Sep 2016



Production, Titration and Imaging of Zika Virus in Mammalian Cells

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Since the outbreak of Zika virus (ZIKV) in Latin America and the US in 2016, this flavivirus has emerged as a major threat for public health. Indeed, it is now clear that ZIKV is vertically transmitted from the infected mother to the fetus and this may lead to severe neurological development defects including (but not restricted to) neonate microcephaly. Although ZIKV has been identified in the late 1940s, very little was known about its epidemiology, symptoms and molecular biology before its reemergence 60 years later. Recently, tremendous efforts have been made to develop molecular clones and tools as well as cell culture and animal models to better understand ZIKV fundamental biology and pathogenesis and to develop so-far-unavailable antiviral drugs and vaccines. This bio-protocol describes basic experimental procedures to produce ZIKV stocks and to quantify their concentration in infectious virus particles as well as to image and study this pathogen within infected cells using confocal microscopy-based imaging.

Keywords: Zika virus (寨卡病毒), NS4B (NS4B), Double-stranded RNA (双链RNA), Virus titration (病毒滴度测定), Immunofluorescence microscopy (免疫荧光显微术), Plaque assay (蚀斑形成测定)


Zika virus (ZIKV), a mosquito-borne Flavivirus within the Flaviviridae virus family, was first isolated from a sentinel Rhesus monkey in 1947 in the Ziika Forest in Uganda and is closely related to dengue virus (DENV) (World Health Organization, 2018). Since then, it became famous in the last decade for its outbreaks in some Pacific islands (2007 and 2013) and in the Americas (2015). Symptomatic patients (25%-20% of the cases) may usually show rather mild clinical manifestations such as fevers, rashes, conjunctivitis, muscle/joint pains and/or headaches. However, since ZIKV re-emergence, severe neurological symptoms including (but not restricted to) Guillain-Barre syndrome in adults and congenitally transmitted newborn microcephaly were identified in infected individuals (Lazear and Diamond, 2016; Grubaugh et al., 2018). Unfortunately, there are currently no available treatments or vaccines against Zika virus, and this is partly due to our poor understanding of its biology.

In terms of phylogeny, there are 2 distinct lineages of Zika virus (Haddow et al., 2012). The so-called “historical” African lineage including the prototype strain MR766 and the “contemporary” Asian lineage derived from the African lineage after decades of mutations. For instance, the Asian strain H/PF/2013 is responsible for the outbreak in French Polynesia in 2013. Similarly, the virus strain responsible for the Brazilian outbreak also phylogenetically derives from the Asian lineage. Indeed, experiments in mouse and mosquito infection models support the idea that the Asian strain has acquired several key mutations which led to microcephaly in infected newborns in Brazil (Yuan et al., 2017) and to enhanced viral infectivity in the arthropod transmission vector Aedes aegypti (Liu et al., 2017).

Upon entry in the host cell, the viral genome, a positive single-stranded RNA, is translated into a single polyprotein at the endoplasmic reticulum (ER), which is subsequently cleaved into 3 structural proteins and 7 non-structural proteins by host and viral proteases. After replication, the genome is encapsidated into neosynthesized virions which are subsequently released from the cell (Neufeldt et al., 2018). Like all other tested flaviviruses, ZIKV remodels the endomembranes of the infected cell to generate a cytoplasmic environment which is favorable to ZIKV life cycle (Cortese et al., 2017). These membranous compartments, called viral replication factories are all ER-derived and can be sub-divided into three classes of ultrastructures (Cortese et al., 2017): 1. vesicle packets, which are ER invaginations believed to be the site of viral RNA replication, 2. virus bags in which assembled immature viruses accumulate in regular arrays, and 3. convoluted membranes whose roles remain unclear although it was proposed for DENV that they modulate host processes for the benefit of replication (Chatel-Chaix et al., 2016).

Before 2016, little was known about the general biology of ZIKV, and most of our knowledge relied on a direct transposition of fundamental discoveries about other flaviviruses closely related to this pathogen like DENV. However, it remains unclear what makes ZIKV so unique in terms of neuropathogenesis as compared to the other members of the Flavivirus genus. Hence, since ZIKV re-emergence and the recent outbreaks, the scientific community around the world has begun to decipher the mysteries surrounding this virus and to develop tools such as molecular clones as well as cell culture and animal models (Schwarz et al., 2016; Shan et al., 2016; Xie et al., 2016; Morrison and Diamond, 2017; Mutso et al., 2017; Munster et al., 2018). Knowing how to culture ZIKV and to measure its infectivity constitutes key methods to perform any descriptive or functional studies about this virus both in cellulo and in vivo. Moreover, given that Asian and African lineages may differ in terms of symptom severity in infected mouse fetuses (Cugola et al., 2016), it is sometimes relevant to compare model strains of these two lineages. In this bio-protocol, we describe basic cell culture methods to produce ZIKV stocks from Asian and African lineages and to quantify their concentration in infectious virus particles (more generally referred to as “titer”) as well as to image this pathogen inside infected cells using immunofluorescence-based microscopy.

Materials and Reagents

  1. Materials
    1. 1.5 ml microtubes (Ultident Scientific, catalog number: 87-B150-C)
    2. 10 cm cell culture dishes (Falcon, Fisher Scientific, catalog number: 08-772-E) 
    3. 15 cm cell culture dishes (Falcon, Fisher Scientific, catalog number: 08-772-6)
    4. 24-well polystyrene culture plates (Corning Life Sciences, Fisher Scientific, catalog number: 08-772-1) 
    5. MCE 0.45 μm filters, 30 mm diameter (Ultident Scientific, catalog number: 229753) 
    6. 20 ml sterile syringes (Ultident Scientific, catalog number: BD-302830) 
    7. 15 ml sterile conical tubes (Falcon, Fisher Scientific, catalog number: 352096) 
    8. Coverslips No. 1 (diameter: 12 mm, thickness: 0.13 to 0.17 mm), sterilized by autoclaving (Fisher Scientific, catalog number: 12-545-80)
    9. Microscope glass slides, frosted clear glass 26 mm x 76 mm, 1-1.2 mm thick (Ultident Scientific, catalog number: 170-8105-W)
    10. Absorbent paper
    11. Parafilm
    12. Aluminum foil

  2. Viruses
    1. ZIKV MR766 (African lineage) (EVAg, catalog number: 001v-EVA143) (under material and transfer agreement. Passage history: P5. Store original desiccated stocks at -80 °C)
    2. ZIKV H/PF/2013 (Asian lineage) (EVAg, catalog number: 001v-EVA1545) (under material and transfer agreement. Passage history: P6. Store original desiccated stocks at -80 °C)

  3. Cell lines
    1. Vero E6 monkey epithelial cells (a kind gift from Drs. Tom Hobman and Anil Kumar, University of Alberta) (ATCC, catalog number: CRL-1586)
    2. Huh7 hepatocarcinoma cells (a kind gift from Dr. Patrick Labonté, INRS) (Creative Bioarray, catalog number: CSC-C9441L)
      Alternatively, this cell line is typically available from most laboratories working on hepatitis C virus or DENV.

