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Nov 2017

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Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies
抗病毒物质和抗体的杀灭病毒活性及中和活性检测   

Chie Aoki-UtsuboChie Aoki-Utsubo*Ming ChenMing Chen*Hak  HottaHak Hotta  (*共同第一作者)
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Abstract

In a narrow definition, virucidal activity represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of the viral particles. The viral infectivity can be measured in cell culture system by means of plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID50) assay, etc. Morphologically, disruption of viral particles can be demonstrated by negative staining electron microscopic analysis of viral particles. In this article, we describe methods to assess virucidal activity in a broad definition.

Keywords: Virucidal activity (杀病毒活性), Neutralizing activity (中和活性), Viral particle (病毒颗粒), Antiviral substance (抗病毒物质), Antibody (抗体), Viral infectivity assay (病毒感染力测定), Negative staining electron microscopic analysis (负染色电镜分析)

Background

Viruses are small intracellular parasites that hijack host cell machinery to replicate their own genome. At the initial step of the viral life cycle, infectious viral particles attach (bind) to particular host proteins, called viral receptors, on the surface of the target cells, followed by viral penetration (internalization and/or fusion) into intracellular compartments of the host cells, where the subsequent steps of the viral life cycle proceed to produce progeny virions (Scheel and Rice, 2013).

Virucidal activity in a narrow definition represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to interact with and functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of viral particles, as in the case of antibody-mediated neutralization.

We have recently reported that an isoform of secreted phospholipase A2 obtained from snake venom (Chen et al., 2017) and a peptide from scorpion venom (El-Bitar et al., 2015) possess strong virucidal activity against viruses that belong to the family Flaviviridae by targeting the lipid bilayer of the viral envelope, which is acquired from the endoplasmic reticulum membrane of the host cells. It was also reported that one of the host defense peptides from the skin of the South Indian frog has a strong virucidal activity against H1 hemagglutinin-bearing human influenza virus by targeting the conserved stalk of H1 hemagglutinin (Holthausen et al., 2017). In this article, we describe a number of useful methods by which to measure virucidal activity in a broad definition, such as plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID50) assay and negative staining electron microscopic analysis.

Materials and Reagents

  1. 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 )
  2. 100 mm culture dish (Corning, Falcon®, catalog number: 353003 )
  3. 6-well culture plate (Corning, Falcon®, catalog number: 353046 )
  4. 12-well culture plate (Corning, Falcon®, catalog number: 353043 )
  5. 24-well culture plate (Corning, Falcon®, catalog number: 353047 )
  6. 96-well culture plate (Corning, Falcon®, catalog number: 353072 )
  7. 1.5 ml microcentrifuge tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
  8. 15 ml tube (Corning, Falcon®, catalog number: 352196 )
  9. Cover slip (13 x 13 mm; Matsunami Glass, catalog number: C013001 )
  10. Microscope slide (Matsunami Glass, catalog number: S2215 )
  11. 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 )
  12. Filter paper (ATTO, catalog number: CB-06A-20A )
  13. 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)
  14. 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).
  15. 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).
  16. 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 )
  17. High glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, catalog number: 044-29765 )
  18. Phospholipase A2 from Naja mossambica snake venom (Sigma-Aldrich, catalog number: P7778 ) (Chen et al., 2017)
  19. Trypsin-EDTA solution (Wako Pure Chemical Industries, catalog number: 209-16941 )
  20. Crystal violet (Wako Pure Chemical Industries, catalog number: 038-04862 )
  21. MEM with non-essential amino acids (Thermo Fisher Science, GibcoTM, catalog number: 10370021 )
  22. Fetal bovine serum (FBS; Biowest, catalog number: S1820 )
  23. Penicillin-Streptomycin solution (Wako Pure Chemical Industries, catalog number: 168-23191 )
  24. Methyl Cellulose 4000 (Wako Pure Chemical Industries, catalog number: 136-02155 )
  25. 4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries, catalog number: 163-20145 )
  26. Formaldehyde solution (AppliChem, catalog number: A3592,0500 )
  27. Gram Hacker’s Stain Solution I (MUTO PURE CHEMICALS, catalog number: 41162 )
  28. Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
  29. Bovine serum albumin (BSA; Wako Pure Chemical Industries, catalog number: 015-21274 )
  30. Hoechst 33342 solution (Thermo Fisher Scientific, Molecular Probes, catalog number: H3570 )
  31. Vectashield mounting solution (Vector Laboratories, catalog number: H-1000 )
  32. Formvar-coated nickel grid (Electron Microscopy Sciences, catalog number: FF200-Ni )
  33. 2% phosphotungstic acid (Wako Pure Chemical Industries, catalog number: 582-66852 )
  34. Sodium chloride (NaCl; Wako Pure Chemical Industries, catalog number: 191-01665 )
  35. Potassium chloride (KCl; Wako Pure Chemical Industries, catalog number: 163-03545 )
  36. Disodium Hydrogen Phosphate (Na2HPO4·12H2O, NACALAI TESQUE, catalog number: 31722-45 )
  37. Potassium phosphate monobasic (KH2PO4; Wako Pure Chemical Industries, catalog number: 169-04245 )
  38. Complete medium for cell culture (see Recipes)
  39. 10x phosphate-buffered saline (PBS[-]) (see Recipes)
  40. Overlay medium (see Recipes)

