and a wide variety of interferon stimulated genes (ISG) (216, 217) . A recent study has suggested that IFNα may be primarily involved in inhibiting viral growth in early stages of viral infection, while IFNβ may control viral growth in the later stages of infection (218) . IFN1 is then secreted and bound by their receptors (IFNAR) on the surface of either the same cell or the neighboring cells, which activates the IFN signaling cascade in a positive feedback loop to produce more of the IFN1 and the antiviral gene products in order to confer virus resistance (219, 220) .

OW arenaviral infection (e.g., LASV) in patients with moderate to severe symptoms is associated with inhibition of the innate immune response with decreased levels of IFN1 and pro-inflammatory cytokines (184, 221, 222) (Figure 5) . The importance for IFN1 signaling for control of arenaviruses appears to be conserved in mammals, as wildtype (WT) mice do not exhibit symptoms during arenaviral infection but IFN receptor (IFNAR) knockout (KO) mice have been found to succumb to disease (223, 224) . Additionally, in vivo infection of non-human primates (NPHs) and guinea pigs with the LASV Soromba strain isolated from local rodents in Mali, Africa, shows a significant increase in cytokine signaling and decrease in mortality compared to other LASV strains (8). Mice infected with the OW LCMV, which is capable of maintaining persistent infection with low levels of IFN1 signaling (225, 226) , display FIGURE 6 | Arenaviral proteins (NP and Z) inhibit the RIG-I/MDA5 and NF-κB pathways. RIG-I and MDA5 are activated by PAMP dsRNAs during virus replication and are potentiated by PACT. Following the activation of MAVS (on the mitochondria) by RIG-I/MDA5, a molecular cascade involves the interaction of IKKε and DDX3, which is followed by phosphorylation of the transcription factors IRF3 and IRF7 to translocate them into the nucleus, where they dimerize and bind to transcription factor binding sites of the IFNα and IFNβ genes to activate their transcriptions. Expression and exportation of these gene products into the cellular milieu trigger the IFN1 signaling cascade in an autocrine or paracrine fashion to induce expression of hundreds of interferon-stimulated genes (ISGs) and inflammatory genes to confer virus resistance. The NF-κB pathway is activated by recognition of certain features of the virus particles by the membrane receptors TLR 2 or TLR 6. This initiates a molecular cascade resulting in the translocation of the two functional NF-κB units (p50 and p65) into the nucleus, resulting in more production of NF-κB. Arenaviral proteins (NP or Z shown in red) are known to inhibit different steps of the RIG-I/MDA5 and NF-κB pathways by either degrading the PAMP dsRNAs (through the NP RNase function) or directly inhibiting the normal function of different cellular proteins (RIG-I, MDA5, PACT, IKKε, or p50/p65). an IFN1 burst 6-48 h after infection and 2-4 days before a peak in viral titers, suggesting that IFN1 signaling is most important for inhibiting viral proliferation in the earliest stages of infection (209, 227) . The difference between the immune responses in rodents and humans is one of the key aspects of active investigations. Rodent reservoirs generally do not experience severe symptoms from OW arenaviral infections (65, 228, 229) , and mice must be immunodeficient to experience significant symptoms. LASV infected mice also experience an early upregulation of cell adhesion molecules in the peripheral blood mononuclear cells (PBMCs) consistent with immune cells being recruited to the site of infection (230) . NW arenaviruses differ from OW strains in that they tend to produce increased levels of IFN1 and cytokines, and a pro-inflammatory response is favored for inhibiting viral proliferation (222, (231) (232) (233) (234) (Figure 5) .

Virus-infected A549 cell cultures (235, 236) as well as fatally infected patients (237) (238) (239) have been shown to have consistently up-regulated levels of IFN1 and TNFα. However, macrophages infected in vitro with NW arenaviruses fail to show this upregulation (240) .

The arenaviral NPs have been found to inhibit the RIG-I/MDA5 pathway. The crystal structure of the LASV NP and subsequent functional studies have revealed a 3 ′ -5 ′ exoribonuclease domain located within its C-terminal domain (241) (242) (243) that preferentially degrades dsRNA (242, 244) . The N-terminus of NP contains a unique domain that binds ssRNA (245) and has been proposed to bind to the m7GpppN mRNA cap structure (241) . NP directly interacts with RIG-I and MDA5 (246) , and recent work from our lab has demonstrated that NP prevents potentiation of RIG-I function by the cellular protein PACT and that the exoribonuclease domain of NP is required for this inhibition (247) , the molecular mechanism of which will be discussed in detail in the next section. The exoribonuclease domain is also required for NP inhibition of the cellular protein kinase IKKε (248) and other cellular proteins in the IRF pathway (249, 250) . Abrogation of the exoribonuclease domain has allowed for IFN1 expression in normally inactivated macrophages and dendritic cells (251, 252) and subsequent activation of natural killer (NK) cells resulting in antigen-presentation (253) . The exception to NP-mediated IFN1 inhibition appears to be Tacaribe virus (TCRV) NP (254) , which was initially thought to be due to variance in 4 exoribonuclease residues found in this viral protein (255) . However, both structural and functional studies have later demonstrated that TCRV NP can effectively degrade dsRNA and inhibit the IFN1 pathway (244, 255) . These studies collectively indicate the importance of PAMP dsRNA degradation by arenaviral NPs for inhibition of the IFN1 pathway.

One important consideration in determining the larger role of NP in arenaviral pathogenesis is the fact that IFN1 inhibition by NPs is also present in strains that are not known to be pathogenic to humans. For example, NP from the non-pathogenic Pichinde virus (PICV) has been found to inhibit the IFN1 pathway in human cells (254) . Additionally, work from our laboratory has demonstrated that each of the 5 exoribonuclease (RNase) catalytic residues is necessary for IFN1 inhibition by Pichinde NP as well as for optimal PICV growth in vitro (256) . RNase domainmutant viruses have also been found to exhibit diminished viral load and pathogenesis in vivo in guinea pigs (which normally experience high lethality, develop human hemorrhagic fever-like symptoms and support a high viral load when infected by the P18 PICV strain), and even result in generation of WT revertant viruses (256) , implicating the important role of NP RNase function for optimal viral replication. Altogether, the ability of NP to inhibit IFN1 expression by degrading PAMP dsRNA is likely conserved across known arenaviruses. As evidenced by the tight control of gene expression and viral RNA localization within cells, it is clear that regulation of viral RNA levels and localization is a critical mechanism of the virus life cycle to allow for maximum replication without being quenched early in the cycle by the innate immune response, which may explain the absolute conservation of IFN1 inhibition by arenaviral NP proteins.

