Novel Oliveros-like Clade C Mammarenaviruses from Rodents in Argentina, 1990-2020.

Following an Argentine Hemorrhagic Fever (AHF) outbreak in the early 1990s, a rodent survey for Junín virus, a New World Clade B arenavirus, in endemic areas of Argentina was conducted. Since 1990, INEVH has been developing eco-epidemiological surveillance of rodents, inside and outside the Argentine Hemorrhagic Fever endemic area. Samples from rodents captured between 1993 and 2019 that were positive for Arenavirus infection underwent Sanger and unbiased, Illumina-based high-throughput sequencing, which yielded 5 complete and 88 partial Mammarenaviruses genomes. Previously, 11 genomes representing four species of New World arenavirus Clade C existed in public records. This work has generated 13 novel genomes, expanding the New World arenavirus Clade C to 24 total genomes. Additionally, two genomes exhibit sufficient genetic diversity to be considered a new species, as per ICTV guidelines (proposed name Mammarenavirus vellosense). The 13 novel genomes exhibited reassortment between the small and large segments in New World Mammarenaviruses. This work demonstrates that Clade C Mammarenavirus infections circulate broadly among Necromys species in the Argentine Hemorrhagic Fever endemic area; however, the risk for Clade C Mammarenavirus human infection is currently unknown.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

Nuclease Activity of the Junín Virus Nucleoprotein C-Terminal Domain.

The mammarenavirus Junín (JUNV) is the causative agent of Argentine hemorrhagic fever, a severe disease of public health concern. The most abundant viral protein is the nucleoprotein (NP), a multifunctional, two-domain protein with the primary role as structural component of the viral nucleocapsids, used as template for viral polymerase RNA synthesis activities. Here, we report that the C-terminal domain (CTD) of the attenuated Candid#1 strain of the JUNV NP can be purified as a stable soluble form with a secondary structure in line with known NP structures from other mammarenaviruses. We show that the JUNV NP CTD interacts with the viral matrix protein Z in vitro, and that the full-length NP and Z interact with each other in cellulo, suggesting that the NP CTD is responsible for this interaction. This domain comprises an arrangement of four acidic residues and a histidine residue conserved in the active site of exoribonucleases belonging to the DEDDh family. We show that the JUNV NP CTD displays metal-ion-dependent nuclease activity against DNA and single- and double-stranded RNA, and that this activity is impaired by the mutation of a catalytic residue within the DEDDh motif. These results further support this activity, not previously observed in the JUNV NP, which could impact the mechanism of the cellular immune response modulation of this important pathogen.

The roles of XJ13 and XJ44-specific mutations within the Candid #1 GPC in Junin virus attenuation.

Junin virus (JUNV) is a member of the Arenaviridae family of viruses and is the pathogen responsible for causing Argentine hemorrhagic fever, a potentially lethal disease endemic to Argentina. A live attenuated vaccine for human use, called Candid#1, is approved only in Argentina. Candid#1 vaccine strain of Junin virus was obtained through serial passage in mouse brain tissues followed by passage in Fetal Rhesus macaque lung fibroblast (FRhL) cells. Previously, the mutations responsible for attenuation of this virus in Guinea pigs were mapped in the gene encoding for glycoprotein precursor (GPC) protein. The resulting Candid#1 glycoprotein complex has been shown to cause endoplasmic reticulum (ER) stress in vitro resulting in the degradation of the GPC. To evaluate the attenuating properties of specific mutations within GPC, we created recombinant viruses expressing GPC mutations specific to key Candid#1 passages and evaluated their pathogenicity in our outbred Hartley guinea pig model of Argentine hemorrhagic fever. Here, we provide evidence that early mutations in GPC obtained through serial passaging attenuate the visceral disease and increase immunogenicity in guinea pigs. Specific mutations acquired prior to the 13th mouse brain passage (XJ13) are responsible for attenuation of the visceral disease while having no impact on the neurovirulence of Junin virus. Additionally, our findings demonstrate that the mutation within an N-linked glycosylation motif, acquired prior to the 44th mouse brain passage (XJ44), is unstable but necessary for complete attenuation and enhanced immunogenicity of Candid#1 vaccine strain. The highly conserved N-linked glycosylation profiles of arenavirus glycoproteins could therefore be viable targets for designing attenuating viruses for vaccine development against other arenavirus-associated illnesses.

