Design and characterization of BSA-mycophenolic acid nanocomplexes: Antiviral activity exploration.

The interactions between bovine serum albumin (BSA) and mycophenolic acid (MPA) were investigated in silico through molecular docking and in vitro, using fluorescence spectroscopy. Dynamic light scattering and scanning electron microscopy were used to figure out the structure of MPA-Complex (MPA-C). The binding affinity between MPA and BSA was determined, yielding a Kd value of (12.0 ± 0.7) µM, and establishing a distance of 17 Å between the BSA and MPA molecules. The presence of MPA prompted protein aggregation, leading to the formation of MPA-C. The cytotoxicity of MPA-C and its ability to fight Junín virus (JUNV) were tested in A549 and Vero cell lines. It was found that treating infected cells with MPA-C decreased the JUNV yield and was more effective than free MPA in both cell line models for prolonged time treatments. Our results represent the first report of the antiviral activity of this type of BSA-MPA complex against JUNV, as assessed in cell culture model systems. MPA-C shows promise as a candidate for drug formulation against human pathogenic arenaviruses.

Chemically modified galactans of Grateloupia indica: From production to in vitro antiviral activity.

Herpes simplex viruses (HSVs) have an affinity for heparan sulfate proteoglycans on cell surfaces, which is a determinant for virus entry. Herein, several sulfated galactans that mimic the active domain of the entry receptor were employed to prevent HSV infection. They were produced from Grateloupia indica using chlorosulfonic acid-pyridine (ClSO3H.Py)/N,N-dimethylformamide reagent (fraction G-402), SO3.Py/DMF reagent (G-403), or by aqueous extraction (G-401). These galactans contained varied molecular masses (33-55 kDa), and sulfate contents (12-20 %), and have different antiviral activities. Especially, the galactan (G-402) generated by using ClSO3H.Py/DMF, a novel reagent, exhibited the highest level of antiviral activity (EC50 = 0.36 µg/mL) compared to G-403 (EC50 = 15.6 µg/mL) and G-401 (EC50 = 17.9 µg/mL). This most active sulfated galactan possessed a linear chain containing ß-(1 â†’ 3)- and α-(1 â†’ 4)-linked Galp units with sulfate group at the O-2/4/6 and O-2/3/6 positions, respectively. The HSV-1 and HSV-2 strains were specifically inhibited by this novel 33 ± 15 kDa galactan, which also blocked the virus from entering the host cell. These results highlight the significant potential of this sulfated galactan for antiviral research and drug development. Additionally, the reagent used for the effective conversion of galactan hydroxy groups to sulfate during extraction may also be useful for the chemical transformation of other natural products.

Effects of the Natural Flavonoid Quercetin on Arenavirus Junín Infection.

There is no specific chemotherapy approved for the treatment of pathogenic arenaviruses that cause severe hemorrhagic fever (HF) in the population of endemic regions in America and Africa. The present study reports the effects of the natural flavonoid quercetin (QUER) on the infection of A549 and Vero cells with Junín virus (JUNV), agent of the Argentine HF. By infectivity assays, a very effective dose-dependent reduction of JUNV multiplication was shown by cell pretreatment at 2-6 h prior to the infection at non-cytotoxic concentrations, with 50% effective concentration values in the range of 6.1-7.5 µg/mL. QUER was also active by post-infection treatment but with minor efficacy. Mechanistic studies indicated that QUER mainly affected the early steps of virus adsorption and internalization in the multiplication cycle of JUNV. Treatment with QUER blocked the phosphorylation of Akt without changes in the total protein expression, detected by Western blot, and the consequent perturbation of the PI3K/Akt pathway was also associated with the fluorescence redistribution from membrane to cytoplasm of TfR1, the cell receptor recognized by JUNV. Then, it appears that the cellular antiviral state, induced by QUER treatment, leads to the prevention of JUNV entry into the cell.

Modulation of the Aryl Hydrocarbon Receptor Signaling Pathway Impacts on Junín Virus Replication.