  4. Reagents
    1. UltraPure distilled water (Life Technologies, catalog number: 10977-015) 
    2. Dulbecco's modified Eagle medium (DMEM) (Life Technologies, catalog number: 111965-092) 
    3. Fetal bovine serum (FBS) performance (Wisent, catalog number: 098150) 
    4. Penicillin-streptomycin (Life Technologies, catalog number: 15140-122) 
    5. MEM non essential amino acids solution (100x) (Life Technologies, catalog number: 11140-050)
    6. Phosphate buffered saline (PBS) (Life Technologies, catalog number: 14190-144)
    7. 1 M HEPES buffer, pH range: 7.2-7.5 (Life Technologies, catalog number: 15630-080)
    8. 0.25% Trypsin- EDTA (Life Technologies, catalog number: 25200-072) 
    9. Minimum Essential Medium (MEM) with L-Glutamine (Life Technologies, catalog number: 11095-080) 
    10. Carboxymethylcellulose (CMC) sodium salt, medium viscosity (Sigma-Aldrich, catalog number: 21902-100G )
    11. 37% formaldehyde (Fisher Scientific, catalog number: BP531-500) 
    12. Crystal violet (Fisher Scientific, catalog number: C581-100) 
    13. 95% ethanol (Commercial Alcohols, catalog number: 1011C)
    14. Triton X-100 (Mallinktrot, catalog number: 3555)
    15. 4% paraformaldehyde (Sigma-Aldrich, catalog number: P6148-1KG)
    16. Normal goat serum (Thermo-Fisher, catalog number: 01-6201)
    17. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A-9647)
    18. Sodium azide (Fisher Scientific, catalog number: BP922I-500)
    19. Rabbit polyclonal anti-ZIKV NS4B (GeneTex, catalog number: GTX133311; dilution 1:200)
    20. Mouse monoclonal anti-dsRNA, clone J2 (Scicons, catalog number: 10010200; dilution 1:400)
    21. Rabbit polyclonal anti-DENV NS4B (GeneTex, catalog number: GTX124250; dilution 1:1,000)
    22. Mouse monoclonal anti-DENV NS3, clone GT2811 (GeneTex, catalog number: GTX629477; dilution 1:100)
    23. Mouse monoclononal panflaviviral anti-E, clone 4G2 (Sigma-Aldrich, catalog number: MAB10216, dilution 1:200)
    24. Goat anti-Rabbit AlexaFluor 488 (Life Technologies, catalog number: A11034)
    25. Goat anti-Mouse AlexaFluor 568 (Life Technologies, catalog number: A11004)
    26. 4',6-Diamidino-2-Phenylindole (DAPI) (Life Technologies, catalog number: D1306)
    27. Fluoromount G (Southern Biotech, catalog number: 0100-01)
    28. Complete DMEM (see Recipes)
    29. Plaquing medium (MEM-CMC) (see Recipes)
    30. Formaldehyde fixative (see Recipes)
    31. Paraformaldehyde fixative (see Recipes)
    32. Crystal violet staining solution (see Recipes)
    33. Triton X-100 permeabilization solution (see Recipes)
    34. BSA Blocking solution (see Recipes)


  1. -80 °C freezer
  2. Vortexer
  3. Pipette
  4. Beaker
  5. CO2 incubator
  6. Hemacytometer
  7. Magnetic stirrer
  8. BSL2 cell culture cabinet
  9. 2D rocking platform shaker
  10. Zeiss LSM780 confocal microscope
  11. Chemical hood
  12. Tweezers
  13. 4 °C refrigerator
  14. Autoclavable glass bottle
  15. Autoclave


IMPORTANT NOTE: All experiments with live viruses (i.e., all pre-fixation steps) should be performed inside a biosafety level 2 (BSL2) or a BSL2+ tissue culture laboratory according to the country and institution regulations and required permits regarding Zika virus handling and storage.

  1. Virus stock production
    1. Virus preparation
      1. Resuspend with 200 μl ultraPure sterile water the desiccated virus stocks by pipetting up and down, at room temperature in a BSL2 cell culture cabinet. 
      2. Aliquot 50 μl of virus in sterile microtubes and store at -80 °C.
    2. Virus amplification
      Day -1: Preparation of cells for infection
      1. Prepare cells from 15 cm culture stock dishes of Vero E6 cells showing 80%-100% confluence. PBS, complete DMEM and trypsin-EDTA should be pre-warmed at 37 °C. It is worth mentioning that we never use Vero E6 cells “older” than 50 passages since this may affect cell permissiveness to ZIKV and yields of virus production. 
      2. Remove the culture medium.
      3. Wash the cells with 10 ml PBS.
      4. Remove PBS and add 7 ml trypsin-EDTA.
      5. Incubate for 2 min at 37 °C in the CO2 incubator.
      6. Remove trypsin-EDTA. 
      7. Thoroughly tap the cell culture dish in order to detach the cells. 
      8. Resuspend the cells in 10 ml complete DMEM (see Recipes section for composition). 
      9. Transfer the cells into a 15 ml sterile tube.
      10. Using a hemacytometer, determine the number of cells per ml. 
      11. Seed 2 x 106 Vero E6 cells in 8 ml of complete DMEM in 10 cm cell culture dishes and incubate overnight in the 37 °C incubator. 

      Day 0: Infection
      1. In a 15 ml sterile tube, dilute 50 μl of virus stock in 6 ml of complete DMEM.
      2. Remove by aspiration the medium of Vero E6 cells. Cells should show a confluence of approximately 70%-80%). 
      Note: Gently add the virus dilution on the cells and slowly shake the cell culture dish. Especially for the first pilot experiments, we recommend including a control dish of uninfected cells (mock infection) that will be cultured in parallel (see important note below).
      1. Incubate for 2 h at 37 °C. 
      2. After incubation, remove the virus inoculum. 
      3. Add 8 ml of pre-warmed complete DMEM. 
      4. Incubate for 3 days at 37 °C.
      Days 3, 4, 5, 6 and 7 post-infection: virus harvest
      1. At each day, harvest the supernatant with a sterile 20 ml syringe directly from the cell culture dish. Place an MCE 0.45 μm filter at the tip of the syringe.
      2. Filter the supernatant into a sterile 15 ml tube. 
      3. Add 8 ml of complete DMEM in the cell culture dish and place it back into the incubator. 
      4. Add 80 μl HEPES buffer 1 M, pH 7.2-7.5 (final concentration of 10 mM) in the filtered supernatant. This will prevent acidification of virus stocks following freeze/thawing and contribute maintaining optimal viral infectivity. 
      5. Aliquot virus supernatants into sterile 1.5 ml microtubes (1 ml per tube). 
      6. Store aliquots at -80 °C.
      7. Proceed with viral titration of each harvest using plaque assays. 
      IMPORTANT NOTE: During viral amplification, ZIKV-induced cytopathic effects which lead to cell death can be observed and increase over time. This is generally a good indication that virus infection and production was successful. As a reference for healthy cells, the mock infection control dish may be observed in parallel. 

  2. Viral titration using plaque assays
    Day -1: Preparation of cells for infection
    1. Cells should be prepared from 15 cm dishes of Vero E6 with 80%-100% confluence. 
    2. One confluent 15-cm dish of cells is generally enough to prepare five 24-well plates corresponding to 10 samples to titer. PBS, trypsin-EDTA and culture media should be pre-warmed at 37 °C. 
    1. Remove by aspiration the medium from the culture dishes.
    2. Wash the cells with 10 ml of PBS.
    3. Remove PBS and add 7 ml trypsin-EDTA.
    4. Incubate for 2 min in the incubator. 
    5. Remove the trypsin by aspiration. 
    6. Thoroughly tap the cell culture dish in order to detach the cells.
    7. Resuspend the cell in 10 ml complete DMEM. 
    8. Transfer the cells into a sterile 15 ml tube.
    9. Determine cell concentration with a hemacytometer.
    10. Dilute the cells with complete DMEM to a final concentration of 4 x 105 Vero E6 cells/ml.
    11. Seed 24-well plates with 500 μl diluted cells per well. One 24-well plate allows the titration of two virus samples. 
    12. Incubate overnight at 37 °C. 

    Day 0: Infection
    Note: The day of infection, the cells should show a confluence of 90%-100%.
    1. Prepare 6 microtubes per sample to titer and identify them (10-1 to 10-6). Add 450 μl complete DMEM in each microtube. 
    2. Dilute 10 times the sample in series: 
      1. Add 50 μl of virus sample in the first microtube (dilution 10-1). Vortex the tube for 5 s. 
      2. Change the tip to avoid any cross-contamination between samples. Pipet 50 μl of the 10-1 dilution and transfer into the second tube (dilution 10-2). Vortex the tube for 5 s. 
      3. Repeat these steps for the other dilutions (10-3 to 10-6).
    3. Remove medium from rows A and B of one 24-well plate (Figure 1).
    4. Pipet up-and-down twice the 10-6 dilution and add 200 μl with the same tip on the side of the last wells of rows A and B according to the plate layout shown in Figure 1. Thus, infection is performed in duplicates. 
    5. Proceed similarly with other sample dilutions (10-5 to 10-1 dilutions) according to the plate layout (Figure 1).
      IMPORTANT NOTE: It is critical to change used tips between each dilution to avoid cross-contamination between wells. Moreover, always start dispensing the samples into the plate starting with the most diluted samples. To avoid that cells dry, always wait to have processed one sample before aspirating the medium for another dilution series (e.g., rows C and D).

