Protocol to evaluate the antiviral effect of FDA-approved drugs against dengue virus in Huh7 cells and AG129 mice

Summary Finding an effective therapy against diseases caused by flaviviruses remains a challenge. Here, we present a protocol to test Food and Drug Administration-approved drugs that inhibit host nuclear protein import, promoting a reduction of dengue infection. We describe steps for analyzing the drug effect on nuclear import inhibition of cellular and viral proteins by confocal microscopy or western blotting. We then describe procedures for measuring the antiviral drug effects on virus-infected cells by flow cytometry and testing drug efficacy in dengue-infected AG129 mice by survival assays. For complete details on the use and execution of this protocol, please refer to Palacios-Rápalo et al.1


Highlights
Protocol for measuring the antiviral effect of FDA-approved drugs on DENV infection Procedures for measuring the FDAapproved drugs effect on DENV proteins nuclear import

Measurement of effect of nuclear import inhibitors on DENV proteins localization in vivo
Note: Gently pipette the cells up and down until a homogeneous suspension is obtained and placed in conical tubes for centrifugation.
d. Centrifuge at 1,900 3 g for 7 min at 25 C.
Note: Eliminate the clarified supernatant, preserving the integrity of the cell pellet.
e. Resuspend the cells with 5 mL of DMEM complete medium.f.Wash the cells once with 1X PBS.Repeat step d).
Note: Depending on the planned experiment, dilute harvested cells to the desired concentration using the DMEM complete medium.

Timing: 2 days
This procedure is based on the protocol of Hashemi et al. 2 7. Preparation of cells prior to transfection.a. Starting from the cell suspension of a 100 mm plate at 80% confluence (8 3 10 6 cells), centrifuge at 1,900 3 g for 7 min at 25 C. b.Discard the clarified supernatant.k.Allow treatment for a further 24-48 h. 9. Collection of slides with transfected cells.
a. Remove the transfection medium.i. Wash with 500 mL of cold 1X PBS once in constant agitation at low speed.b.Prepare the slides with your transfected cells for indirect immunofluorescence (see section 1:  in vitro measurement of the molecular antiviral effect of nuclear import inhibitor drugs).
ii. Infected with DENV-2 and treated with FDA-approved drugs.
Pause point: after obtaining the mice tissues of interest, freeze them on dry ice and store them at À80 C until use.Once you are ready, continue the process from step b).b.Add 1 mL of TRIzol to the sample and disaggregate the 100 mg of tissue using a homogenizer (TissueRuptor II, QIAGEN).c. Extract total RNA from 2 or 3 mice per condition using the TRIzol reagent manufacturer's procedure.
Note: Each mouse is considered one experimental unit.
d. Treat samples with DNase I to avoid DNA contamination.11.Determine the concentration of total RNA using the nanodrop (Nandodrop 2000, Thermo Scientific).12. Perform reverse transcription of 1 mg of total RNA to cDNA using the Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase kit.
Pause point: cDNA can be stored at 4 C for 1 day or À20 C for a few weeks.Once you are ready, continue the process from step 13a).
a. Use primers corresponding to the C protein region (see key resources table ).b.For each PCR reaction, use: CRITICAL: It is important to thoroughly mix the reaction mixture before adding RNA while on ice.Please protect it from light.

Note:
The plasmid containing the 151 bp corresponding to a region of the C protein of DENV was made by Angel-Ambrocio et al. 3 Note: To obtain the initial volume of the stock plasmid, use the following formula: ii. Mass of one plasmid molecule.
To calculate the mass of one plasmid molecule, the following formula is used: m=(n) (1.096 3 10 -21 grams/base pairs).m = mass.n = number of base pairs (bp) of the plasmid (vector + insert).The pJETL2/blunt vector has a total of 2974 bp, and the insert of the C protein region is 151 bp, so the "n" of our plasmid is 3125 bp.The mass of 1 plasmid would be (3125 bp) (1.096 3 10 -21 g/bp) = 3.425310 -21 g/copy.iii.Calculation of plasmid copy number.
To calculate the grams that correspond to the number of copies of interest, the following formula is used: g=(#copies) (m).
Note: From this initial volume with 10 10 copies, perform serial dilutions.d.Use the thermal cycling conditions below in the Eco Ilumina System equipment: Note: Any equivalent RT-PCR equipment would be good for obtaining the number of viral copies.
a. Adjust the threshold with a mock-infected mouse sample and the non-templated control.
Note: In this protocol, the results were analyzed with EcoStudy software.Any equivalent software would be good for analyzing RT-PCR results.
b. Express data as viral copies in 100 mg of tissue.
Note: To express data as viral copies in 100 mg of tissue, use the following formula: i. Copies in 100 mg of tissue= (#copies) (Total RNA) O (cDNA input).