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 5 years 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.

Potential and action mechanism of favipiravir as an antiviral against Junin virus.

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.

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.

Machupo Virus with Mutations in the Transmembrane Domain and Glycosylation Sites of the Glycoprotein Is Attenuated and Immunogenic in Animal Models of Bolivian Hemorrhagic Fever.

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuates MACV in an interferon alpha/beta and gamma (IFN-α/ß and -γ) receptor knockout (R-/-) mouse model. However, some capability to induce neurological pathology still remained. The highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. An F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in the GPC TMD. The MACV mutant is fully attenuated in IFN-α/ß and -γ R-/- mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV completely protected guinea pigs from wild-type MACV lethal challenge. Last, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which provide insight into the rational design of potential vaccine candidates for highly pathogenic arenaviruses. IMPORTANCE For arenaviruses, the only vaccine available is the live attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

Structural basis for recognition and regulation of arenavirus polymerase L by Z protein.

Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.

Second-Generation Live-Attenuated Candid#1 Vaccine Virus Resists Reversion and Protects against Lethal Junín Virus Infection in Guinea Pigs.

Live-attenuated virus vaccines are highly effective in preventing viral disease but carry intrinsic risks of residual virulence and reversion to pathogenicity. The classically derived Candid#1 virus protects seasonal field workers in Argentina against zoonotic infection by Junín virus (JUNV) but is not approved in the United States, in part due to the potential for reversion at the attenuating locus, a phenylalanine-to-isoleucine substitution at position 427 in the GP2 subunit of the GPC envelope glycoprotein. Previously, we demonstrated facile reversion of recombinant Candid#1 (rCan) in cell culture and identified an epistatic interaction between the attenuating I427 and a secondary K33S mutation in the stable signal peptide (SSP) subunit of GPC that imposes an evolutionary barrier to reversion. The magnitude of this genetic barrier is manifest in our repeated failures to rescue the hypothetical revertant virus. In this study, we show that K33S rCan is safe and attenuated in guinea pigs and capable of eliciting potent virus-neutralizing antibodies. Immunized animals are fully protected against lethal challenge with virulent JUNV. In addition, we employed a more permissive model of infection in neonatal mice to investigate genetic reversion. RNA sequence analysis of the recovered virus identified revertant viruses in pups inoculated with the parental rCan virus and none in mice receiving K33S rCan (P < 0.0001). Taken together, our findings support the further development of K33S rCan as a safe second-generation JUNV vaccine. IMPORTANCE Our most successful vaccines comprise weakened strains of virus that initiate a limited and benign infection in immunized persons. The live-attenuated Candid#1 strain of Junín virus (JUNV) was developed to protect field workers in Argentina from rodent-borne hemorrhagic fever but is not licensed in the United States, in part due to the likelihood of genetic reversion to virulence. A single-amino-acid change in the GPC envelope glycoprotein of the virus is responsible for attenuation, and a single nucleotide change may regenerate the pathogenic virus. Here, we take advantage of a unique genetic interaction between GPC subunits to design a mutant Candid#1 virus that establishes an evolutionary barrier to reversion. The mutant virus (K33S rCan) is fully attenuated and protects immunized guinea pigs against lethal JUNV infection. We find no instances of reversion in mice inoculated with K33S rCan. This work supports the further development of K33S rCan as a second-generation JUNV vaccine.

A single mutation (V64G) within the RING Domain of Z attenuates Junin virus.

Junin virus (JUNV) is a New World arenavirus that is the causative agent of Argentine hemorrhagic fever (AHF). Candid#1 (Can) is a live-attenuated vaccine strain of JUNV that since its introduction has resulted in a marked decrease in AHF incidence within the endemic regions of the Pampas in Argentina. Originally, the viral determinants and mechanisms of Can attenuation were not well understood. Recent work has identified the glycoprotein as the major attenuating factor for Can. The establishment of attenuating strategies based on any of the other viral proteins, however, has not been pursued. Here, we document the role of Can Z resulting in incompatibilities with wild type JUNV that results in decreased growth in vitro. In addition, this incompatibility results in attenuation of the virus in the guinea pig model. Further, we identify a single mutation (V64G) in the Z protein that is able to confer this demonstrated attenuation. By establishing and characterizing a novel attenuation strategy for New World mammarenaviruses, we hope to aid future vaccine development for related emerging pathogens including Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV).

