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).

Comparison of the Innate Immune Responses to Pathogenic and Nonpathogenic Clade B New World Arenaviruses.

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

Junín Virus Promotes Autophagy To Facilitate the Virus Life Cycle.

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

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.

Inhibition of the PI3K/Akt pathway by Ly294002 does not prevent establishment of persistent Junín virus infection in Vero cells.

In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.

Potent inhibition of Junín virus infection by interferon in murine cells.

The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice.

[Optimization of conditions of quantitative evaluation of argentine hemorrhagic fever causative agent].

AIM: Optimization of conditions of quantitative evaluation of Argentine hemorrhagic fever causative agent. MATERIALS AND METHODS: Junin virus (XJ P37 strain) was obtained from National collection of viral hemorrhagic fever causative agents of the 1st pathogenicity group of the 33rd Central Research Testing Institute. Junin virus (XJ P37 strain) culture with biological activity of 5.2 lg PFUxm(-1) was used in the experiments. Vero B, 6619-1(D) and GMK-AH-1(D) were obtained from collection of cell culture of the Research Scientific Centre of the 33rd Central Research Testing Institute. Calculation of biological activity of Junin virus during titration in cell cultures was carried out by Kerber method with modification by I.P. Ashmarin. RESULTS: During incubation for 4 - 7 days after the infection of cell monolayer the determined biological activity was 4.4 - 6.4 lg PFUxml(-1); the size of the formed negative colonies--(1.5 +/- 0.5) mm. CONCLUSION: The conditions of quantitative evaluation of Argentine hemorrhagic fever were optimized by negative colonies method (using 5 - 7 day Vero B cell culture monolayer with staining of monolayer on day 5 of secondary incubation, recording of results at day 7 after the infection).

Junin virus infects mouse cells and induces innate immune responses.

Junín virus is the causative agent for Argentine hemorrhagic fever, and its natural host is the New World rodent Calomys musculinus. The virus is transmitted to humans by aerosolization, and it is believed that many of the clinical symptoms are caused by cytokines produced by sentinel cells of the immune system. Here we used the Junín virus vaccine strain Candid 1 to determine whether mouse cells could be used to study virus entry and antiviral innate immune responses. We show that Candid 1 can infect and propagate in different mouse-derived cell lines through a low-pH-dependent, transferrin receptor 1-independent mechanism, suggesting that there is a second entry receptor. In addition, Candid 1 induced expression of the antiviral cytokines tumor necrosis factor alpha and beta interferon in macrophages, and this induction was independent of viral replication. Using Candid 1, as well as virus-like particles bearing the viral glycoprotein, to infect different primary cells and established macrophage cell lines with deletions in the Toll-like receptor (TLR) pathway, we show that TLR2 is a cellular sensor of both the Parodi and Candid 1 viral glycoproteins. Because Junín virus is highly lethal in humans, the use of an experimentally tractable model system, such as the mouse, could provide a better understanding of the antiviral innate cellular responses to Junín virus and the role of these responses in pathogenesis.

Inhibitory effect of thiosemicarbazone derivatives on Junin virus replication in vitro.

The inhibitory effect of several thiosemicarbazones (TSCs), synthesized from aromatic ketones and terpenones, and their heterocyclic thiadiazoline (TDZ) derivatives, was investigated against Junin virus (JUNV), an arenavirus agent of Argentine haemorrhagic fever. From the 25 compounds tested, six compounds belonging to the TSC group were found to be selective inhibitors of JUNV, with EC50 values determined by a virus yield inhibition assay in the range 3.4-12.5 microM, and selectivity indices greater than 10. By contrast, most of the TDZs obtained by heterocyclization of the TSCs were not active against JUNV. No conclusive structure-activity relationships could be established but systematically higher activity was associated to TSCs derived from aromatic ketones. The mode of action of one of the most active compound, the 3,4-dihydronaphtalen-1(2H)one thiosemicarbazone (tetralone thiosemicarbazone), was studied further. This TSC lacked virucidal effects on JUNV virions. Results from time of addition experiments and viral protein expression assays suggest that tetralone thiosemicarbazone inhibited a late stage in the replicative cycle of JUNV.

Differential inhibitory action of two azoic compounds against arenaviruses.