STEP-BY-STEP METHOD DETAILS
This protocol focuses on two sections: 1) the confocal immunofluorescence and flow cytometry approach to measure the antiviral effect of nuclear import inhibitors FDA-approved drugs such as Ivermectin (IVM) or Atorvastatin (ATV) and 2) mice survival assays to measure the antiviral efficacy of these drugs (Figure 1).

Protocol
Section 1: In vitro measurement of the molecular antiviral effect of nuclear import inhibitor drugs

Timing: 4-7 days
This section describes the protocol to measure, in cell monolayers, the effect of nuclear import inhibitor drug treatment on the subcellular localization of host and viral proteins by confocal microscopy or Western blot.In addition, we measured the percentage of DENV-2 infected cells by flow cytometry after treatment with both drugs.
Nuclear import inhibitor FDA-approved drug treatment The following procedure is described based on Huh7 cells grown in 24-well dishes on coverslips at 70%-80% confluence to analyze the nuclear import of DENV-2 proteins early in the infection during treatment with nuclear import inhibitor FDA-approved drugs.The drugs must be prepared in sterile conditions, aliquoted, and kept at À20 C until use.We used a plasmid constructed with an SV40 NLS (RKRK) tagged with four GFP to validate the drug's ability to inhibit nuclear import.This NLS is recognized by the importin a/b pathway, which IVM or ATV4 can inhibit.c.Add 300 mL of cold 4% paraformaldehyde (PFA) per well and fix the cells at 4 C for 30 min.
Pause point: If samples cannot be processed immediately, replace PFA with 500 mL per well of sterile 1X PBS and keep at 4 C up to a maximum of 1 week.Seal the plate well to avoid drying out.Once you are ready, continue the process from step 3, c).Note: Use minimally 200 mL of Hank's for 24-well dishes to prevent wells from drying out during infection.Please adjust the volume if you use different culture dishes to perform this assay.

Note:
The multiplicity of infection (MOI) is calculated depending on the experiment.For early infection assays (8 h or 12 h), use an MOI of 10; for assays longer than 24 h, use an MOI of 0.5 or 1.
d. Remove Hank's medium.e. Add a complete medium supplemented with drugs or vehicles to mock-infected or infected cells.f.Allow infection in the presence of treatment for 12 h for early and 24 h for late infection assays.
CRITICAL: If you observe loss of monolayers from the coverslips after treatment, see troubleshooting in problem 1.
Note: Use 500 mL of complete DMEM medium for 24-well dishes.Please adjust the volume if you use different culture dishes to perform this assay.

Timing: 2 days
In this step, after drug treatment, antibodies are added sequentially to study the subcellular localization of host nuclear transport receptors (NTR) and DENV proteins that have nuclear localization (non-structural protein 3 and 5).c.Add 300 mL of cold 4% PFA per well and fix the cells at 4 C for 30 min.
Pause point: If samples cannot be processed immediately, replace PFA with 500 mL per well of PBS 1X sterile and keep at 4 C for one week.Seal the plates well to avoid them from drying out.Once you are ready, continue the process from step d).
d. Remove PFA or 1X PBS from step c).
i. Wash with 1X PBS three times, each 500 mL per well in slow stirring for 5 min.e.For each well, add 300 mL of permeabilization buffer (0.2% saponin with 1% fetal bovine serum) in low agitation for 30 min at 25 C.

