Suppression of TGF-ß/Smad2 signaling by GW788388 enhances DENV-2 clearance in macrophages.

Dengue fever, caused by the dengue virus (DENV-1, -2, -3, and -4), affects millions of people in the tropical and subtropical regions worldwide. Severe dengue is correlated with high viraemia and cytokine storm, such as high levels of transforming growth factor-ß1 (TGF-ß1) in the patient's serum. Here, the TGF-ß1 signaling was investigated in the context of in vitro viral clearance. Macrophages were infected with DENV-2 at MOI 5 and treated with the TGF-ß receptor 1 and 2 inhibitor, GW788388. TGF-ß1 expression, signal transduction and viral load were evaluated 48 h after DENV-2 infection by enzyme-linked immunoassay, immunofluorescence, and RT-qPCR assays. Total TGF-ß1 level was reduced in 15% after DENV-2 infection, but the secretion of its biologically active form increased threefold during infection, which was followed by the phosphorylation of Smad2 protein. Phosphorylation of Smad2 was reduced by treatment with GW788388 and it was correlated with reduced cytokine production. Importantly, treatment led to a dose-dependent reduction in viral load, ranging from 6.6 × 105 RNA copies/ml in untreated cultures to 2.3 × 103 RNA copies/ml in cultures treated with 2 ng/ml of GW788388. The anti-TGF-ß1 antibody treatment also induced a significant reduction in viral load to 1.6 × 103 RNA copies/ml. On the other hand, the addition of recombinant TGF-ß1 in infected cultures promoted an increase in viral load to 7.0 × 106 RNA copies/ml. These results support that TGF-ß1 plays a significant role in DENV-2 replication into macrophages and suggest that targeting TGF-ß1 may represent an alternative therapeutic strategy to be explored in dengue infection.

ZIKV replication is differential in explants and cells of human placental which is suppressed by HSV-2 coinfection.

During the Zika fever outbreak in Brazil in 2015-2016, only some babies from infected mothers had teratogenic effects, suggesting that cofactors may influence congenital transmission. We investigated the ZIKV infection profile in explants and isolated cells from full-term human placenta to infection with the Brazilian Zika virus strain (ZIKVBR) and the effect of coinfection with the Brazilian Human alphaherpesvirus 2 strain (HSV-2BR) on ZIKV replication. We found that the ZIKVBR infect the explants of amniotic and chorionic membranes, as well as chorionic villi core, but not the trophoblasts layer. It was also observed that ZIKV replication was higher in amniotic cells than chorionic and trophoblasts cells. Upon coinfection, the replication of ZIKVBR was reduced according to exposed HSV-2BR load in trophoblasts cells and the levels of TNF-α and IL-6 cytokines were also reduced. These findings suggest that the placental cell types and HSV-2BR coinfection may impact on ZIKV replication.

Chloroquine and Sulfadoxine Derivatives Inhibit ZIKV Replication in Cervical Cells.

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 µM of C-Sds and observed CC50 values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV-envelope glycoprotein expression in infected cells as compared to 36.8% ± 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 ± 0.7 × 108 copies/mL in vehicle control. The dose-response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC50 values ranged from 3.2 ± 0.1 to 5.0 ± 0.2 µM, and the EC90 values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity.