Junin Virus Infection Research Articles

Coadministration of LHF-535 and favipiravir protects against experimental Junín virus infection and disease

Argentine hemorrhagic fever, caused by Junín virus (JUNV), is the most common of the South American arenaviral hemorrhagic fevers. The disease has a case fatality rate of 15–30% in untreated patients. Although early intervention with immune plasma is effective, diminishing stocks and limited availability outside of Argentina underscores the need for new therapeutics. Ideally, these would be broadly active agents effective against all the pathogenic arenaviruses. The fusion inhibitor LHF-535 and the nucleoside analog favipiravir have shown promise in animal models of Lassa fever, a disease endemic in parts of Africa and the most prominent of the arenaviral hemorrhagic fevers. Against JUNV, a high dose of favipiravir is required to achieve protection in the gold-standard guinea pig infection model. Here, we demonstrate a synergistic effect by the coadministration of LHF-535 with a sub-optimal dose of favipiravir in guinea pigs challenged with JUNV. Administered individually, LHF-535 and sub-optimal favipiravir only delayed the onset of severe disease. However, combined dosing of the drugs afforded complete protection against lethal JUNV infection in guinea pigs. The benefits of the drug combination were also evident by the absence of viremia and infectious virus in tissues compared to guinea pigs treated with only the placebos. Thus, combined targeting of JUNV-endosomal membrane fusion and the viral polymerase with pan-arenaviral LHF-535 and favipiravir may expand their indication beyond Lassa fever, providing a significant barrier to drug resistance.

Modulation of the Aryl Hydrocarbon Receptor Signaling Pathway Impacts on Junín Virus Replication.

Junín virus (JUNV), a member of the family Arenaviridae, is the etiological agent of the Argentine hemorrhagic fever, an endemic disease in the rural region of Argentina lacking a specific chemotherapy. Aryl hydrocarbon receptor (AHR) is expressed in several mammalian tissues and has been indicated as a sensor of ligands from variable sources and a modulator of the cell immune response. Interestingly, recent studies have suggested that the activation or depression of the AHR signaling pathway may play a role in the outcome of diverse human viral infections. In the present report, the effect of the pharmacological modulation of AHR on JUNV in vitro infection was analyzed. An initial microarray screening showed that the AHR pathway was overexpressed in JUNV-infected hepatic cells. Concomitantly, the infection of Vero and Huh-7 cells with the JUNV strains IV4454 and Candid#1 was significantly inhibited in a dose-dependent manner by treatment with CH223191, a specific AHR antagonist, as detected by infectivity assays, real-time RT-PCR and immunofluorescence detection of viral proteins. Furthermore, the pro-viral role of AHR in JUNV infection appears to be independent of the IFN-I pathway. Our findings support the promising perspectives of the pharmacological modulation of AHR as a potential target for the control of AHF.

Recent developments on Junin virus, a causative agent for Argentine haemorrhagic fever.

Junin virus consists of ribonucleic acid as the genome and is responsible for a rapidly changing tendency of the virus. The virus is accountable for ailments in the human body and causes Argentine Haemorrhagic Fever (AHF). The infection is may be transmitted through contact between an infected animal/host and a person, and later between person to person. Prevention of outbreaks of AHF in humans can be a tough practice, as their occurrence is infrequent and unpredictable. In this review, recent information from the past 5years available on the Junin virus including the risk of its emergence, infectious agents, its pathogenesis in humans, available diagnostic and therapeutic approaches, and disease management has been summarised. Altogether, this article would be highly significant in understanding the mechanistic basis behind virus interaction and other processes during the life cycle. Currently, no specific therapeutic options are available to treat the Junin virus infection. The information covered in this review could be important for finding possible treatment options for Junin virus infections.

The Virus-Host Interplay in Junín Mammarenavirus Infection.