Junín virus (JUNV), a member of the family Arenaviridae, is the etiological agent of the Argentine hemorrhagic fever, an endemic disease in the rural region of Argentina lacking a specific chemotherapy. Aryl hydrocarbon receptor (AHR) is expressed in several mammalian tissues and has been indicated as a sensor of ligands from variable sources and a modulator of the cell immune response. Interestingly, recent studies have suggested that the activation or depression of the AHR signaling pathway may play a role in the outcome of diverse human viral infections. In the present report, the effect of the pharmacological modulation of AHR on JUNV in vitro infection was analyzed. An initial microarray screening showed that the AHR pathway was overexpressed in JUNV-infected hepatic cells. Concomitantly, the infection of Vero and Huh-7 cells with the JUNV strains IV4454 and Candid#1 was significantly inhibited in a dose-dependent manner by treatment with CH223191, a specific AHR antagonist, as detected by infectivity assays, real-time RT-PCR and immunofluorescence detection of viral proteins. Furthermore, the pro-viral role of AHR in JUNV infection appears to be independent of the IFN-I pathway. Our findings support the promising perspectives of the pharmacological modulation of AHR as a potential target for the control of AHF.

Inhibitors of Nucleotide Biosynthesis as Candidates for a Wide Spectrum of Antiviral Chemotherapy.

Emerging and re-emerging viruses have been a challenge in public health in recent decades. Host-targeted antivirals (HTA) directed at cellular molecules or pathways involved in virus multiplication represent an interesting strategy to combat viruses presently lacking effective chemotherapy. HTA could provide a wide range of agents with inhibitory activity against current and future viruses that share similar host requirements and reduce the possible selection of antiviral-resistant variants. Nucleotide metabolism is one of the more exploited host metabolic pathways as a potential antiviral target for several human viruses. This review focuses on the antiviral properties of the inhibitors of pyrimidine and purine nucleotide biosynthesis, with an emphasis on the rate-limiting enzymes dihydroorotate dehydrogenase (DHODH) and inosine monophosphate dehydrogenase (IMPDH) for which there are old and new drugs active against a broad spectrum of pathogenic viruses.

Inhibition of dengue virus infection by trifluoperazine.

Dengue virus (DENV), a member of the genus Flavivirus, family Flaviviridae, is the most widespread viral pathogen transmitted to humans by mosquitoes. Despite the increased incidence of DENV infection, there are no antiviral drugs available for treatment or prevention. Phenothiazines are heterocyclic compounds with various pharmacological properties that are very adaptable for drug repurposing. In the present report, we analyzed the antiviral activity against DENV and the related Zika virus (ZIKV) of trifluoperazine (TFP), a phenothiazine derivative in clinical use as an antipsychotic and antiemetic agent. TFP exhibited dose-dependent inhibitory activity against the four DENV serotypes and ZIKV in monkey Vero cells at non-cytotoxic concentrations with 50% effective concentration values in the range 1.6-6.4 µM. A similar level of antiviral efficacy was exhibited by TFP against flavivirus infection in the human cell lines A549 and HepG2. Mechanistic studies, performed using time-dependent infectivity assays, real-time RT-PCR, Western blot, and immunofluorescence techniques, indicated that uncoating of the virus during penetration into the cell was the main target for TFP in infected cells, but the compound also exerted a minor effect on a late stage of the virus multiplication cycle. This study demonstrates that TFP, a pharmacologically active phenothiazine, is a selective inhibitor of DENV multiplication in cell culture. Our findings open perspectives for the repositioning of phenothiazines like TFP with a wide spectrum of antiviral efficacy as potential agents for the control of pathogenic flaviviruses.

Murine models of dengue virus infection for novel drug discovery.

