Chorionic cells were obtained from placentas at term (38–40 weeks) of pregnant women with no TORCH group infections or genetic and clinical alterations, who received care at the National Institute of Women, Child and Adolescent Health Fernandes Figueira-FIOCRUZ (CAAE 88642218.1.0000.52-69). After placenta collection (n = 5), the amnionchorionic membrane was mechanically separated and the maternal face (chorionic cells) was washed in phosphate buffered saline (PBS) to remove blood clots, followed by an enzymatic dissociation protocol adapted from Kliman et al.24 Briefly, the membrane was chopped with a scalpel and subjected to enzymatic dissociation cycles with 0.25% trypsin and 100 µg/mL of a DNase solution (Gibco, Waltham, MA, USA), under agitation at 37 °C. The chorionic cell suspension was centrifuged and filtered through a 100 μm mesh, and the pellet was resuspended in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM F-12; Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Cultilab, Campinas, SP, BR), 2% l-glutamine (Gibco, Waltham,MA, USA) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). The chorionic cells were plated in culture flasks and maintained at 37 °C under a 5% CO2 atmosphere. The human cervical cell line (C33-A) and Vero cell line (kidney epithelial cells from African green monkey) were maintained at 37 °C under a 5% CO2 atmosphere in RPMI 1640 Medium (Sigma-Aldrich, St. louis, MO, USA) supplemented with 10% FBS, 2% l-glutamine and 1% penicillin/streptomycin. The C6/36 cell line from Aedes albopictus mosquito was maintained at 28 °C in Leibovitz's l-15 Medium (L-15; Gibco, Waltham, MA, USA) supplemented with 10% FBS, 10% tryptose phosphate (Sigma-Aldrich, St. louis, MO, USA), 1% l-glutamine, 1% nonessential amino acids (Gibco, Waltham, MA, USA) and 1.5% sodium bicarbonate (Sigma-Aldrich, St. louis, MO, USA) and 1% penicillin and streptomycin.

The Zika virus BRZIKV_AB_ES strain was isolated from a patient's serum in Vero cells (GenBank Accession No. KX212103; Espírito Santo) and the dengue virus DENV-2 44/2 strain was isolated in C6/36 cells (56344; Vitória, Espírito Santo).25 The stocks of both viruses were grown on C6/36 cells through three passages and subsequently titrated in Vero cell cultures by plaque forming unit assay (3,3 × 109 PFU/mL of ZIKV and 4 × 108 of DENV-2 PFU/mL) and stored at –70 °C. All experiments were carried out with the same viruses’ stocks.

Nitazoxanide (NTZ) is a drug available in many countries worldwide, approved by the FDA in the USA and ANVISA in Brazil (Fig. 1S). Powdered form of the drug (Annita®) was used to prepare stock solutions (20 mg/mL) in an 100% dimethyl sulfoxide solution (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored at 4 °C. Fresh dilutions were prepared in culture medium immediately prior to the assays.

Non-infected cell cultures seeded in 96-well plates were treated with 2-fold serially diluted NTZ (ranging from 12.5 to 1600 µg/mL) or 0.5–2% DMSO (control) and maintained at 37 °C under a 5% CO2 atmosphere for 48 h. Subsequently, cellular viability was measured by incubation with 5 mg/mL methyl thiazolyl tetrazolium reagent (MTT; Sigma-Aldrich, St. louis, MO, USA) for 4 h at 37 °C. After this time, the formazan crystals formed from viable cell metabolism were dissolved by the addition of 10% DMSO. Absorbances were determined in a spectrophotometer at 570 nm, allowing for determination of the percentage of viable cells and cytotoxic concentration that reduces 50% of cell culture viability (CC50) though a nonlinear regression analysis (dose-response curve) using the GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA).