Junín virus (JUNV) belongs to the Arenaviridae family and is the causative agent of Argentine hemorrhagic fever (AHF), a severe human disease endemic to agricultural areas in Argentina. At this moment, there are no effective antiviral therapeutics to battle pathogenic arenaviruses. Cumulative reports from recent years have widely provided information on cellular factors playing key roles during JUNV infection. In this review, we summarize research on host molecular determinants that intervene in the different stages of the viral life cycle: viral entry, replication, assembly and budding. Alongside, we describe JUNV tight interplay with the innate immune system. We also review the development of different reverse genetics systems and their use as tools to study JUNV biology and its close teamwork with the host. Elucidating relevant interactions of the virus with the host cell machinery is highly necessary to better understand the mechanistic basis beyond virus multiplication, disease pathogenesis and viral subversion of the immune response. Altogether, this knowledge becomes essential for identifying potential targets for the rational design of novel antiviral treatments to combat JUNV as well as other pathogenic arenaviruses.

Junin Virus Activates p38 MAPK and HSP27 Upon Entry.

Junín virus (JUNV), a New World arenavirus, is a rodent-borne virus and the causative agent of Argentine hemorrhagic fever. Humans become infected through exposure to rodent host secreta and excreta and the resulting infection can lead to an acute inflammatory disease with significant morbidity and mortality. Little is understood about the molecular pathogenesis of arenavirus hemorrhagic fever infections. We utilized Reverse Phase Protein Microarrays (RPPA) to compare global alterations in the host proteome following infection with an attenuated vaccine strain, Candid#1 (CD1), and the most parental virulent strain, XJ13, of JUNV in a human cell culture line. Human small airway epithelial cells were infected with CD1 or XJ13 at an MOI of 10, or mock infected. To determine proteomic changes at early timepoints (T = 1, 3, 8 and 24 h), the JUNV infected or mock infected cells were lysed in compatible buffers for RPPA. Out of 113 proteins that were examined by RPPA, 14 proteins were significantly altered following JUNV infection. Several proteins were commonly phosphorylated between the two strains and these correspond to entry and early replication events, to include p38 mitogen-activated protein kinase (MAPK), heat shock protein 27 (HSP27), and nuclear factor kappa B (NFκB). We qualitatively confirmed the alterations of these three proteins following infection by western blot analysis. We also determined that the inhibition of either p38 MAPK, with the small molecule inhibitor SB 203580 or siRNA knockdown, or HSP27, by siRNA knockdown, significantly decreases JUNV replication. Our data suggests that HSP27 phosphorylation at S82 upon virus infection is dependent on p38 MAPK activity. This work sheds light on the nuances of arenavirus replication.

Inhibitors of the p38 cell signaling pathway as antiviral compounds against Junín virus.

In the present study, we analyzed the modulation of p38 cell signaling by Junín virus (JUNV) and evaluated the antiviral activity of p38 inhibitors against JUNV. While JUNV induced a progressive activation of p38 throughout the infection in Vero cells, a partial downregulation of p38 phosphorylation was observed in HEK293 and HeLa cells. The compounds SB203580 and SB202190, which are selective inhibitors of p38, significantly reduced viral protein expression and viral yield in the cell lines examined, indicating that the p38 signaling pathway might be a promising antiviral target against JUNV infection.

Type I interferon underlies severe disease associated with Junín virus infection in mice.

Junín virus (JUNV) is one of five New World mammarenaviruses (NWMs) that causes fatal hemorrhagic disease in humans and is the etiological agent of Argentine hemorrhagic fever (AHF). The pathogenesis underlying AHF is poorly understood; however, a prolonged, elevated interferon-α (IFN-α) response is associated with a negative disease outcome. A feature of all NWMs that cause viral hemorrhagic fever is the use of human transferrin receptor 1 (hTfR1) for cellular entry. Here, we show that mice expressing hTfR1 develop a lethal disease course marked by an increase in serum IFN-α concentration when challenged with JUNV. Further, we provide evidence that the type I IFN response is central to the development of severe JUNV disease in hTfR1 mice. Our findings identify hTfR1-mediated entry and the type I IFN response as key factors in the pathogenesis of JUNV infection in mice.

Human BST-2/tetherin inhibits Junin virus release from host cells and its inhibition is partially counteracted by viral nucleoprotein.

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

Lassa Virus, but Not Highly Pathogenic New World Arenaviruses, Restricts Immunostimulatory Double-Stranded RNA Accumulation during Infection.