      Figure 1. Serial dilution plan and plate layout for plaque assays. Schematic representation of the virus stock 10-fold dilutions and of 24-well plates used for plaque assays. Two samples per plate can be titered. Each dilution is assessed in biological duplicates.

    6. Once a 24-well plate has been entirely processed, put it back to the incubator and proceed with the next plate and two new samples to titer.
    7. Incubate all plates with gentle agitation at 37 °C for at least one hour using a 2D rocking platform shaker. Incubation period can be extended up to several hours without impacting titers.
    8. After this incubation, take the first plate and aspirate the supernatant of the row A and B starting by the 10-6 dilution to avoid cross-contamination.
    9. Add on the side of wells, starting from the highest dilutions, 1 ml of MEM-CMC (see Recipes) with a 10 ml pipette. Discard the pipette. It is important to change the pipette between each sample series to avoid contamination of the stock bottle of MEM-CMC with virus. Especially, contamination with fast-growing viruses may result in future experiments in the appearance of very large plaques in the wells which will render impossible to count smaller expected plaques. 
    10. Repeat for rows C and D and then for all plates. 
    11. Incubate for 5 days at 37 °C.
      IMPORTANT NOTE: Do not move the plates during the 5-day incubation since this will result in the formation of “comet-shaped” plaques which might render them difficult to count.

    Day 5 post-infection: Fixation and staining
    1. Add 1 ml 10% formaldehyde per well.
    2. Incubate for at least 2 h at room temperature in the BSL2 laboratory. Incubation period can be extended up to several days. 
    3. Discard the liquid by inversion in a large beaker in a chemical hood. Formaldehyde-containing liquid wastes should be handled according to the chemical safety regulations of the research institution. 
    4. Wash plates by vigorously rinsing them with tap water to remove all the methylcellulose. Remove the excess of water by taping the plates on an absorbent paper. 
    5. Add 500 μl crystal violet-containing staining solution (see Recipes) on cell monolayers.
    6. Incubate for 15 min at room temperature. Incubation period can be extended up to several hours. 
    7. Remove crystal violet solution by inversion in a reusable plastic container. Collected crystal violet solution can be transferred back to the stock bottle and be reused for future staining. 
    8. Wash the plates extensively with tap water to remove the excess of staining solution. 
    9. Dry the plates on absorbent paper to remove excess of water. 
    10. Count the number of plaques in the appropriate dilution and determine infectious titers (see Figure 2 and Data analysis).

  3. Typical infection experiments for imaging
    Day -1: Preparation of cells for infection
    1. Using tweezers which have been cleaned with 70% ethanol, transfer sterile coverslips in each well of a 24-well culture dish.
    2. Seed Huh7 cells (or any cell line of interest to be tested): 20,000 cells in 500 µl complete DMEM. Vigorously shake the plate to homogenously disperse the cells throughout the well. Of note, we never use Huh7 cells “older” than 50 passages since we have noticed a significant change of morphology and growth properties beyond this passage.
    3. Incubate overnight at 37 °C with 5% CO2.

    Day 0: Viral infection
    1. For infection, appropriate multiplicity of infection (MOI) and virus dilution must be chosen according to stock infectious titers (see Procedure B and Data analysis). For immunofluorescence purposes, MOI of 1 is typically used. MOI of 1 means that on average, each target cell is inoculated with 1 infectious virus particle. 
    2. In this case (20,000 cells) with an MOI of 1, 20,000 viruses must be diluted in a final volume of 500 µl DMEM in each well. Prepare a master mix for all the wells.
    3. Remove culture medium and add gently on the side of each well 500 µl of the ZIKV/DMEM master mix.
    4. Incubate for at least 2 h at 37 °C with 5% CO2 and gentle agitation.
    5. Remove virus inoculum and add 1 ml complete DMEM per well.
    6. Incubate for 48 or 72 h at 37 °C with 5% CO2.

    Day 2 or 3: Fixation
    1. Remove media.
    2. Wash the cells twice with cold PBS.
    3. Add in each well 500 µl 4% paraformaldehyde/PBS solution.
    4. Incubate for 20 min at room temperature with gentle shaking.
    5. Wash once with 500 µl PBS and add 1 ml PBS. 
    6. Seal the plate with parafilm to avoid that cells dry because of PBS evaporation. Store fixed cells at 4 °C protected from light with an aluminum foil. The cells can be stored at 4 °C for a few months if no staining is immediately planned.

  4. Immunofluorescence staining for confocal microscopy
    1. Remove PBS.
    2. Add 500 µl PBS/Triton X-100 permeabilization solution (see Recipes) and incubate for 15 min at room temperature with gentle shaking.
    3. Wash the cells once with PBS.
    4. During permeabilization, freshly prepare a premix of blocking solution (see Recipes). Plan 300 µl of blocking solution per coverslip to be labeled.
    5. Add 300 µl complete blocking solution per well.
    6. Incubate for 1 h at room temperature with gentle shaking.
    7. Quickly wash the cells three times with 500 µl PBS.
    8. Prepare the primary antibodies
      1. Dilute rabbit anti-ZIKV NS4B and mouse anti-dsRNA in PBS/5% BSA/0.05% sodium azide (i.e., blocking solution without normal goat serum) with dilution of 1/200 and 1/400, respectively. Plan 30 µl antibody solution per coverslip.
      2. Alternatively, antibodies against dengue virus NS4B and NS3 may also be used to detect ZIKV since there is a cross-reaction between these two flaviviruses for the antibody epitopes. Using panflaviviral antibodies against E protein is also an option to be considered (see antibody reference and dilutions in the Materials and Reagents).
    9. To avoid coverslips to dry and maintain a sufficient level of humidity, prepare an airtight container with wet pieces of paper at the bottom.
    10. On a plastic support inside the box, place a piece of parafilm cleaned with 70% ethanol.
    11. For each coverslip to label, add 30 µl of primary antibody as drops.
    12. With tweezers, remove the excess of liquid on a piece of absorbent paper and put each coverslip on the antibody drop, with the cells facing parafilm.
    13. Incubate for at least 2 h at room temperature, protected from light.
    14. Take back coverslips and transfer them in a sterile 24-well plate which contains 500 µl PBS per well. Cells must face the top of the plate.
    15. Wash the coverslips three times for 5 min with 500 µl PBS.
    16. During the last wash, prepare the secondary fluorescently labeled antibodies at a dilution of 1:1,000 in PBS/5% BSA/0.05% sodium azide. Plan 300 µl per coverslip.
    17. Cover the coverslips with 300 µl of the secondary antibody solution. Alternatively, in order to spare reagents, coverslips may be incubated on an antibody drop exactly as the primary antibody incubation.
    18. Incubate for 1 h at room temperature protected from light on a 2D rocking shaker.
    19. Quickly wash once and then, three times for at least 10 min with gentle agitation with 500 µl PBS.
    20. Dilute 10,000 fold the DAPI stock into PBS. Plan 500 µl per coverslip. 
    21. Remove PBS from the plate and add 500 µl DAPI/PBS per well. 
    22. Incubate for 10 min with gentle agitation at room temperature protected from light.
    23. Quickly wash three times each with 500 µl PBS.
    24. Wash once with 500 µl sterile water.
    25. Clean a microscope glass slide with 70% ethanol. Add a 5 µl drop of Fluoromount G per coverslip. Depending on the microscope used for imaging, each slide can accommodate 3-4 coverslips. 
    26. Remove the excess of water on coverslip to-be-mounted by touching its side with a piece of absorbent paper. Place the coverslip on a Fluoromount G drop on the glass slide, with cells facing the drop.
    27. Keep slides protected from light at 4 °C overnight before imaging.
    28. Image the cells with a confocal or epifluorescence microscope in order to determine the % of infection, i.e., the % of NS4B- or dsRNA-positive cells.