According to cell type, the cultures were infected with ZIKV at different multiplicities of infection (MOI) and maintained for 24–72 h at 37 °C under a 5% CO2 atmosphere. Previous data indicate that Vero cells are very susceptible to ZIKV infection. Thus, the cultures were infected at a MOI of 1 for screening treatments with NTZ. Regarding DENV-2 infection, Vero cell cultures are more resistant and were infected at a MOI of 10 to assess whether NTZ is effective against other Flaviviruses. C6/36 cells infected with ZIKV at a MOI 1 exhibited low levels of infection, so the cultures were infected at a MOI of 10 to assess antiviral efficacy of NTZ in non-mammalian cells. As previous data indicate that chorionic cells were highly resistant to ZIKV infection at MOI 1 and MOI 10, the cultures were infected at a MOI of 20 for antiviral evaluation. On the other hand, C33-A cells are moderately resistant and were infected at a MOI of 10 for antiviral evaluation. After two hours, all cultures were washed with PBS to remove non-internalized viruses and fresh RPMI or DMEM-F12 media supplemented with 2% FBS was added.

Infected cell cultures were treated with 2-fold serially diluted non-toxic concentrations of NTZ (12.5 µg/mL to 100 µg/mL), defined through a cytotoxicity assay, or with 0.5% DMSO solution without NTZ (vehicle control) in two regimens: (i) 24 h before or (ii) two hours after viral inoculum. Regarding antiviral assays, the cultures were monitored during 24 and 48 h after infection to calculate the percentage of infected cells and determine the half-maximal effective concentration (EC50) through a nonlinear regression analysis (dose-response curve) using the GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). This analysis allows the calculation of the selective index (SI), a ratio that measures the window between cytotoxicity and antiviral effect (CC50/EC50). The viral load was measured in the culture supernatants and the plaque assay was performed to qualitatively analyze plaque-forming reduction upon treatment.

The viral envelope protein (E) of Flaviviruses (ZIKV and DENV-2) was assessed in infected and NTZ treated cell cultures to evaluate the percentage of infected cells and antiviral activity. The cultures were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. louis, MO, USA) for 20 min at 4 °C, washed three times with PBS, permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, Waltham, MA, USA) for five minutes, washed once more and then saturated with 3% bovine serum albumin (BSA; Sigma-Aldrich, St. louis, MO, USA) for 30 min. Samples were incubated with 4G2 mouse antibody (LATAM/Biomanguinhos) for 1 h/37 °C and, after washing twice with PBS, incubated with AlexaFluor 488-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h/37 °C. For host cell nucleus visualization, the samples were incubated with 10 µg/mL 4, 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA, USA) and mounted with 2.5% 1.4-diazabicyclo- (2.2.2)- octane (DABCO; Sigma-Aldrich, St. louis, MO, USA) to prevent fading and analyzed under a Axio Observer Z1 Motorized Inverted Fluorescence Microscope (Carl Zeiss, Oberkochen, Germany). Images were processed using the ImageJ software (ImageJ Image Processing and Analysis in Java) and CellProfiler (CellProfilerTM cell image analysis software) software for infected cell quantification.

Monolayers of Vero cells in 24-well plates were exposed to 100 µL/well of the supernatant from infected and treated cultures for one hour at 37 °C to assess plaque forming by released infectious viral particles. Next, cells were washed with PBS and RPMI containing 1% FBS and 3% carboxymethylcellulose (CMC; Sigma-Aldrich, St. Louis, MO, USA) (overlay medium) was added. After seven days at 37 °C, the monolayers were fixed with 10% formaldehyde (Sigma-Aldrich, St. louis, MO, USA) in PBS and stained with a 0.04% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA), for qualitative evaluations regarding plaque-forming reduction.

Viral RNA was extracted from the supernatant of ZIKV-infected and NTZ-treated cultures using the High Pure Viral Nucleic Acid Extraction Commercial Kit (Roche Diagnostics Brazil Ltda., São Paulo, SP, BR) ZIKV cDNA quantification was performed by the PCR technique through AgPath-ID One Step RT-PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA), using primers and probes (Table 1S) published previously by Corman and collaborators.26 A synthetic standard curve (Table 1S) was designed to detect and quantify viral load, with detection limit of 10² copies/mL, R2 = 0.989 and Slope = 3.453.

