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.

Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.

Introduction: Drug repurposing is the promptest way to obtain an effective drug during a global public health emergency as the spread of Zika virus that is associated with the congenital syndrome. Why the virus reaches the fetus is still unclear, however the placenta represents an important route of transmission, since the virus was detected in vivo and in vitro human and murine placenta. The fetal infection may occur by passing the virus through spaces created by lesions or inflammation that may break the placental barrier. Another hypothesis is that the virus crosses the placenta through infection of host cells, being possible the transmission of ZIKV to the fetus occur from sexual transmission. Despite the emerging severity caused by the ZIKV infection, there still no specific treatment for this disease. The antiparasitic and antiviral drug Annita® already approved through the Food and Drug Admistration and safe for pediatric use and for administration in pregnant women, being included in category B, could have activity against ZIKV due to broad aspect and affordable price. Objective: With this in mind our group evaluated the antiviral effect this drug in chorionic cells of primary culture human placenta and in cervix human cells, two important targets of infection, in comparing to Vero cells from African green monkey and C6/36 cells from Aedes albopictus. Methodology: The antiviral effect was assessed by immunofluorescence, Plaque assay and RT-qPCR in cultures infected with ZIKV and treated with non-toxic concentrations of the drug for 48h. Results: Previous results using Vero cultures showed antiviral effect of drug only when the treatment was performed after the adsorption step. This treatment scheme revealed a dosedependent reduction of infection in chorionic cells at 79% at the concentration of 50 μg/ mL. Interestingly, in the cervix cells, at the concentration of 12 μg/mL already had a 95% of infection reduction. The EC50 values were 23±5μg/mL for chorionic cells and 6±0.4μg/mL for cervix cells. Analyzing the reduction of infectious particles, the concentration of 25 μg/ mL in the chorionic cells and 12 μg/mL in the cervix cells inhibited 100% of the progeny. In addition, the quantification of viral RNA copies/mL in the supernatant of the treated cultures with 50 μg/mL showed a reduction of 93% in chorionic cells and 87% in cervix cell cultures. However, in cells of the ZIKV-infected C6/36 mosquito line, the treatment did not reduce the number of infectious particles in the cultures supernatant, suggesting that the activity of this drug is related to the response of the host cells. Conclusion: The Annita® drug demonstrated good antiviral activity, which may be related to host cell response. Studies aimed at reusing drugs should be encouraged because they accelerate the discovery of drugs for the treatment of ZIKV infection, especially for infected pregnant women.

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