Recent studies have also been focused on the role of the viral Z protein in inhibiting the IFN1 pathway. Originally, the Z proteins from 4 tested NW arenaviral strains [JUNV, Machupo virus (MACV), TCRV, and Sabia virus (SABV)] but not from the OW LASV or LCMV strains were found to directly interact with RIG-I and inhibit IFN1 expression (257) . More recently, our laboratory has demonstrated that the Z proteins from both OW and NW arenaviral strains that are known to be pathogenic in humans can directly interact with RIG-I to inhibit IFN1 expression, but Z proteins from nonpathogenic arenaviral viruses cannot (258) . These results have been recapitulated in primary human macrophages infected with WT and chimeric PICVs to show that the N-terminus of LCMV Z was sufficient to inhibit macrophage activation when expressed as a chimeric Z protein with the non-pathogenic PICV (259) . As a whole, the Z protein is emerging as another key target in investigating the mechanism of innate immune suppression by arenaviruses, and an encouraging potential contributor to disease pathogenesis (i.e., a potential virulence factor) that will need further characterization (105, 260) .

One area of study with increasing attention is the role of innate immune cells and cytokines in initiating the adaptive immune response against mammarenaviral infection. A key pattern that has emerged from preliminary studies is that an arenavirus's ability to suppress the innate immune response appears to be positively correlated with its ability to suppress any subsequent adaptive immune response. The correlation of an inhibited innate immune response with an attenuated and delayed T cell response in those succumbing to fatal arenaviral infection has been demonstrated in humans (210, 261, 262) , non-human primates (221) , guinea pigs (47, 190, 211, (263) (264) (265) , immunodeficient mice (223, 224, 266) as well as in immunodeficient mice transplanted with cells from WT mouse bone-marrow (193) . Accordingly, individuals that survive arenaviral infections manage to keep viral loads low, peaking 10-20 days after infection for LASV, while individuals that succumb have viral loads that continually increase exponentially (205, 221, 261, 262, (267) (268) (269) . In WT mice, LCMV induces robust expression of key cytokines such as the type I and II IFNs as well as IL-18, the production of type II IFN being dependent on CD8 T cells (270) . This not only illustrates the close inter-connectedness of the innate and adaptive immune responses, but also further illustrates how an active immune response is key to the natural resistance against arenaviral infection in mice. The importance of the innateadaptive interface was also recapitulated in a recent co-infection model of LCMV and E. Coli, demonstrating that an LPS-induced inhibitory mechanism of NK cells significantly attenuated the LCMV specific CD8 T cell response and the vitality of the infected mice (271) .

CD8 T cells have been found to be indispensable for clearing arenaviruses (221, (272) (273) (274) (275) (276) (277) (278) (279) , while CD4 T cell (277, 280) and antibody (184) responses have been found to be dispensable. In fact, a potential contributing factor toward arenaviral pathogenicity as well as the failure of most previous arenaviral vaccine candidates in protecting against pathogenic strains may be a skewed CD4+ T cell response. STAT1 KO mice were only able to survive LCMV infection when CD4+ T cells were depleted (266) , suggesting that a lack of IFN signaling could somehow overwhelmingly favor lethal CD4+ T cell proliferation. The determination of the mechanism of CD4+ T cell lethality, whether it is simply due to less CD8 T cells being activated or through some novel mechanism of CD4 T cells, has yet to be determined.

Myeloid dendritic cells (mDCs) are vital constituents in the innate-adaptive immune response interface due in combination to their large population sizes (281) , their robust activation of CD8 T-cells (282) , and their production of IFN1 as a CD8 Tcell activation enhancer under certain conditions, such as during LCMV infection (283) . Dendritic cells (DCs) are a key target of infection by LASV (204, 205) , but like macrophages, they are not activated by the infection (204, 205) . However, this feature isn't consistent among arenaviral strains, as demonstrated by the well-documented comparative model of mammarenaviral immunogenicity of LASV and the Mopeia virus (MOPV). Both are OW arenaviruses and share 75% amino acid identity and the Mastomys natalensis African rat as a viral reservoir (23). However, MOPV is non-pathogenic in humans and has been considered as a potential LASV vaccine candidate in non-human primates (284) , and no human cases of MOPV infection have ever been recorded (285) . Initial studies found that MOPV infection of monocyte-derived dendritic cells (MoDCs) induced moderate activation and significant CD8 T-cell proliferation in the MoDC/T-cell co-culture system (286, 287) , whereas LASV infected MoDCs induced low activation and weak and delayed T-cell responses (204, 287) .

The same group of researchers have more recently demonstrated that both LASV and MOPV could not replicate well in myeloid dendritic cells (mDCs) isolated directly from PBMCs, and as a result, induced very low levels of IFN1 production, and that LASV-infected cells could not activate T-cells in the mDC/T-cell co-culture system (288). However, MOPV was able to induce higher levels of IFN1 and secreted cytokines known for inducing T-cell activation than in LASV-infected cells in the coculture system, though the involvement of monocytes and potentially other cell types present in the co-culture system as a result of impurity could not be completely ruled out. Regardless, the observation seems to corroborate earlier findings indicating that DCs are dispensable for T-cell activation during LCMV infection (289) . Like LASV, LCMV is known to inhibit innate immune response as demonstrated in another study (290) .

Using chimeric viruses carrying different genes from either LASV or MOPV on either the LASV reverse genetics (RG) or MOPV RG backbone, the same group of investigators have shown that LASV NP is the main determinant in suppressing IFN1 production by mDCs (288) , which is consistent with the known ability of all known arenaviral NPs to degrade PAMP RNA via their encoded RNase domain and thereby suppressing IFN1 production (105, 241, 244, 247, (252) (253) (254) (255) (256) 291) . Due to the fact that mDCs did not appear to support productive replication of LASV and MOPV, it was difficult to ascertain whether the IFN1 suppressive effect was due to the expression of viral proteins in the infected cells by the different chimeric viruses. Moreover, the data could not explain the differentiation of mDC phenotype in the mDC/T-cell co-culture system between LASV and MOPV chimeric virus infections. In an attempt to provide a potential explanation for this observation, the authors have posited that various efficiencies of the NP RNase domains of different arenaviruses to suppress IFN1 production may explain differences among arenaviruses to induce DC-mediated T-cell activation. However, this hypothesis needs to be formally tested.

A chimeric LASV with a substitution of its GP with that of MOPV has been found to significantly increase IFN1 production (288) , which may reflect different mechanisms of viral detection based on the different innate immune receptors (106) . As an example, the Toll-like Receptor TLR2/TLR6 heterocomplex has been found to be responsible for innate immune activation by recognizing the GP of the NW JUNV upon virus infection for robust induction of the innate (292) and adaptive (293) immune responses (Figure 6) , while only TLR2 has been implicated in the anti-viral activity of OW arenaviruses (294, 295) . JUNV and other NW arenaviruses have also been found to initiate caspase-dependent apoptosis (233, (296) (297) (298) , while OW arenaviruses fail to induce apoptosis (204, 298) or induce significantly delayed apoptosis (299) (Figure 5) . The relative contributions of pathways directly mediated by the specific cellular receptor and other viral signaling mechanisms needs to be further characterized in future studies.

Contrary to earlier findings (258, 259) , the Z protein was not found in the LASV/MOPV chimeric virus model to exhibit a significant effect on immune suppression (288) , which might be attributed to different infectious models being utilized. It is worth noting that the experiments with the chimeric viruses did not take into consideration the potential differential gene expression levels of the viral proteins in virus-infected cells under the context of the chimeric virus backbones. Additionally, as mDCs did not appear to support productive viral replication, it raised a question of whether any of the viral proteins were synthesized in the virus-infected mDC model (288) . It is therefore difficult to definitively discern the roles of the individual viral protein in modulating antiviral immunity in this unique in vitro viral infection model (288) .

Another mammarenaviral-host interaction that has an impact on IFN1 production is that with the cellular protein PACT (Figure 6) . PACT is a 313-aa protein that contains 3 conserved dsRNA binding motifs (dsRBMs) with dsRBM1 and 2 binding to dsRNA and dsRBM3 mediating activation of the dsRNAdependent kinase PKR (300) . PACT has been shown to activate RIG-I via interaction with the C-terminal repression domain of RIG-I and activates its ATPase function to potently enhance IFN1 production upon viral infection (301, 302) . Even though PACT has initially been identified as a cellular binding partner and protein activator of PKR (303), activation of RIG-I by PACT does not require PKR (302) . Likewise, while PACT has also been shown to interact with and stimulate the cellular Dicer protein (303, 304) , Dicer does not appear to play an important role in PACT-mediated regulation of antiviral response (302) .

While PACT and RIG-I pull down together in coimmunoprecipitation assays, it is less clear whether this interaction occurs strictly through protein-protein interactions or by both proteins associating with viral and/or cellular RNAs or some combination of both. The potential role of RNAs in activation of the cellular innate immune proteins (RIG-I and PACT) has been strongly implicated in recent findings that arenaviral NPs require an intact dsRNA binding domain as well as its 3 ′ -5 ′ exoribonuclease activity to block PACT enhancement of RIG-I function (189) (190) (191) (Figure 6) . A number of other viral nucleoproteins can block PACT enhancement of RIG-I function, which also requires an intact dsRNA binding domain (305) (306) (307) , or in the case of herpes simplex virus 1 (HSV1), is independent of the RNA binding capacity of the nucleoprotein (308) . Recently, our laboratory has shown that arenaviral NPs block RIG-I potentiation by PACT, and that the exoribonuclease domain is required (247) . However, NP does not appear to negatively impact PACT/RIG-I direct interaction, suggesting that NP may inhibit PACT indirectly by degrading dsRNA that may be associated with the complex and is essential for its proper function. Taken together, it appears that most if not all known viral protein partners of PACT (as well as PACT itself and its cellular protein partner RIG-I) have RNA-binding properties, yet it still isn't entirely clear whether dsRNA binding is an absolute requirement to inhibit PACT-induced RIG-I activation. These studies illustrate the current lack of detail understanding of the roles of the different viral proteins, including arenaviral NPs, to inhibit PACT-mediated RIG-I activation, and the nature of the dsRNAs (viral, cellular, or both) that may intimately be involved in this process. Future studies into the exact role and molecular mechanism (s) of virus-host interactions via the PACT/RIG-I pathway can serve as an appealing pan-antiviral therapeutic target for development. Additionally, antivirals targeting PACT can influence PACT-PKR mediated NF-κB and p53 stress signaling (300, (309) (310) (311) (312) (313) (314) (315) (316) to impact not only antiviral control but also other known cellular processes involved in cancer and other physiological and metabolic conditions.

Currently, the only clinically successful anti-arenaviral vaccine is the anti-JUNV Candid #1 strain, which is currently manufactured by the Argentinian government but is not being considered for large-scale use due to its limited target population (222) as only Argentina is endemic for JUNV infection. To generate Candid #1, a human viral isolate (317) was used to passage twice in guinea pigs followed by additional passages in suckling mice and cell cultures (318) (319) (320) (321) (322) (323) . A number of unique mutations in Candid #1 were originally thought to attenuate Candid #1 compared to its parental WT strain (323, 324) , and one such mutation (F427I in the transmembrane domain of the GP2 subunit) was consistently found to be sufficient for attenuation (325, 326) . While the mechanism for this attenuation has yet to be elucidated, it is thought that the mutation may affect viral fusion or maturation efficiency (325) . The equivalent mutation (F438I) in Machupo virus (MACV) was also found to be attenuated in mice (327) , suggesting that this attenuation mechanism may be highly conserved. Additionally, the presence of a mutation in the SSP subunit along with the F427I mutation in GP2 is thought to prevent the virus from reverting to its wild type sequence (326) . It is tempting to assume that similar attenuation techniques can be applied toward the development of other arenaviral vaccines. However, they may be limited to the JUNV vaccine for several reasons. While the IFN1 pathway (particularly IFNβ) pathway and subsequent T-cell response have been found to be critical in controlling arenaviral infection in mice (193, 209, 223, 224, 231, 266, 270, 328) , arenaviral-associated immunosuppression results in limited T-cell responses (184, 204, 221, 222, 279, 287) . Furthermore, the JUNV GP has been found to contain fewer glycans than the GPs from other mammarenaviruses (320, 329) , and glycan residues on the glycan-rich LASV GP has been shown to promote neutralization antibody evasion (330) . This is strengthened by recent observations that anti-JUNV antibodies from infected patients can neutralize other JUNV strains but cannot offer neutralization against other NW arenaviruses (331) .

Partly due to the aforementioned reasons, the development of effective vaccines against other pathogenic mammarenaviruses (besides JUNV) has proven to be much more challenging. An early attempt was to use gamma-irradiated LASV particles, which produced considerable humoral responses in NHPs but failed to protect against fatal LASV challenge (332). A similar theme has been noted across subsequent studies where adaptive immunity [and even cross-protective immunity (333) ] has been induced in vaccinated subjects, but has failed to protect against lethal viral challenge. Novel reverse genetic strategies are being explored to overcome some of these obstacles, such as codon optimization to increase the expression of viral protein antigens (334), trisegmented arenaviral vectors on LCMV (335) (336) (337) (338) (339) or PICV (340, 341) backbones to increase viral attenuation, and single-cycle arenaviral vectors (342, 343) to minimize immunosuppression through exponential virus replication.