Note:
The permeabilization buffer permeabilizes and blocks non-specificity in the cells prior to labeling with antibodies.
Note: Cells transfected with SV40 NLS-containing plasmid do not need to be stained with any antibody; skip to step m).
CRITICAL: To prevent the cells from not staining with the antibody, avoid leaving bubbles when placing the coverslip on top.
h. Incubate at 4 C (16-18 h).Seal the plate well to avoid drying out.i.Return the coverslips to the 24-well plate and wash 3 times with 1X PBS in low agitation for 5 min.
Pause point: If samples cannot be processed immediately, add 500 mL per well of PBS 1X sterile and keep at 4 C for a few days.Seal the plate well to avoid drying out.Once you are ready, continue the process from step j).j.Prepare secondary antibodies, anti-mouse Alexa 488 (1:1000) or anti-rabbit Alexa 555 (1:300), in the permeabilization buffer.k.In a humidified chamber covered with parafilm, drop 15 mL of secondary antibodies per well and leave the coverslip on top (Figures 2 and 3).l.Incubate 2 h at 37 C. Seal the plate well to avoid drying out.Then repeat step i).
Pause point: If samples cannot be processed immediately, add 500 mL per well of PBS 1X sterile and keep at 4 C for a few days.Seal the plate well to avoid drying out and cover it from light.Once you are ready, continue the process from step m).
CRITICAL: From this step, protect the coverslips from light.m.Prepare Hoechst's stain (1:1000) in PBS 1X and add 300 mL per well in low agitation for 10 min.
Then repeat step i).n.Finally, mount the coverslips with 3 mL of vectashield on clean slides and seal the edges (example: nail varnish) (Figure 3).
Pause point: If the samples cannot be visualized, store the slides protected from light at 4 C for short-term storage or at À20 C for long-term storage.Note: We used the Leica TCS SP8 Confocal Microscope (Leica Microsystems) in this protocol.The objective used was 633 oil lens, and the resolution of images is 1024 3 1024 pixels.Any equivalent confocal microscope with similar settings would be suitable for producing confocal images for later quantification analysis.

Confocal image analysis.
Note: The images were analyzed with the Leica Application Suite X Core Offline v3.3.0 software in this protocol.

Note:
The images obtained were imported into Icy software, and we determined the mean fluorescence intensity (MFI) in the regions of interest (ROI) of at least 30 cells per condition.
Flow cytometry to measure the antiviral effect of nuclear import inhibitor drugs Timing: 2 days  k.Remove the clarified supernatant, preserving the integrity of the cell pellet.l.Add 1 mL of 1X PBS.Resuspend gently.m.Repeat step j) and resuspend in 300 mL of 1X PBS.
Pause point: Cells in 1X PBS can be stored for further analysis.Store the cells at 4 C for future analysis for 1-2 weeks maximum.Once you are ready, continue the process from step 7a).CRITICAL: Do not store at À80 C. CRITICAL: The cells must be constantly stirred to ensure the primary and secondary antibodies are correctly incorporated.However, it is possible that cells may remain associated with the tube walls, so it is essential to recover as many cells as possible by detaching with 1X PBS solution.
m. Add 1 mL of 1X PBS and centrifuge at 500 3 g for 8 min at 4 C. n.Remove the clarified supernatant, preserving the integrity of the cell pellet.o.Add 300 mL of 1X PBS.
Pause point: Cells in 1X PBS can be stored for further analysis.Store the cells at 4 C for future analysis for 1-2 weeks maximum.

Analyze the samples in the flow cytometer.
Note: In this protocol, we use the BD LSR Fortessa.The parameters used for sample analysis were as follows.A Threshold of 15,000 was used, and voltages of FSC (240 V), SSC (260 V), Alexa Flour 488 (375 V), and Pacific Blue (320 V) were used to determine cell autofluorescence.Any equivalent flow cytometer with similar settings should be suitable to produce data for later quantification analysis.9. Cytometry data analysis.

Note:
The data were analyzed using FlowJo v10 software.The total population (SSC-A vs. FSC-A) and single events (FSC-H vs FSC-A) were adjusted by analyzing 10,000 events.

Protocol
Section 2: Survival assay to measure the antiviral efficacy of nuclear import inhibitor drugs in DENV-infected AG129 mice

Timing: 4-8 weeks
This section describes the protocol to test nuclear import inhibitor FDA-approved drugs in DENV-2infected AG129 mice by survival assays and the procedure to obtain tissue sections to analyze the nuclear localization of viral proteins in vivo.

Note:
We use six-to eight-week-old female and male AG129 mice (La Jolla Institute for Allergy and Immunology, Strain: 129/Sv).