Equipment

  1. Micropipette (Gilson, P20, P200, P1000)
    1. P20 (Gilson, catalog number: F123600 )
    2. P200 (Gilson, catalog number: F123601 )
    3. P1000 (Gilson, catalog number: F123602 )
  2. Multichannel micropipette (10-100 μl) (Eppendorf, catalog number: 3125000036 )
  3. Hemocytometer chamber (e.g., Erma, catalog number: 03-303-1 )
  4. Biosafety cabinet (e.g., PHC, model: MHE-S1301A2 )
  5. CO2 incubator (e.g., PHC, model: MCO-20AIC )
  6. Autoclave (e.g., TOMY DIGITAL BIOLOGY, model: SX-500 )
  7. Refrigerated tabletop centrifuge (e.g., Eppendorf, model: Centrifuge 5424 )
  8. Vortex (e.g., Scientific Industries, model: Vortex-Genie 2 )
  9. -80 °C freezer (e.g., PHC, model: MDF-384 )
  10. Inverted microscope (e.g., Olympus, model: CKX53 )
  11. Fluorescent microscope (e.g., ZEISS, model: Axio Vert. A1 )
  12. Multilabel Plate Counter (PerkinElmer, model: 1420 ALBOSX )
  13. Transmission electron microscope (Hitachi, model: HT7700 TEM )

Procedure

Part I: Virucidal and neutralization reactions (Figure 1)

  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.
  2. Dilute HCV stock in DMEM to a concentration of 2 x 104 focus-forming unit (FFU) per 200 μl in a 15 ml tube.
  3. 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.
  4. Incubate for 1 h at 37 °C.
  5. 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.

  1. 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).

  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).

  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).

  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.

  1. Plot data using ImageJ software and draw a sigmoid curve (Figure 5).
  2. 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.
  3. 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

  1. 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)
  2. 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
  3. 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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
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  6. Hayashi, K., Iwasaki, Y. and Yanagi, K. (1986). Herpes simplex virus type 1-induced hydrocephalus in mice. J Virol 57(3): 942-951.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.