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

Monoclonal Antibodies with Neutralizing Activity and Fc-Effector Functions against the Machupo Virus Glycoprotein

Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever (BHF), is a New World arenavirus that was first isolated in Bolivia from a human spleen in 1963. Due to the lack of a specific vaccine or therapy, this virus is considered a major risk to public health and is classified as a category A priority pathogen by the U.S. National Institutes of Health. In this study, we used DNA vaccination against the MACV glycoprotein precursor complex (GPC) and murine hybridoma technology to generate 25 mouse monoclonal antibodies (MAbs) against the GPC of MACV. Out of 25 MAbs, five were found to have potent neutralization activity in vitro against a recombinant vesicular stomatitis virus expressing MACV GPC (VSV-MACV) as well as against authentic MACV. Furthermore, the five neutralizing MAbs exhibited strong antibody-dependent cellular cytotoxicity (ADCC) activity in a reporter assay. When tested in vivo using VSV-MACV in a Stat2-/- mouse model, three MAbs significantly lowered viral loads in the spleen. Our work provides valuable insights into epitopes targeted by neutralizing antibodies that could be potent targets for vaccines and therapeutics and shed light on the importance of effector functions in immunity against MACV.IMPORTANCE MACV infections are a significant public health concern and lead to high case fatality rates. No specific treatment or vaccine for MACV infections exist. However, cases of Junin virus infection, a related virus, can be treated with convalescent-phase serum. This indicates that a MAb-based therapy for MACV could be effective. Here, we describe several MAbs that neutralize MACV and could be used for this purpose.

Guinea Pig Transferrin Receptor 1 Mediates Cellular Entry of Junín Virus and Other Pathogenic New World Arenaviruses.

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

Junín virus induces autophagy in human A549 cells.

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

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species.

Spatial difference in the incidence of Argentine Hemorrhagic Fever and composition and abundance of rodents in the assemblage

The Argentine Hemorrhagic Fever (AHF) is a zoonotic disease endemic in a wide area of the humid pampa of Argentina. The etiologic agent is the Junin virus that is maintained in the wild by the rodent Calomys musculinus and transmitted to humans, mainly, through aerosols generated from secretions and excretions. To characterize and compare the assemblages of small rodent composition and diversity inside the epidemic, historic and non-endemic zone of AHF and to register C. musculinus abundance in each zone and in each area within each zone, registering the prevalence of infection in rodent populations. One central and two peripheral areas were delimited to sample rodents in each zone with different incidence of AHF. Thus, 18 localities were selected to do the sampling in two years. Host abundance between zones and among areas inside each zone and among nearby areas between zones were compared applying nested ANOVA's. In each zone, the rodent assemblage showed differences in composition, diversity and numeric representation of C. musculinus. The epidemic zone was the richest of the three, registering also great host abundance; meanwhile in the historic zone, A. azarae was the dominant numeric species with less number of other species. Regarding the non-endemic zone, the assemblage composition and C. musculinus abundance varied respect the sampled year. Junin virus infection was only detected in C. musculinus individuals corresponding to the epidemic zone, with a prevalence of 2.7 and 1.1% for the years 2007 and 2008, respectively. In this system, the abundance of C. musculinus could be impacting over the pathogen dynamic, rather than the assemblage diversity or the A. azarae presence.