The statistical analysis was carried out using the GraphPad Prisma 8.0 software (GraphPad Software Inc., San Diego, CA, USA), with the following levels of significance: **p < 0.0013, ***p = 0.0009, and ****p < 0.0001. All analyses were performed using one-way analysis of variance ANOVA with Dunnett’s multiple comparisons test. The data are representative of 3–5 experiments run in duplicate.

Infection kinetics were determined to assess the ZIKV infection profile of each cell type (Fig. 1). Vero cell cultures infected with MOI 1 exhibited decreased infection percentage in a time-dependent manner, with values of 58 ± 6.8, 30 ± 7.4 and 17 ± 1.9% at 24 h, 48 h and 72 h, respectively (Fig. 1D). Decreased infection was a consequence of cellular death resulting from cytopathic viral effect. On the other hand, chorionic cells showed increased values in a time-dependent manner of 21 ± 0.3% at 24 h, 57 ± 5.4% at 48 h and 81 ± 3.0% at 72 h of infection (Fig. 1E). The same infection profile was observed in cervical cell cultures, with increasing values from 3.1 ± 3.1 to 49 ± 8.1 and 69 ± 1.9 during kinetic infection (Fig. 1F). Intense labeling of the viral envelope protein (E) were observed in Vero cells infected at a MOI of 1 for 24 h (Fig. 1A), chorionic cells infected at a MOI of 20 (Fig. 1B) and cervical cells infected at an MOI of 10 (Fig. 1C) for 48 h, whose infection rates were similar, at about 58%, 57% and 49%, respectively. These experimental conditions were employed to evaluate treatment regimens in Vero cell cultures and antiviral activity in chorionic and cervical cells.

Fig. 1.

Kinetics of ZIKV infection in cell cultures. The detections of the viral antigen (green) and nucleus of host cells (blue) were performed by immunofluorescence: (A) Vero cells were infected with ZIKV at a MOI of 1 for 24 h, (B) chorionic cells at a MOI of 20 and (C) cervical cells at a MOI of 10 for 48 h. The graphs represent the means ± standard deviations of the percentage of infected cells during the infection kinetics in (D) Vero, (E) chorionic and (F) cervical cells. The data are representative of 3–5 experiments run in duplicate. Bars (A and B) 100 µm, (C) 50 µm.

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In order to rule out toxic effects of the drug on host cells, uninfected cells were incubated with different NTZ concentrations and cell viability was determined by the MTT assay (Fig. 2). After 48 h of treatment, C33-A cells (Fig. 2) and C6/36 (Fig. 2S E) were found to be more sensitive to NTZ toxicity than chorionic and Vero cells (Fig. 2). CC50 values for C6/36 and C33A cells were 134 ± 4.9 µg/mL and 156 ± 2.9 µg/mL, respectively (Fig. 2S E, Fig. 2, Table 1), while the CC50 value for Vero cells was of 379 ± 1.8 µg/mL and of 1086 ± 66 µg/mL for chorionic cells (Fig. 2, Table 1). Due to different levels of sensitivity to NTZ treatment observed between host cells, doses of 12.5 µg/mL to 50 µg/mL were selected for the antiviral assays for C6/36 and C33A cells and up to 100 µg/mL for Vero and chorionic cells, which maintained about 80% of cellular viability.

Fig. 2.

Cellular viability evaluated by the MTT assay. The C33-A cell line was treated with 0 to 200 μg/mL of the drug. The Vero cell line and the primary culture of chorionic cells were treated with 0 to 1600 μg/mL of the drug for 48 h. The graph represents the means ± standard deviations of the percentage of cellular viability. The data are representative of 3–5 experiments run in duplicate.

