Materials and Reagents

Disposable tips
1. 10 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3510-05 )
2. 200 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3900 )
3. 1 ml capacity (FUKAEKASEI and WATSON, catalog number: 110-502C )
100 mm culture dish (Corning, Falcon^®, catalog number: 353003 )
6-well culture plate (Corning, Falcon^®, catalog number: 353046 )
12-well culture plate (Corning, Falcon^®, catalog number: 353043 )
24-well culture plate (Corning, Falcon^®, catalog number: 353047 )
96-well culture plate (Corning, Falcon^®, catalog number: 353072 )
1.5 ml microcentrifuge tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
15 ml tube (Corning, Falcon^®, catalog number: 352196 )
Cover slip (13 x 13 mm; Matsunami Glass, catalog number: C013001 )
Microscope slide (Matsunami Glass, catalog number: S2215 )
Disposable serological pipette
1. 1 ml capacity (Corning, Falcon^®, catalog number: 356521 )
2. 5 ml capacity (IWAKI, catalog number: 7153-005 )
3. 10 ml capacity (IWAKI, catalog number: 7154-010 )
Filter paper (ATTO, catalog number: CB-06A-20A )
Viruses (Chen et al., 2017):
1. Hepatitis C virus (HCV, J6/JFH-1 strain)
2. Dengue virus (DENV, Trinidad 1751 strain)
3. Japanese encephalitis virus (JEV, Nakayama strain)
4. Influenza A virus (FLUAV, A/Udorn/307/72[H3N2])
5. Sendai virus (SeV, Fushimi strain)
6. Herpes simplex virus type 1 (HSV-1, CHR3 strain)
7. Coxsackievirus B3 (CV-B3, Nancy strain)
8. Vesicular stomatitis New Jersey virus (VSNJV)
9. Sindbis virus (SINV)
10. Encephalomyocarditis virus (EMCV, DK-27 strain)
Huh7it-1 cells (Apriyanto et al., 2016)
Note: Huh7it-1 cells are susceptible to all viruses described above (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, VSNJV, SINV and EMCV).
Vero cells (ATCC, catalog number: CCL-81 )
Note: Vero cells are susceptible to all viruses described above (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, VSNJV, SINV and EMCV).
Antibodies
1. Rabbit polyclonal antibody against DENV PrM (Gene Tex, catalog number: GTX128093 )
2. Mouse monoclonal antibody against DENV type 2 (3H5; Hotta et al., 1984)
3. UV-inactivated anti-HCV human serum (Bungyoku et al., 2009)
4. Anti-HCV E2 neutralizing antibody #55 (Shimizu et al., 2013)
5. Rabbit antiserum against CV-B3 (DENKA SEIKEN, catalog number: 300638 )
6. Rabbit antiserum against FLUAV (Shimizu et al., 1985)
7. Rabbit antiserum against SeV (Hayashi et al., 1991)
8. Rabbit antiserum against HSV-1 (Hayashi et al., 1986)
9. FITC-conjugated goat anti-human IgG (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 104AG )
10. Alexa Flour488-conjugated goat anti-mouse IgG (Thermo Fisher Science, catalog number: A-11001 )
11. Alexa Flour488-conjugated goat anti-rabbit IgG (Thermo Fisher Science, catalog number: A-11008 )
High glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, catalog number: 044-29765 )
Phospholipase A₂ from Naja mossambica snake venom (Sigma-Aldrich, catalog number: P7778 ) (Chen et al., 2017)
Trypsin-EDTA solution (Wako Pure Chemical Industries, catalog number: 209-16941 )
Crystal violet (Wako Pure Chemical Industries, catalog number: 038-04862 )
MEM with non-essential amino acids (Thermo Fisher Science, Gibco^TM, catalog number: 10370021 )
Fetal bovine serum (FBS; Biowest, catalog number: S1820 )
Penicillin-Streptomycin solution (Wako Pure Chemical Industries, catalog number: 168-23191 )
Methyl Cellulose 4000 (Wako Pure Chemical Industries, catalog number: 136-02155 )
4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries, catalog number: 163-20145 )
Formaldehyde solution (AppliChem, catalog number: A3592,0500 )
Gram Hacker’s Stain Solution I (MUTO PURE CHEMICALS, catalog number: 41162 )
Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
Bovine serum albumin (BSA; Wako Pure Chemical Industries, catalog number: 015-21274 )
Hoechst 33342 solution (Thermo Fisher Scientific, Molecular Probes, catalog number: H3570 )
Vectashield mounting solution (Vector Laboratories, catalog number: H-1000 )
Formvar-coated nickel grid (Electron Microscopy Sciences, catalog number: FF200-Ni )
2% phosphotungstic acid (Wako Pure Chemical Industries, catalog number: 582-66852 )
Sodium chloride (NaCl; Wako Pure Chemical Industries, catalog number: 191-01665 )
Potassium chloride (KCl; Wako Pure Chemical Industries, catalog number: 163-03545 )
Disodium Hydrogen Phosphate (Na₂HPO₄·12H₂O, NACALAI TESQUE, catalog number: 31722-45 )
Potassium phosphate monobasic (KH₂PO₄; Wako Pure Chemical Industries, catalog number: 169-04245 )
Complete medium for cell culture (see Recipes)
10x phosphate-buffered saline (PBS[-]) (see Recipes)
Overlay medium (see Recipes)

