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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.

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BACKGROUND: Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1-2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33-40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection. CONCLUSIONS/SIGNIFICANCE: The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses.

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The efficacy of the antiviral agents nucleoside analogues manufactured in Russia and foreign countries against Bolivian hemorrhagic fever in the treatment and rapid prevention regimens was studied in guinea pigs. In rapid prevention and treatment of guinea pigs, the intraperitoneal injection of 1/10 of the equivalent dose of vero-ribavirin for this animal species was ascertained to protect 70% of the animals infected with Machupo virus strain Carvallo in doses 8 and 10 LD50.

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Se comparó en cobayos la seguridad, inmunogenicidad y eficácia protectora de um lote de vacuna Candid #1 (C#1) fabricada en Estados Unidos de América (EE.UU.) y distintos lotes de la misma vacuna fabricados en Argentina (Arg.). El lote TSI 5-1-92 (EE.UU) y los lotes Exp N3, 7A y 8A (Arg) fueron inoculados (0.5ml, IM) en cobayos de 250400g. Para cada ensayo diez animales recibieron solución fisiológica y sirvieron como control. Todos fueron desafiados con la cepa patógena P23790 de vírus Junin. Se registro: a) temperatura rectal, b) peso corporal , c) presencia de anticuerpos neutralizantes (AcNT) pré y post-vacunación, d) respuesta al desafio . Todos los animales vacunados desarrollaron AcNT anti vírus Junin (rango= 4081920 y sobrevivieron al desafio. En cada grupo control 810 animales murieron (dia 23.3+_ 5.4 post- desaportada y los diferentes lotes de C#1 producidos en Argentina.

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Se comparó en cobayos la seguridad, inmunogenicidad y eficácia protectora de um lote de vacuna Candid #1 (C#1) fabricada en Estados Unidos de América (EE.UU.) y distintos lotes de la misma vacuna fabricados en Argentina (Arg.). El lote TSI 5-1-92 (EE.UU) y los lotes Exp N3, 7A y 8A (Arg) fueron inoculados (0.5ml, IM) en cobayos de 250400g. Para cada ensayo diez animales recibieron solución fisiológica y sirvieron como control. Todos fueron desafiados con la cepa patógena P23790 de vírus Junin. Se registro: a) temperatura rectal, b) peso corporal , c) presencia de anticuerpos neutralizantes (AcNT) pré y post-vacunación, d) respuesta al desafio . Todos los animales vacunados desarrollaron AcNT anti vírus Junin (rango= 4081920 y sobrevivieron al desafio. En cada grupo control 810 animales murieron (dia 23.3+_ 5.4 post- desaportada y los diferentes lotes de C#1 producidos en Argentina. (AU)

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Bolivian hemorrhagic fever (BHF) is a potentially severe febrile illness caused by Machupo virus (family Arenaviridae). Initial symptoms include headache, fever, arthralgia, and myalgia. In the later stages of this illness, patients may develop hemorrhagic manifestations including subconjunctival hemorrhage, epistaxis, hematemesis, melena, and hematuria, as well as neurological signs including tremor, seizures, and coma. During the BHF epidemics of the 1960s, convalescent-phase immune plasma from survivors of BHF was administered to selected patients infected with Machupo virus. However, there is currently a paucity of survivors of BHF who can donate immune plasma, and there is no active program for collection and storage of BHF immune plasma; therefore, we had the opportunity to offer intravenous ribavirin to two of three patients with this potentially life-threatening infection. One patient with laboratory-confirmed Machupo virus infection who received ribavirin recovered without sequelae, as did a second patient with suspected BHF whose epidemiological and clinical features were similar to those of the first patient. This report describes the first use of intravenous ribavirin therapy for BHF in humans, and the results suggest the need for more extensive clinical studies to assess the usefulness of ribavirin for treating BHF.

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Administration of cyclophosphamide (CY) to guinea pigs infected with a lethal strain of Junin virus (JV) delayed the time of death, with survival of a small number of animals. Virological studies showed a temporary decrease of virus concentration in blood and viscera shortly after the CY injection. In the pathological study no differences were found in the organic lesions present in CY-treated and nontreated animals, with the exception of the pulmonary alterations. In CY-treated guinea pigs the lungs appeared almost normal, but in the control, nontreated animals severe alterations with the pattern of the "respiratory distress syndrome of the adult" were consistently present. In in vitro experiments, incorporation of serum collected from guinea pigs injected 30 minutes before exsanguination with CY to cell cultures, infected with JV, prevented virus replication. On the basis of these results it is suggested that the delay of time of death and eventual survival of CY-treated guinea pigs after JV infection depends on a direct antiviral effect of the drug rather than on its known immunosuppressive action. In addition, the absence of pulmonary alterations in CY-treated animals was tentatively considered to be dependent on the marked polymorphonuclear leukocyte depletion induced by the drug.

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Junin virus, the etiological agent of Argentine hemorrhagic fever, produces in man a disease mainly characterized by hemorrhagic alterations, commonly accompanied by neurological symptoms, and leading to 10% mortality. Intracerebral inoculation in 10-day-old rats or intraperitoneal inoculation in 2-day-old rats leads to high mortality due to severe encephalitis. Here, the effect of Ribavirin on these experimental models was tested in order to evaluate the degree of protection achieved against neuropathological manifestations. In intracerebrally infected 10-day-old rats the drug was administered 2 hr before virus inoculation. Doses ranged from 30 to 90 mg/kg body weight. Protection reached 40% for the 60 and 90 mg doses. Intraperitoneally infected 2-day-old rats received the drug in five 30-mg daily doses, starting the same day as virus inoculation. Survival was 73%. Viral replication within peritoneal macrophages dropped markedly, leading to much lower CNS viral titres. Together with results reported in primates, our findings support further studies on Ribavirin, with a view to eventual trials in humans.

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