To produce recombinant ZIKV, we first constructed a DNA-based replicon system pZIKVrepdCME (Fig. 1A). The first quarter of the ZIKV genome, containing 5'-UTRC-prM-E-NS1134, was synthesized and cloned into the pCAGneo vector under transcriptional control of the CMV promoter and resulted in pZCME-NS1134. To generate the GFP-expressing ZIKV reporter, plasmid pC38GFP-CMENS1134 was derived from pZCME-NS1134, in which the GFP reporter gene followed by the sequence encoding FMDV 2A was cloned in-frame downstream of sequences encoding the additional first 38 amino acids of the C protein and upstream of the complete C protein (Fig. 1A). The 2A protease ensured the authentic N-terminus of the C protein. DNA fragment A, containing the CMV promoter and 5'-UTR-C-prM-E-NS1134 of ZIKV, was amplified by PCR and was then used, in addition to linearized replicon plasmids, to transfect 293T cells (Fig. 1B). And DNA fragment B, which contained sequences of the CMV promoter and 5'-UTR-C38-GFP-2A-C-prM-E-NS1134, was amplified by PCR. Cells of 293T were transfected with linearized replicon plasmids and fragment B (Fig. 1B).
Figure 1. Schematic representation of the strategy for plasmid construction and the production of recombinant ZIKV and ZIKV-GFP. A Illustration of the ZIKV genome and constructs used in this study. In the pZIKVrepdCME construct, the GFP- and FMDV-2A-coding sequences were inserted downstream of sequences encoding the first 20 amino acids of the C gene and upstream of sequences encoding the last 22 amino acids of the E gene. The 30 untranslated region was flanked with HDVr to ensure the authenticity of the 30 terminus of the transcribed RNA. B Schematic diagrams for the production of recombinant ZIKV and ZIKV-GFP marker viruses. DNA fragments containing the CMV promoter and the coding sequences of structural proteins were amplified by PCR. The purified PCR products and enzymelinearized ZIKV replicon plasmid DNA were pooled and transfected into HEK-293T cells to generate infectious ZIKVs.
At 3 days post transfection, the supernatants were inoculated onto BHK-21 cells and infectious viruses were detected using an immunofluorescence assay (IFA) with pan-flavivirus E protein-specific mouse MAb 4G2 (Fig. 2A). The recombinant ZIKV was cytopathic and formed plaques in BHK-21 cells (Fig. 2B) and Vero cells (data not shown). Recombinant ZIKV grown on BHK-21 cells reached an infectious titer up to 5.7 log PFU/mL at 72 h post-infection (hpi) and then the virus titer decreased at 96 hpi and 120 hpi. Viral growth dynamics were then analyzed on Vero cells and the virus titer reached 6.7 log PFU/mL at 48 hpi and was maintained at 7.0 log PFU/mL from 72 to 120 hpi (Fig. 2C). These results showed that the replication of recombinant ZIKV in Vero cells was highly efficient. For rescuing ZIKV-GFP, at 3 days post transfection, the supernatants were inoculated onto BHK-21 and Vero cells to detect the titer of infectious virus. Positive GFP-expressing BHK-21 cells (Fig. 2D) and Vero cells were observed following inoculation with supernatants of transfected 293T cells. The infection of Vero cells with the recombinant reporter virus ZIKV-GFP caused cytopathic effects and led to plaque foramtion (data not shown). The infectious titer of ZIKV-GFP grown in Vero cells reached 6.5 log TCID50/mL at 72 hpi (Fig. 2E). However, the GFP signal in infected Vero cells was mainly observed as distinct foci of green fluorescence signal, which did not conform to the shape of cells (Fig. 2F). This observation weakens the utility of of ZIKV-GFP as a reporter virus. In infected BHK-21 cells, the green fluorescence signal was evenly distributed throughout the cytoplasm and the cell outline was apparent (Fig. 2D and 2F). However, the replication of ZIKV-GFP in BHK-21 cells was attenuated compared with that of wild-type recombinant ZIKV growth in BHK-21 cells. In BHK-21 cells infected with ZIKVGFP, the green fluorescence foci spread very slowly (Supplementary Fig. S1) and ZIKV-GFP did not cause cytopathic effects in BHK-21 cells even after incubation for 6 dpi or longer (Fig. 2D).