Another method to produce candidate vaccine in development is based on a reassorted virus known as ML29, which contains the L genomic segment of MOPV and the S genomic segment of LASV (269, 291, (344) (345) (346) 418 -420) ( Figure 3C) . It was determined that the LASV NP and GP antigen expressions from this reassorted virus were essential to provide strong protective immunity in rodents (e.g., guinea pigs) and NHPs (e.g., marmosets and Rhesus macaques) against lethal disease caused by LASV infection (344-347). DI particles produced by persistently infected cells with ML29 have also been found to interfere with the replication of LASV, MOPV and LCMV, and to induce strong cell-mediated immunity in STAT1 KO mice (348) , implicating the potential for formulation of ML29 with its DIs toward the development of a pan-arenaviral vaccine. Other LASV candidate vaccines include those expressing LASV GP or NP antigens from the genome of different recombinant viruses, such as Vesicular Stomatitis Virus (VSV) (349, 350) , vaccinia virus (351, 352) , MOPV (291), Yellow Fever Virus 17D (353, 354) , alphavirus replicons (355, 356) , and inactivated rabies virus (357) , all of which have been shown to exhibit various protective efficacies against LASV infection in different animal models. Other LASV vaccine development efforts, which are being supported by a non-profit international organization called Coalition for Epidemic Preparedness Innovations (CEPI), include those that are based on new vaccine platforms, such as a recombinant measles-virus-based vaccine (358) and DNA-based vaccine (359, 360) . CEPI has also supported two other LASV vaccine candidates that are based on recombinant rVSV-based vectors. The first is a rVSV construct expressing the LASV GP antigen based on the same platform applied to express the EBOV GP as a vaccine antigen that was used in a ring vaccination trial in Guinea, Africa (361, 362) . The second is another rVSV-LASV GP construct with a translocated VSV-N gene and a truncated VSV-G cytoplasmic tail designed for increased vaccine safety. Similar attenuated rVSV vectors expressing single or multiple EBOV GPs have been shown to be immunogenic in NHPs (363) and successfully protected NHPs against EBOV and Marburg virus challenge (364) (365) (366) , but there are currently no published pre-clinical data to support the development of a Lassa vaccine based on this platform. CEPI has also recently awarded a contract for pre-clinical development of yet another LASV-GP construct on the backbone of a non-replicating simian adenovirus, but again no pre-clinical information is available about the suitability of this viral platform for Lassa vaccine development (367) . It remains to be seen how well the CEPI-supported vaccines will perform in the coming years. A formidable challenge of Lassa fever vaccine development is the requirement for a development of predominantly T-cell-mediated mechanism of protection, as strong memory CD4+ T-cell responses to LASV NP and GP2 of different strains of LASV have been demonstrated in some survivors of LASV infection in Guinea (368, 369) . This is consistent with observations that, while CD4+ T-cells can provide a partial protection of ML29-vaccinated animals against lethal LASV challenge, depletion of CD8+ T-cells completely abolishes protection (370) . Additionally, NP-specific CD8+ T-cells play a major protective role in mice infected with LCMV (279) .

In addition to vaccine development as a preventative measure, the discovery of anti-viral drugs is a burgeoning area of arenaviral research. Currently, the only anti-viral treatment clinically in use that is specific to mammarenaviruses is convalescent plasma therapy against JUNV (175) . Standard anti-viral nucleoside analogs, such as Ribavirin and Favipiravir (69, (371) (372) (373) (374) have seen moderate clinical success, but are only effective when given in the earliest stages of infection when symptoms are primarily non-specific (173) . Peptideconjugated morpholino oligomers have also been tested as alternative nucleoside analogs, reducing the titers of several arenaviruses in cell culture and LCMV-infected mice (375) . Nevertheless, recovery has been documented with symptom management in place of nucleoside analogs due to a delay in diagnosis being reached after the resolution of symptoms (376) , limiting the desire for using nucleoside analogs as anti-viral compounds.

Recent advances in anti-arenaviral drug discovery have focused on identifying compounds that specifically target viral proteins or that modulate the activity of host proteins (377) . The bulk of arenaviral drug discovery has focused on small molecule inhibitors of the mammarenaviral glycoprotein and cell entry, which has seen some successes with several different approaches. Amphipathic DNA polymers have been found to block LCMV GP-α-dystroglycan interaction by the virtue of its hydrophobicity rather than nucleotide sequence (378) . Clotrimazole derivatives, which traditionally target the calcium-activated potassium channel KCa3.1, have also recently been found to inhibit arenaviral membrane fusion (379) . However, its mechanism of action was found to be independent of KCa3.1, and thus currently unclear.

Conversely, a number of other compounds have been found to stabilize the GP-α-dystroglycan prefusion complex formation, thereby blocking pH-mediated endocytosis (380) (381) (382) (383) (384) . One of these compounds has been found to reduce LASV titers in a guinea pig infection model (385) . Another small molecule (LHF-535) has recently been found to inhibit a wide variety of arenaviruses with the exception of strains that contained a V434I mutation, which corresponds to the mutation responsible for attenuation in Candid #1, suggesting that it could also affect the prefusion stability complex (386) . Finally, it has also been shown that a small molecule inhibitor of the cellular site 1 protease (PF-429292) can block GP processing (387, 388) as well as can serve as a general antiviral by inhibiting lipid and cholesterol synthesis needed for virus replication (389) (390) (391) (392) .

Some pharmaceutical compounds have also been found to disrupt the normal functions of mammarenaviral proteins. Given that myristoylation is necessary for Z-mediated arenavirus budding (160, 161) , compounds that inhibit myristoylation enzymes (161, 393) have been found to change the cellular localization of Z and therefore inhibit virion budding from the infected cells. Aromatic compounds targeting the zincfinger motifs of Z have also been found to inhibit arenaviral proliferation (394, 395) . A variety of mechanistic effects have been characterized for one of these compounds. For example, the NSC20625 compound has been found to induce Z to unfold and accumulate in oligomeric structures (394) and thereby blocking the interaction of Z with the host PML protein and allowing nuclear bodies to form (396) . The arenaviral L polymerase and NP have arguably experienced the least progress as drug targets. For the former case, one appealing potential pharmaceutical target is the cap-snatching mechanism of arenaviral polymerases. A similar drug has already been demonstrated to reduce titers of the West Nile virus (397) . The specificity of viral cap snatching mechanisms (398) and the suspicion that arenaviral cap snatching mechanisms may be conserved across species (399, 400) make cap snatching a promising therapeutic target for antiviral drug development (401) . Some metal chelators have indeed been found to potently inhibit the LCMV L endonuclease domain and its subsequent cap snatching activity, demonstrating the potential for future antivirals targeting this virus feature (402) . Lethal viral mutagenesis has also been identified as a possible mechanism for viral control (403, 404) . While few studies have found drugs that target arenaviral NPs, one recent paper has identified a compound from a pyridine library that can reduce LCMV titers post-cell entry and is thought to affect NP and Z by unknown mechanisms (405) .

Drugs that target host proteins are also being explored as potential pharmaceutical therapeutics. The tyrosine kinase inhibitor genistein is currently one of the most supported compounds, as ATF-2 phosphorylation has been found to be critical for viability of the NW PICV, which is susceptible to genistein in vitro (406, 407) and in vivo (408) . Larger scale drug screens seem to implicate cellular kinase inhibition as a promising anti-viral target in general, with compounds affecting arenaviruses at multiple stages of the virus life cycle (409) (410) (411) . Inhibitors of nucleoside production have also been tested in a variety of in vitro contexts (412) (413) (414) and may have additive effects when used in conjunction with nucleoside analogs (412) . While these compounds didn't appear to negatively affect the vitality of cell cultures, they should be cautiously explored due to possible adverse effects on some rapidly growing cells in vivo. Finally, it may be possible to target immune signaling proteins as anti-viral control, with one anti-TLR inhibitor showing limiting LCMV-initiated cytokine responses in cells and viral replication in mice (294) . For all potential anti-viral drugs, severe side effects (e.g., in neurological and embryonic development) due to known cytotoxicity of these compounds can be a great impediment to the widespread application of these compounds in treating arenavirus infections. Future anti-arenaviral drug and vaccine developments are still urgently needed to combat these deadly human pathogens.

