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.

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.

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.

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 natural and synthetic ß-carbolines against dengue virus.

Dengue virus (DENV) is the most prevalent mosquito borne viral pathogen worldwide. In this work we first evaluated the antiviral activity of natural and synthetic ß-carbolines against DENV-2 multiplication in cell cultures. We determined that the natural ß-carboline harmol and a synthetic harmine derivative, 9N-methylharmine, exhibit inhibitory effect on DENV-2 production without virucidal activity. The active compounds were inhibitory of all DENV serotypes, being DENV-2 the more susceptible to their antiviral action. The mode of action of 9N-methylharmine against DENV-2 was further explored. We determined that the derivative neither affects viral adsorption-internalization events nor viral RNA synthesis. The quantification of intracellular and extracellular viral genomes and infectious virus particles indicated that 9N-methylharmine would impair the maturation and release of virus particles to the extracellular medium affecting the spreading of the infection. Furthermore, we also determined that 9N-methylharmine antiviral activity is not related to the ability of the compound to downregulate p38 MAPK phosphorylation.

Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection.

The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.

Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.

The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.