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First, NTZ antiviral activity was screened in Vero cells by plaque forming (Fig. 3), immunofluorescence (Fig. 4A and B) and RT-qPCR assays (Fig. 4C). The treatment of infected cultures 24 h before the viral inoculum did not inhibit the production of infectious viruses (Fig. 3A). The same cytopathic effect was observed compared to untreated cultures (0 µg/mL), even at the highest tested concentration (100 µg/mL) (Fig. 3A). However, when treating after viral entry, the results showed a time and dose-dependent effect against ZIKV (Fig. 3B and C, Figs. 4A and B). Antiviral activity was stronger 48 h post-infection, where complete inhibition of infectious virus production was reached at 100 μg/mL NTZ, observed by the absence of plaque forming (Fig. 3C). Immunofluorescence applied to detect the intracellular viral antigen (E protein) indicated a time-dependent effect, with an important reduction in positively stained cells in the culture treated with 25 μg/mL NTZ compared to untreated cultures (Fig. 4A). The percentage of infected cells was significantly reduced by 66.9 ± 0.5% at 24 h post-infection when treated with 50 μg/mL NTZ, and by 72.8 ± 0.8% at 48 h post-infection when treated with 12.5 μg/mL NTZ, while at 25 μg/mL NTZ at 48 h post-infection 100% of infection was inhibited as compared to untreated cultures (Fig. 4B, Table 1). The IC50 value was 11.7 ± 0.2 μg/mL and the selective index (SI) was 32.4 after 48 h of infection (Table 1). When 100% of intracellular infection was eliminated at 25 μg/mL (Fig. 4B), RNA accumulation was quantified in the culture supernatants by RT-qPCR (Fig. 4C). The results indicate a greatly reduced viral load to 1.2 ± 1.0 × 106 copies/mL compared to 3.3 ± 1.1 × 108 copies/mL in untreated cultures (Fig. 4C), resulting in a significant 2-log amplitude reduction in viral load. Whereas efficacy of NTZ treatment in non-mammalian cells was not observed, C6/36 insect cells infected with ZIKV did not reduce the production of infectious viral particles after NTZ treatment (Fig. 2S F). On the other hand, NTZ activity against DENV-2 was observed in Vero cells (Fig. 3S), with a dose-dependent decrease in the percentage of infection (Fig. 3S C) and plaque formation (Fig. 3S D). Infection reductions at 50 μg/mL and 100 μg/mL NTZ of 96.4 ± 4.1% and 99.5 ± 0.7% were observed, respectively (Fig. 3S C). Taken together, these results indicate a strong effect of NTZ in inhibiting ZIKV and DENV-2 infection in Vero cells after viral entry, probably at the viral replication step.

Fig. 3.

Antiviral effect of Nitazoxanide determined by the plaque assay. Vero cell cultures were treated or not (Mock) with 12.5, 25, 50 and 100 μg/mL of the drug and infected with ZIKV at a MOI of 1. Treatment was performed (A) 24 h before infection and (B and C) after the viral inoculum. (A and B) the infection was followed-up until 24 h and (C) until 48 h. The data are representative of 3–5 experiments run in duplicate.

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Fig. 4.

Antiviral effect of Nitazoxanide detected by immunofluorescence and RT-qPCR. Vero cell cultures were infected with ZIKV at a MOI of 1 and subsequently treated with 12.5, 25, 50 and 100 μg/mL of the drug for 24 h and 48 h. (A) The viral antigen (green) and the host cell nucleus (blue) detections were performed by immunofluorescence. (B) The graph represents the means ± standard deviations of the percentage of infected cells after 24 h and 48 h of treatment. (C) The supernatant from cultures treated with 25 μg/mL was collected 48 h after infection to quantify the viral load by the RT-qPCR assay. The graph represents the means ± standard deviations of the copies of the ZIKV genome/mL. Statistical significance was determined by a one-way ANOVA, followed by Dunnett’s multiple-comparisons test. **p < 0.0013, ***p = 0.0009, ****p < 0.0001. The data are representative of 3-5 experiments run in duplicate. Bars 50 µm.