It is important to reiterate the important roles of the IFN1 pathway and T-cell-mediated immunity in controlling mammarenavirus infections (209) . These pathways are also known to be critical for both the innate and the adaptive immune responses to other RNA virus infection models (415, 416) as well as during vaccination (417), further illustrating the importance for future studies of each of these mechanisms separately and in concert. In addition, continuation of ecological efforts to characterize the phylogeny and spread of arenaviruses (2, 9, 18, 21, 46) , socioeconomic efforts to increase the public's awareness and capacity to contain disease (49-53), and vaccine and therapeutic developments are needed in order to optimize preventative and control measures for mammarenaviruses.

MB and HL contributed to the literature review and writing of the manuscript. MB prepared all Figures and Table. FUNDING This work was supported in part by NIH NIAID grant R01 AI131586 to HL and by a pre-doctoral NIH fellowship T32 DA007097 to MB.

The authors wish to thank Dr. Y. Liang for her helpful comments and suggestions about the manuscript. 

Past, present, and future of arenavirus taxonomy

The evolutionary history of vertebrate RNA viruses

Mutagenesis-induced, large fitness variations with an invariant arenavirus consensus genomic nucleotide sequence

Phylogeny and evolution of old world arenaviruses

Phylogeny of the genus Arenavirus

Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa

Efficient rescue of recombinant Lassa virus reveals the influence of S

When viruses don't go viral: the importance of host phylogeographic structure in the spatial spread of arenaviruses

Lassa serology in natural populations of rodents and horizontal transmission. Vector-Borne Zoonot Dis

Shedding dynamics of Morogoro virus, an African arenavirus closely related to Lassa virus, in its natural reservoir host Mastomys natalensis

Arenavirus infection correlates with lower survival of its natural rodent host in a long-term capture-mark-recapture study

Historical Lassa fever reports and 30-year clinical update

Imported Lassa fever

A case of Lassa fever diagnosed at a Community Hospital-Minnesota

Favipiravir and Ribavirin treatment of epidemiologically linked cases of Lassa fever

Imported Lassa fever: a report of 2 cases in Ghana

Imported case of Lassa fever in Sweden with encephalopathy and sensorineural hearing deficit

Management of a Lassa fever outbreak

Control measures following a case of imported Lassa fever from Togo

Lassa fever in travelers from West Africa

Viral agents as biological weapons and agents of bioterrorism

Current and emerging strategies for the diagnosis, prevention and treatment of Lassa fever

Lassa fever in post-conflict sierra leone

Transmission of lymphocytic choriomeningitis virus by organ transplantation

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P

Site 1 protease is required for proteolytic processing of the glycoproteins of the South American hemorrhagic fever viruses Junin, Machupo, and Guanarito

Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication

The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain

Structural basis for antibody-mediated neutralization of Lassa virus

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor

Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins

The curious case of arenavirus entry, and its inhibition

Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C

The signal peptide of the Junín arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex

Myristoylation of the arenavirus envelope glycoprotein stable signal peptide is critical for membrane fusion but dispensable for virion morphogenesis

Role of the stable signal peptide and cytoplasmic domain of G2 in regulating intracellular transport of the Junín virus envelope glycoprotein complex

Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor

Maturation cleavage within the ectodomain of Lassa virus glycoprotein relies on stabilization by the cytoplasmic tail

Dissection of the role of the stable signal peptide of the arenavirus envelope glycoprotein in membrane fusion

Characterization of the glycoprotein stable signal peptide in mediating pichinde virus replication and virulence

The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C

Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Molecular recognition by LARGE is essential for expression of functional dystroglycan

Posttranslational modification of alpha-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding

Dystroglycan function requires xylosyl-and glucuronyltransferase activities of LARGE

Old World and Clade C New World arenaviruses mimic the molecular mechanism of receptor recognition used by-dystroglycan's host-derived ligands

Genome-wide detection and characterization of positive selection in human populations

Genome-wide scans provide evidence for positive selection of genes implicated in Lassa fever

Human hemorrhagic Fever causing arenaviruses: molecular mechanisms contributing to virus virulence and disease pathogenesis

Lassa virus entry requires a trigger-induced receptor switch

Crystal structure of the prefusion surface glycoprotein of the prototypic arenavirus LCMV

Mutational analysis of lassa virus glycoprotein highlights regions required for alpha-dystroglycan utilization

Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity

Evolution of recombinant lymphocytic choriomeningitis virus/Lassa virus in vivo highlights the importance of the GPC cytosolic tail in viral fitness

Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells

Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan

Differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics

Emerging intracellular receptors for hemorrhagic fever viruses

Molecular mechanism for LAMP1 recognition by Lassa virus

Lamp1 increases the efficiency of lassa virus infection by promoting fusion in less acidic endosomal compartments

Sodium hydrogen exchangers contribute to arenavirus cell entry

Axl can serve as entry factor for Lassa virus depending on the functional glycosylation

TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus

Virus-host interactions involved in lassa virus entry and genome replication

NRP2 and CD63 are host factors for lujo virus cell entry

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses

Human and host species transferrin receptor 1 use by North American arenaviruses

Receptor determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses

Envelope glycoprotein of arenaviruses

Utilization of human DC-SIGN and L-SIGN for entry and infection of host cells by the New World arenavirus, Junín virus

siRNA screen for genes that affect Junín virus entry uncovers voltage-gated calcium channels as a therapeutic target

Involvement of cellular proteins in Junin arenavirus entry

TRIM2, a novel member of the antiviral family, limits New World arenavirus entry

Receptor use by the Whitewater Arroyo virus glycoprotein

Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses

Old World arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport

Acidic pH triggers LCMV membrane fusion activity and conformational change in the glycoprotein spike

Kinetics and pH dependence of acid-induced structural changes in the lymphocytic choriomeningitis virus glycoprotein complex

Identification of residues in lassa virus glycoprotein subunit 2 that are critical for protein function

NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs

Transcription and RNA replication of tacaribe virus genome and antigenome analogs require N and L proteins: Z protein is an inhibitor of these processes

Replicon system for Lassa virus

A highly conserved leucine in mammarenavirus matrix Z protein is required for Z interaction with the virus L polymerase and Z stability in cells harboring an active viral ribonucleoprotein

Arenavirus Z protein controls viral RNA synthesis by locking a polymerase-promoter complex

The completed sequence of lymphocytic choriomeningitis virus reveals a unique RNA structure and a gene for a zinc finger protein

Characterization of the genomic promoter of the prototypic arenavirus lymphocytic choriomeningitis virus

