Modified ribavirin analogues as antiviral agents against Junín virus.

In this work, several ribavirin analogues were synthesized and incorporated into a multivalent arrangement. Both were subsequently modified by the addition of polyhydroxylated residues. Their antiviral activity was tested against Junín virus, etiological agent responsible of Argentine hemorrhagic fever. Some compounds inhibited Junín virus in the range of 13.2-389.1 µM. Two modified ribavirin analogues presented an effective concentration comparable to ribavirin but with a higher selectivity index.

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus.

The aim of this study was to investigate the effect of A771726, the active metabolite of leflunomide, (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad against the infection with Junín virus (JUNV), agent of Argentine hemorrhagic fever (AHF). The treatment with non-cytotoxic concentrations of A771726 of Vero and A549 cells infected with JUNV inhibited virus replication in a dose-dependent manner, as determined by virus yield reduction assay. The antiviral effectiveness of A771726 was not importantly affected by the multiplicity of infection and the virus strain. Moreover, the combination of A771726 and ribavirin had a significantly more potent antiviral activity than each single drug treatment. Mechanistic studies showed that the main action of A771726 is exerted before 6 h of JUNV infection. Accordingly, inhibition of viral RNA synthesis was detected in treated infected cells by real time RT-PCR. The exogenous addition of uridine or orotic acid produced a partial reversal of the inhibitory effect of A771726 on infective virus production whereas a total reversion was detected on JUNV RNA synthesis, probably by restoration of the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and the intracellular pyrimidine pools. In conclusion, these results suggest that the antiviral target would be viral RNA synthesis through pyrimidine depletion, but any other effect of the compound on JUNV infection cannot be excluded. This study opens the possibility of the therapeutic application of a wide spectrum host-targeted compound alone or in combination with ribavirin to combat AHF as well as other human pathogenic arenaviruses.

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.

Favipiravir (T-705) inhibits Junín virus infection and reduces mortality in a guinea pig model of Argentine hemorrhagic fever.

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.

S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection.

It has been previously described that S-layer binds to the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209). It was also shown that DC-SIGN is a cell-surface adhesion factor that enhances viral entry of several virus families. Among those, Junin virus (JUNV) entry is enhanced in cells expressing DC-SIGN and for that reason surface-layer protein (S-layer) of Lactobacillus acidophilus ATCC 4365 was evaluated as a possible JUNV inhibitor. Experiments using 3T3 cells stably expressing DC-SIGN, showed an almost complete inhibition of JUNV infection when they were treated with S-layer in a similar extend as the inhibition shown by mannan. However no inhibition effect was observed in 3T3 wild type cells or in 3T3 cells expressing liver/lymph node-specific ICAM-3 grabbing nonintegrin (L-SIGN or DC-SIGNR or CD209L). Treatments with S-layer during different times in the infection demonstrated that inhibition was only observed when S-layer was presented in early stages of the viral infection. This inhibition does not involve the classic recognition of mannose by this C-type lectin as the S-layer showed no evidence to be glycosylated. In fact, the highly basic nature of the S-layer (pI>9.5) seems to be involved in electrostatic interactions between DC-SIGN and S-layer, since high pH abolished the inhibitory effect on infection cause by the S-layer. In silico analysis predicts a Ca(2+)-dependant carbohydrate recognition domain in the SlpA protein. This novel characteristic of the S-layer, a GRAS status protein, contribute to the pathogen exclusion reported for this probiotic strain and may be applied as an antiviral agent to inhibit several kinds of viruses.

Inhibition of Junin virus RNA synthesis by an antiviral acridone derivative.

There are no specific approved drugs for the treatment of agents of viral hemorrhagic fevers (HF) and antiviral therapies against these viruses are urgently needed. The present study characterizes the potent and selective antiviral activity against the HF causing arenavirus Junin virus (JUNV) of the compound 10-allyl-6-chloro-4-methoxy-9(10H)-acridone, designated 3f. The effectiveness of 3f to inhibit JUNV multiplication was not importantly affected by the initial multiplicity of infection, with similar effective concentration 50% (EC(50)) values in virus yield inhibition assays performed in Vero cells in the range of 0.2-40 plaque forming units (PFU)/cell. Mechanistic studies demonstrated that 3f did not affect the initial steps of adsorption and internalization. The subsequent process of viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR in compound-treated cells relative to non-treated cells. The addition of exogenous guanosine rescued the infectivity and RNA synthesis of JUNV in 3f-treated cells in a dose-dependent manner, but the reversal was partial, suggesting that the reduction of the GTP pool contributed to the antiviral activity of 3f, but it was not the main operative mechanism. The comparison of 3f with two other viral RNA inhibitors, ribavirin and mycophenolic acid, showed that ribavirin did not act against JUNV through the cellular enzyme inosine monophosphate dehydrogenase (IMPDH) inhibition whereas the anti-JUNV activity of mycophenolic acid was mainly targeted at this enzyme.

