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

Two non-virogenic Vero cell lines persistently infected with the arenavirus Junin (JUNV), named V3 and V7, were characterized with respect to their resistance to superinfection with homologous and antigenically related viruses. Both lines were refractory to JUNV multiplication and partially resistant to other arenaviruses. JUNV was able to adsorb and penetrate persistently infected cells and, although V3 and V7 were able to support synthesis of antigenomic sense viral RNA, protein production of superinfecting virus was totally blocked. This resistance was not mediated by defective interfering particles but rather by a "cell-associated factor" that could be cell to cell transmitted. Prolonged thermal treatment of V3 and V7 abrogated expression of the viral nucleoprotein (N) and turned persistently infected cells permissive to JUNV multiplication. Thermal treated cells cultured at 37 degrees C resumed the expression of N in association to the recovery of resistance. Results strongly suggest a correlation between the presence of the viral nucleoprotein and superinfection exclusion.

Two Vero cell lines persistently infected with XJCl3 and Cl67 strains of Junin virus and named V3 and V7, respectively, have been characterized with respect to the presence and expression of the nucleoprotein (N) and the glycoprotein precursor (GPC) viral genes. After the acute phase of infection, where a marked CPE and high titers of virus were obtained, JV persistently infected cells became morphologically undistinguishable from Vero cells and virus production dropped to undetectable levels. V3 and V7 were resistant to the superinfection with antigenically related viruses. This fact could not be attributed to the presence of defective interfering particles or non-infectious virus in the supernatant. Expression of N was consistently detected in both cultures and accumulation of two degradation products of N was evident during the late passages. Although no G1 (main surface glycoprotein) expression was observed, a marked fusogenic capacity was detected in both cultures indicating at least, the synthesis of a GPC derived fusogenic glycoprotein. Cell lysates from V3 and V7 subjected to RT-PCR, using specific primers for N gene, or to a nested RT-PCR using specific primers for GPC (G1 region) confirmed the presence of both viral genes. No viral DNA sequences could be detected in JV persistently infected cells.

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.

Summary The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryoctye formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.

Summary To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10–15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

The XJCl3 strain of Junin virus (JV) and the mouse-attenuated mutant Cl67 showed different GP 38 peptide mapping after limited proteolysis with ficin and papain; viral infectivity of both viruses also exhibited a different susceptibility to protease treatment. A correlation between envelope glycoprotein alteration and JV virulence in neonatal mice is proposed.

The role of vacuolar-proton ATPase (V-H+ ATPAse) on Junin virus (JV) replication was evaluated by analyzing the effect of specific inhibitors of the enzyme activity on different steps of virus multiplication cycle. The presence of the macrolide antibiotics bafilomycin A1 and concanamycin A during the first two hours of infection caused a significant reduction of extracellular infectious virus production and viral protein expression in Vero and BHK-21 cells. The inhibitory action of the compounds was mainly exerted at an early stage of the JV multiplication cycle, without affecting virus attachment to the cell but preventing virus penetration. A correlation between the inhibitory action of the compounds on intracellular compartments acidification and the reduction of JV yield was observed. The addition of concanamycin A at different times after infection indicated that the compound also interferes with the release of infectious particles to the extracellular medium. Although, intracellular transport of JV glycoproteins to the cell membrane, seems not to be affected as revealed by immunofluorescence staining. The results confirm that JV enters into the cell through the endocytic pathway as previously suggested by using lysosomotropic compounds.

The action of reducing, oxidizing and thiol-alkylating agents on early steps of Junin virus (JV) multiplication in Vero cells was investigated. The presence of reducing agents during virus adsorption as well as incubation of viral particles with these compounds before infection enhanced JV infectivity. On the contrary, the thiol-alkylating agent 5, 5′ dithiobis (2-nitrobenzoic acid) and the oxidizing compound potassium periodate showed an inhibitory effect, suggesting that sulfhydryl groups and certain sugar moieties of viral glycoproteins play an important role in the first steps of JV infection. Also enzymatic treatment of cell monolayers and addition of concanavalin A to cultures prior to infection suggest that cellular glycoproteins are involved in virus attachment.

Summary Junin virus (JV) infected Vero cells were used to investigate virus capacity to induce cell-cell fusion. Polykaryocyte formation due to JV was found to be pH and temperature-dependent. A reduced fusion activity was detected on BHK-21 cells. Different JV-strains exhibited a similar extent and pH dependence of their fusion activity. Neutralizing antibodies against the main viral glycoprotein (GP38) inhibited syncytium production and GP38 conformational changes in response to acid treatment were detected by an immunoprecipitation assay.

The establishment of an experimental persistent infection with Junin virus, the aetiological agent of argentine hemorrhagic fever, involves the emergence of antigenic variants in brain and blood of the cricetid Calomys musculinus. We demonstrate that antigenic variants can also be isolated in vitro under the selective pressure of polyclonal antibodies and from a long-term infected C. musculinus primary embryo fibroblast culture. The participation of neutralizing antibodies and host cells in the appearance of viral variants in vivo is discussed.

Otherwise resistant adult mice were rendered susceptible to intracerebral Junin virus (JV) infection only when a staggered cyclophosphamide (CY) schedule was used. Forty-five-day old Balb/c mice, intracerebrally JV-infected and immunosuppressed with four 50 mg/kg body weight CY doses at days -1, +1, +4, +6 (day 0: viral infection) developed a lethal disease (86.6 per cent mortality) with high CNS viral titers and brain lesions. Neutralizing antibodies were absent throughout, while immunofluorescent antibody levels were considerably diminished. The transfer of hyperimmune serum conferred partial though significant protection on CY-treated animals but no correlation was found between CNS viral titers and mortality since in both infected CY-treated and untreated mice similar brain viral content was found. This was also confirmed by immune spleen cell transfer at day 0 where the clearance achieved was unable to modify the time course of the disease. Feasible mechanisms explaining recovery from JV infection by means of the protective effect of antibodies and the cell-mediated clearance are discussed.

Junin virus establishes a long-term persistent infection in its natural host, Calomys musculinus. Virus recovered from blood of infected animals from 14 to 61 days p.i. behaved antigenically distinct to parental virus, as shown by cross neutralization assays. The emergence of antigenic viral variants occurs during both the acute and persistent state of infection.

Vero cell cultures persistently infected with Junin virus and subjected to different cultural conditions were established. The production of infectious plaque-forming virus, ts mutants and interfering viral particles was determined at different times during 110 days after infection. Carrier cultures maintained in stationary conditions continuously released PFU while proliferating persistent cultures exhibited a cyclical pattern which tends to a rapid PFU disappearance. Concomitantly, in stationary cultures the production of interfering particles was delayed and was lower than in actively growing persistent cells. The metabolic state of the infected cells did not affect the release of ts mutants. The results suggest that a cellular function is involved on the regulation of Junin virus persistent infections.

Vero cells grown in Leighton tubes were infected with blood taken from Argentine Hemorrhagic Fever patients. In 11 out of 12 cases, and between the 2nd–8th days p.i. of the monolayers, Junin viral antigen was detected by the PAP method.