Regulation of tacaribe mammarenavirus translation: positive 5 ′ and negative 3 ′ elements and role of key cellular factors

Arenavirus nucleocapsid protein displays a transcriptional antitermination activity in vivo

A single stem-loop structure in Tacaribe arenavirus intergenic region is essential for transcription termination but is not required for a correct initiation of transcription and replication

General molecular strategy for development of arenavirus live-attenuated vaccines

The high degree of sequence plasticity of the arenavirus noncoding intergenic region (IGR) enables the use of a nonviral universal synthetic IGR to attenuate arenaviruses

Tacaribe arenavirus RNA synthesis in vitro is primer dependent and suggests an unusual model for the initiation of genome replication

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys

Arenavirus infection induces discrete cytosolic structures for RNA replication

Visualization of double-stranded RNA colocalizing with pattern recognition receptors in arenavirus infected cells

Visualization of arenavirus RNA species in individual cells by singlemolecule fluorescence in situ hybridization (smFISH) suggests a model of cyclical infection and clearance during persistence

Arenaviruses: genomic RNAs, transcription, and replication

The RING domain and the L79 residue of Z protein are involved in both the rescue of nucleocapsids and the incorporation of glycoproteins into infectious chimeric arenavirus-like particles

A role for the C terminus of Mopeia virus nucleoprotein in its incorporation into Z protein-induced virus-like particles

Arenavirus Z-glycoprotein association requires Z myristoylation but not functional RING or late domains

Cis-and cell-type-dependent trans-requirements for Lassa virus-like particle production

Mapping of the tacaribe arenavirus Z-protein binding sites on the L protein identified both amino acids within the putative polymerase domain and a region at the N terminus of L that are critically involved in binding

Cellular factors required for Lassa virus budding

Myristoylation of the RING finger Z protein is essential for arenavirus budding

The role of myristoylation in the membrane association of the Lassa virus matrix protein

Recent advances in filovirusand arenavirus-like particles

The lymphocytic choriomeningitis virus matrix protein PPXY late domain drives the production of defective interfering particles

Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles

The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies

Defective viral particles and viral disease processes

Defective interfering viruses

A novel type of defective viral genome suggests a unique strategy to establish and maintain persistent lymphocytic choriomeningitis virus infections

Determinants of lymphocytic choriomeningitis interference

Defective interfering particles in mice infected with lymphocytic choriomengingitis virus

Glycoproteins of the arenaviruses

Viral persistence: mechanisms and consequences

Lassa virus

Treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy

Efficiacy of immune plasma in treatment of Argentine haemorrhagic fever and association between treatment and a late neurological syndrome

Innate immune response to arenaviral infection: a focus on the highly pathogenic new world hemorrhagic arenaviruses

NCDC/Government of Nigeria, WHO) (ECHO Daily Flash of 14

Nigeria Centre for Disease Control. An Update of Lassa Fever Outbreak in Nigeria

Nigeria hit by unprecedented Lassa fever outbreak

A prospective study of the epidemiology and ecology of Lassa fever

A prospective study of maternal and fetal outcome in acute Lassa fever infection during pregnancy

Congenital lymphocytic choriomeningitis virus: a case of prenatal diagnosis

Experimental lymphocytic choriomeningitis of monkeys and mice produced by a virus encountered in studies of the 1933 St

Clinical virology of Lassa fever in hospitalized patients

Clinical features and patient management of Lujo hemorrhagic fever

Pathogenesis of Lassa fever

The role of the vascular endothelium in arenavirus haemorrhagic fevers

Pathology of experimental machupo virus infection, chicava strain, in cynomolgus macaques (Macaca fascicularis) by intramuscular and aerosol exposure

Persistence of lassa virus associated with severe systemic arteritis in convalescing guinea pigs (Cavia porcellus)

Pathogenesis of a pichinde virus strain adapted to produce lethal infections in guinea pigs

Temporal progression of lesions in guinea pigs infected with Lassa virus

Cholestasis induced liver pathology results in dysfunctional immune responses after arenavirus infection

Chimeric mice with competent hematopoietic immunity reproduce key features of severe Lassa fever

Involvement of pro-inflammatory macrophages in liver pathology of pirital virus-infected syrian hamsters

Metabolomics analyses identify platelet activating factors and heme breakdown products as Lassa fever biomarkers

A case-control study of the clinical diagnosis and course of Lassa fever

Acute sensorineural deafness in Lassa fever

Early-onset sensorineural hearing loss in Lassa fever

Lassa fever-induced sensorineural hearing loss: a neglected public health and social burden

Animal model of sensorineural hearing loss associated with Lassa virus infection

Immune-mediated systemic vasculitis as the proposed cause of sudden-onset sensorineural hearing loss following lassa virus exposure in cynomolgus macaques

Diseases of the central nervous system caused by lymphocytic choriomeningitis virus and other arenaviruses

Lymphocytic choriomeningitis virus: an underrecognized cause of neurologic disease in the fetus, child, and adult

Lassa virus infection of human dendritic cells and macrophages is productive but fails to activate cells

Pathogenesis of Lassa fever in cynomolgus macaques

Lymphatic tissue in Argentine hemorrhagic fever. Pathologic features

Fields Virology

Exotic emerging viral diseases: progress and challenges

Inhibition of innate immune responses is key to pathogenesis by arenaviruses

Pathologic and virologic study of fatal Lassa fever in man

Experimental guinea pigs with venezuelan henorrhagic fever virus (Guanarito): a model of human disease

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures

Defective interfering particles with covalently linked [+/-]RNA induce interferon

Interferon induction by viruses. X. A model for interferon induction by Newcastle disease virus

Mechanisms of RIG-I-like receptor activation and manipulation by viral pathogens

The NF-kappaB family of transcription factors and its regulation

The IRF family transcription factors at the interface of innate and adaptive immune responses

Blockade of interferon beta, but not interferon alpha, signaling controls persistent viral infection

The interferon response circuit: induction and suppression by pathogenic viruses

Differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor-7

Early and strong immune responses are associated with control of viral replication and recovery in lassa virus-infected cynomolgus monkeys

Comparative analysis of disease pathogenesis and molecular mechanisms of New World and Old World arenavirus infections

Functional interferon system is required for clearance of Lassa virus

Infection of type i interferon receptordeficient mice with various old world arenaviruses: a model for studying virulence and host species barriers

Persistent LCMV infection is controlled by blockade of type I interferon signaling

Blockade of chronic type I interferon signaling to control persistent LCMV infection

Persistent virus infection inhibits type I interferon production by plasmacytoid dendritic cells to facilitate opportunistic infections

No measurable adverse effects of Lassa, Morogoro and Gairo arenaviruses on their rodent reservoir host in natural conditions Joachim. Parasit Vectors

Reservoir host immune responses to emerging zoonotic viruses

Lassa and Marburg viruses elicit distinct host transcriptional responses early after infection

Mice lacking alpha/beta and gamma interferon receptors are susceptible to junin virus infection