Imidazo[2,1-b]thiazole carbohydrate derivatives: Synthesis and antiviral activity against Junin virus, agent of Argentine hemorrhagic fever.

Herein, we describe the syntheses of 3,5-disubstituted imidazo[2,1-b]thiazole. The cyclization step was performed in two different conditions by using either thermal or microwave heating. Comparing the results of both methodologies, we found that the microwave assistance is an improved alternative to obtain this family of heterocyclic compound. Compounds were first evaluated for cytotoxicity in Vero cells by MTT method and then, the antiviral activity was assayed by a virus yield inhibition assay in the range of concentrations lower than the corresponding CC(50), using JUNV strain IV4454 as the model system. The most active compounds (3 and 4), showed a level of antiviral activity against JUNV in monkey Vero cells better than the reference substance ribavirin. Then, they are promising lead compound for further analysis and characterization to establish their therapeutic potential against hemorrhagic fever viruses.

Virucidal activity and chemical composition of essential oils from aromatic plants of central west Argentina.

The essential oils of seven aromatic plants from central west Argentina were isolated by steam distillation and analyzed by a gas chromatography/mass spectrometry technique. The oils were screened for cytotoxicity and in vitro inhibitory activity against herpes simplex virus type 1 (HSV-1), dengue virus type 2 (DENV-2) and Junin virus (JUNV). The oils showed a variable virucidal action according to the virus. JUNV was the least susceptible virus in comparison with HSV-1 and DENV-2. The better relationship between cytotoxicity and inhibitory activity was observed for the essential oil of Lantana grisebachiii (Seckt.) var. grisebachii against DENV-2 and HSV-1 with IC50 (inhibitory concentration 50%) values of 21.1 and 26.1 ppm, respectively. This effect was specific since the selectivity indices (ratio cytotoxicity/virucidal activity) were > 23.7 and > 19.1 for DENV-2 and HSV-1, respectively. Furthermore, the oil from L. grisebachii was also an effective inhibitor of HSV-2 and acyclovir resistant variants of herpes virus. This study demonstrates the effective and selective inhibitory activity of the essential oil from Lantana grisebachii against HSV and DENV by direct virus inactivation.

Inhibition of arenavirus infection by thiuram and aromatic disulfides.

A selected group of aromatic disulfides, thiuram disulfides and thiosulfones, provided by the National Cancer Institute, were evaluated in vitro for their inhibitory activity against Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. The aromatic disulfides NSC4492 and NSC71033 and the thiuram disulfide NSC14560 were, respectively, the more potent virucidal and antiviral agents against JUNV, with inactivating concentration 50% (IC(50)) values of 0.2-0.5 microM for virucidal compounds and antiviral effective concentration 50% (EC(50)) of 8.5 microM for NSC14560. Both types of compounds exhibited inhibitory activity against three arenaviruses. Additionally, a comparable efficacy in the antiviral action of NSC14560 was observed in monkey, hamster or human cells with selectivity indices in the range 55.9-85.7. Time of addition experiments showed that the main antiviral activity of NSC14560 was situated before 5h of infection, but a significant inhibition was still observed when the compound was added up 9h p.i. This compound did not induce a refractory state to infection by cell pretreatment. Nor did it prevent viral entry, but the cytoplasmic and membrane expression of the main viral proteins was inhibited. The possible involvement of the RING finger motif of arenavirus Z protein as target for the thiuram disulfide is discussed.

Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junín virus replication in vitro.