Survival assay to measure the antiviral efficacy of nuclear import inhibitors
Timing: 25 days 10.Determine treatment groups.
a. Group three female or three male mice for each treatment group.The groups are: i.Control (uninfected and untreated mice).
ii. Vehicle (infected mice treated with water).
iii.Independent treatment (infected and treated with IVM or ATV).iv.Combined treatment (infected and treated with IVM+ATV).

Note:
In this protocol, we used 0.6 mg/kg/day of IVM and 20 mg/kg/day of ATV.
11. Set up AG129 mice infection with DENV-2.a. Measure the initial weight of each mouse.b.Using an insulin syringe, inoculate DENV-2 intraperitoneally in 100 mL of sterile 1X PBS.c. Allow infection for 4 days.
Note: For the control group, inoculate only sterile 1X PBS.
CRITICAL: Make sure not to puncture any mouse organ during the injection of the virus.
12. Measure the mice's weight and clinical score daily (see Table 1).
CRITICAL: Exclude mice that die from causes other than infection.
13. Set up the treatment scheme for AG129 mice.a. Prepare commercial drugs (IVM or ATV) for humans in sterile water for mice.b.Four days post-infection, using a gastric tube, administer 50-60 mL of the vehicle or treatment orally to simulate the treatment used in humans.

2
Mild signs of lethargy.

5
Moribund: Lethargic, very bristly hair, not able to stand up, no mobility, and no response to stimuli.
CRITICAL: Based on the initial weight of each mouse, the concentrations of each drug should be prepared in sterile water in accordance with the doses chosen.
Note: For this protocol, mice were treated with ten doses of ATV once per day and with 5 doses of IVM every other day (see Figure 4).
14. Monitor the development of the disease during and after treatment.a. Treatment will end 13 days post-infection, and surviving mice will be euthanized until day 25 (12 days after the end of treatment).
CRITICAL: If you have problems with this step, please go to the troubleshooting section in problem 4.
b. Measure each mouse's weight and clinical score (see Table 1) during and after treatment.
CRITICAL: Exclude mice that die from causes other than infection.

Obtaining tissue sections to analyze the nuclear localization of DENV proteins
Timing: 4 days 15. Preparation of gelatinized slides a.In a glass flask, dissolve 0.5% of porcine skin gelatin in distilled water at 60 C with gentle stirring in a stirrer/hot plate.
Note: Use a clean thermometer inside the flask to monitor temperature.
Note: Gelatin solution can be stored for no more than 1 month at 4 C or before if contamination or debris are not detected.e. Place the clean and degreased microscope slides in the Coplin jar with the gelatin solution.f.Incubate for 1 min.
i. Drain the gelatin excess, and place them in the stainless-steel basket.
CRITICAL: The microscope slides must be perfectly clean and degreased for the proper adhesion of the gelatin.CRITICAL: The gelatin should be at RT prior to use.
g. Gelatinized slides placed in the basket are covered with aluminum foil to protect them from dust.
h. Store gelatinized slides in a microscope slide holder box at 25 C until use.
Note: Prepare only the necessary slides for the complete experiment.
16. Histological Processing.a. Immediately after obtaining the mice tissues of interest: i. Freeze them on dry ice.
ii. Cover them with parafilm and aluminum foil.
iii.Store them at À80 C until use.
Note: In this protocol, brains and livers were obtained from DENV-2-infected AG129 mice treated with IVM, ATV, or IVM+ATV.
Note: Stored mouse tissues can be analyzed by RT-qPCR to quantify DENV RNA copy number (see RT-qPCR procedure).
Note: Pre-fixed tissues (i.e., 4% PFA solution) can also be processed in the same way but must be cryoprotected with a 10% sucrose PBS solution for 24 h at 4 C before freezing.
b. Adhere the frozen tissue to the cryostat sample holder with a tissue freezing medium.i. Align tissue samples to cut in the axis of interest: sagittal, coronal, or transversal (axial).
c.In cryostat (Leica CM1100 with low profile blades, Leica Microsystems), obtain 8 mm thick slices and mount them into gelatin-coated slides.d.Store the slides in a holder box at À20 C until use.17.Tissue immunofluorescence preparation.
a. Before fixing the tissue sections: i. Surround the tissue with a hydrophobic barrier pen to prevent the antibodies from diffusing throughout the slide.
b. Transfer the slides to a Coplin jar.i. fix tissue sections using 4% PFA for 30 min.c.Wash three times with 1X PBS.
CRITICAL: If the tissue section detaches from the slice, please see the troubleshooting section in problem 5.
d. Transfer the slides to a Coplin jar with the permeabilizing solution for 30 min.e. Prepare the primary antibody (anti-NS3) in the permeabilizing solution.f.Place the slides in a humid chamber.
i. Add 30-50 mL of primary antibody on each tissue section.g.Incubate 16-18 h at 4 C. h.Wash three times with 1X PBS.i. Prepare the secondary antibody (Alexa Fluor 555) in the permeabilizing solution.j.Place the slides in a humid chamber.
i. Add 30-50 mL of secondary antibody to each tissue section.k.Incubate at 37 C for 2 h.Then repeat step h).
CRITICAL: From this step, protect the slides from light.l.Prepare Hoechst's stain (1:1000) in PBS 1X and cover the tissue sections.Then, repeat step h).m.Finally, mount the tissue sections with vectashield.
i. Seal the edges (for example, nail varnish).
Pause point: If the samples cannot be observed, store the slides protected from light at 4 C for short-term storage or at À20 C for long-term storage.
18. Visualize the samples under a confocal microscope.
Note: We used the Leica TCS SP8 Confocal Microscope (Leica Microsystems) in this protocol.The objective used was 633 oil lens, and the resolution of images is 1024 3 1024 pixels.Any equivalent confocal microscope with similar settings would be good for producing confocal images for later quantification analysis.