Potent inhibition of Junín virus infection by interferon in murine cells

RIG-I enhanced interferon independent apoptosis upon Junin virus infection

The interplay between viperin antiviral activity, lipid droplets and Junín mammarenavirus multiplication

Junín virus infection activates the type I interferon pathway in a RIG-I-dependent manner

Highly pathogenic New World and Old World human arenaviruses induce distinct interferon responses in human cells

Increased tumor necrosis factor-alpha levels in Argentine hemorrhagic fever

Correlation between endogenous interferon and the clinical evolution of patients with Argentine hemorrhagic fever

Proinflammatory cytokines and elastase-alpha-1-antitrypsin in Argentine hemorrhagic fever

Tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and macrophages

Cap binding and immune evasion revealed by Lassa nucleoprotein structure

Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3 ′ to 5 ′ exonuclease activity essential for immune suppression

Structure of the LCMV nucleoprotein provides a template for understanding arenavirus replication and immunosuppression

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

Crystal structure of the Lassa virus nucleoprotein-RNA complex reveals a gating mechanism for RNA binding

Induction and inhibition of type I interferon responses by distinct components of lymphocytic choriomeningitis virus

Arenaviral nucleoproteins suppress PACT-induced augmentation of RIG-I function to inhibit type I interferon production

Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKK

Ollmann Saphire E. Structural basis for the dsRNA specificity of the lassa virus NP exonuclease

Arenavirus nucleoproteins prevent activation of nuclear factor kappa B

Lassa virus nucleoprotein mutants generated by reverse genetics induce a robust type I interferon response in human dendritic cells and macrophages

Exonuclease domain of the Lassa virus nucleoprotein is critical to avoid RIG-I signaling and to inhibit the innate immune response

The exonuclease domain of Lassa virus nucleoprotein is involved in antigen-presenting-cell-mediated NK cell responses

Differential inhibition of type I interferon induction by arenavirus nucleoproteins

Identification of critical amino acids within the nucleoprotein of tacaribe virus important for anti-interferon activity

In vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions

Z Proteins of New World arenaviruses bind RIG-I and interfere with type I interferon induction

The Z proteins of pathogenic but not nonpathogenic arenaviruses inhibit RIG-i-like receptor-dependent interferon production

Differential inhibition of macrophage activation by lymphocytic choriomeningitis virus and pichinde virus is mediated by the Z protein N-terminal domain

Differential immune responses to new world and old world mammalian arenaviruses

The pathology of lassa fever

Hematologic dysfunction in Lassa fever

Comparative pathogenesis of passaged Pichinde virus variants in outbred guinea pigs

Cytokine patterns in a comparative model of arenavirus haemorrhagic fever in guinea pigs

Pathogenesis of bolivian hemorrhagic fever in guinea pigs

Mice deficient in STAT1 but not STAT2 or IRF9 develop a lethal CD4+ T-cell-mediated disease following infection with lymphocytic choriomeningitis virus

Decoding arenavirus pathogenesis: essential roles for alpha-dystroglycan-virus interactions and the immune response

Improving the breadth of the host's immune response to Lassa virus

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

Bacterial coinfection restrains antiviral CD8 T-cell response via LPS-induced inhibitory NK cells

Induction or prevention of immunopathological disease by cloned cytotoxic T cell lines specific for lymphocytic choriomeningitis virus

The acute inflammatory process in murine lymphocytic choriomeningitis is dependent on Lyt-2+ immune T cells

Immunogenetic analysis of cellular interactions governing the recruitment of T lymphocytes and monocytes in lymphocytic choriomeningitis virus-induced immunopathology

Dissection of an inflammatory process induced by CD8+ T cells

Immune response against lymphocytic choriomeningitis virus infection in mice without CD8 expression

The role of CD4+ T cells in cell-mediated immunity to LCM V: studies in MHC class I and class II deficient mice

Use of a high-affinity peptide that aborts mhc-restricted cytotoxic T lymphocyte activity against multiple viruses in vitro and virusinduced immunopathologic disease in vivo

Absence of CTL responses to early viral antigens facilitates viral persistence

Functional analysis of T lymphocyte subsets in antiviral host defense

Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody

Human dendritic cell subsets

Conventional but not plasmacytoid dendritic cells foster the systemic virus-induced type I IFN response needed for efficient CD8 T cell priming

Protection of rhesus monkeys from Lassa virus by immunisation with closely related arenavirus

Isolation of an arenavirus closely related to Lassa virus from Mastomys natalensis in South-East Africa

Human macrophages, but not dendritic cells, are activated and produce alpha/beta interferons in response to Mopeia virus infection

Human dendritic cells infected with the nonpathogenic Mopeia virus induce stronger T-cell responses than those infected with Lassa virus

Lassa virus activates myeloid dendritic cells but suppresses their ability to stimulate T cells

Dendritic cells are dispensable for T cell priming and control of acute lymphocytic choriomeningitis virus infection

The effects of dendritic cell hypersensitivity on persistent viral infection

A vaccine platform against arenaviruses based on a recombinant hyperattenuated mopeia virus expressing heterologous glycoproteins

Junin virus infects mouse cells and induces innate immune responses

Toll-like receptor 2-mediated innate immune responses against Junín virus in mice lead to antiviral adaptive immune responses during systemic infection and do not affect viral replication in the brain

Discovery of a novel TLR2 signaling inhibitor with anti-viral activity

Pathogenic old world arenaviruses inhibit TLR2/Maldependent proinflammatory cytokines in vitro

Cleavage of the Junin virus nucleoprotein serves a decoy function to inhibit the induction of apoptosis during infection

The New World arenavirus Tacaribe virus induces caspase-dependent apoptosis in infected cells

Apoptosis during arenavirus infection: mechanisms and evasion strategies

Delayed inflammatory and cell death responses are associated with reduced pathogenicity in Lujo virus-infected cynomolgus macaques

PACT, a protein activator of the interferoninduced protein kinase, PKR

PACT, a double-stranded RNA binding protein acts as a positive regulator for type I interferon gene induced by newcastle disease virus

The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA

Approaching the RNA ligand for RIG-I?