In this paper we demonstrate that infection of cell cultures with the arenavirus Junín (JUNV), agent of the argentine haemorrhagic fever, leads to the activation of PI3K/Akt signalling pathway. Phosphorylation of Akt occurs early during JUNV infection of Vero cells and is blocked by the PI3K inhibitor, Ly294002. Infection of cells with UV-irradiated JUNV redeemed the pattern of stimulation observed for infectious virus indicating that an early stage of multiplication cycle would be enough to trigger activation. Treatment of cells with chlorpromazine abrogated phosphorylation of Akt upon JUNV infection suggesting virus internalization as responsible for activation. Inhibition of Akt phosphorylation by Ly294002 impaired viral protein synthesis and expression leading to a reduced infectious virus yield without blocking the onset of persistent stage of infection. This impairment is linked to a reduced amount of virus bound to cells probably due to a blockage on the recycling of transferrin cell-receptor, employed by the virus to adsorb to the cell surface. Early Akt activation was also observed in BHK-21 and A549 JUNV infected cells suggesting an important role of PI3K/Akt signalling in JUNV multiplication in vitro.

Characterization of Junín virus particles inactivated by a zinc finger-reactive compound.

Our previous studies reported the inhibitory action against arenaviruses of antiretroviral zinc finger-reactive compounds provided by the National Cancer Institute (USA). These compounds were able to inactivate virions as well as to reduce virus yields from infected cells. Here, the inactivation of the arenavirus Junín (JUNV), agent of Argentine hemorrhagic fever, by the aromatic disulfide NSC20625 was analyzed. The treatment of purified JUNV with this compound eliminated infectivity apparently through irreversible modifications in the matrix Z protein detected by: (a) alterations in the electrophoretic migration profile of Z under non-reducing conditions; (b) an electrodense labeling in the internal layer beneath the envelope and around the matrix Z protein, in negatively stained preparations; (c) changes in the subcellular localization of Z in cells transfected with a recombinant fusion protein JUNVZ-eGFP. The infection of Vero cells with JUNV inactivated particles was blocked at the uncoating of viral nucleocapsid from endosomes, providing new evidence for a functional role of Z in this stage of arenavirus cycle. Furthermore, the inactivated JUNV particles retained the immunoreactivity of the surface glycoprotein GP1 suggesting that this disulfide may be useful in the pursuit of an inactivating agent to obtain a vaccine antigen or diagnostic tool.

Involvement of cytoskeleton in Junín virus entry.

The early events in Junín virus (JUNV) infection are not thoroughly understood. We have previously shown that JUNV enter cells by clathrin-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early virus infection. Inhibitory effects of drugs affecting main cytoskeletal components on JUNV entry into Vero cells were analyzed. Drugs that disrupted microfilaments or stabilized microtubules inhibited early steps of virus entry. In contrast, drugs that stabilized microfilaments or depolymerized microtubules were not able to block virus entry very efficiently. Furthermore, real time PCR was performed to detect viral entry and we found more than 10-fold less RNA when microfilaments were depolymerized while a 100-fold diminution was seen when microtubules were stabilized. Taken together our results demonstrate that JUNV relies on an intact actin network during early infection in Vero cells while a dynamic microtubule network is also needed. This represents an important contribution to the characterization of arenavirus multiplication cycle.

Synthesis and antiviral activity of azoles obtained from carbohydrates.

Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and Junin virus (the etiological agent of Argentine hemorrhagic fever). The 3-(p-bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-alpha-d-xylofuranos-5-ulos-5-yl)imidazo[2,1-b]thiazole was able to inhibit the replication of both viruses in Vero cells at concentration significantly lower than the CC(50).

Synthesis and evaluation of N-substituted acridones as antiviral agents against haemorrhagic fever viruses.

BACKGROUND: In the present study, a series of N-substituted acridone derivatives was synthesized and evaluated against two haemorrhagic fever viruses (HFV). METHODS: Compounds were tested against Junin virus (JUNV), an arenavirus agent of Argentine haemorrhagic fever, and dengue virus (DENV), a flavivirus agent of the most prevalent arthropod-borne viral disease in humans. RESULTS: Among tested compounds, two N-allyl acridones (derivatives 3c and 3f) elicited a potent and selective antiviral activity against JUNV (strain 1V4454) and DENV-2 (strain NGC) with 50% effective concentration values between 2.5 and 5.5 microM, as determined by virus yield inhibition. No cytotoxicity was detected at concentrations up to 1,000 microM, resulting in selectivity indices >181.8-400.0. Both acridones were effective against a wide spectrum of arenaviruses and the four serotypes of DENV. Furthermore, 3c and 3f failed to inactivate virus before cell infection as well as to induce a refractory state by cell pretreatment, indicating that the inhibitory effect was exerted through a blockade in virus multiplication during the infectious process. CONCLUSION: These data are the first demonstration that acridone derivatives have a potent antiviral activity that block in vitro multiplication of HFV belonging to Arenaviridae and Flaviviridae, such as JUNV and DENV.