Equipment

Micropipette (Gilson, P20, P200, P1000)
1. P20 (Gilson, catalog number: F123600 )
2. P200 (Gilson, catalog number: F123601 )
3. P1000 (Gilson, catalog number: F123602 )
Multichannel micropipette (10-100 μl) (Eppendorf, catalog number: 3125000036 )
Hemocytometer chamber (e.g., Erma, catalog number: 03-303-1 )
Biosafety cabinet (e.g., PHC, model: MHE-S1301A2 )
CO₂ incubator (e.g., PHC, model: MCO-20AIC )
Autoclave (e.g., TOMY DIGITAL BIOLOGY, model: SX-500 )
Refrigerated tabletop centrifuge (e.g., Eppendorf, model: Centrifuge 5424 )
Vortex (e.g., Scientific Industries, model: Vortex-Genie 2 )
-80 °C freezer (e.g., PHC, model: MDF-384 )
Inverted microscope (e.g., Olympus, model: CKX53 )
Fluorescent microscope (e.g., ZEISS, model: Axio Vert. A1 )
Multilabel Plate Counter (PerkinElmer, model: 1420 ALBOSX )
Transmission electron microscope (Hitachi, model: HT7700 TEM )

Procedure

Part I: Virucidal and neutralization reactions (Figure 1)

Prepare serial dilutions of PLA₂ (or anti-HCV E2 neutralizing antibody) in DMEM in 1.5 ml tubes.
Note: In our study, the PLA₂ concentrations in each tube are 2, 20, 200 and 2,000 ng/ml and the anti-HCV E2 antibody concentrations are 0.2, 2, 20, 200 and 2,000 μg/ml.
Dilute HCV stock in DMEM to a concentration of 2 x 10⁴ focus-forming unit (FFU) per 200 μl in a 15 ml tube.
Mix 100 μl of each dilution of PLA₂ (or anti-HCV E2 antibody) with 100 μl of the HCV solution in a 1.5 ml tube and gently vortex.
Note: After being mixed with the HCV solution, the final concentration of PLA₂ (or anti-HCV E2 antibody) in each tube is 1, 10, 100 and 1,000 ng/ml (or anti-HCV E2 antibody: 0.1, 1, 10, 100 and 1,000 μg/ml), respectively.
Incubate for 1 h at 37 °C.
Determine remaining viral infectivity by fluorescent antibody (FA) method, plaque assay or TCID₅₀ assay. See Part II for detailed procedure.

Figure 1. Illustration of the flowchart of virucidal and neutralizing activity tests. A fixed amount of virus is incubated with serial dilutions of an antiviral compound (e.g., PLA₂) (A. virucidal activity test) or an antibody (B. neutralizing activity test) at 37 °C for 1 h before inoculation to cultured cells. Antiviral activities of the test samples are assessed by appropriate procedures, such as plaque assay, focus assay and TCID₅₀ assay.

Part II: Determination of viral infectivity

Virus titers are expressed as plaque-forming unit (PFU)/ml, focus-forming unit (FFU)/ml, cell-infectious unit (CIU)/ml and 50% tissue culture infectious dose (TCID₅₀)/ml.

Plaque assay (for SINV, VSNJV and EMCV)
Plaque assay is one of the standard methods to determine infectious titers of viruses that cause strong cytopathic effect (CPE). A confluent monolayer of cells are infected with virus at various dilutions and cultured in a solid or semisolid overlay medium containing agarose or methylcellulose. This minimizes subsequent viral spread only to neighboring cells in the monolayer. Virus-infected cells undergo cell death by CPE and an area where a group of dead cells have detached is called a plaque (Figure 2). The infectivity titer is expressed as PFU/ml.