Data analysis

  1. Infectious titer determination for virus stocks using plaque assays
    1. Count the number of plaques in the well where they can be discriminated. Select the well duplicate showing the highest number of individual plaques. See Figure 2 for a typical example. 
    2. Calculate the infectious viral titers according to the following formula:

    3. Values are multiplied by 5 because the virus inoculum is 200 µl and the titers are normalized to 1 ml of the corresponding ZIKV stock.
    The unit of the viral titers is plaque forming units (PFU) per ml of virus stock. One PFU corresponds to one viral particle which has initially infected one target cell on Day 0. During the 5-day incubation, newly produced viruses have only locally spread because of the semi-solid CMC-containing medium. At its late time points, virus infection ultimately leads to cell death in Vero E6 cells resulting in the formation of a plaque. Plaques are not stained by the crystal violet because dead cells have been washed off. It is important to note that in Vero E6 cells, plaque size is larger for ZIKV H/PF/2013 than for MR766 because of higher cytopathic effects (see Figure 2). If the plaques are so big that they overlap and are not distinguishable for counting, one may fix the cells one day earlier to reduce plaque size. Another alternative is to use 6- or 12-well plates to increase the cell culture area and to better separate the plaques, hence facilitating the counting.

    Figure 2. Typical plaque assay for ZIKV MR766 and H/PF/2013. Vero E6 cells were infected with 10-fold serial dilutions of ZIKV MR766 or H/PF/2013 stocks (Day 3 harvests). Five days post-infection, cells were fixed and stained with crystal violet. In this specific example, plaques can be counted in the 10-3 and 10-4 dilution well of ZIKV MR766 and H/PF/2013 (indicated in red), respectively. According to the formula described above, infectious titers are 1.3 x 105 PFU/ml for MR766 and 1.45 x 104 PFU/ml for H/PF/2013.

    Typically, starting from ~104-105 PFU/ml depending on the experiment to ~107 PFU/ml (Figure 3), infectious titers for both strains logarithmically increase during the five first days of amplification and then reach a plateau. We never collect viruses at 1 and 2 days post-infection because titers are too low to perform subsequent experiments. In our hands, infectious titers over 5 x 107 PFU/ml were never achieved, even if the amplification period is extended beyond 7 days. It is worth mentioning that plaque morphology, and the profiles of amplification kinetics may change if high passages of viruses as well as of cells are used for virus stock production. This should be carefully considered and addressed if relevant.

    Figure 3. Virus amplification kinetics of ZIKV strains MR 766 and H/PF/2013 in Vero E6 cells. Vero E6 cells were infected with ZIKV MR766 or H/PF/2013 strains at an MOI of 0.01. Virus supernatants were collected at 3, 4, 5, 6 and 7 days post-infection. Infectious virus titers were determined using plaque assays in Vero E6 cells.

  2. Imaging
    Observe slides at a confocal microscope and set up the detection parameters with the “uninfected” condition coverslip. Any signal observed in this condition is non-specific. For NS4B antibody, it may be diffuse while it generally appears for anti-dsRNA as small dots with a very weak signal throughout the cell. Although anti-dsRNA is not supposed to recognize RNA helixes smaller than 40 base pairs (which are presumably inexistent or rare in cells), it most likely detects cellular highly structured RNAs with low affinity, explaining the observed background in uninfected cells. If non-specific signals are too high, detergent concentration in permeabilization buffer and/or primary antibody dilution may be increased. The ZIKV-specific signal for dsRNA (corresponding to the ZIKV RNA replication intermediate), typically shows a perinuclear accumulation of small intense puncta which presumably correspond to replication complexes within ER-derived vesicle packets (Chatel-Chaix et al., 2016; Cortese et al., 2017) (Figure 4). It was recently shown that dsRNA is localized in the vicinity of the microtubule organization center (MTOC) surrounded by a "cage-like” structure constituted of rearranged cytoskeleton (Cortese et al., 2017). NS4B labeling for both ZIKV strains shows a diffuse distribution, which colocalizes with dsRNA signal (Figure 4), consistent with the fact that DENV NS4B was shown to be a component of vesicle packets as shown by immunogold labeling followed by electron microscopy (Welsch et al., 2009). In addition, ZIKV H/PF/2013 NS4B also accumulates at the periphery of dsRNA clusters within large and intense punctated structure (diameter of ~1-5 µm) which by analogy with DENV NS4B most likely correspond to convoluted membranes, another replication factory sub-structure (Chatel-Chaix et al., 2016).
      To estimate the infection rate, count at least a hundred cells in five different fields of the coverslip and count how many are infected, i.e., NS4B- and dsRNA-positive.

    A 100% infection rate is reproducibly achieved within 48 h with ZIKV MR766 in Huh7 cells at an MOI of 1. At 72 h post-infection, cells show obvious signs of cytopathic effect. In contrast, with the same infection conditions, ZIKV H/PF/2013 is generally detected in only 10% of the cells (in infected clusters of 10-20 cells) with no signs of cell death at 48 h post-infection. This infection rate can be improved by fixing the cells one day later. Moreover, we regularly achieve ~100% infection with this strain in Huh7 cells when we increase the MOI to 50. No obvious signs of cytopathic effects were noticed in Huh7 cells in these conditions.

    Figure 4. Imaging of Zika virus infection in Huh7 cells. Huh7 cells were infected with ZIKV MR766 or H/PF/2013 strains at an MOI of 1 or left uninfected. Forty-eight hours post-infection, cells were fixed, permeabilized and labeled with anti-dsRNA and anti-ZIKV NS4B antibodies. Nuclei were visualized with DAPI staining. Cells were observed with a Zeiss LSM780 confocal microscope. For ZIKV H/PF/2013, an infected cluster is shown. Scale bars: 20 μm.

  3. Relevant remarks
    It is important to note that the infection procedure may be adapted for other end-point readouts such as Western blotting, qPCR or RNA interference combined to any functional assay. For those purposes, the number of cells and volume may be up- or down-scaled according to dish or plate format. Cell density for microscopy experiments is kept rather low so that cells are well individualized for imaging. Hence, it is worth mentioning that more cells may be used as compared to microscopy experiments so that sufficient biological material is available. Typically, those ratios/formats may be used:
    1. 10 cm dish: 1,000,000 Huh7 cells. Infection in 5 ml
    2. 6-well plate: 200,000 Huh7 cells. Infection in 1 ml
    3. 12-well plate: 100,000 Huh7 cells. Infection in 500 µl
    4. 24-well plate: 50,000 Huh7 cells. Infection in 250-300 µl
    5. 96-well plate: 10,000 Huh7 cells. Infection in 50 µl
      When performing functional assays such as evaluating the impact of a drug or host gene expression modulation on ZIKV titers, a low MOI (typically between 0.01 and 0.1 for Huh7 cells) should be selected. Indeed, some phenotypes may be masked by the fact that titers are saturated over 106 PFU/ml if a high MOI is used. Using low MOI ensures that virus replication is in its logarithmic dynamic window when samples are collected.
      Finally, any cell line or primary cells may theoretically be permissive to ZIKV infection. To test this, we typically perform replication kinetics experiments in which we infect cells with ZIKV at an MOI of 1. Cell supernatants are collected and filtered 1, 2, 3 and 4 days post-infection similarly to ZIKV stock harvest. Putative supernatant-associated infectivity is then assessed using plaques assays. The % of infection may be determined using the protocol described in this manuscript. Of note, it should be always kept in mind that the infectious titers determined using plaque assays are relative to Vero E6 cells permissiveness to ZIKV. For many possible reasons that are intrinsic to a given tested cell line (e.g., virus receptor availability/expression, high induction of antiviral responses…) and/or virus strain, the infection rate may be low even with an MOI of 1. To circumvent this limitation, the MOI may be increased up to 10-50 and the infection procedure optimized (e.g., time of infection, presence of serum in the inoculum…) in future experiment to eventually reach values close to 100%.