INTRODUCTION: Dengue virus (DENV) is the causative agent of the most prevalent human disease transmitted by mosquitoes in tropical and subtropical regions worldwide. At present, no antiviral drug is available and the difficulties to develop highly protective vaccines against the four DENV serotypes maintain the requirement of effective options for dengue chemotherapy. AREAS COVERED: The availability of animal models that reproduce human disease is a very valuable tool for the preclinical evaluation of potential antivirals. Here, the main murine models of dengue infection are described, including immunocompetent wild-type mice, immunocompromised mice deficient in diverse components of the interferon (IFN) pathway and humanized mice. The main findings in antiviral testing of DENV inhibitory compounds in murine models are also presented. EXPERT OPINION: At present, there is no murine model that fully recapitulates human disease. However, immunocompromised mice deficient in IFN-α/ß and -γ receptors, with their limitations, have shown to be the most suitable system for antiviral preclinical testing. In fact, the AG129 mouse model allowed the identification of celgosivir, an inhibitor of cellular glucosidases, as a promising option for DENV therapy. However, clinical trials still were not successful, emphasizing the difficulties in the transition from preclinical testing to human treatment.

Antiviral activity against Zika virus of a new formulation of curcumin in poly lactic-co-glycolic acid nanoparticles.

OBJECTIVES: In the search of an effective antiviral formulation, the natural product curcumin (CUR) was encapsulated into poly(lactic-co-glycolic acid) nanoparticles, a non-toxic bioresorbable and biocompatible copolymer. The resulting CUR containing particles (PLGA-CUR NPs) were characterized and analysed for antiviral activity against Zika virus (ZIKV) infection. METHODS: The PLGA-CUR NPs were characterized by Fourier transform infrared, differential scanning calorimetry, dynamic light scattering, scanning electron microscopy and thermogravimetric analysis and release profile. Cytotoxicity of PLGA-CUR and the antiviral activity against ZIKV were determined in Vero cells. The effect of PLGA-CUR NPs on viral RNA synthesis and protein expression was analysed by RT-qPCR and immunofluorescence staining, respectively. KEY FINDINGS: The PLGA-CUR NPs showed an appropriate in vitro drug release profile. Our studies of the antiviral activity of PLGA-CUR NPs and CUR against ZIKV by virus yield reduction as well as viral RNA synthesis and protein expression have shown that PLGA-CUR formulation is more effective than free CUR to inhibit ZIKV infection of Vero cells. CONCLUSIONS: Our results demonstrate for the first time the antiviral activity against ZIKV of PLGA nanoparticles charged with CUR, suggesting that PLGA-CUR NPs are promising candidates for a drug formulation against human pathogenic flaviviruses.

Chemically sulfated arabinoxylans from Plantago ovata seed husk: Synthesis, characterization and antiviral activity.

Limited options for the treatments of diseases triggered through viral infections revealed the quest for novel antiviral drugs. Polysaccharide sulfates owing to their unique mode of action are prominent antiviral drug candidates. Herein, the arabinoxylan of Plantago ovata seed husk was simultaneously extracted and chemically sulfated using sulphur trioxide-pyridine reagent in N,N-dimethylformamide solvent (SO3⋅Py/DMF). Thus, three arabinoxylan sulfates (IS1201-IS1203) holding variable degrees of sulfation (DS: 0.1-0.9), molar masses (18.4-31.3 kDa) and glycosyl makeup (Ara: Xyl::10-19:81-90; molar ratio) were produced and then characterized. According to the results, these polymers displayed anti-herpes simplex virus type 1 activity and their potency depends upon DS. The utmost effective compound (IS1203, IC50: 2.9 µg mL-1) was a 18.4 kDa arabinoxylan possessing sulfate groups at O-3 and O-2,3 positions of xylopyranosyl (Xylp), and O-5 of arabinofuranosyl (Araf) residues. Besides, this polymer showed no cytotoxicity at concentration up to 1000 µg mL-1. Given that polysaccharide sulfates have antiviral activities, synthesis of new molecules possessing diverse structures will be a useful addition to the arsenal of antivirals.

Antibody-independent and dependent infection of human myeloid cells with dengue virus is inhibited by carrageenan.