简介

在狭义定义中,杀病毒活性表示与病毒颗粒相互作用和物理破坏的活性。 在一个广义的定义中,它包括了功能上抑制(中和)病毒感染性而没有病毒颗粒的明显形态改变的活性。 可以通过噬菌斑测定,感染性聚焦测定,50%组织培养感染剂量(TCID 50)测定,等等在细胞培养系统中测量病毒感染性。 形态学上,病毒颗粒的破坏可以通过病毒颗粒的负染色电子显微镜分析来证明。 在这篇文章中,我们描述了用广义定义评估杀病毒活性的方法。

【背景】病毒是细胞内寄生虫,它们劫持宿主细胞机制来复制它们自己的基因组。在病毒生命周期的最初阶段,感染性病毒颗粒附着(结合)到靶细胞表面上的称为病毒受体的特定宿主蛋白,然后病毒渗透(内化和/或融合)到细胞的细胞内区室宿主细胞,其中病毒生命周期的后续步骤继续产生后代病毒体(Scheel和Rice,2013)。

狭义定义中的杀病毒活性表示与病毒颗粒相互作用和物理破坏的活性。在一个广义的定义中,它包括与病毒感染性相互作用并在功能上抑制(中和)病毒感染性而没有病毒颗粒的明显形态改变的活性,如抗体介导的中和的情况。

最近我们报道了从蛇毒(Chen等,2017)获得的分泌型磷脂酶Aβ的异构体和来自蝎毒(El-Bitar >)等人,2015)通过靶向病毒包膜的脂双层(其从内质网膜获得)具有对属于黄病毒科的病毒的强杀病毒活性宿主细胞。还有报道说,来自南印度青蛙皮肤的一种宿主防御肽通过靶向H1血凝素的保守茎而具有对抗H1血球凝集素的人流感病毒的强杀病毒活性(Holthausen等人, 2017)。在这篇文章中,我们描述了许多有用的方法来衡量杀病毒活性的广义定义,如噬菌斑测定,感染性聚焦测定,50%组织培养感染剂量(TCID 50)测定和负染色电子显微镜分析。

关键字:杀病毒活性, 中和活性, 病毒颗粒, 抗病毒物质, 抗体, 病毒感染力测定, 负染色电镜分析

材料和试剂

  1. 一次性提示
    1. 10μl容量(Thermo Fisher Scientific,分子生物制品,分类号:3510-05)
    2. 200μl容量(Thermo Fisher Scientific,Molecular BioProducts,目录号:3900)
    3. 1毫升容量(FUKAEKASEI和WATSON,目录号:110-502C)
  2. 100mm培养皿(Corning,Falcon ,目录号:353003)
  3. 6孔培养板(Corning,Falcon ,目录号:353046)
  4. 12孔培养板(Corning,Falcon ,目录号:353043)
  5. 24孔培养板(Corning,Falcon ,目录号:353047)
  6. 96孔培养板(Corning,Falcon ,目录号:353072)
  7. 1.5 ml微量离心管(FUKAEKASEI和WATSON,目录号:131-715C)
  8. 15ml试管(Corning,Falcon ,产品目录号:352196)
  9. 封面纸(13 x 13毫米;松浪玻璃,产品目录号:C013001)
  10. 显微镜幻灯片(Matsunami Glass,目录号:S2215)
  11. 一次性血清移液器
    1. 1毫升容量(Corning,Falcon ,目录号:356521)
    2. 5毫升容量(IWAKI,目录号:7153-005)
    3. 10毫升容量(IWAKI,目录号:7154-010)
  12. 滤纸(ATTO,产品目录号:CB-06A-20A)
  13. 病毒(Chen et。,2017):
    1. 丙型肝炎病毒(HCV,J6 / JFH-1株)
    2. 登革热病毒(DENV,特立尼达1751株)
    3. 日本脑炎病毒(JEV,Nakayama株)
    4. 甲型流感病毒(FLUAV,A / Udorn / 307/72 [H3N2])
    5. 仙台病毒(SeV,伏见菌株)
    6. 单纯疱疹病毒1型(HSV-1,CHR3株)
    7. 柯萨奇病毒B3(CV-B3,Nancy株)
    8. 水泡性口炎新泽西州病毒(VSNJV)
    9. 辛德比斯病毒(SINV)
    10. 脑心肌炎病毒(EMCV,DK-27株)
  14. Huh7it-1细胞(Apriyanto等人,2016)
    注:Huh7it-1细胞易受上述所有病毒(HCV,DENV,JEV,FLUAV,SeV,HSV-1,CV-B3,VSNJV,SINV和EMCV)的影响。
  15. Vero细胞(ATCC,目录号:CCL-81)
    注意:Vero细胞易受上述所有病毒(HCV,DENV,JEV,FLUAV,SeV,HSV-1,CV-B3,VSNJV,SINV和EMCV)的影响。
  16. 抗体
    1. 针对DENV PrM的兔多克隆抗体(Gene Tex,目录号:GTX128093)
    2. 抗DENV 2型的小鼠单克隆抗体(3H5; Hotta等人,1984)
    3. UV灭活的抗HCV人血清(Bungyoku等人,2009)
    4. 抗HCV E2中和抗体#55(Shimizu et al。 ,2013)