  1. Complete DMEM
    500 ml DMEM
    50 ml FBS Performance
    5 ml penicillin/streptomycin
    5 ml non essential amino acids solution
    Store at 4 °C
  2. Plaquing medium (MEM-CMC)
    1. Put 7.5 g CMC in an autoclavable glass bottle which contains a magnetic bar
    2. Autoclave the bottle to sterilize the powder and stirrer (121 °C, 30 min, 20 bars; drying for 25 min)
      c. Add into the bottle 500 ml MEM within the sterile environment of a cell culture cabinet. Close the bottle without touching the bottlenecks with the hands. Mix by inversion. Keep at 4 °C the original MEM bottle for further use (see below)
      d. Incubate the MEM/CMC bottle at 4 °C above a magnetic stirring plate until complete solubilization of CMC. This generally takes between two and three days.
      e. Transfer the reconstituted medium from the glass bottle into the original MEM plastic bottle inside the sterile environment of a cell culture cabinet. This will avoid introducing into the BSL2 area any glass-made material
      f. Store at 4 °C. The final concentration of CMC is 1.5%
  3. Formaldehyde fixative
    270 ml distilled water
    100 ml 37% formaldehyde (10% final concentration)
    Store at room temperature
  4. Paraformaldehyde fixative
    1 L 1x PBS
    40 g paraformaldehyde (4% final concentration)
    Facilitate dissolving the paraformaldehyde by heating (~60-70 °C) the preparation inside a chemical hood for ~30 min
    Prepare 12 ml aliquots in 15 ml tubes and store at -20 °C
  5. Crystal violet staining solution
    5 g crystal violet
    Dissolve in 52.6 ml of 95% ethanol
    Add 445 ml of double-distilled water
    Final concentrations of crystal violet and ethanol are 1% and 10%, respectively
    Store at room temperature
  6. Triton X-100 permeabilization solution
    500 ml PBS
    1 ml Triton X-100 (final concentration 0.2%)
    Store at room temperature
  7. BSA Blocking solution
    1. Dissolve 10 g BSA in 200 ml PBS (5% [w/v] final concentration)
    2. Add 1 ml 10% (w/v) sodium azide solution (NaN3; final concentration: 0.05%)
    3. Store at 4 °C
    1. OPTIONAL: The solution can be sterilized by filtration in order to avoid contamination and to maintain the quality of the solution over several months.
    2. The day of immunofluorescence staining, supplement the solution with normal goat serum to a final concentration of 10% (v/v).


LCC is receiving a research scholar (Junior 2) salary support from Fonds de la Recherche du Québec-Santé (FRQS). LCC’s research is supported by grants from Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2016-05584), the Canadian Institutes of Health Research (CIHR; PJT153020; ICS154142), Fonds de la Recherche du Québec-Nature et Technologies (FRQNT; 2018-NC-205593), Armand-Frappier Foundation and Institut National de la Recherche Scientifique. This protocol is mostly adapted from the experimental procedures used in our previous work (Chatel-Chaix et al., 2016) in which we have optimized the production and detection of ZIKV. We thank the European Virus Archive goes Global (EVAg) and Dr. Xavier de Lamballerie (Emergence des Pathologies Virales, Aix-Marseille University) for providing ZIKV MR766 and H/PF/2013 original stocks. We are grateful to Drs Patrick Labonté (Institut National de la Recherche Scientifique), Tom Hobman (University of Alberta) and Anil Kumar (University of Alberta) for generously providing Huh7 and Vero E6 cells.

Competing interests

The authors declare that they do not have any conflicts of interests or competing interests.


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  2. Cortese, M., Goellner, S., Acosta, E. G., Neufeldt, C. J., Oleksiuk, O., Lampe, M., Haselmann, U., Funaya, C., Schieber, N., Ronchi, P., Schorb, M., Pruunsild, P., Schwab, Y., Chatel-Chaix, L., Ruggieri, A. and Bartenschlager, R. (2017). Ultrastructural characterization of Zika virus replication factories. Cell Rep 18(9): 2113-2123.
  3. Cugola, F. R., Fernandes, I. R., Russo, F. B., Freitas, B. C., Dias, J. L., Guimaraes, K. P., Benazzato, C., Almeida, N., Pignatari, G. C., Romero, S., Polonio, C. M., Cunha, I., Freitas, C. L., Brandao, W. N., Rossato, C., Andrade, D. G., Faria Dde, P., Garcez, A. T., Buchpigel, C. A., Braconi, C. T., Mendes, E., Sall, A. A., Zanotto, P. M., Peron, J. P., Muotri, A. R. and Beltrao-Braga, P. C. (2016). The Brazilian Zika virus strain causes birth defects in experimental models. Nature 534(7606): 267-271.
  4. Grubaugh, N. D., Faria, N. R., Andersen, K. G. and Pybus, O. G. (2018). Genomic insights into Zika virus emergence and spread. Cell 172(6): 1160-1162.
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  8. Morrison, T. E. and Diamond, M. S. (2017). Animal models of Zika virus infection, pathogenesis, and immunity. J Virol 91(8): e00009-17.
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【背景】Zika病毒(ZIKV)是黄病毒科病毒科中一种由蚊子传播的黄病毒,于1947年在乌干达的Ziika森林中首次从哨兵恒河猴中分离出来,并且非常接近与登革病毒有关(DENV)(世界卫生组织,2018年)。从那时起,它在过去十年中因在一些太平洋岛屿(2007年和2013年)和美洲(2015年)爆发而闻名。有症状的患者(25%-20%的病例)通常可能表现出相当温和的临床表现,如发烧,皮疹,结膜炎,肌肉/关节疼痛和/或头痛。然而,自从ZIKV再次出现以来,在感染者中发现了严重的神经系统症状,包括(但不限于)成人的吉兰 - 巴利综合征和先天性传播的新生小头畸形(Lazear and Diamond,2016; Grubaugh et al。,2018)。不幸的是,目前没有针对寨卡病毒的治疗或疫苗,部分原因是我们对其生物学认识不足。

在系统发育方面,有2种不同的寨卡病毒谱系(Haddow et al。,2012)。所谓的“历史”非洲血统,包括原型菌株MR766和经过数十年突变后衍生自非洲血统的“当代”亚洲血统。例如,亚洲菌株H / PF / 2013是造成2013年法属波利尼西亚爆发的原因。同样,负责巴西爆发的病毒株也在系统发育上来自亚洲血统。事实上,在小鼠和蚊子感染模型中的实验支持这样的观点,即亚洲株已经获得了几个关键突变,这些突变导致巴西感染新生儿的小头畸形(Yuan et al。,2017)和增强的病毒感染性在节肢动物传播载体埃及伊蚊(Liu et al。,2017)。

进入宿主细胞后,病毒基因组即正单链RNA在内质网(ER)中翻译成单个多蛋白,随后通过宿主和病毒切割成3种结构蛋白和7种非结构蛋白。蛋白酶。复制后,将基因组衣壳化为新合成的病毒粒子,随后从细胞中释放出来(Neufeldt et al。,2018)。与所有其他测试的黄病毒一样,ZIKV重塑感染细胞的内膜,以产生有利于ZIKV生命周期的细胞质环境(Cortese 等,,2017)。这些膜质区室,称为病毒复制工厂,都是ER衍生的,可以细分为三类超微结构(Cortese et al。,2017):1。囊泡包,这是ER内陷被认为病毒RNA复制的位点,2。病毒袋,其中组装的未成熟病毒在规则阵列中累积;和3.复杂的膜,其作用仍然不清楚,尽管为DENV提出它们调节宿主过程以利于复制(Chatel) -Chaix et al。,2016)。