Figure 2. Representative image of a plaque assay plate of VSNJV. Plaques are visualized by staining with Gram Hacker’s Stain Solution I. Cell monolayers infected with serial 10-fold dilutions (10^-2, 10^-3, 10^-4, 10^-5 and 10^-6) of VSNJV and mock-infected are shown.
1. Seed Huh7it-1 cells (5 x 10⁵ cells) in a final volume of 3 ml of complete medium in each well of a 6-well plate.
2. Incubate for 20-24 h at 37 °C in a 5% CO₂ incubator. (Cells should be 90-100% confluent.)
3. To prepare virus solution, dilute 100 μl of virus-PLA₂ (or neutralizing antibody) mixture from Part I in 900 μl culture medium (10^-1 dilution) and make subsequent 10-fold serial dilutions of the virus (10^-2 to 10^-6 dilution).
4. Remove culture medium from each well.
5. Inoculate virus solution (10^-2 to 10^-6 dilution) to the cells (300 μl/well).
6. Incubate for 1 h at 37 °C in a 5% CO₂ incubator.
7. Remove the inoculum and rinse with medium (3 ml/well).
8. Gently add overlay medium containing 1% methylcellulose (3 ml/well).
  Note: Overlay medium contains 2% FBS.
9. Incubate at 37 °C in a 5% CO₂ incubator for 2 to 4 days – VSNJV and EMCV(2 days), SINV (3 to 4 days).
10. Aspirate the overlay medium.
  Note: As overlay medium is viscous, aspirate it slowly.
11. Fix the cells with 10% formaldehyde solution in PBS(-) (1 ml/well) for 20 min at room temperature.
12. Add Gram Hacker’s Stain Solution I (1 ml/well).
13. Incubate for 30 min.
14. Discard the staining solution, rinse with PBS (3 ml/well) and dry the plate.
15. Count the number of plaques (Figure 2).
Focus forming assay by FA method (for HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3)
Focus forming assay is particularly useful to measure infectivity of viruses that do not cause strong CPE. Virus-infected cells are incubated for a duration of a single round of the viral life cycle (one-step growth). Virus-infected cells are detected by an FA method using antibodies specific to the respective viral antigens (Figure 3). The infectivity titer is expressed as FFU/ml or CIU/ml.

Figure 3. Visualization of virus-infected cells by FA method. Virus-infected cells are stained in green. Nuclei are stained in blue. Scale bar = 10 μm.
1. Seed Huh7it-1 cells (1 x 10⁵ cells per 1ml) in each well of a 24-well plate containing a sterile glass slip.
2. Incubate for 20-24 h at 37 °C in a 5% CO₂ incubator.
3. To prepare virus solution, dilute 100 μl of virus-PLA₂ (or neutralizing antibody) mixture from Part I in 900 μl culture medium (10^-1 dilution) and make subsequent 10-fold serial dilutions of the virus (10^-2 to 10^-6 dilution).
4. Remove culture medium from each well.
5. Inoculate virus solution to the cells (200 μl/well).
6. Incubate for 1 h at 37 °C in a 5% CO₂ incubator.
7. Remove the inoculum and rinse with medium (1 ml/well).
8. Add complete medium (500 μl/well).
9. Incubate for 24 h at 37°C in a 5% CO₂ incubator.
10. Remove culture medium and rinse twice with PBS (200 μl/well).
11. Fix the cells with 200 μl of 4% paraformaldehyde solution.
12. Incubate for 20 min at room temperature.
13. Rinse cells three times with PBS (200 μl/well).
14. Add 200 μl of 0.1% Triton X-100 in PBS.
15. Incubate for 20 min at room temperature.
16. Add 200 μl of 1% BSA in PBS.
17. Incubate for 1 h at room temperature.
18. Prepare primary antibodies in PBS.
  Note: We use UV-inactivated anti-HCV human serum (1:500 dilution), rabbit polyclonal antibody against DENV PrM (1:500 dilution), mouse monoclonal antibody against DENV type 2 (1:500 dilution), rabbit antiserum against CV-B3 (1:100 dilution), rabbit antiserum against FLUAV (1:1,000 dilution), rabbit antiserum against SeV (1:1,000 dilution) and rabbit antiserum against HSV-1 (1:1,000 dilution).
19. Remove 1% BSA solution.
20. Add primary antibodies against the respective viruses (200 μl/well).
21. Incubate for 1 h at room temperature.
22. Prepare FITC-conjugated secondary antibodies in PBS (1:800 dilution, 200 μl/well).
23. Rinse cells three times with PBS.
24. Add secondary antibodies (200 μl/well).
25. Incubate for 1 h at room temperature in a dark box.
26. Rinse cells three times with PBS (500 μl/well).
27. Add Hoechst 33342 solution (1 mg/ml) in PBS (200 μl/well).
28. Incubate for 15 min at room temperature.
29. Rinse cells three times with PBS (500 μl/well).
30. Mount the glass coverslip on a microscope slide using Vectashield mounting solution.
31. Observe under a fluorescence microscope and count the number of virus-infected (stained in green) cells (Figure 3).
TCID₅₀ assay (for DENV, JEV, HSV-1, SINV, VSNJV and EMCV)
TCID₅₀ assay can be used to measure infectivity of viruses that cause strong CPE. TCID₅₀ represents a dilution of virus that makes 50% of the test wells show cell detachment (Figure 4). The infectivity titer is expressed as TCID₅₀/ml.