    5. 抗CV-B3的兔抗血清(DENKA SEIKEN,目录号:300638)
    6. 针对FLUAV的兔抗血清(Shimizu et al。 ,1985)
    7. 对抗SeV的兔抗血清(Hayashi等人,1991)
    8. 抗HSV-1的兔抗血清(Hayashi et al。,1986)
    9. FITC缀合的山羊抗人IgG(MEDICAL& BIOLOGICAL LABORATORIES,目录号:104AG)
    10. Alexa Flour488结合山羊抗小鼠IgG(Thermo Fisher Science,目录号:A-11001)
    11. Alexa Flour488结合山羊抗兔IgG(Thermo Fisher Science,目录号:A-11008)
  17. 高糖Dulbecco改良的Eagle's培养基(DMEM; Wako Pure Chemical Industries,目录号:044-29765)
  18. 来自蛇吸血管蛇毒(Sigma-Aldrich,目录号:P7778)(Chen等人,2017)的磷脂酶A 2, >
  19. 胰蛋白酶-EDTA溶液(Wako Pure Chemical Industries,目录号:209-16941)
  20. 结晶紫(Wako Pure Chemical Industries,目录号:038-04862)
  21. 具有非必需氨基酸的MEM(Thermo Fisher Science,Gibco TM,目录号:10370021)
  22. 胎牛血清(FBS; Biowest,目录号:S1820)
  23. 青霉素 - 链霉素溶液(Wako Pure Chemical Industries,目录号:168-23191)
  24. 甲基纤维素4000(Wako Pure Chemical Industries,目录号:136-02155)
  25. 4%多聚甲醛磷酸缓冲液(和光纯药工业,目录号:163-20145)
  26. 甲醛溶液(AppliChem,目录号:A3592,0500)
  27. 格兰姆黑客染色液I(MUTO PURE CHEMICALS,目录号:41162)
  28. Triton X-100(Wako Pure Chemical Industries,目录号:169-21105)
  29. 牛血清白蛋白(BSA; Wako Pure Chemical Industries,目录号:015-21274)
  30. Hoechst 33342溶液(Thermo Fisher Scientific,Molecular Probes,目录号:H3570)
  31. Vectashield安装解决方案(Vector Laboratories,目录号:H-1000)
  32. Formvar涂层镍网格(Electron Microscopy Sciences,目录号:FF200-Ni)
  33. 2%磷钨酸(Wako Pure Chemical Industries,目录号:582-66852)
  34. 氯化钠(NaCl; Wako Pure Chemical Industries,目录号:191-01665)
  35. 氯化钾(KCl; Wako Pure Chemical Industries,目录号:163-03545)
  36. 磷酸氢二钠(Na 2 HPO 4·12H 2 O,NACALAI TESQUE,目录号:31722-45)
  37. 磷酸二氢钾(KH 2 PO 4; Wako Pure Chemical Industries,目录号:169-04245)
  38. 细胞培养的完整培养基(见食谱)
  39. 10倍磷酸盐缓冲盐水(PBS [ - ])(见食谱)
  40. 覆盖媒体(见食谱)