This study demonstrated that the λ-carrageenan is a potent and selective inhibitor of the primary infection of human myeloid U937 and K562 cells with the four DENV serotypes, achieving a higher than 99 % reduction in virus production at the highest tested concentration of 20 µg/mL, without affecting cell viability at concentrations up to 1000 µg/mL. Since antibody-dependent enhancement (ADE) is thought to play a main role in the aggravation of severe DENV disease, we also evaluated the activity of carrageenan against ADE of DENV infection. The λ-carrageenan was also effective to block the antibody dependent infection mediated by Fcγ-RII in both cell lines, causing 96-99 % inhibition in virus production from cells infected with immune complexes of DENV-2 and DENV-3. Moreover, the inhibitory effectiveness of carrageenan was similar against prM-mediated ADE or E-mediated ADE. Mechanistic studies indicated that DENV-2 entry is the main antiviral target for carrageenan in DENV or DENV-Ab infected human myeloid cells since a strong inhibitory effect was observed when the carrageenan was present only during adsorption at 4 °C or internalization at 37 °C, whereas the infection was not altered when the compound was added after virus internalization. Thus, our findings have shown that carrageenan may be considered an interesting antiviral agent able to block DENV entry during both primary and antibody-dependent infection of human myeloid cells.

Cellular Organelles Reorganization During Zika Virus Infection of Human Cells.

Zika virus (ZIKV) is an enveloped positive stranded RNA virus belonging to the genus Flavivirus in the family Flaviviridae that emerged in recent decades causing pandemic outbreaks of human infections occasionally associated with severe neurological disorders in adults and newborns. The intracellular steps of flavivirus multiplication are associated to cellular membranes and their bound organelles leading to an extensive host cell reorganization. Importantly, the association of organelle dysfunction with diseases caused by several human viruses has been widely reported in recent studies. With the aim to increase the knowledge about the impact of ZIKV infection on the host cell functions, the present study was focused on the evaluation of the reorganization of three cell components, promyelocytic leukemia nuclear bodies (PML-NBs), mitochondria, and lipid droplets (LDs). Relevant human cell lines including neural progenitor cells (NPCs), hepatic Huh-7, and retinal pigment epithelial (RPE) cells were infected with the Argentina INEVH116141 ZIKV strain and the organelle alterations were studied by using fluorescent cell imaging analysis. Our results have shown that these three organelles are targeted and structurally modified during ZIKV infection. Considering the nuclear reorganization, the analysis by confocal microscopy of infected cells showed a significantly reduced number of PML-NBs in comparison to uninfected cells. Moreover, a mitochondrial morphodynamic perturbation with an increased fragmentation and the loss of mitochondrial membrane potential was observed in ZIKV infected RPE cells. Regarding lipid structures, a decrease in the number and volume of LDs was observed in ZIKV infected cells. Given the involvement of these organelles in host defense processes, the reported perturbations may be related to enhanced virus replication through protection from innate immunity. The understanding of the cellular remodeling will enable the design of new host-targeted antiviral strategies.

Author Correction: AHR is a Zika virus host factor and a candidate target for antiviral therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

AHR is a Zika virus host factor and a candidate target for antiviral therapy.

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity.

Synthesis and antiviral activity of some imidazo[1,2-b][1,3,4]thiadiazole carbohydrate derivatives.

Herein we describe the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles from carbohydrates with D-ribo and D-xylo configuration. The antiviral activity of these compounds was tested against Junín virus (the etiological agent of Argentine hemorrhagic fever). The p-chlorophenyl derivatives showed antiviral activity in a range of micromolar concentration.

Fucoidans from the phaeophyta Scytosiphon lomentaria: Chemical analysis and antiviral activity of the galactofucan component.

The brown seaweed Scytosiphon lomentaria produces moderate amounts of fucoidans. By cetrimide fractionation, typical heavily sulfated galactofucans are obtained, with no major signs of chemical heterogeneity, together with fractions with higher proportions of xylose, mannose and uronic acids. Anyway, fucose is the most important monosaccharide in most of the subfractions of the subsequent extracts. The fucan moieties appear to be mostly as 3-linked α-l-fucopyranosyl units, with several patterns of sulfate and branching. Galactose is mostly 6-linked, whereas mannose appears to be 2-linked, and xylose appears mostly as terminal stubs. Small amounts of 2-O-acetylated fucose units appear. A high and selective antiviral activity against HSV-1 and HSV-2 was determined for the galactofucan fractions whereas the uronofucoidans were inactive.