Confocal image analysis.
Note: The images were analyzed with the Leica Application Suite X Core Offline v3.3.0 software.

Note:
The images obtained were imported into Icy software and we determined the mean fluorescence intensity (MFI) in the regions of interest (ROI) of at least 30 cells per condition.

EXPECTED OUTCOMES Measurement of nuclear import during IVM or ATV treatment by IIF assay
The subcellular distribution of cellular and DENV-2 protein during treatment with FDA-approved drugs such as IVM and ATV, which affect the nuclear import of viral and host cellular proteins, 1 was analyzed by confocal microscopy.Nuclear import of the host cellular protein KPNA1 and NS3 or NS5 proteins of DENV-2 was measured by fluorescence nucleus-cytoplasm ratio (Fn/C).The nuclear dye Hoechst was used to define nuclei, commercial anti-KPNA1 antibody was used to identify NTR of the host cell, and anti-NS3 or anti-NS5 antibodies were used to identify NS3 and NS5 proteins of DENV-2 (Figures 5A-5D and 5F).In mock-infected cells treated with the vehicle, KPNA1 The ordinary one-way ANOVA with Tukey multiple comparisons post hoc test was used to determine significant differences among means of each condition.

Statistical analysis
For in vitro and in vivo studies, experimental data are presented as the mean G standard error of the mean (SEM).For in vivo assays, Wilcoxon and Mantel-Cox tests were used to compare the survival rate between treated and untreated groups, and the ANOVA-LSD test was used to compare the average survival time between treated and untreated groups.All statistical analysis was performed using the GraphPad Prism 6.0 software.Differences were considered statistically significant when P values were less than 0.05.

LIMITATIONS
This protocol includes steps and details of methods developed for the study of treatment with IVM and ATV on the nuclear transport of proteins, replication, and pathogenesis of dengue virus in vitro and AG129 mice.This standardized protocol for FDA-approved drug analysis against DENV is intended to provide a basis for wider use in studies, including both observational studies and randomized clinical trials.We believe that this protocol will be useful for research laboratories worldwide.Although the protocol works well for us, it can easily be adjusted and improved among the scientific community to the same objective.The effect of nuclear import inhibitory FDA-approved drugs during viral infection has been extensively evaluated in in vitro studies.However, one of the limitations of this protocol was to analyze the effect of nuclear import inhibitor drugs on the nuclear transport of DENV proteins in a mouse model due to the difficulty of controlling the time and number of infected cells in the tissues, addition to the fact that there are few methodologies to evaluate nuclear transport in murine models.Furthermore, it is important to note that this protocol was performed in AG129 mice in an immunodeficient murine model, so if it is performed in an immunocompetent murine model, the treatment scheme would have to be readjusted because these mice resolve virus infections.Not every aspect of the protocol has been optimized in detail, and we provided comments and notes whenever other researchers recommended further optimizations and testing.