Figure 4. Schematic presentation of a result of TCID₅₀ assay. White circles indicate the wells in which the cells have undergone cell death to detach from the plastic plate due to strong CPE. Blue circles indicate the wells in which the cells remain intact to be stained with crystal violet.
1. Seed Vero cells (2 x 10⁴ cells in 100 μl) in complete medium in each well of a 96-well plate.
2. Incubate for 20-24 h at 37 °C in a 5% CO₂ incubator.
3. To prepare virus solution, dilute 100 μl of virus-PLA₂ (or neutralizing antibody) mixture from Part I in 100 μl culture medium (2^-1 dilution) and make subsequent 2-fold serial dilutions of the virus (2^-2 to 2^-10 dilution).
4. Remove culture medium from each well.
5. Inoculate the virus solution to the cells (100 μl/well)
  Note: Aliquots of the same sample should be inoculated to 4 to 8 wells.
6. Incubate for 2 h at 37 °C in a 5% CO₂ incubator.
7. Remove the inoculum and add overlay medium.
  Note: Overlay medium contains 2% FBS.
8. Incubate at 37 °C in a 5% CO₂ incubator.
  Note: DENV (5 to 8 days), JEV (5 to 6 days), HSV-1 and SINV (4 days), VSNJV and EMCV (2 days).
9. Count the number of wells with or without CPE under an inverted microscope.
10. Remove culture medium and rinse with PBS (100 μl/well).
11. Fix the cells with 10% formaldehyde solution in PBS(-) (100 μl/well) for 20 min at room temperature.
12. Rinse the cells with PBS (100 μl/well).
13. Add crystal violet solution to each well (100 μl /well).
14. Incubate for 10 min at room temperature.
15. Discard the crystal violet solution, rinse with PBS (100 μl/well), and dry the plate.
16. Count the number of wells with or without CPE by the naked eye (Figure 4).
Negative staining electron microscopic analysis (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, SINV, VSNJV and EMCV)
Negative-staining electron microscopy of viruses requires adequate concentrations of virus particle (> 10⁸/ml).
1. Add 5 to 10 μl of a purified virus solution onto a Formvar-coated nickel grid.
2. Wait for 5 min at room temperature so that the viral particles are adsorbed to the grid.
3. Remove the solution using the tip of a strip of filter paper.
4. Add 5 to 10 μl of 2% phosphotungstic acid in distilled water onto the grid and incubate for 2 min.
5. Remove the solution using a piece of filter paper.
6. Add 10 μl of PBS to wash the grid. (Repeat the Steps D5 and D6 three times.)
7. Air-dry the grid.
8. Observe under a transmission electron microscope.

Data analysis

Determination of 50% inhibitory concentration (IC₅₀): IC₅₀ of an antiviral substance against a given virus can be obtained based on the percent inhibition of viral infectivity mediated by serial dilutions of the antiviral substance. This model can be used for typical dose-response curves and receptor-ligand binding assays in pharmacological studies.

Plot data using ImageJ software and draw a sigmoid curve (Figure 5).
Four parameter (A, B, C, D) logistic equation is obtained.
y = D + (A - D)/(1 + (x/C)^B)
A = minimum asymptote; B = slope factor; C = concentration corresponding to the response midway between A and D; and D = maximum asymptote.
Parameter C is calculated as the estimate of IC₅₀.

Figure 5. A typical sigmoid curve showing inhibition of viral infectivity by serial dilutions of an antiviral drug. The red line shows the IC₅₀ value of the drug.

Notes

Manipulation of infectious viruses requires special biosafety laboratories. The degree of biocontainment is revised in accord with laws in the country/region where research will be conducted.