The action of five azo-based compounds against the arenaviruses Junin (JUNV) and Tacaribe (TCRV) was evaluated in vitro by a virus yield inhibition assay in Vero cells and a cell-free virion inactivation assay. The compound 2-azo-(1'-(2'-nitroso)naphthyl)-benzoate (ANNB) was the most effective inhibitor of arenavirus production in Vero cells with EC(50) (effective concentration 50%) values in the range 6.5-26.2 microM and without inactivating properties. By contrast, the azodicarbonamide (ADA) was very effective in inactivating both arenaviruses with IC(50) (inactivating concentration 50%) values of 7.6 and 5.3 microM against JUNV and TCRV, respectively. The virucidal activity of ADA was time- and temperature-dependent. ANNB had no inhibitory action on virus binding or penetration of target cells and did not affect the synthesis of viral proteins. The most likely event susceptible to ANNB would be the process of intracellular virion assembly.

Mode of inactivation of arenaviruses by disulfide-based compounds.

Several disulfide-based compounds, including intermolecular aromatic disulfides of the type Ph-S-S-Ph and dithianes with the sulfur atoms tethered in a ring structure, have shown effective inhibitory activity against the arenaviruses Junin (JUNV), agent of Argentine hemorrhagic fever, and Tacaribe (TCRV). These compounds showed a strong virucidal effect with inactivating concentration 50% (IC(50)) values in the range 0.6-5.0 microM, and also were effective to reduce virus yields from infected cells. The mode of inactivating action of two active compounds, the aromatic bis disulfide NSC20625 and the dithiane NSC624152, was further studied. Both compounds were able to inactivate arenaviruses after a few minutes of direct contact with virions, in a concentration- and time-dependent manner. The ability of drug-treated virus to perform several steps of the replication cycle was analyzed. The killed virus particles were found to bind and enter to Vero cells with the same efficacy as infectious native virions, but the ability of inactivated virions to synthesize viral proteins in Vero cells was abolished. Thus, treatment of JUNV and TCRV with these compounds destroyed virion infectivity, generating particles which entered the host cell but were unable to complete the viral biosynthetic processes.

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Junin virus in rodents.

Junin virus is the etiological agent of Argentine hemorrhagic fever, a serious rodent-borne disease. An enzyme-linked immunosorbent assay (ELISA) to detect Junin virus IgG antibodies in rodents was evaluated using sera from 27 Calomys musculinus and five Calomys laucha, inoculated experimentally with a live attenuated strain of this arenavirus. The test performance was compared against an indirect immunofluorescence assay (IFA). The ELISA had a sensitivity and specificity of 100% and a reproducibility of 87.9% for samples with titers above the selected cut-off value. IFA had lower sensitivity (53%) with the same specificity. The ELISA results were similar, whether carried out on whole blood or serum samples, thus eliminating the need for serum separation. A high correlation (K=0.86) between ELISA and IFA results was obtained from 1011 wild sigmodontine and murine rodents collected within and outside of the Argentine hemorrhagic fever endemic area. These results indicate that Junin virus IgG ELISA is the most suitable assay for detection of Junin virus antibodies in rodent samples.

Evaluation of the antiviral activity against Junin virus of macrocyclic trichothecenes produced by the hypocrealean epibiont of Baccharis coridifolia.

Four macrocyclic trichothecenes, roridin A, roridin E, verrucarin A and verrucarin J, produced by the hypocrealean epibiont of Baccharis coridifolia, were evaluated for their inhibitory activity against the arenavirus Junin (JUNV), the etiological agent of Argentine hemorrhagic fever. The trichothecenes achieved a dose-dependent inhibition of JUNV multiplication at concentrations not affecting cell viability. The 50 % inhibitory concentration (IC50) values determined by a virus yield inhibition assay were in the range 1.2 - 4.9 ng/ml. The most active compound was verrucarin J which reduced JUNV yield more than 2 log units and had a similar effect against the arenavirus Tacaribe. The trichothecenes lacked virucidal effects on JUNV virions. From time of addition and removal experiments, it can be concluded that verrucarin J inhibited a late stage in the replicative cycle of JUNV, after 5 h of adsorption.