Figure 2. Production and characterization of recombinant ZIKV and ZIKV-GFP reporter viruses. A Immunofluorescence analysis of recombinant ZIKV. The supernatants of transfected 293T cells were inoculated into BHK-21 cells. Forty-eight hours later, infected cells were fixed and probed with pan-flavivirus E proteinspecific MAb 4G2. Nuclei were stained with DAPI. B Representative images of plaque assays of BHK-21 and/or BHK-DR cells infected with ZIKV and ZIKV-GFP viruses and fixed and stained at the indicated days postinfection. C Replication kinetics of recombinant wild-type ZIKV in BHK-21, Vero and BHK-DR cells. Cells were infected at an MOI of 0.1. Supernatants were harvested at 24 hpi to 120 hpi and analyzed using a plaque form assay in BHK-21 cells. D ZIKV-GFP reporter virus infection in BHK-21 and BHK-DR cells. Cells infected with ZIKV-GFP at the indicated time were analyzed by fluorescence microscopy for GFP expression and by light microscopy for cytopathic effects. E Replication kinetics of recombinant ZIKVGFP in Vero and BHK-DR cells. Cells were infected at an MOI of 0.1. Supernatants were harvest at 24 hpi to 120 hpi and analyzed using a TCID assay in BHK-DR cells by observation of fluorescent foci. F GFP fluorescence in Vero and BHK-DR cells infected with ZIKVGFP. One representative experiment out of three is shown. At 48 hpi, cells were stained with DAPI and visualized using a high-content screening microplate imaging reader (Operetta; PerkinElmer). The GFP fluorescence intensity G and GFP-positive ratio H were analyzed using Acapella high-content imaging and analysis software. The results are the mean of three independent experiments performed in triplicate. Statistical significance was determined by the Student's t-test (** P < 0.01, ns P > 0.05).
DC-SIGNR as receptor of flavivirus could increase the susceptibility of cells to ZIKV infection. To make ZIKVGFP a more readable and stable genetic reporting tool for viral replication studies and inhibitor screening, we decided to establish a cell line that stably expressed DC-SIGNR, to enhance the replication efficiency of ZIKV-GFP. Following transfection, selection with G418 and IFA identification, one clone was selected to further evaluate its ability to enhance the replication of ZIKV-GFP and was named BHK-DR. DC-SIGNR was expressed on cell membranes and in the cytoplasm (Supplementary Fig. S1). As expected, ZIKV-GFP replicated and the foci of GFP fluorescence spread more efficiently in BHK-DR cells than in BHK-21 cells (Supplementary Fig. S1). Infection with ZIKV-GFP caused cytopathic effects (Fig. 2D) and formed plaques (Fig. 2B) in BHK-DR cells. The virus titer reached a plateau at 6.5 log TCID50/mL at 72 hpi. The growth kinetic of ZIKV-GFP in BHK-DR cells was similar to that in Vero cells (Fig. 2E). We further assessed the infectivity of ZIKV-GFP in BHK-DR and Vero cells with a high-content screening system. Unlike the dot-like fluorescence observed in Vero cells, GFP fluorescence was observed throughout the cytoplasm of BHK-DR cells (Fig. 2F) and the cell outline was clearly identifiable. The GFP fluorescence intensity in infected BHK-DR cells was significantly higher than that in Vero cells (Fig. 2G), but the frequency of GFP-positive cells did not differ significantly between Vero and BHK-DR cells (Fig. 2H). These results indicate that the BHK-DR cell line was suitable for the propagation and visualization of the ZIKV-GFP reporter virus.