Mutual antagonism between the ebola virus VP35 protein and the RIG-I activator PACT determines infection outcome

Middle East respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response

Quantitative proteomic analysis of the influenza A virus nonstructural proteins NS1 and NS2 during natural cell infection identifies PACT as an NS1 target protein and antiviral host factor

Suppression of PACT-induced type I interferon production by herpes simplex virus 1 Us11 protein

PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR

Modular structure of PACT: distinct domains for binding and activating PKR

Molecular basis for PKR activation by PACT or dsRNA

The role of PACT in mediating gene induction, PKR activation, and apoptosis in response to diverse stimuli

TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress

Stress-induced phosphorylation of PACT reduces its interaction with TRBP and leads to PKR activation

Increased interaction between PACT molecules in response to stress signals is required for PKR activation

Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures

The glycoprotein precursor gene of the attenuated junin virus vaccine strain (Candid #1)

Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group

Junin virus vaccines

Immune response to vaccination against argentine hemorrhagic fever in an area where different arenaviruses coexist

Genomic features of attenuated junín virus vaccine strain candidate

Molecular analysis of the virulence attenuation process in Junín virus vaccine genealogy

The major determinant of attenuation in mice of the Candid1 vaccine for Argentine hemorrhagic fever is located in the G2 glycoprotein transmembrane domain

The glycoprotein precursor gene of Junin virus determines the virulence of the Romero strain and the attenuation of the Candid #1 strain in a representative animal model of Argentine hemorrhagic fever

A substitution in the transmembrane region of the glycoprotein leads to an unstable attenuation of Machupo virus

Interferon regulatory factor 7 (IRF7) is required for the optimal initial control but not subsequent clearance of lymphocytic choriomeningitis virus infection in mice

Junín virus pathogenesis and virus replication

Arenavirus glycan shield promotes neutralizing antibody evasion and protracted infection

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies

Inactivated Lassa virus elicits a non protective immune response in rhesus monkeys

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Development of live-attenuated arenavirus vaccines based on codon deoptimization of the viral glycoprotein

Expanded potential for recombinant trisegmented lymphocytic choriomeningitis viruses: protein production, antibody production, and in vivo assessment of biological function of genes of interest

Arenavirus reverse genetics for vaccine development

Generation of recombinant arenavirus for vaccine development in FDA-approved Vero cells

Arenavirus genome rearrangement for the development of live attenuated vaccines

Reverse genetics approaches to control arenavirus

A novel live pichinde virus-based vaccine vector induces enhanced humoral and cellular immunity after a booster dose

Recombinant tri-segmented pichinde virus as a novel live viral vaccine platform

Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses

Lymphocytic choriomeningitis virus (LCMV) infection of macaques: a model for Lassa fever

An attenuated Lassa vaccine in SIV-infected rhesus macaques does not persist or cause arenavirus disease but does elicit Lassa virus-specific immunity

A ML29 reassortant virus protects guinea pigs against a distantly related Nigerian strain of Lassa virus and can provide sterilizing immunity

Attenuated replication of lassa virus vaccine candidate ML29 in STAT-1-/-mice

Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses

Development of a new vaccine for the prevention of Lassa fever

Vaccinia recombinant expressing lassa-virus internal nucleocapsid protein protects guinea pigs against lassa fever

Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection

A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins

Non-neutralizing antibodies elicited by recombinant Lassa-Rabies vaccine are critical for protection against Lassa fever

Immunogenicity, safety, and tolerability of a recombinant measles-virusbased chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial

Evaluating the immunogenicity and protective efficacy of a DNA vaccine encoding Lassa virus nucleoprotein

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever

Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial

Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!)

Neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates

Vaccination with a highly attenuated recombinant vesicular stomatitis virus vector protects against challenge with a lethal dose of ebola virus

Singledose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus

Single dose trivalent vesiculovax vaccine protects macaques from lethal ebolavirus and marburgvirus challenge

Awards Contract Worth Up To USD$19 Million to Oxford University and Janssen Vaccines to Develop MERS, Lassa, and Nipah Vaccines

Characterization of human CD4+ T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein

Old and New World arenaviruses share a highly conserved epitope in the fusion domain of the glycoprotein 2, which is recognized by Lassa virus-specific human CD4+ T-cell clones

Evaluation of Lassa virus vaccine immunogenicity in a CBA/J-ML29 mouse model

T-705 (favipiravir) inhibition of arenavirus replication in cell culture

Effective oral favipiravir (T-705) therapy initiated after the onset of clinical disease in a model of arenavirus hemorrhagic Fever

Favipiravir pharmacokinetics in nonhuman primates and insights for future efficacy studies of hemorrhagic fever viruses

Favipiravir as a potential countermeasure against neglected and emerging RNA viruses

Development of peptide-conjugated morpholino oligomers as panarenavirus inhibitors

Lassa fever-full recovery without ribavarin treatment: a case report

Targeting virulence mechanisms for the prevention and therapy of arenaviral hemorrhagic fever

Inhibition of cellular entry of lymphocytic choriomeningitis virus by amphipathic DNA polymers

Identification of clotrimazole-derivatives as specific inhibitors of Arenavirus fusion

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

pH-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors

Identification of a broad-spectrum arenavirus entry inhibitor

Identification and mechanism of action of a novel small-molecule inhibitor of arenavirus multiplication

Screening and identification of lassa virus entry inhibitors from an FDA-approved running title: drug repurposing for LASV entry

Evaluation of Lassa antiviral compound ST-193 in a guinea pig model

A potent Lassa virus antiviral targets an arenavirus virulence determinant

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242

Human subtilase SKI-1/S1P is a master regulator of the HCV lifecycle and a potential host cell target for developing indirect-acting antiviral agents

SKI-1/S1P inhibition: a promising surrogate to statins to block Hepatitis C virus replication

SKI-1/S1P inhibitor PF-429242 impairs the onset of HCV infection

Suppressive effects of the site 1 protease (S1P) inhibitor, PF-429242, on dengue virus propagation

Myristic acid analogs are inhibitors of Junin virus replication

Arenavirus Z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds

Inhibition of arenavirus infection by thiuram and aromatic disulfides

An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism

Comparative structural and functional analysis of bunyavirus and arenavirus cap-snatching endonucleases

Role of the C terminus of Lassa virus L protein in viral mRNA synthesis

Structural insights into reptarenavirus cap-snatching machinery

The viral RNA capping machinery as a target for antiviral drugs

Metal chelators for the inhibition of the lymphocytic choriomeningitis virus endonuclease domain

Arenavirus extinction through lethal mutagenesis

Prospects for new Ribavirin-based interventions

Mining a kröhnke pyridine library for anti-arenavirus activity

Genistein treatment of cells inhibits arenavirus infection

Inhibition of Lassa virus and Ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin

Genistein, a general kinase inhibitor, as a potential antiviral for arenaviral hemorrhagic fever as described in the Pirital virus-Syrian golden hamster model

Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junín virus replication in vitro

The PI3K/Akt pathway contributes to arenavirus budding

Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses

Inhibition of arenavirus by A3, a pyrimidine biosynthesis inhibitor

Lifecycle modelling systems support inosine monophosphate dehydrogenase (IMPDH) as a pro-viral factor and antiviral target for New World arenaviruses

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus

The microRNA miR-155 controls CD8+ T cell responses by regulating interferon signaling

RIG-I-like receptor activation by dengue virus drives follicular T helper cell formation and antibody production

Virus-derived immunostimulatory RNA induces type I IFN-dependent antibodies and T-cell responses during vaccination

Safety, immunogenicity, and efficacy of the ML29 reassortant vaccine for Lassa fever in small non-human primates

Lukashevich IS. Generation of reassortants between African arenaviruses

Supplemental Table 1 | Summary of documented cases of international importation of Lassa fever.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Copyright © 2019 Brisse and Ly. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.