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.

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.

Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity.

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

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.

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.

A Proteomics Survey of Junín Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication.

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

Protocol for the Production of a Vaccine Against Argentinian Hemorrhagic Fever.

Argentinian hemorrhagic Fever (AHF) is a febrile, acute disease caused by Junín virus (JUNV), a member of the Arenaviridae. Different approaches to obtain an effective antigen to prevent AHF using complete live or inactivated virus, as well as molecular constructs, have reached diverse development stages. This chapter refers to JUNV live attenuated vaccine strain Candid #1, currently used in Argentina to prevent AHF. A general standardized protocol used at Instituto Nacional de Enfermedades Virales Humanas (Pergamino, Pcia. Buenos Aires, Argentina) to manufacture the tissue culture derived Candid #1 vaccine is described. Intermediate stages like viral seeds and cell culture bank management, bulk vaccine manufacture, and finished product processing are also separately presented in terms of Production and Quality Control/Quality Assurance requirements, under the Adminitracion Nacional de Medicamentos, Alimentos y Tecnología Medica (ANMAT), the Argentine national regulatory authority.

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.

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties.

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9µM) and MPA (0.8-2.5µM). 6-Azauridine toxicity was 6.1-17.5µM with NR, VTG, and WST-1, compared to 48-92µM with ALB-A and ALB-F (P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83-93µM with ALB-F versus 2.4-7.0µM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).

The Ectodomain of Glycoprotein from the Candid#1 Vaccine Strain of Junin Virus Rendered Machupo Virus Partially Attenuated in Mice Lacking IFN-αß/γ Receptor.

Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC.

Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells.

Many viruses have evolved strategies of so-called "superinfection exclusion" to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.

Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains.

The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.

Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles.

The nature and concentration of lipids and proteins at the surface of viruses are essential parameters for determining particle infectiveness. Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations. Here we analyse the properties of Junin virus particles using a sensitive flow virometry assay and further sort virions while conserving their infectiveness. This method allows us to characterize the relationship between infectivity, virus size and RNA content and to compare particles secreted by Vero cells with those from physiologically relevant human primary macrophages. Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site. Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.

[Study of sensitivity of laboratory animals to a causative agent of argentine hemorrhagic fever].

AIM: Study sensitivity of laboratory animals to a causative agent ofArgentine hemorrhagic fever. MATERIALS AND METHODS: Junin virus strain XJ P37 was obtained from the State Collection of Causative Agents of Viral Hemorrhagic Fevers of the Pathogenicity Group I of Scientific Research Center of the 33rd Central Scientific Research Test Institute (SRC of the 33rd CSRTI). Junin virus strain XJ P37 culture with biological activity of 5.2 1g PFU x ml was used in the experiments. Mice (2 - 4 and 7 - 14 days old), guinea pigs (250 - 300 g), 1.8 - 2.5 kg shinshilla breed rabbits, 2.0 - 3.0 kg javanese macaque monkeys were obtained from vivarium of the SRC of the 33rd CSRTI. Vero (B) and GMK-AH-1 (D) cell cultures were obtained from cell culture collection of the SRC of the 33rd CSRTI. Biological activity calculation of Junin virus was carried out by Kerber in I.P. Amsharin modification. RESULTS: Lethality in animals was from 12.5 to 50% after intranasal and intraperitoneal infection of guinea pigs, intramuscular, intraperitoneal and subcutaneous infection of rabbits, intracerebral and intranasal infection of mice at the doses from 0.4 to 1.0 x 10(5) PFU. Death of infected monkeys after intramuscular administration of the virus at 1.0 x 10(4) PFU dose was not observed. In 100% of surviving animals formation of virus-neutralizing antibodies was registered. CONCLUSION: Evaluation of sensitivity of laboratory animals to Junin virus has shown that intracerebrally infected mice may be used to maintain causative agent culture, infected guinea pigs - to prepare virus-containing cultures and modelling infection exacerbation in humans. Intramuscularly infected rabbits may be used to obtain hyper-immune sera.

[Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain]. / Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1 / Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.(AU)

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.(AU)

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1 / Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain

La Fiebre Hemorrágica Argentina es una enfermedad producida por el virus Junín. Para la prevención de esta enfermedad se obtuvo una vacuna efectiva denominada Candid#1. Durante un ensayo clínico realizado en el INEVH, dos cepas de virus Junín fueron aisladas de sangre periférica de dos voluntarios mediante co-cultivo de células mononucleares. El objetivo de este trabajo fue comparar las características fenotípicas de atenuación de esas dos cepas recuperadas de humanos con las de la vacuna Candid#1 utilizando los indicadores de atenuación desarrollados por Contigiani y Sabattini en 1977. A tal fin se midieron los índices de letalidad, infección y protección en cobayos y ratones de diferentes edades. Las tres cepas investigadas resultaron letales para ratones recién nacidos pero no para ratones de 10 a 12 días, ratones adultos ni cobayos, aun a la más baja dilución inoculada. Los cobayos inoculados con las cepas recuperadas de humanos y con la cepa Candid#1 no presentaron síntomas de enfermedad y mostraron estar protegidos cuando fueron desafiados con una cepa patógena. Los índices de infección y de protección hallados indican que estas cepas poseen elevada capacidad infectante y protectora en las especies animales aquí estudiadas. Estos resultados demuestran que las cepas de virus Junín aisladas de voluntarios inmunizados con Candid#1 mantienen el mismo fenotipo atenuado de la vacuna Candid#1 después de un pasaje por humanos.

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.

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

The regulation of apoptosis during infection is an important factor for host survival and, in some cases, also for the virus life cycle. At the same time, mechanisms to prevent the induction of apoptosis have been observed in numerous viral pathogens, but until now the role of apoptosis during arenavirus infection has not been investigated. Junin virus (JUNV) belongs to the New World arenavirus serogroup of the Arenaviridae and is the causative agent of Argentine hemorrhagic fever. We have demonstrated that infection with JUNV in cell culture does not induce apoptosis but leads to cleavage of the nucleoprotein (NP) into discrete products resembling caspase cleavage events. Similar specific NP degradation patterns were also observed in NP-transfected cell lines, and a closer examination of the sequence of NP showed several putative caspase cleavage motifs. Point mutations that abolished these cleavage motifs were consistent with the loss of certain cleavage products. Consistent with these data, further studies showed that treatment with a caspase inhibitor also reduced NP cleavage, indicating that the observed cleavage events were occurring as a result of caspase activity with NP as a substrate. Finally, we showed that expression of NP suppresses the cleavage of caspase 3 in cells treated with an apoptosis activator. Based on these findings, we propose that NP functions as a decoy substrate for caspase cleavage in order to inhibit the induction of apoptosis in JUNV-infected cells.

Marcadores fenotípicos de atenuación en cepas de virus Junín recuperadas de individuos vacunados con la cepa Junín Candid#1. / [Phenotypic markers of attenuation in Junin virus strains recently isolated from individuals vaccinated with Junin Candid#1 strain].

Argentine hemorrhagic fever is a severe acute disease caused by Junin virus. For prevention of this disease an effective vaccine called Candid#1 has been developed, composed of a live attenuated Junin virus strain. During a clinical trial conducted at Instituto Nacional de Enfermedades Virales Humanas (INEVH) in 2005, Junin virus was isolated from two vaccinated volunteers by co-culture of peripheral mononuclear blood cells. The aim of this study was to compare the strains isolated from these human volunteers with Candid#1 strain regarding phenotypic characteristics of attenuation according to the indicators developed by Contigiani and Sabattini in 1977. The three strains were lethal to suckling mice but not to 10-12 days old mice and guinea pigs. Surviving guinea pigs from primary infection were protected when challenged by intra-muscular inoculation with lethal doses of a virulent strain. Infection and protection rates indicate that these strains are highly infective and protective in the hosts studied herein. These results demonstrate that Junin virus strains isolated from volunteers immunized with Candid#1 maintain the same attenuated phenotype of Candid#1 vaccine after one passage in humans.