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.

Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies.

BACKGROUND: Dengue is the most prevalent arthropod-borne viral human disease in tropical and subtropical regions, caused by four dengue virus (DENV) serotypes. In spite of the increasing global incidence, no specific antiviral therapy is available. Cells of the mononuclear phagocyte lineage are the main targets either for direct antibody (Ab)-independent or Ab-mediated human DENV infection, usually associated to the severe forms of disease. Since the virus entry may be a convenient therapeutic alternative, this study aimed to investigate the mode of DENV internalization into myeloid cells in the absence and presence of DENV Ab and evaluate the inhibitory activity of diverse biochemical inhibitors of endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: By infectivity assays and quantitative RT-PCR determinations, it was demonstrated that DENV-2 entry into U937 and K562 cells in the absence of Ab was highly inhibited by the early treatment with ammonium chloride, chlorpromazine and dynasore, but it was not affected by methyl-ß-cyclodextrin, indicating that DENV-2 utilizes a low pH-dependent, clathrin- and dynamin-mediated endocytic infectious pathway for the direct entry into both human myeloid cells. To study the Ab-mediated entry of DENV, the experimental conditions for enhancement of infection were established by inoculating immune complexes formed with DENV-2 and the Ab 2H2 or 3H5. The internalization of DENV-2-2H2 or DENV-2-3H5 complexes in both myeloid cells was also dependent on acid pH and dynamin but a differential requirement of the clathrin-mediated endocytic route was observed depending on the FcγR involved in the complex uptake: the infection through FcγRII was dependent on clathrin-coated vesicles whereas the internalization pathway mediated by FcγRI was independent of clathrin. This property was not serotype-specific. CONCLUSIONS/SIGNIFICANCE: DENV entry into myeloid cells in the absence or presence of Ab can be blocked by diverse biochemical inhibitors affecting the cellular factors involved in endocytosis. The identification of the virus-host interactions involved in virus penetration may allow the finding of host-targeted antivirals widely active against diverse pathogenic flaviviruses with similar requirements for virus entry.

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.

Determining the Virus Life-Cycle Stage Blocked by an Antiviral.

Among the members of the Arenaviridae family, Junín virus and Lassa virus represent important human health threats generating annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of Argentina and Western Africa, respectively. Given the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. During the last two decades, academic research studies originated important results identifying novel molecules to be considered for further in vivo characterization. This chapter summarizes experimental in vitro approaches used to determine the possible mechanism of action of these antiviral agents.

The Role of Heterotypic DENV-specific CD8+T Lymphocytes in an Immunocompetent Mouse Model of Secondary Dengue Virus Infection.

Dengue is the most prevalent arthropod-borne viral disease worldwide and is caused by the four dengue virus serotypes (DENV-1-4). Sequential heterologous DENV infections can be associated with severe disease manifestations. Here, we present an immunocompetent mouse model of secondary DENV infection using non mouse-adapted DENV strains to investigate the pathogenesis of severe dengue disease. C57BL/6 mice infected sequentially with DENV-1 (strain Puerto Rico/94) and DENV-2 (strain Tonga/74) developed low platelet counts, internal hemorrhages, and increase of liver enzymes. Cross-reactive CD8+ T lymphocytes were found to be necessary and sufficient for signs of severe disease by adoptively transferring of DENV-1-immune CD8+T lymphocytes before DENV-2 challenge. Disease signs were associated with production of tumor necrosis factor (TNF)-α and elevated cytotoxicity displayed by heterotypic anti-DENV-1 CD8+ T lymphocytes. These findings highlight the critical role of heterotypic anti-DENV CD8+ T lymphocytes in manifestations of severe dengue disease.