设备

  1. 微量移液器(Gilson,P20,P200,P1000)
    1. P20(Gilson,目录号:F123600)
    2. P200(Gilson,目录号:F123601)
    3. P1000(Gilson,目录号:F123602)
  2. 多通道微量移液器(10-100μl)(Eppendorf,目录号:3125000036)
  3. 血细胞计数器室(例如,Erma,目录号:03-303-1)
  4. 生物安全柜( ,PHC,型号:MHE-S1301A2)
  5. CO 2培养箱(例如PHC,型号:MCO-20AIC)。
  6. 高压灭菌器(例如,,TOMY DIGITAL BIOLOGY,型号:SX-500)
  7. 冷藏台式离心机(例如Eppendorf,型号:离心机5424)
  8. Vortex(例如,Scientific Industries,型号:Vortex-Genie 2)
  9. -80°C冷冻机(,例如,PHC,型号:MDF-384)
  10. 倒置显微镜(,例如,奥林巴斯,型号:CKX53)
  11. 荧光显微镜(例如,ZEISS,型号:Axio Vert A1)
  12. 多标记板计数器(PerkinElmer,型号:1420 ALBOSX)
  13. 透射电子显微镜(日立,型号:HT7700 TEM)

程序

第一部分:杀病毒和中和反应(图1)

  1. 准备在1.5ml管中的DMEM中的PLA2(或抗HCV E2中和抗体)的系列稀释液。
    注:在我们的研究中,每管中PLA2浓度分别为2,20,200和2,000 ng / ml,抗HCV E2抗体浓度分别为0.2,2,20,200和2000μg/ ml。
  2. 在15ml试管中,将DMEM中的HCV原液稀释至每200μl2×10-4焦点形成单位(FFU)的浓度。
  3. 将100μlPLA2(或抗HCV E2抗体)的每种稀释液与100μlHCV溶液在1.5ml管中混合并轻轻涡旋。
    注:与HCV溶液混合后,每管中PLA2(或抗HCV E2抗体)的终浓度分别为1,10,100和1,000 ng / ml(或抗HCV E2抗体:0.1,1,10,100和1,000μg/ ml)。

  4. 在37°C孵育1小时
  5. 通过荧光抗体(FA)方法,空斑测定或TCID 50测定来确定剩余的病毒感染性。有关详细步骤,请参阅第II部分。


    图1.杀病毒和中和活性测试流程图的图解将固定量的病毒与连续稀释的抗病毒化合物(例如,PLA <2> (A.杀病毒活性试验)或抗体(B.中和活性试验)在37℃下接种1小时至培养细胞。通过适当的程序评估测试样品的抗病毒活性,例如噬菌斑测定,焦点测定和TCID 50测定。


第二部分:病毒感染性的测定&nbsp;

病毒滴度以斑块形成单位(PFU)/ ml,聚焦形成单位(FFU)/ ml,细胞感染单位(CIU)/ ml和50%组织培养感染剂量(TCID <50> >)/毫升。

  1. 噬斑测定(用于SINV,VSNJV和EMCV)
    噬斑测定是确定导致强细胞病变效应(CPE)的病毒的传染性滴度的标准方法之一。汇合的单层细胞用各种稀释度的病毒感染,并在含有琼脂糖或甲基纤维素的固体或半固体覆盖培养基中培养。这最大限度地减少了后续的病毒传播,只有单层中的相邻细胞。病毒感染的细胞通过CPE进行细胞死亡,并且将一组死细胞脱落的区域称为斑块(图2)。传染性滴度表示为PFU / ml。


    图2. VSNJV的噬菌斑测定板的代表性图像通过用Gram Hacker的染色溶液I进行染色来显现噬菌斑。用10倍稀释系列(10-2-2)感染细胞单层, 10 -3 -3,-10 -4,10 -5和10 -6)和显示模拟感染。