Antiviral activity of Tripartite-Motif 2 protein against New World Arenaviruses

Background: New World Arenaviruses (NWA) are single-stranded bisegmented RNA viruses that cause viral hemorrhagic fevers in humans. NWAs are endemic in New World rodents and are transmitted to humans by aerosol. Host factors that alter entry and infection by Junín virus (JUNV), a NWA found in Argentina, were identified through a high throughput RNAi screen. Among them was an antiviral restriction factor, TRIM2, a member of the tripartite-motif (TRIM) family, many of which have effector roles responses to viral infections. TRIM proteins share a RING finger-B box-Coiled coil (RBCC) motif at the N-terminus but have diverse C-terminal domains; TRIM2 contains FIL/NHL domain. TRIMs are E3 ubiquitin ligases whose activity is conferred by the RING domain. Our data suggests that TRIM2 acts at the viral entry step, which would be unique for TRIM proteins. Methods & Materials: Using CRISPR/Cas9 technology we generated different TRIM2 mutant mice with different deletions. Bone Marrow-derived macrophages (BMDMs) were isolated from the mutant mice, and infected with the JUNV vaccine strain Candid 1 for 24 hours. RNA was extracted from the cells and subjected to RT-qPCR to assess the levels of infection. JUNV was also detected by immunoblotting using an anti NP monoclonal antibody. Eight old weeks mice were infected intracranially with 1 × 104 PFU of Candid 1 for 5 days and the RNA was assayed as described above. Constructs containing different domains of TRIM2 were cloned into expression vectors and transfected into 293T cells followed by JUNV infection to assess the domain involved in the antiviral activity. TRIM2-interacting proteins were identified by co-immunoprecipitation from BMDMs and JUNV-infected brains using an anti-TRIM2 monoclonal antibody. Results: We observed higher levels of JUNV infection in the TRIM2 mutant cells and mice in comparison to wild-type. We found that the FIL domain, but not the RING domain, was involved in the antiviral activity and that TRIM2 interacted with signal regulatory protein alpha (SIRPA), which had anti-NWA activity. Conclusion: TRIM2 is an anti-arenaviral host factor and might work through SIRPA by a mechanism not fully elucidated yet. This type of restriction has not been previously described for any TRIM protein.

Novel neutralizing monoclonal antibodies against Junin virus

Junin virus (JUNV) is the pathogen of Argentine haemorrhagic fever which results in high lethality, while there is limited therapeutics available. Here, a series of mouse monoclonal antibodies (mAbs) were isolated through immunization with DNA and screening against glycoprotein complex (GPC). Finally, five mAbs were found to effectively neutralize JUNV. Further research indicated that they were capable of binding conformational GPC and strongly binding glycoprotein 1 which is responsible for receptor recognition. Epitope mapping revealed that they targeted loop 3 of GP1, and Tyr122 and Asp123 in loop 3 were identified as their common binding sites, which may account for their neutralizing activity. This study presents a new strategy for developing neutralizing antibodies against JUNV and provides therapeutic candidates for protection against JUNV infection.

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

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

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

Junín arenavirus infections are associated with high levels of interferons in both severe and fatal cases. Upon Junín virus (JUNV) infection a cell signaling cascade initiates, that ultimately attempts to limit viral replication and prevent infection progression through the expression of host antiviral proteins. The interferon stimulated gene (ISG) viperin has drawn our attention as it has been highlighted as an important antiviral protein against several viral infections. The studies of the mechanistic actions of viperin have described important functional domains relating its antiviral and immune-modulating actions through cellular lipid structures. In line with this, through silencing and overexpression approaches, we have identified viperin as an antiviral ISG against JUNV. In addition, we found that lipid droplet structures are modulated during JUNV infection, suggesting its relevance for proper virus multiplication. Furthermore, our confocal microscopy images, bioinformatics and functional results also revealed viperin-JUNV protein interactions that might be participating in this antiviral pathway at lipid droplet level. Altogether, these results will help to better understand the factors mediating innate immunity in arenavirus infection and may lead to the development of pharmacological agents that can boost their effectiveness thereby leading to new treatments for this viral disease.

Highly Pathogenic New World Arenavirus Infection Activates the Pattern Recognition Receptor Protein Kinase R without Attenuating Virus Replication in Human Cells.

The arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication.IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide new evidence for a distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provides insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.

Enhanced protection against experimental Junin virus infection through the use of a modified favipiravir loading dose strategy

A collection of Old and New World arenaviruses are etiologic agents of viral hemorrhagic fever, a syndrome that features hematologic abnormalities, vascular leak, hypovolemia, and multi-organ failure. Treatment is limited to ribavirin for Lassa fever and immune plasma for Argentine hemorrhagic fever. Improved therapeutic options that are safe, more effective and widely available are needed. Here, we show that modification of favipiravir treatment to include a high-dose loading period achieves complete protection in a guinea pig model of Argentine hemorrhagic fever when treatment was initiated two days following challenge with Junin virus (JUNV). This loading dose strategy also protected 50% of animals from lethal disease when treatment was delayed until 5 days post-infection and extended the survival time in those that succumbed. Consistent with the survival data, dramatic reductions in serum and tissue virus loads were observed in animals treated with favipiravir. This is the first report demonstrating complete protection against uniformly lethal JUNV infection in guinea pigs by administration of a small molecule antiviral drug.