Development of Reverse Genetics for the Prototype New World Mammarenavirus Tacaribe Virus.

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

The Glycoprotein of the Live-Attenuated Junin Virus Vaccine Strain Induces Endoplasmic Reticulum Stress and Forms Aggregates prior to Degradation in the Lysosome.

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

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

Infection with Junín virus (JUNV) is currently being effectively managed in the endemic region using a combination of targeted vaccination and plasma therapy. However, the long-term sustainability of plasma therapy is unclear and similar resources are not available for other New World arenaviruses. As a result, there has been renewed interest regarding the potential of drug-based therapies. To facilitate work on this issue, we present the establishment and subsequent optimization of a JUNV minigenome system to a degree suitable for high-throughput miniaturization, thereby providing a screening platform focused solely on factors affecting RNA synthesis. Using this tool, we conducted a limited drug library screen and identified AVN-944, a non-competitive inosine monophosphate dehydrogenase (IMPDH) inhibitor, as an inhibitor of arenavirus RNA synthesis. We further developed a transcription and replication competent virus-like particle (trVLP) system based on these minigenomes and used it to screen siRNAs against IMPDH, verifying its role in supporting arenavirus RNA synthesis. The antiviral effect of AVN-944, as well as siRNA inhibition, on JUNV RNA synthesis supports that, despite playing only a minor role in the activity of ribavirin, exclusive IMPDH inhibitors may indeed have significant therapeutic potential for use against New World arenaviruses. Finally, we confirmed that AVN-944 is also active against arenavirus infection in cell culture, supporting the suitability of arenavirus lifecycle modelling systems as tools for the screening and identification, as well as the mechanistic characterization, of novel antiviral compounds.

Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses.

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1-Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern.

Epistastic Interactions within the Junín Virus Envelope Glycoprotein Complex Provide an Evolutionary Barrier to Reversion in the Live-Attenuated Candid#1 Vaccine.

The Candid#1 strain of Junín virus was developed using a conventional attenuation strategy of serial passage in nonhost animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but it has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and we demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I thus may minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine.IMPORTANCE The live-attenuated Candid#1 vaccine strain of Junín virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile reversion to pathogenicity. Here, we characterize a genetic interaction between GPC subunits that evolutionarily forces retention of the attenuating mutation. By incorporating this secondary mutation into Candid#1 GPC, we hope to minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. A similar approach may guide the design of live-attenuated vaccines against Lassa and other arenaviral hemorrhagic fevers.

Hemorrhagic Fever Virus Budding Studies.

Independent expression of the VP40 or Z matrix proteins of filoviruses (marburgviruses and ebolaviruses) and arenaviruses (Lassa fever and Junín), respectively, gives rise to the production and release of virus-like particles (VLPs) that are morphologically identical to infectious virions. We can detect and quantify VLP production and egress in mammalian cells by transient transfection, SDS-PAGE, Western blotting, and live cell imaging techniques such as total internal reflection fluorescence (TIRF) microscopy. Since the VLP budding assay accurately mimics budding of infectious virus, this BSL-2 assay is safe and useful for the interrogation of both viral and host determinants required for budding and can be used as an initial screen to identify and validate small molecule inhibitors of virus release and spread.

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.

Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics.

BACKGROUND: We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. METHODOLOGY: An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. PRINCIPAL FINDINGS: The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. CONCLUSIONS: Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.

Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis.

UNLABELLED: Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. IMPORTANCE: Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that nonmyristoylated GPC mutants of the Candid #1 strain of Junín virus display a commensurate deficiency in their infectivity, albeit without additional defects in virion assembly and budding. These results suggest that SSP myristoylation may function late in the fusion process to facilitate merging of the viral and cellular membranes. Antiviral agents that target this novel aspect of GPC membrane fusion may be useful in the treatment of arenavirus hemorrhagic fevers.