TROUBLESHOOTING Problem 1
Monolayer loss from coverslips after treatment in IIF or flow cytometry assays.Some drugs, such as ATV (statins), affect the cytoskeleton distribution of treated cells, affecting cell morphology and favoring the loss of the monolayer easily.

Potential solution
To avoid detachment of the cells in the monolayer during washes, slowly add 1X PBS against the wall of the well.Carefully perform this step before fixing the cells with 4% PFA in IIF assays.During trypsinization, to obtain cells for flow cytometry, remove trypsin quickly (less than 1 min) to avoid damaging cells and losing cells from the monolayer.

Problem 2
Antibodies or Hoechst stains have other locations than usual.
During handling and marking of slides, there may be a misplaced mark due to a high concentration of salts or bacterial contamination.The antibody concentration and permeabilization solution were not prepared correctly.On the other hand, it may be due to lack of washing.

Potential solution
The 1X PBS used to prepare the solutions used in the methodology should be filtered to avoid the appearance of a misplaced mark.Excess salts can cause noise in the images.Increasing the number of washes caring for the cell monolayer can help reduce the image noise.

Problem 3
Low performance of cell collection for flow cytometry analysis.
Appropriate flow cytometry results are obtained with good cell collection efficiency.Cell aggregates or cell loss may prevent proper analysis of the results.

Potential solution
Allow good cell homogenization before adding the fixation solution.Poor homogenization before adding the paraformaldehyde leads to the fixation of cell clumps, favoring cell rupture and formation of cell debris, and can be recognized as double events in the flow cytometer, decreasing single events.During manipulation, some cells may adhere to the tube walls.Recover as many cells as possible by detachment with 1X PBS.
The high toxicity of some drugs may affect the survival of infected mice, resulting in a lower mean survival time compared to untreated infected mice.

Potential solution
To avoid accelerated drug-induced death of DENV-infected mice, decrease the drug concentration and administer one dose every other day.This will reduce the cellular toxicity of the drugs while preserving the antiviral activity (see Figure 8 as an example).

Problem 5
Detachment of tissue sections from gelatinized slides during the immunofluorescence staining process.
Usually, during fixation or washings, tissue sections are detached from the slide.

Potential solution
To avoid detachment of tissue sections, degrease and clean the slides properly.
Immerse the slides in a 2N NaOH solution for 2 h at room temperature in a Coplin jar.Rinse thoroughly with running tap water approximately 10 times.Rinse thoroughly with distilled water approximately 10 times.Place the slides in 100% ethanol for 30 min.Air dry the slides at room temperature, protect them from dust, and proceed with gelatinization.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rosa Marı ´a del A ´ngel (rmangel@cinvestav.mx).

Technical contact
Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Selvin Palacios (selvin.palacios@cinvestav.mx).

Materials availability
This study did not generate new unique reagents.

Figure 1 . 1 : 1 .
Figure 1.The starting materials are Huh7 cells (in vitro assays) and AG19 mice (in vivo assays) Section 1: In vitro measurement of the molecular antiviral effect of nuclear import inhibitor drugs, IIF to evaluate nuclear import during treatment, and flow cytometry to measure the antiviral effect of nuclear import inhibitor drugs.Section 2: Survival assay to measure the antiviral efficacy of nuclear import inhibitor drugs in DENV-infected AG129 mice and obtaining tissue sections to analyze the nuclear localization of DENV proteins.
Huh7 cell infection with DENV-2.a. Remove the complete DMEM medium.b.Add DENV-2 of desired MOI suspended in Hank's medium.c.Allow infection for 2 h at 37 C in CO2 incubation.

3 .
Set up IIF to analyze nuclear import.a. Remove the complete DMEM medium.b.Wash once with 500 mL per well of 1X PBS.

4 .
Visualize the samples under a confocal microscope.CRITICAL: If you observe antibodies or the Hoechst mark has a different location than usual, see troubleshooting, problem 2.