[Neutralization test for lymphocytic choriomeningitis virus for distinguishing between two arenavirus infections in Argentina]. / Prueba de neutralización del virus de la coriomeningitis linfocítica para diferenciar infecciones con dos arenavirus en Argentina.

The active coexistence of two pathogenic arenaviruses, Junin (JUNV) and lymphocytic choriomeningitis (LCMV), in the same region of Argentina, has been known since the early 70's, and records of clinical and subclinical human infections by one and/or the other agent have been continuously produced for the last 25 years. Anti-LCMV antibody is currently searched only by indirect immunofluorescence, a test that shows cross reactions among a number of arenaviruses yielding, in the cases of LCMV and JUNV consecutive infections, a concomitant seroconversion for both viruses, as an inconclusive diagnostic result. In contrast, neutralization (NT) tests reveal arenavirus antibodies directed to unique epitopes on these virus envelopes, thus allowing to disclose the sequence in the cases of consecutive infections. In this paper, the characteristics of neutralization (NT) test for LCMV in cell cultures are described, as well as its performance in the field diagnosis of LCMV human infections. The native LCMV strain Cba An 13065 was inoculated on L-929 cell (ATCC CCL 1), and procedures were followed to perform a constant virus-variable serum NT test. Final points of sera titrations were expressed as the maximal serum dilution that yielded 75% of pfu inhibition. This NT test was assayed on paired serum samples of 36 patients with confirmed Argentine hemorrhagic fever (AHF) (a disease caused by JUNV), who had had a known previous contact with LCMV through IFI. The use of this one test led to confusing diagnosis of the disease due to concomitant seroconversion for JUNV and LCMV. By using NT test, it was shown that: some of them were possibly not infected by LCMV, and that 30/36 cases (83.3%) had a pre-existing level of LCMV antibody, with titers in the range of 5 to 640, remaining unchanged 60 days after the clinical AHF. This shows that NT antibodies to LCMV are not influenced by the outcome of the immune response to JUNV, thus confirming the efficiency of NT test as identificator among arenaviruses. To assess the performance of this NT test in individuals having only IFI antibodies to LCMV, 126 serum samples obtained through serological surveillance in a rural area of Argentina, were used. It was found that NT had improved coincidence with IFI as IFI titers increased. Interpretations were based on the pan-arenavirus antibody response obtained by using IFI as the only test. Results presented herein prove that the described NT test is a valuable tool for the detection of LCMV infections, particularly when a previous infection with LCMV has to be demonstrated during the acute phase of Argentine hemorrhagic fever.

Antiviral and virucidal activities against arenaviruses of zinc-finger active compounds.

Fifteen antiretroviral Zn-finger active compounds with diverse chemical structures, including azoic compounds, hydrazide derivatives, disulphide-based reagents and others were screened in vitro against Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever, by a virus yield inhibition assay in Vero cells. Cytotoxicity was evaluated simultaneously by the MTT method. Of the compounds, three were totally inactive as antivirals, nine presented moderate anti JUNV-activity and three were truly active with EC50 (effective concentration 50%) values in the range 6.5-9.3 microM and with selectivity indices greater than 10. The most active inhibitors, named NSC20625, 3-7 and 2-71, demonstrated a broad range of action against arenaviruses, including several attenuated and pathogenic strains of JUNV as well as the antigenically related Tacaribe virus (TACV) and Pichinde virus (PICV). The direct treatment of JUNV and TACV virions with the compounds showed two types of behaviour: the aromatic disulphide NSC20625 was a very potent virucidal agent, whereas the other two compounds exhibited moderate or negligible virus-inactivating properties.

Effect of inhibitors of the intracellular exocytic pathway on glycoprotein processing and maturation of Junin virus.

The effect of glycoprotein processing and transport on Junin virus (JV) maturation was studied by using brefeldin A (BFA) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), two inhibitors affecting different steps of the intracellular exocytic pathway, combined with low temperature incubation. Both compounds inhibited the multiplication of JV, strain IV4454, in Vero cells in a dose dependent manner. The addition of the compounds after several cycles of infection for a very short treatment period produced an immediate arrest on the formation of JV infectious particles, due to their effect on a late event in JV infective cycle. Extracellular and cell-associated virus yields were reduced to the same extent, suggesting that the assembly of virus particles was the blocked stage. In infected cells treated with CCCP and BFA an altered subcellular distribution of partially processed viral glycoproteins was observed, with an accumulation of JV glycoproteins at the endoplasmic reticulum and a blockade of their transport to the site of envelopment in the plasma membrane. Concomitantly, the cleavage of the precursor GPC to the mature glycoprotein GP38 was strongly inhibited when the exocytic pathway was affected. Thus, results obtained with BFA allow to conclude that the proteolytic processing of GPC probably occurs at the trans-Golgi network and this cleavage together with the glycoprotein presence at the cell surface is required for maturation of infectious JV particles.