To assess whether the recombinant ZIKV-GFP reporter virus and BHK-DR cell system could be applied to the high-throughput screening for inhibitors of ZIKV, a reported inhibitor of ZIKV, chloroquine (CQ) (Delvecchio et al. 2016; Li et al. 2017a), was tested for its ability to inhibit infection of BHK-DR cells by ZIKV-GFP. Treatment with CQ inhibited ZIKV-GFP replication in BHK-DR cells in a dose-dependent manner (Supplementary Fig. S2). The number of infected BHK-DR cells was clearly reduced by CQ at a concentration of 4 μmol/L (Supplementary Fig. S2A). This observation was confirmed by quantitatively analyzing the infection rate using Acapella highcontent imaging and analysis. The relative infectivity of cells treated with CQ at 4 μmol/L or a higher dose was significantly lower than that of vehicle-treated cells (Supplementary Fig. S2C). The calculated IC50 value of CQ was 3.96 μmol/L, which was similar to previously published results with diverse screening systems (Li et al. 2017a).
The high-throughput system to screen ZIKV-GFP and BHK-DR cells was further evaluated using another reported flavivirus inhibitor, 6-azauridine (6-Az) (Lo et al. 2003; Adcock et al. 2017). Infection by ZIKV-GFP was inhibited by 6-Az at an IC50 of 7.16 μmol/L (Supplementary Fig. S2F), which was relatively consistent with the previously reported IC50 of 11 μmol/L against WNV (Lo et al. 2003) and an IC50 of 3.18 μmol/L against ZIKV strain MR766 (Adcock et al. 2017). A 6-Az concentration of 4 μmol/L significantly decreased the virus infectivity of treated cells (P < 0.001) compared with that of vehicletreated cells (Supplementary Fig. S2G). These results demonstrate that the HTS assay with ZIKV-GFP and BHKDR cells was effective and reliable.
To discover novel anti-ZIKV compounds or to repurpose approved drugs, we screened a library of 974 selected plant-sourced compounds for anti-ZIKV activity using a high-throughput system schematically depicted in Fig. 3A. The first round of screening was conducted at a compound concentration of 10 μmol/L. At this concentration, most compounds showed little or no inhibition effect on ZIKV infection. The subset of compounds with a relative infection inhibition rate from –40% to 40% accounted for 76.4% of the total (Fig. 3B). Cell viabilities were evaluated by normalizing cell counts to those of vehicle-treated cells. In total, 95 compounds showed a relative infection inhibition rate of > 80%. Among these 95 compounds, 31 (indicated by red circles in Fig. 3C) were selected for further evaluation. The 31 hits included dihydroartemisinin (DHA), a well-known anti-malarial drug, and homoharringtonine (HHT), a drug used to treat myeloid leukemia (Chen et al. 2009, 2019).
Figure 3. High-throughput screening for inhibitors of ZIKV infection from a selected plant-sourced compound library. A High-throughput screening (HTS) assay timeline. BHK-DR cells were seeded in CellCarrier-96 microplates. After incubation for 16 h (usually overnight), cells were treated with compounds. One hour later, cells were infected with ZIKV-GFP for 48 h. The final concentration of compounds was 10 μmol/L. The final concentration of vehicle (DMSO) was 1%. Cell nuclei were stained with Hoechst for 20 min. The plates were scanned using a high-content-screening microplate imaging reader. B High-throughput screening of a library of 974 selected plant-sourced compounds. Each circle represents the percentage inhibition achieved with each compound at a concentration of 10 μmol/L. The circles within the blue box represent an inhibition > 80%. In total, 103 compounds causing a virus infection inhibition rate of > 80% were selected for further analysis of relative cell viability. C Out of 103 compounds from the primary selection, 31 compounds represented by red hollow circles within the blue-dotted square passed the criterion of relative cell viability > 80% at a concentration of 10 μmol/L. These compounds were selected for a confirmatory screen. D IC50 and CC50 of the top four selected compounds.