Figure 2 .
Figure 2. Incubation of primary and secondary antibodies in a humidity chamber (A) Humidity chamber.(B) Drop the antibody in a permeabilizing solution into the center of the well.(C) Lift the coverslip with long-tipped forceps (or a needle with a bent tip).(D) Remove the coverslips with the help of long-tipped forceps.(E) Leave the coverslip on top of the drop (the cell monolayer should touch the drop).

7.
Set up cell labeling.a. Add 1 mL of 1X PBS and centrifuge at 500 3 g for 8 min at 4 C. b.Remove the clarified supernatant, preserving the integrity of the cell pellet.c.Add 500 mL of permeabilizing solution.d.Resuspend the cells and incubate for 30 min under constant agitation at RT. e. Add 1 mL of 1X PBS and centrifuge at 500 3 g for 8 min at 4 C. f.Remove the clarified supernatant, preserving the integrity of the cell pellet.g.Add 50 mL of 2H2 antibody (this antibody recognizes the prM protein and part of the E protein) (1:50) in a permeabilizing solution.h.Incubate at 4 C under constant agitation.i. Add 1 mL of 1X PBS and centrifuge at 500 3 g for 8 min at 4 C. j.Remove the clarified supernatant, preserving the integrity of the cell pellet.k.Add 50 mL of Alexa 488 secondary antibody (1:750) in a permeabilizing solution.l.Incubate for 2 h at 25 C with constant agitation.Note: The sample should be protected from light due to the fluorescence of the secondary antibody.

Figure 6 .
Figure 6.Antiviral effect of FDA-approved nuclear import inhibitor drugs on the percentage of infected cells and DENV-2 viral titer (A and B) The percentage of DENV-2 infection at 48 h was analyzed by flow cytometry from three independent experiments in duplicate.The analyzed population evaluates 10,000 events from the total population's single events (SSC-H vs. SSC-A).The events are represented as SSC-A vs. Alexa Fluor 488::2H2.ns, not significant; ***p < 0.001; ****p < 0.0001.(C) The viral titer of supernatants from DENV-2 infected cells was determined by plaque assay from three independent experiments.ns, not significant; **p < 0.01; ****p < 0.0001.Figure reprinted with permission from Palacios-Ra ´palo et al. 1

Figure 7 .
Figure 7.In vivo antiviral effect of FDA-approved nuclear import inhibitor drugs (A-C) Mean survival time of AG129 mice treated with IVM, ATV, and the combination IVM+ATV (n = 6 (2 _, 4 \), 7 (3 _, 4 \) and 8 (4 _, 4 \), respectively).Vehicle-treated mice infected with DENV-2 were used as controls (n = 8 (4 _, 4 \)).ns, not significant; *p 0.05.(D) Mean clinical scores of AG129 male mice infected with DENV-2 treated with Vehicle, IVM, ATV, and IVM+ATV.The morbidity scale is shown in Table 1.(E) Kaplan-Meier survival curves represent the percentage survival of male AG129 mice treated with Vehicle, IVM, ATV, and IVM+ATV.The continuous line highlights the treatment that most increased the survival rate compared to the Vehicle.(F) Brain tissue sections from AG129 male mice.Mice received half of the treatment schedule (five days of treatment).Frozen tissue sections were prepared and labeled with anti-NS3 and Hoechst (nuclei).Scale bar: 40 mm.(G) Effect of IVM, ATV, and IVM+ATV treatments on viral load in brains of DENV-2 infected AG129 mice eight days post-infection (n = 3, for each group) compared to vehicle-treated mice (n = 2).Treatment started 4 days post-infection.The independent treated groups received three doses of IVM and five doses of ATV, and the combined group received three doses of IVM+ATV every other day.ns, not significant; *p < 0.05.(H and I) Quantification of the NS3 distribution signal.The nucleus-cytoplasmic fluorescence ratio was used to show the distribution of NS3 protein at the indicated condition.The Mean Fluorescence Intensity (MFI) was determined for selected regions of interest (ROIs) in the nucleus and cytoplasm from mice-infected cells of the indicated condition.n = 30 cells.ns, not significant; *p 0.05; **p < 0.01. Figure reprinted with permission from Palacios-Ra ´palo et al. 1

Table 1 .
Morbidity scale in AG129 mice