Evaluation of an enzyme immunosorbent assay for the diagnosis of Argentine haemorrhagic fever.

To elaborate a set of serological tests for the diagnosis of Argentine haemorrhagic fever (AHF), an enzyme-linked immunosorbent assay (ELISA) for detection of specific anti-Junin virus (JV) IgG is described, and its performance is compared with that of the plaque reduction neutralization test (PRNT). The reproducibility, sensitivity, specificity, and confidence limits for positive and negative results for ELISA were statistically analysed. The value of 800 was demonstrated as the lowest positive titer. Titers > or = 800 varied within one (two-fold) dilution in 95.6% of the tests, while the sensitivity and specificity were 99.2% and 98.8%, respectively. The assay yielded 1% of false positives and 0.05% of false negatives. A comparison of ELISA to PRNT in detecting the seroconversion for JV was studied by the chi square test (comparison of proportions in paired samples) and the K parameter for agreement proportion. Comparison of ELISA to PRNT showed no significant difference in the proportions of positive and negative results of these assays (P < 0.01), demonstrating an equivalent performance (K = 0.98) in the diagnosis of AHF. In addition, the simplicity and safety of the procedures involved make this ELISA the most suitable test to detect natural human JV infections.

Calomys musculinus, the natural reservoir of Argentine haemorrhagic fever, is a semipermissive host for a mouse-attenuated mutant of Junin virus.

C167, a mouse-attenuated strain of Junin virus derived from the XJC13 strain, also displayed reduced virulence for the South American cricetid Calomys musculinus, a natural reservoir of this virus in nature. Intracerebral inoculation of C. musculinus with 500 PFU of C167 produced only 25% mortality, whereas the parental XJC13 killed 85% of the animals. The attenuation of C167 for this cricetid was lower than for albino mice. The multiplication of C167 in C. musculinus-derived embryo or kidney fibroblasts was diminished with respect to XJC13, allowing us to define C. musculinus cells as a semipermissive system for C167, whereas murine and Vero cells were restrictive and permissive cultures, respectively. As a consequence, C167 as well as XJC13 were able to establish a persistent infection in C. musculinus embryo fibroblasts and Vero cells, but the mutant could not induce a carrier state in murine cells. Thus, the degree of susceptibility of C. musculinus to C167 was linked to the semipermissiveness of cricetid cells to virus multiplication.

[Present status of zoonotic hemorrhagic fevers of South America]. / Actualité des fièvres hémorragiques zoonotiques d'Amérique du sud.

Since 1958, the geographical distribution of Argentine hemorrhagic fever (AHF) has especially extended non only into the province of Buenos Aires but also towards the provinces of Santa Fe and Cordoba, leading to an estimated population at risk of about 1.2 M inhabitants. Recent epidemiological field studies has confirmed the major role of Calomys musculinus and C. laucha rodents in both transmission to man and conservation of Junin virus in nature. However, the human infection may result essentially from contacts with infected C. musculinus. Clinical condition of patients with AHF was greatly improved using AHF convalescent plasma and additional administration of vidarabin may still improve the results of treatment. A live attenuated vaccine, Candid No 1, is presently under evaluation in endemic foci of AHF. On the contrary Bolivian hemorrhagic fever (BHV) appears at present quite silent. A new disease resembling both AHF and BHF, the Venezuelan hemorrhagic fever, appeared in 1989 in the rural areas of central Llanos of Venezuela. The mortality was very high, reaching 23% or more among severely ill patients. The wild small rodents responsible for the disease were identified as Sigmodon alstoni and Zygotontomys brevicauda. Recent extension of agricultural practices and massive immigration may probably explain the recent emergence of this new viral zoonosis.