    1. 种子Huh7it-1细胞(5×10 5个细胞)在6孔板的每个孔中终体积为3ml完全培养基中。
    2. 在37℃,5%CO 2培养箱中孵育20-24小时。 (细胞应该是90-100%汇合。)
    3. 为了制备病毒溶液,在900μl培养基(10 -1稀释度)中稀释100μl来自部分I的病毒-PLA 2(或中和抗体)混合物并随后制备10倍系列稀释的病毒(10 -2 -2至10 -6稀释度)。

    4. 移除每口井的培养基
    5. 将细胞(300μl/孔)接种病毒溶液(10 -2 -2至10 -6稀释)。
    6. 在37℃,5%CO 2培养箱中孵育1小时。
    7. 去除接种物并用培养基(3ml /孔)冲洗。
    8. 轻轻地添加含有1%甲基纤维素(3毫升/孔)的覆盖介质。
      注意:覆盖媒体包含2%FBS。
    9. 在37℃,5%CO 2培养箱中培养2至4天 - VSNJV和EMCV(2天),SINV(3至4天)。
    10. 吸取重叠介质。
      注意:由于覆盖介质是粘性的,请慢慢吸入。

    11. 在室温下用含10%甲醛的PBS( - )(1ml /孔)溶液固定细胞20分钟。
    12. 添加革兰染色黑色染液I(1ml /孔)。
    13. 孵育30分钟。
    14. 弃去染色溶液,用PBS(3ml /孔)冲洗并干燥板。
    15. 计数斑块的数量(图2)。

  2. 通过FA方法进行焦点形成测定(对于HCV,DENV,JEV,FLUAV,SeV,HSV-1,CV-B3)
    焦点形成测定对于测量不会导致强CPE的病毒的传染性特别有用。将病毒感染的细胞温育一轮病毒生命周期(一步生长)。用FA方法检测病毒感染的细胞,使用对各病毒抗原特异性的抗体(图3)。
    传染性滴度表示为FFU / ml或CIU / ml

    图3.用FA法显示病毒感染细胞病毒感染细胞染成绿色。核被染成蓝色。比例尺= 10微米。

    1. 在含有无菌玻璃浆的24孔板的每个孔中种入Huh7it-1细胞(每1ml含1×10 5个细胞)。
    2. 在37℃,5%CO 2培养箱中孵育20-24小时。
    3. 为了制备病毒溶液,在900μl培养基(10 -1稀释度)中稀释100μl来自部分I的病毒-PLA 2(或中和抗体)混合物并随后制备10倍系列稀释的病毒(10 -2 -2至10 -6稀释度)。

    4. 移除每口井的培养基
    5. 将病毒溶液接种到细胞(200μl/孔)。
    6. 在37℃,5%CO 2培养箱中孵育1小时。
    7. 去除接种物并用培养基(1ml /孔)冲洗。
    8. 添加完整培养基(500μl/孔)。
    9. 在37℃,5%CO 2培养箱中孵育24小时。
    10. 去除培养基,并用PBS(200μl/孔)冲洗两次。
    11. 用200μl4%多聚甲醛溶液固定细胞。
    12. 在室温下孵育20分钟。
    13. 用PBS(200μl/孔)冲洗细胞三次。

    14. 加入200μl0.1%Triton X-100的PBS溶液。
    15. 在室温下孵育20分钟。
    16. 在PBS中加入200μl的1%BSA。
    17. 在室温下孵育1小时。
    18. 在PBS中制备一抗。
      注意:我们使用UV灭活的抗HCV人血清(1:500稀释),抗DENV PrM的兔多克隆抗体(1:500稀释),抗DENV 2型(1:500稀释)的小鼠单克隆抗体,兔抗CV-B3的抗血清(1:100稀释度),抗FLUAV的兔抗血清(1:1,000稀释),抗SeV的兔抗血清(1:1,000稀释度)和抗HSV-1的兔抗血清(1:1,000稀释)。
    19. 删除1%BSA解决方案。
    20. 添加针对相应病毒的一级抗体(200μl/孔)。