In a primary screen, 31 compounds were selected for follow-up analysis based on the inhibition efficiency of ZIKV-GFP infection and the effect on cell counts. Each compound was used to pre-treat cells at concentrations of 0.1–100 μmol/L. As expected, most compounds displayed pronounced antiviral activity at a concentration of 10 μmol/L, which was consistent with the primary screen results. The selected 31 compounds included 19 with an IC50 ≤ 4 μmol/L (Table 1) and the most potent of these were homoharringtonine (HHT), bruceine D (BD), dihydroartemisinin (DHA) and digitonin (DGT) (Fig. 3D). All these compounds inhibited viral infectivity in a dosedependent manner (Fig. 4). The four compounds all possessed IC50 values less than 1 μmol/L. The virus infectivity of cells treated with HHT and BD at a concentration of 1 μmol/L was reduced by up to 90%; DHA and digitonin reduced virus infectivity by about 80%, without greatly affecting cell number, and showed CC50 values above 100 μmol/L (Fig. 4A). Cell number was significantly reduced by HHT and BD at a concentration of 100 μmol/L and the CC50 values were 50.66 and 25.66 μmol/L, respectively.
Compounds IC50 μmol/L CC50 μmol/L Homoharringtonine 0.19 50.66 Bruceine D 0.36 25.66 Cucurbitacin B 0.26 15.66 Dihydroartemisinin 0.47 > 100 Digitonin 0.62 > 100 Plumbagin 0.41 12.56 Schizandrin A 0.42 21.53 Dihydrochelerythrine 0.49 > 100 Betulonic acid 0.52 44.54 Artesunate 0.57 > 100 Sodium aescinate 0.69 > 100 Escin 0.76 > 100 Oleanonic acid 1.99 32.61 Cepharanthine 2.25 29.85 Corosolic acid 2.62 9.99 Bergamotine 2.98 23.76 Harringtonine 3.02 21.34 Schisanhenol 3.02 21.34 Fangchinoline 3.78 38.58 Fangchinoline 4.39 40.10 Dihydroactinidiolide 4.66 > 100 Schizandrin B 6.00 41.18 Tetrandrine 6.37 19.35 Liriope muscari baily saponins C 7.78 28.32 Ophiopogonin D 7.78 28.32 Saikosaponin A 9.22 27.72 Berbamine dihydrochloride 9.79 40.32 Daurisoline 10.13 29.28 Reserpine 10.20 33.33 Curcumin 11.23 > 100 Magnolol 31.37 > 100 After primary HTS, 31 compounds were selected for follow-up analysis. Each compound was used to pre-treat cells at concentrations of 0.1 μmol/L, 0.25 μmol/L, 0.5 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L, 10 μmol/L and 100 μmol/L. The vehicle was used as negative control. After 48 h of infection, cell nuclei were stained with Hoechst. Then the plates were scanned by using a high-contentscreening microplate imaging reader. Fifty nine fields per well were scanned using 209 objective and analysed for percentage of infection and cell number. The infection positive cell rates and total cell counts were all normalized to that of vehicle control wells.
Table 1. Evaluation of the primary HTS selected compounds.
Figure 4. Validation of the antiviral effects of the hit compounds. A Top: The chemical structures of the hit compounds homoharringtonine (HHT), dihydroartemisinin (DHA), digitonin (DGT) and bruceine D (BD). Bottom: Dose–response curves showing the effect of compound treatment on virus infection inhibition (red) and cell viability (blue) in BHK-DR cells infected with ZIKV-GFP. Values represent the means ± SD from two independent experiments performed in triplicate. The data are normalized to those of DMSO-treated cells. B Representative fluorescence images of BHK-DR cells treated with varying concentrations of hit compounds. BHK-DR cells were treated with the indicated compounds at the indicated final concentrations and were infected after 1 h with reporter virus at an MOI of 0.1. Cells treated with DMSO at a final concentration of 1% were used as a vehicle control. At 48 hpi, cell nuclei were stained with Hoechst and cell plates were scanned using a high-content-screening microplate imaging reader.