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As observed in Vero cell cultures, treatment was effective only after the viral inoculum. This treatment regimen was employed in placental (chorionic) and cervical (C33A) cells cultures and strong antiviral activity was confirmed after 48 h of treatment (Figs. 5–7). The antiviral activity of NTZ in chorionic cells led to a significant dose-dependent reduction in the number of infected cells (Fig. 5, Table 1), as well as in fluorescence intensity at 12.5 μg/mL (Fig. 5 B), 25 μg/mL (Fig. 5C) and 50 μg/mL (Fig. 5D) compared to untreated cells (Fig. 5A). At 50 μg/mL, the reduction of infected cells was of 79.2 ± 3% (Figs. 5D and E, Table 1). Regarding cervical cells, treatment with 12.5 μg/mL exhibited a drastic reduction of 95.3 ± 6.5% in the number of infected cells (Figs. 6B and E, Table 1). At 25 μg/mL (Fig. 6C) and 50 μg/mL (Fig. 6D), only about 0.3 ± 0.5% of infected cells were observed. EC50 values were 17.5 ± 0.1 μg/mL for chorionic cells and 4.1 ± 1.0 μg/mL for C33A cells (Table 1) and SI values were 62.1 and 38.0, respectively (Table 1). In addition, RT-qPCR and plaque forming assays were performed (Fig. 7). Quantification of viral loads in the culture supernatants treated with 50 μg/mL revealed a significant reduction to 3.2 ± 2.8 × 106 copies/mL and 5.9 ± 3.2 × 106 copies/mL in chorionic and cervical cells, respectively, compared with 4.6 ± 1.2 × 107 copies/mL in untreated cultures (Fig. 7C). To confirm these results, the production of infectious viruses by treated cells was analyzed. Of importance, a 25 μg/mL concentration in chorionic cells inhibited 100% of viral progeny (Fig. 7A). However, despite the observed reduction in infectious virus production by the treated cervical cells, complete inhibition was not observed (Fig. 7B).

Fig. 5.

Antiviral effect of Nitazoxanide on a primary chorionic cell culture determined by immunofluorescence. The cultures were infected or not (inset A) with ZIKV at a MOI of 20 and subsequently treated or not (A) with (B) 12.5 μg/mL, (C) 25 μg/mL or (D) 50 μg/mL of the drug for 48 h. The fluorescence staining shows the viral antigen (green) and the host cell nucleus (blue). (E) The graph represents the means ± standard deviations of the percentage of infected cells. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple-comparisons test. ****p < 0.0001. The data are representative of 3-5 experiments run in duplicate. Bars 100 µm.

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Fig. 6.

Antiviral effect of Nitazoxanide on cervical cells (C33-A). Cultures were infected or not (inset A) with ZIKV at a MOI of 10 and subsequently treated or not (A) with (B) 12.5 μg/mL, (C) 25 μg/mL or (D) 50 μg/mL of NTZ for 48 h. The fluorescence staining shows the viral antigen (green). (E) The graph represents the means ± standard deviations of the percentage of infected cells. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple-comparisons test. ****p < 0.0001. The data are representative of 3-5 experiments run in duplicate. Bars 50 µm.

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Fig. 7.

Evaluation of anti-ZIKV effect of Nitazoxanide on host cells. (A) The primary cultures of chorionic cells were infected or not (Mock) with ZIKV at a MOI of 20 and (B) cervical cells (C33-A) at a MOI of 10 and subsequently treated with 12.5, 25, 50 or 100 μg/mL of NTZ for 48 h. Furthermore, (A and B) the culture supernatants were collected for the detection of infectious viruses by the plaque assay and (C) quantification of viral loads. The graph represents the means ± standard deviations of the copies of the ZIKV genome/mL. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s multiple-comparisons test. **p < 0.0013, ***p = 0.0009. The data are representative of 3–5 experiments run in duplicate.

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