    21. 在室温下孵育1小时
    22. 在PBS中制备FITC偶联的二抗(1:800稀释,200μl/孔)。
    23. 用PBS冲洗细胞三次。
    24. 加入二抗(200μl/孔)。

    25. 在室温下在暗箱中孵育1小时
    26. 用PBS(500μl/孔)冲洗细胞三次。

    27. 加入Hoechst 33342溶液(1 mg / ml),PBS(200μl/孔)
    28. 在室温下孵育15分钟。
    29. 用PBS(500μl/孔)冲洗细胞三次。

    30. 使用Vectashield安装解决方案将玻璃盖玻片安装在显微镜载玻片上
    31. 在荧光显微镜下观察并计数病毒感染(绿色染色)细胞的数量(图3)。

  3. TCID 50测定法(对于DENV,JEV,HSV-1,SINV,VSNJV和EMCV)
    TCID50测定可用于测量导致强CPE的病毒的感染性。 TCID 50表示使50%的测试孔显示细胞分离的病毒稀释度(图4)。感染滴度表示为TCID 50 / ml。


    图4.TCID50测定结果的示意图白色圆圈表示由于强CPE而使细胞已经经历细胞死亡从而与塑料板分离的孔。蓝色的圆圈表示细胞保持完整,可以结晶紫染色的孔。

    1. 在96孔板的每个孔中的完全培养基中接种Vero细胞(100μl中2×10 4个细胞)。
    2. 在37℃,5%CO 2培养箱中孵育20-24小时。
    3. 为了制备病毒溶液,在100μl培养基(2μl稀释液)中稀释100μl来自部分I的病毒-PLA 2(或中和抗体)混合物并且随后制备病毒的2倍连续稀释液(2〜2〜2〜10稀释)。
    4. 从每个孔中取出培养基。
    5. 将病毒溶液接种到细胞(100μl/孔)
      注:同一样本的等分试样应接种到4至8孔。

    6. 在37℃,5%CO 2培养箱中培养2小时
    7. 去除接种物并添加覆盖介质。
      注意:覆盖媒体包含2%FBS。
    8. 在37℃下在5%CO 2培养箱中孵育。
      注:DENV(5至8天),JEV(5至6天),HSV-1和SINV(4天),VSNJV和EMCV(2天)。
    9. 在倒置显微镜下计数有或没有CPE的孔数。
    10. 去除培养基并用PBS(100μl/孔)冲洗。

    11. 在室温下用含10%甲醛的PBS溶液( - )(100μl/孔)固定细胞20分钟。
    12. 用PBS(100μl/孔)冲洗细胞。

    13. 每孔加入结晶紫溶液(100μl/孔)
    14. 在室温下孵育10分钟。
    15. 丢弃结晶紫溶液,用PBS(100μl/孔)冲洗,并干燥板。
    16. 用肉眼计数有或没有CPE的孔数(图4)。

  4. 阴性染色电镜分析(HCV,DENV,JEV,FLUAV,SeV,HSV-1,CV-B3,SINV,VSNJV和EMCV)

    病毒的阴性电子显微镜检查需要足够浓度的病毒颗粒(> 10 8 / ml)。
    1. 将5至10μl的纯化病毒溶液加入Formvar涂层的镍网格中。
    2. 在室温下等待5分钟,以使病毒颗粒吸附在电网上。

    3. 使用滤纸条的尖端去除溶液。

    4. 在蒸馏水中加入5至10μl2%磷钨酸到电网上并孵育2分钟。

    5. 使用一张滤纸去除溶液。
    6. 加入10μlPBS清洗电网。 (重复步骤D5和D6三次。)
    7. 风干网格。
    8. 透射电子显微镜下观察。