The inhibition efficiency of the four selected compounds was further confirmed using BHK-21 cells and the human cell line A549 infected with recombinant wild-type Zika virus (Fig. 5). As expected, most compounds displayed inhibitory activity: 0.5 μmol/L HHT inhibited the E protein level by over 90% in both BHK-21 and A549 cells (Fig. 5A, 5E). For BD, ZIKV E protein levels in both cell types were significantly decreased at concentrations of 0.5 μmol/L (Fig. 5B, 5F). Treatment with BHK-21 cells at a DGT concentration of 8 μmol/L and A549 cells with 2 μmol/L DGT significantly inhibited the expression of ZIKV E protein (Fig. 5C, 5G). DHA treatment efficiently reduced the ZIKV E protein level in BHK-21 cells but only slightly inhibited that in A549 cells (Fig. 5D, 5H).
Figure 5. Confirmation of the inhibitory effect of selected compounds on ZIKV in BHK-21 and A549 cells. Each compound was used to pretreat indicated cells at concentrations of 8 μmol/L, 4 μmol/L, 2 μmol/L, 1 μmol/L and 0.5 μmol/L for 1 h prior to infection with wild-type ZIKV at an MOI of 0.01. Vehicle-treated cells and uninfected cells were used as controls. At 48 hpi, the cell lysates were analyzed by immunoblotting for ZIKV E protein expression, and the virus titer in the culture supernatants was measured on BHK-21 cells. BHK-21 cells treated with A HHT, B BD, C DGT and D DHA. Top: Representative western blot images. Bottom: Quantification of E protein band intensities relative to those for GAPDH. Data were normalized to those of DMSO-treatment cells. The data were pooled from two independent experiments. Values represent the mean ± SD. Statistical significances were determined by one-way ANOVA, compared to DMSO-treated cells (***P < 0.001). A549 cells treated with E HHT, F BD, G DGT and H DHA. Top: Representative western blot images. Bottom: Quantification of E protein band intensities relative to those for GAPDH. Data were normalized to those of DMSO-treatment cells. The data were pooled from two independent experiments. Values represent the mean ± SD. Statistical significances were determined by one-way ANOVA compared to DMSO-treated cells (*P < 0.05; **P < 0.01; ***P < 0.001).
To investigate the underlying cellular mechanism of the hit compounds that inhibited ZIKV replication, time-of-addition experiments were performed (Fig. 6A). The HHT (Fig. 6B), BD (Fig. 6C) and DHA (Fig. 6D) compounds all inhibited ZIKV infection at the post-entry stage, but not at the pre-entry stage or during infection. However, DGT reduced ZIKV E protein expression at all stages, including virus pretreatment, cells pretreatment, during infection and at the post-entry stage of ZIKV (Fig. 6E). Digitonin is a natural plant detergent, which affects the membrane proteins of viruses or cells and might inhibit the binding of viruses or their entry into host cells. Therefore, DGT exhibits an inhibitory effect at the early stage of ZIKV infection. This hypothesis requires further testing.
Figure 6. Time-of-addition analysis of the antiviral activity of the hit compounds. A Schematic illustration of the time-of-addition experiment. B, C, D and E BHK-21 cells were infected with ZIKV at an MOI of 0.01 for 1 h (0 to 1 h). Either 1 μmol/L HHT (B), 4 μmol/L BD (C), 4 μmol/L DHA (D), or 8 μmol/L DGT (E) were introduced at different time points of ZIKV infection, designated virucidal, pretreatment (pre), during treatment (during), or posttreatment (post). The inhibitory effect of the compounds in each group was determined by immunoblotting for E protein expression. Top: Representative western blot images. Bottom: Quantification of E protein band intensities relative to those for GAPDH. Data were normalized to those of DMSO-treatment cells. The data were pooled from two independent experiments. Values represent the mean ± SD. Statistical significances were determined by one-way ANOVA compared to DMSO-treated cells (***P < 0.001).