在2016年之前,对ZIKV的一般生物学了解甚少,我们的大部分知识依赖于与DENV等与该病原体密切相关的其他黄病毒的基本发现的直接转换。然而,与 Flavivirus 属的其他成员相比,仍然不清楚是什么使ZIKV在神经病理发生方面如此独特。因此,自从ZIKV重新出现和最近的爆发以来,世界各地的科学界已经开始破译围绕这种病毒的谜团,并开发分子克隆以及细胞培养和动物模型等工具(Schwarz et al 。,2016; Shan et al。,2016; Xie et al。,2016; Morrison and Diamond,2017; Mutso et al。< / em>,2017; Munster et al。,2018)。了解如何培养ZIKV并测量其感染性是构建关于该病毒的任何描述性或功能性研究的关键方法,包括 in cellulo 和 in vivo 。此外,鉴于亚洲和非洲谱系在感染小鼠胎儿的症状严重程度方面可能不同(Cugola 等,2016),有时比较这两种谱系的模型菌株。在这个生物方案中,我们描述了从亚洲和非洲谱系生产ZIKV原种的基本细胞培养方法,并量化它们在感染性病毒颗粒中的浓度(更通常称为“滴度”)以及在感染细胞内成像这种病原体。使用基于免疫荧光的显微镜。

关键字:寨卡病毒, NS4B, 双链RNA, 病毒滴度测定, 免疫荧光显微术, 蚀斑形成测定


  1. 材料
    1. 1.5 ml microtubes(Ultident Scientific,目录号:87-B150-C)
    2. 10厘米细胞培养皿(Falcon,Fisher Scientific,目录号:08-772-E)&nbsp;
    3. 15厘米细胞培养皿(Falcon,Fisher Scientific,目录号:08-772-6)
    4. 24孔聚苯乙烯培养板(Corning Life Sciences,Fisher Scientific,目录号:08-772-1)&nbsp;
    5. MCE0.45μm过滤器,直径30 mm(Ultident Scientific,目录号:229753)&nbsp;
    6. 20毫升无菌注射器(Ultident Scientific,目录号:BD-302830)&nbsp;
    7. 15毫升无菌锥形管(Falcon,Fisher Scientific,目录号:352096)&nbsp;
    8. 1号盖玻片(直径:12 mm,厚度:0.13至0.17 mm),通过高压灭菌消毒(Fisher Scientific,目录号:12-545-80)
    9. 显微镜载玻片,磨砂透明玻璃,26 mm x 76 mm,厚1-1.2 mm(Ultident Scientific,目录号:170-8105-W)
    10. 吸水纸
    11. 封口膜
    12. 铝箔

  2. 病毒
    1. ZIKV MR766(非洲谱系)(EVAg,目录号:001v-EVA143)(根据原料和转让协议。通过历史:P5。在-80°C下储存原始干燥的原料)
    2. ZIKV H / PF / 2013(亚洲谱系)(EVAg,目录号:001v-EVA1545)(根据材料和转让协议。通过历史:P6。将原始干燥的原料储存在-80°C)

  3. 细胞系
    1. Vero E6猴子上皮细胞(来自阿尔伯塔大学Tom Hobman和Anil Kumar博士的礼物)(ATCC,目录号:CRL-1586)
    2. Huh7肝癌细胞(来自PatrickLabonté博士,INRS的礼物)(Creative Bioarray,目录号:CSC-C9441L)

  4. 试剂
    1. UltraPure蒸馏水(Life Technologies,目录号:10977-015)&nbsp;
    2. Dulbecco的改良Eagle培养基(DMEM)(Life Technologies,目录号:111965-092)&nbsp;
    3. 胎牛血清(FBS)表现(Wisent,目录号:098150)&nbsp;
    4. 青霉素 - 链霉素(Life Technologies,目录号:15140-122)&nbsp;
    5. MEM非必需氨基酸溶液(100x)(Life Technologies,目录号:11140-050)
    6. 磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190-144)
    7. 1 M HEPES缓冲液,pH范围:7.2-7.5(Life Technologies,目录号:15630-080)
    8. 0.25%胰蛋白酶-EDTA(Life Technologies,目录号:25200-072)&nbsp;
    9. 含L-谷氨酰胺的最低必需培养基(MEM)(Life Technologies,目录号:11095-080)&nbsp;
    10. 羧甲基纤维素(CMC)钠盐,中等粘度(Sigma-Aldrich,目录号:21902-100G)
    11. 37%甲醛(Fisher Scientific,目录号:BP531-500)&nbsp;
    12. 结晶紫(Fisher Scientific,目录号:C581-100)&nbsp;
    13. 95%乙醇(商品醇,目录号:1011C)
    14. Triton X-100(Mallinktrot,目录号:3555)
    15. 4%多聚甲醛(Sigma-Aldrich,目录号:P6148-1KG)
    16. 普通山羊血清(Thermo-Fisher,目录号:01-6201)
    17. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A-9647)
    18. 叠氮化钠(Fisher Scientific,目录号:BP922I-500)
    19. 兔多克隆抗ZIKV NS4B(GeneTex,目录号:GTX133311;稀释度1:200)
    20. 小鼠单克隆抗dsRNA,克隆J2(Scicons,目录号:10010200;稀释度1:400)
    21. 兔多克隆抗DENV NS4B(GeneTex,目录号:GTX124250;稀释度1:1,000)
    22. 小鼠单克隆抗DENV NS3,克隆GT2811(GeneTex,目录号:GTX629477;稀释度1:100)
    23. 小鼠单克隆panflaviviral anti-E,克隆4G2(Sigma-Aldrich,目录号:MAB10216,稀释度1:200)
    24. 山羊抗兔AlexaFluor 488(Life Technologies,目录号:A11034)
    25. 山羊抗小鼠AlexaFluor 568(Life Technologies,目录号:A11004)
    26. 4',6-二脒基-2-苯基吲哚(DAPI)(Life Technologies,目录号:D1306)
    27. Fluoromount G(Southern Biotech,目录号:0100-01)
    28. 完整的DMEM(见食谱)
    29. Plaquing medium(MEM-CMC)(见食谱)
    30. 甲醛固定剂(见食谱)
    31. 多聚甲醛固定剂(见食谱)
    32. 结晶紫染色液(见食谱)
    33. Triton X-100透化液(见食谱)
    34. BSA阻断解决方案(见食谱)


  1. -80°C冰箱
  2. 涡流混合器
  3. 吸管
  4. 烧杯
  5. CO 2 培养箱
  6. 血球
  7. 电磁搅拌机
  8. BSL2细胞培养柜
  9. 2D摇摆平台振动筛
  10. 蔡司LSM780共聚焦显微镜
  11. 化学罩
  12. 镊子
  13. 4°C冰箱
  14. 可高压灭菌的玻璃瓶
  15. 高压灭菌器


重要注意事项:所有活病毒实验(即所有预固定步骤)均应在生物安全2级(BSL2)或BSL2 +组织培养实验室内根据国家和机构法规以及寨卡病毒所需许可进行。处理和储存。

  1. 病毒库存生产
    1. 病毒准备
      1. 通过在室温下在BSL2细胞培养箱中上下移液,用200μlUltraPure无菌水重悬干燥的病毒原种。&nbsp;
      2. 在无菌微管中等分50μl病毒并储存在-80℃。
    2. 病毒扩增
      1. 从15cm培养的Vero E6细胞培养皿中制备细胞,显示80%-100%汇合。 PBS,完全DMEM和胰蛋白酶-EDTA应在37°C预热。值得一提的是,我们从未使用Vero E6细胞“老”超过50代,因为这可能会影响ZIKV细胞的放射性和病毒产量。&nbsp;
      2. 取出培养基。
      3. 用10ml PBS洗涤细胞。
      4. 取出PBS,加入7 ml胰蛋白酶-EDTA。
      5. 在37℃下在CO 2 培养箱中孵育2分钟。
      6. 删除胰蛋白酶-EDTA。&nbsp;
      7. 彻底轻拍细胞培养皿,以便分离细胞。&nbsp;
      8. 将细胞重悬于10 ml完全DMEM中(参见食谱部分的组成)。&nbsp;
      9. 将细胞转移到15ml无菌管中。
      10. 使用血细胞计数器,确定每毫升细胞数。&nbsp;
      11. 在10cm细胞培养皿中的8ml完全DMEM中培养2×10 6个 Vero E6细胞,并在37℃培养箱中孵育过夜。&nbsp;