数据分析

基于对连续稀释介导的病毒感染性的抑制百分比,可以获得针对给定病毒的50%抑制浓度(IC 50:IC 50)的抗病毒物质的IC50的抗病毒物质。该模型可用于药理研究中的典型剂量反应曲线和受体 - 配体结合测定。

  1. 使用ImageJ软件绘制数据并绘制S形曲线(图5)。
  2. 四个参数(A,B,C,D)对数方程得到。
    y = D +(A-D)/(1 +(x / C)^ B)
    A =最小渐近线; B =斜率因子; C =与A和D之间的响应对应的浓度; D =最大渐近线。
  3. 参数C被计算为IC 50的估计值。&nbsp;


    的图5示出了由一种抗病毒药物的连续稀释病毒感染的抑制的典型S形曲线。红线表示的IC <子> 50 药物的价值。


笔记

感染性病毒的操作需要特殊的生物安全实验室。根据进行研究的国家/地区的法律修改生物防护程度。

食谱

  1. 完成Dulbecco改良的Eagle's培养基(完全培养基)
    1个非必需氨基酸
    100U / ml青霉素和链霉素
    10%胎牛血清(FBS;在56℃热灭活30分钟)
  2. 10x磷酸盐缓冲盐水(PBS)
    1. 溶解NaCl(80g),KCl(2g),Na 2 HPO 4·12H 2 O(28.8g)和KH 2 (2.4g)在800ml H 2 O中的溶液
    2. 使用dH 2 O
      将音量调节至1 L
    3. 在121℃高压灭菌20分钟。用蒸馏水稀释至1x
  3. 覆盖媒体
    1. 在100°C的高压灭菌玻璃瓶中加热DMEM(500毫升)
    2. 把磁棒放在瓶子里
    3. 将甲基纤维素(5克)加入到热DMEM(500毫升)中并充分搅拌
      注:甲基纤维素尚未溶解(混浊)。
    4. 在121°C高压灭菌20分钟
    5. 当含1%甲基纤维素的DMEM的温度下降到约60℃时(溶液仍然浑浊),用冰浴迅速冷却瓶子,同时用磁力搅拌器搅拌内容物。甲基纤维素开始溶解,介质变成半透明
    6. 添加FBS和其他必需的细胞培养试剂

致谢

这项工作得到了卫生,劳动和福利部以及日本医学研究和开发署(AMED)对病毒性肝炎研究的资助。这项工作也得到了东京都政府特别研究登革热疫苗开发援助的部分支持。该协议是根据Chen等人发表的程序改编的。作者没有任何利益冲突或利益冲突声明。

参考

  1. Apriyanto,DR,Aoki,C.,Hartati,S.,Hanafi,M.,Kardono,LB,Arsianti,A.,Louisa,M.,Sudiro,TM,Dewi,BE,Sudarmono,P.,Soebandrio,A.和Hotta,H。(2016)。 龙眼粗提物的抗丙型肝炎病毒活性( Dimocarpus longan Lour。)leaves。 Jpn J Infect Dis 69(3):213-220。
  2. (2009年),Bungyoku,Y.,Shoji,I.,Makine,T.,Adachi,T.,Hayashida,K.,Nagano-Fujii,M.,Ide,Y. H.,Deng,L.and Hotta,H。(2009)。 在培养的肝癌细胞中高效生产具有适应性突变的传染性丙型肝炎病毒 J Gen Virol 90(Pt 7):1681-1691。
  3. 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.和Hotta,H。(2017)。 广谱抗病毒药物:分泌型磷脂酶A 2靶向病毒包膜脂质双层来源于内质网膜。 Sci Rep 7(1):15931.
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引用:Aoki-Utsubo, C., Chen, M. and Hotta, H. (2018). Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies. Bio-protocol 8(10): e2855. DOI: 10.21769/BioProtoc.2855.
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