Dehydroepiandrosterone, epiandrosterone and synthetic derivatives inhibit Junin virus replication in vitro.

In the present paper the in vitro antiviral activity of dehydroepiandrosterone (DHEA), epiandrosterone (EA) and 16 synthetic derivatives against Junin virus (JUNV) replication in Vero cells was studied. DHEA and EA caused a selective inhibition of the replication of JUNV and other members of the Arenaviridae family such as Pichinde virus and Tacaribe virus. The compounds were not virucidal to cell-free JUNV. The impairment of viral replication was not due to an inhibitory effect of the steroids on virus adsorption or internalization. An inhibitory effect of the compounds on JUNV protein synthesis and both intracellular and extracellular virus production was demonstrated. A partial inhibitory action on cell surface expression of JUNV glycoprotein G1 was also detected on DHEA- and EA-treated cultures. Like DHEA and EA, three compounds obtained from EA by chemical synthesis showed selectivity indexes higher than ribavirin, the only antiviral compound that has shown partial efficacy against arenavirus infections.

Evaluation of chemical and antiviral properties of essential oils from South American plants.

The essential oils of seven aromatic plants from Córdoba, San Luis and San Juan Provinces (Argentina) were isolated by steam distillation and analysed by a gas chromatography/mass spectrometry technique. The oils were screened for cytotoxicity and in vitro inhibitory activity against herpes simplex virus type 1 (HSV-1), dengue virus type 2 (DENV-2) and Junin virus (JUNV) by a virucidal test. The oils showed a variable virucidal action according to the virus. The better relationship between cytotoxicity and antivirus action was observed with the essential oils of Heterothalamus alienus and Buddleja cordobensis against JUNV, with virucidal concentration 50% (VC50) values of 44.2 and 39.0 ppm and therapeutic indices (cytotoxicity to virucidal activity ratio) of 3.3 and 4.0, respectively. The inhibitory action was exerted by a direct interaction of virions with the oils. Virions inactivated with B. cordobensis and H. alienus essential oil were not affected in their ability to bind to the host cell. The therapeutic indices shown by these essential oils in toto were very modest, but given the complexity of their chemical composition the future identification of the precise active principle may allow the elimination of cytotoxic components and increase the selectivity of the effective compound.

Antiviral mode of action of a synthetic brassinosteroid against Junin virus replication.

The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against Junin virus replication in Vero cells was investigated. Time-related experiments showed that 6b mainly affects an early event of virus growth cycle. Neither adsorption nor internalization of viral particles was the target of the inhibitory action. The analysis of the effect of 6b on viral RNA synthesis demonstrated that the presence of the compound adversely affects virus RNA replication by preventing the synthesis of full length antigenomic RNA. Although 6b was most effective the earlier it was added to the cells after infection with JV, a high level of inhibition of JV yield and fusion activity of newly synthesized viral glycoproteins was still detected when the compound was present during the last hours of infection. Therefore, we cannot rule out an inhibitory action of 6b on later events of JV replicative cycle.

Ensayo preclinico de la vacuna candid #1 producida en Argentina contra la fiebre hemorragica Argentina / Preclinical assay of candid #1 vaccine against argentine hemorrhagic fever made in Argentina

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.

Ensayo preclinico de la vacuna candid #1 producida en Argentina contra la fiebre hemorragica Argentina / Preclinical assay of candid #1 vaccine against argentine hemorrhagic fever made in Argentina

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)

Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus.

The in vitro antiviral activity of antimicrobial cationic peptides: cecropin A, melittin, magainin I and II and indolicidin against the arenavirus Junin virus (JV), and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was evaluated. Cecropin A effectively inhibited JV multiplication and failed to affect HSV replication whereas melittin impeded the multiplication of JV and HSV, but was highly toxic for the host cell. Magainins I and II exhibited inhibitory action toward HSV-1 and HSV-2 but were inactive against JV. Only indolicidin showed a direct inactivation effect on cell-free virus stocks. Besides its inhibitory effect on JV replication cecropin A also was active against the arenaviruses Tacaribe and Pichinde, mainly affecting late events of arenavirus multiplication cycle by preventing viral morphogenesis and egress from infected cells.