      1. 在15ml无菌试管中,在6ml完全DMEM中稀释50μl病毒原液。
      2. 通过吸入Vero E6细胞培养基除去。细胞应显示约70%-80%的汇合。&nbsp;
      1. 在37°C孵育2小时。&nbsp;
      2. 孵育后,去除病毒接种物。&nbsp;
      3. 加入8毫升预热的完全DMEM。&nbsp;
      4. 在37°C孵育3天。
      1. 在每天,直接从细胞培养皿中用无菌20ml注射器收集上清液。在注射器尖端放置MCE0.45μm过滤器。
      2. 将上清液过滤到15毫升无菌管中。&nbsp;
      3. 在细胞培养皿中加入8 ml完全DMEM,然后将其放回培养箱中。&nbsp;
      4. 在过滤的上清液中加入80μlHEPES缓冲液1M,pH 7.2-7.5(终浓度10mM)。这将防止冻/融后病毒原种的酸化,并有助于维持最佳的病毒感染性。&nbsp;
      5. 将病毒上清液分装到无菌的1.5ml微管中(每管1ml)。&nbsp;
      6. 将等分试样储存在-80°C。
      7. 使用噬斑测定法对每个收获物进行病毒滴定。&nbsp;

  2. 使用噬斑测定进行病毒滴定
    1. 细胞应从15厘米Vero E6培养皿中制备,80%-100%汇合。&nbsp;
    2. 一个汇合的15-cm细胞培养皿通常足以制备5个24孔板,对应于10个样品滴度。 PBS,胰蛋白酶-EDTA和培养基应在37°C预热
    1. 通过从培养皿中吸出培养基除去。
    2. 用10ml PBS洗涤细胞。
    3. 取出PBS,加入7 ml胰蛋白酶-EDTA。
    4. 在培养箱中孵育2分钟。&nbsp;
    5. 通过抽吸去除胰蛋白酶。&nbsp;
    6. 彻底轻拍细胞培养皿以分离细胞。
    7. 将细胞重悬于10 ml完全DMEM中。&nbsp;
    8. 将细胞转移到无菌的15ml管中。
    9. 用血细胞计数器测定细胞浓度。
    10. 用完全DMEM稀释细胞至终浓度为4×10 5个/ ml Vero E6细胞/ ml。
    11. 种子24孔板,每孔稀释500μl细胞。一个24孔板允许滴定两个病毒样本。&nbsp;
    12. 在37°C孵育过夜。&nbsp;

    1. 每个样品准备6个微管以滴定并鉴定它们(10 -1 至10 -6 )。在每个微管中加入450μl完整的DMEM。&nbsp;
    2. 将系列样品稀释10倍:&nbsp;
      1. 在第一个微管中加入50μl病毒样品(稀释10 -1 )。将管涡旋5秒钟。&nbsp;
      2. 更换尖端以避免样品之间的任何交叉污染。吸取50μl10 -1 稀释液并转移到第二管中(稀释10 -2 )。将管涡旋5秒钟。&nbsp;
      3. 对其他稀释液重复这些步骤(10 -3 至10 -6 )。
    3. 从一个24孔板的A行和B行中取出培养基(图1)。
    4. 根据图1中所示的板布局,在10 -6 稀释液中上下移液两次,并在A行和B行的最后一侧的孔侧添加200μl相同的尖端。 ,感染是重复进行的。&nbsp;
    5. 根据平板布局(图1),与其他样品稀释液(10 -5 至10 -1 稀释液)类似地进行。


    6. 一旦24孔板完全处理完毕,将其放回培养箱中,继续进行下一个培养板和两个新样品滴定。
    7. 使用2D摇摆平台振荡器在37℃温和搅拌至少1小时孵育所有平板。孵化期可延长至数小时而不影响滴度。
    8. 在该温育后,取第一块板并从10 -6 稀释液开始吸出A和B行的上清液以避免交叉污染。
    9. 加入孔的一侧,从最高稀释度开始,用10ml移液管加入1ml MEM-CMC(见食谱)。丢弃移液器。重要的是更换每个样品系列之间的移液管,以避免MEM-CMC储备瓶被病毒污染。特别是,快速生长的病毒污染可能会导致未来在孔中出现非常大的斑块的实验,这将无法计算较小的预期斑块。&nbsp;
    10. 重复行C和D,然后对所有板重复。&nbsp;
    11. 在37°C孵育5天。

    1. 每孔加入1ml 10%甲醛。
    2. 在BSL2实验室中在室温下孵育至少2小时。孵化期可延长至数天。&nbsp;
    3. 通过倒置在化学罩中的大烧杯中丢弃液体。含甲醛的液体废物应根据研究机构的化学品安全规定进行处理。&nbsp;
    4. 通过用自来水剧烈冲洗来清洗板以除去所有甲基纤维素。通过在吸水纸上粘贴板来除去多余的水。&nbsp;
    5. 在细胞单层上加入500μl含结晶紫的染色溶液(见食谱)。
    6. 在室温下孵育15分钟。孵化期可延长至数小时。&nbsp;
    7. 在可重复使用的塑料容器中通过倒置除去结晶紫溶液。收集的结晶紫溶液可以转移回原料瓶中,然后重复使用以备将来染色。&nbsp;
    8. 用自来水彻底清洗平板,去除多余的染色液。&nbsp;
    9. 用吸水纸擦干印版,去除多余的水分。&nbsp;
    10. 计算适当稀释度的斑块数量并确定感染性滴度(参见图2和数据分析)。

  3. 典型的成像感染实验
    1. 使用已用70%乙醇清洁的镊子,在24孔培养皿的每个孔中转移无菌盖玻片。
    2. 种子Huh7细胞(或任何待测试的细胞系):500μl完全DMEM中的20,000个细胞。剧烈摇动平板以使细胞均匀地分散在整个孔中。值得注意的是,我们从未使用过比50代更“老”的Huh7细胞,因为我们注意到超过这段经历的形态和生长特性的显着变化。
    3. 在37℃下用5%CO 2 孵育过夜。

    1. 对于感染,必须根据库存感染滴度选择适当的感染复数(MOI)和病毒稀释度(参见程序B和数据分析)。对于免疫荧光目的,通常使用1的MOI。 MOI为1意味着平均每个靶细胞接种1个感染性病毒颗粒。&nbsp;
    2. 在这种情况下(20,000个细胞),MOI为1,必须在每个孔中以500μlDMEM的最终体积稀释20,000个病毒。为所有井准备主混合物。
    3. 取出培养基,轻轻加入每孔500μlZIKV/ DMEM预混液。
    4. 在37℃下用5%CO 2 孵育至少2小时并轻轻搅拌。
    5. 除去病毒接种物,每孔加入1 ml完全DMEM。
    6. 在37℃下用5%CO 2 孵育48或72小时。

    1. 删除媒体。
    2. 用冷PBS洗涤细胞两次。
    3. 在每个孔中加入500μl4%多聚甲醛/ PBS溶液。
    4. 在室温下孵育20分钟,轻轻摇动。
    5. 用500μlPBS洗一次,加入1 ml PBS。&nbsp;
    6. 用封口膜密封平板以避免细胞因PBS蒸发而干燥。将固定电池存放在4°C,用铝箔避光。如果没有立即染色,细胞可以在4°C下储存几个月。