Recipes

Complete Dulbecco’s modified Eagle’s medium (complete medium)
1x non-essential amino acids
100 U/ml penicillin and streptomycin
10% fetal bovine serum (FBS; heat-inactivated at 56 °C for 30 min)
10x phosphate buffered saline (PBS)
1. Dissolve NaCl (80 g), KCl (2 g), Na₂HPO₄·12H₂O (28.8 g) and KH₂PO₄ (2.4 g) in 800 ml H₂O
2. Adjust volume to 1 L with dH₂O
3. Autoclave at 121 °C for 20 min. Dilute to 1x with distilled water
Overlay medium
1. Heat DMEM (500 ml) at 100 °C in an autoclavable glass bottle
2. Put a magnet bar in the bottle
3. Add methylcellulose (5 g) to the hot DMEM (500 ml) and stir well
  Note: Methylcellulose is not yet dissolved (turbid).
4. Autoclave at 121 °C for 20 min
5. When the temperature of the 1% methylcellulose-containing DMEM drops to about 60 °C (the solution is still turbid), rapidly cool the bottle in ice bath while stirring the content with a magnetic stirrer. Methylcellulose starts to dissolve and the medium will become translucent
6. Add FBS and other necessary reagents for cell culture

Acknowledgments

This work was supported in part by grants-in-aid for Research on Viral Hepatitis from the Ministry of Health, Labour and Welfare, and from the Japan Agency for Medical Research and Development (AMED). This work was also supported in part by a grant-in-aid for Special Research on Dengue Vaccine Development from Tokyo Metropolitan Government. This protocol was adapted from procedures published in Chen et al. (2017). The authors do not have any conflicts of interest or competing interests to declare.

References

Apriyanto, D. R., Aoki, C., Hartati, S., Hanafi, M., Kardono, L. B., Arsianti, A., Louisa, M., Sudiro, T. M., Dewi, B. E., Sudarmono, P., Soebandrio, A. and Hotta, H. (2016). Anti-hepatitis C virus activity of a crude extract from Longan (Dimocarpus longan Lour.) leaves. Jpn J Infect Dis 69(3): 213-220.
Bungyoku, Y., Shoji, I., Makine, T., Adachi, T., Hayashida, K., Nagano-Fujii, M., Ide, Y. H., Deng, L. and Hotta, H. (2009). Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells. J Gen Virol 90(Pt 7): 1681-1691.
Chen, M., Aoki-Utsubo, C., Kameoka, M., Deng, L., Terada, Y., Kamitani, W., Sato, K., Koyanagi, Y., Hijikata, M., Shindo, K., Noda, T., Kohara, M. and Hotta, H. (2017). Broad-spectrum antiviral agents: secreted phospholipase A₂ targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane. Sci Rep 7(1): 15931.
El-Bitar, A. M., Sarhan, M. M., Aoki, C., Takahara, Y., Komoto, M., Deng, L., Moustafa, M. A. and Hotta, H. (2015). Virocidal activity of Egyptian scorpion venoms against hepatitis C virus. Virol J 12: 47.
Hayashi, T., Hotta, H., Itoh, M. and Homma, M. (1991). Protection of mice by a protease inhibitor, aprotinin, against lethal Sendai virus pneumonia. J Gen Virol 72 (Pt 4): 979-982.
Hayashi, K., Iwasaki, Y. and Yanagi, K. (1986). Herpes simplex virus type 1-induced hydrocephalus in mice. J Virol 57(3): 942-951.
Holthausen, D. J., Lee, S. H., Kumar, V. T., Bouvier, N. M., Krammer, F., Ellebedy, A. H., Wrammert, J., Lowen, A. C., George, S., Pillai, M. R. and Jacob, J. (2017). An amphibian host defense peptide is virucidal for human H1 hemagglutinin-bearing influenza viruses. Immunity 46(4): 587-595.
Hotta, H., Wiharta, A. S. and Hotta, S. (1984). Antibody-mediated enhancement of dengue virus infection in mouse macrophage cell lines, Mk1 and Mm1. Proc Soc Exp Biol Med 175(3): 320-327.
Scheel, T. K. and Rice, C. M. (2013). Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat Med 19(7): 837-849.
Shimizu, Y. K., Hijikata, M., Oshima, M., Shimizu, K., Alter, H. J., Purcell, R. H., Yoshikura, H. and Hotta, H. (2013). Isolation of human monoclonal antibodies to the envelope e2 protein of hepatitis C virus and their characterization. PLoS One 8(2): e55874.
Shimizu, K., Mukaigawa, J., Oguro, M., Ono, Y., Nakajima, K. and Kida, H. (1985). Inhibition of transcriptase activity of influenza A virus in vitro by anti-haemagglutinin antibodies. Vaccine 3(3 Suppl): 207-210.