  4. 共聚焦显微镜的免疫荧光染色
    1. 删除PBS。
    2. 加入500μlPBS/ Triton X-100透化溶液(参见配方),在室温下温和振荡孵育15分钟。
    3. 用PBS洗涤细胞一次。
    4. 在透化过程中,新鲜制备阻塞溶液的预混物(参见食谱)。每个要标记的盖玻片计划300μl封闭溶液。
    5. 每孔加入300μl完全封闭液。
    6. 在室温下孵育1小时,轻轻摇动。
    7. 用500μlPBS快速洗涤细胞三次。
    8. 准备一抗抗体
      1. 用PBS / 5%BSA / 0.05%叠氮化钠(即,无正常山羊血清的封闭液)稀释兔抗ZIKV NS4B和小鼠抗dsRNA,稀释度为1/200和1/400,分别。每个盖玻片计划30μl抗体溶液。
      2. 或者,抗登革病毒NS4B和NS3的抗体也可用于检测ZIKV,因为这两种黄病毒之间存在抗体表位的交叉反应。使用抗E蛋白的泛黄病毒抗体也是一种考虑因素(参见材料和试剂中的抗体参考和稀释)。
    9. 为避免盖玻片干燥并保持足够的湿度,请在底部准备一个带有湿纸的密封容器。
    10. 在盒子内的塑料支架上,放置一块用70%乙醇清洗过的封口膜。
    11. 对于要标记的每个盖玻片,添加30μl一抗作为滴剂。
    12. 用镊子取出一块吸水纸上的多余液体,将每个盖玻片放在抗体滴上,细胞面对封口膜。
    13. 在室温下孵育至少2小时,避光。
    14. 取回盖玻片并将其转移到无菌24孔板中,每孔含有500μlPBS。细胞必须面向平板顶部。
    15. 用500μlPBS洗涤盖玻片三次,每次5分钟。
    16. 在最后一次洗涤期间,在PBS / 5%BSA / 0.05%叠氮化钠中以1:1,000的稀释度制备第二荧光标记的抗体。每个盖玻片计划300μl。
    17. 用300μl二抗溶液覆盖盖玻片。或者,为了备用试剂,盖玻片可以在抗体滴上温育,与第一抗体孵育完全一样。
    18. 在室温下孵育1小时,在2D摇摆振动器上避光。
    19. 快速洗涤一次然后,三次至少10分钟,同时用500μlPBS轻轻搅拌。
    20. 将DAPI原液稀释10,000倍至PBS中。每个盖玻片计划500μl。&nbsp;
    21. 从培养板中取出PBS,每孔加入500μlDAPI/ PBS。&nbsp;
    22. 孵育10分钟,在室温下轻轻搅拌,避光。
    23. 用500μlPBS快速洗涤三次。
    24. 用500μl无菌水洗一次。
    25. 用70%乙醇清洁显微镜载玻片。每个盖玻片加入5μlFloromountG滴。根据用于成像的显微镜,每个载玻片可容纳3-4个盖玻片。&nbsp;
    26. 通过用一块吸水纸接触其侧面,去除盖玻片上的多余水分。将盖玻片放在载玻片上的Fluoromount G滴,细胞面向滴。
    27. 在成像前将载玻片保持在4°C保持过夜。
    28. 用共聚焦或落射荧光显微镜对细胞成像,以确定感染的百分比,即,NS4B-或dsRNA-阳性细胞的%。


  1. 使用噬斑测定法测定病毒原种的感染滴度
    1. 计算可以区分的孔中斑块的数量。选择显示最多个体斑块的孔复制品。有关典型示例,请参见图2.&nbsp;
    2. 根据以下公式计算感染性病毒滴度:

    3. 将值乘以5,因为病毒接种物为200μl,滴度标准化为1ml相应的ZIKV原液。
    病毒滴度的单位是每ml病毒原液的噬斑形成单位(PFU)。一个PFU对应于在第0天最初感染一个靶细胞的一个病毒颗粒。在5天孵育期间,由于含有半固体CMC的培养基,新产生的病毒仅局部扩散。在其晚期时间点,病毒感染最终导致Vero E6细胞中的细胞死亡,导致斑块的形成。斑块不会被结晶紫染色,因为已经洗掉了死细胞。值得注意的是,在Vero E6细胞中,ZIKV H / PF / 2013的斑块大小比MR766大,因为细胞病变效应更高(见图2)。如果斑块是如此之大以至于它们重叠并且无法区分计数,可以提前一天固定细胞以减少斑块大小。另一种方法是使用6孔或12孔板来增加细胞培养面积并更好地分离斑块,从而便于计数。

    图2.ZIKV MR766和H / PF / 2013的典型噬斑测定。 Vero E6细胞用10倍连续稀释的ZIKV MR766或H / PF / 2013原种(第3天收获)感染。感染后5天,固定细胞并用结晶紫染色。在这个具体的例子中,斑块可以分别在ZIKV MR766和H / PF / 2013的10 -3 和10 -4 稀释孔中计数(以红色表示) 。根据上述公式,MR766的感染滴度为1.3×10 5 sup / 5 PFU / ml,H / PF / 2013为1.45×10 4 PFU / ml。 />
    通常,从~10 4 -10 5 PFU / ml开始,取决于实验至~10 7 PFU / ml(图3),两种菌株的感染性滴度在扩增的第一天中以对数方式增加,然后达到平台期。我们从未在感染后1天和2天收集病毒,因为滴度太低而无法进行后续实验。在我们的手中,即使扩增期延长超过7天,也从未达到超过5 x 10 7 PFU / ml的感染性滴度。值得一提的是,如果病毒以及细胞的高通道用于病毒原种生产,则斑块形态和扩增动力学的概况可能会改变。如果相关,应仔细考虑并解决这个问题。

    图3.Vero E6细胞中ZIKV毒株MR 766和H / PF / 2013的病毒扩增动力学。 VIK E6细胞用ZIKV MR766或H / PF / 2013株感染,MOI为0.01。在感染后3,4,5,6和7天收集病毒上清液。使用Vero E6细胞中的噬斑测定法测定感染性病毒滴度。

  2. 成像
    在共聚焦显微镜下观察载玻片,并使用“未感染”条件盖玻片设置检测参数。在这种情况下观察到的任何信号都是非特异性的。对于NS4B抗体,它可能是弥漫性的,而它通常表现为抗dsRNA,因为整个细胞中信号非常弱的小点。尽管抗dsRNA不应该识别小于40个碱基对的RNA螺旋(可能在细胞中不存在或罕见),但它很可能检测到具有低亲和力的细胞高度结构化RNA,解释了未感染细胞中观察到的背景。如果非特异性信号太高,则可以增加透化缓冲液和/或一抗稀释液中的洗涤剂浓度。 dsRNA的ZIKV特异性信号(对应于ZIKV RNA复制中间体)通常显示小的强烈斑点的核周积累,其可能对应于ER衍生的囊泡包内的复制复合物(Chatel-Chaix 等。,2016; Cortese et al。,2017)(图4)。最近显示,dsRNA定位于微管组织中心(MTOC)附近,周围是由重排细胞骨架构成的“笼状”结构(Cortese et al。,2017).NS4B标记对于两种ZIKV菌株均显示弥散分布,其与dsRNA信号共定位(图4),与DENV NS4B显示为囊泡包的组分的事实一致,如免疫金标记随后电子显微镜所示(Welsch et此外,ZIKV H / PF / 2013 NS4B也在dsRNA簇的外围积聚,在大而强烈的点状结构(直径约1-5μm)中,与DENV NS4B类似,大多数可能对应于复杂的膜,另一个复制工厂子结构(Chatel-Chaix et al。,2016)。

    使用ZIKV MR766在Huh7细胞中以MOI为1在48小时内可重复地实现100%感染率。在感染后72小时,细胞显示出明显的细胞病变效应迹象。相反,在相同的感染条件下,ZIKV H / PF / 2013通常仅在10%的细胞中检测到(在10-20个细胞的感染簇中),在感染后48小时没有细胞死亡的迹象。通过一天后固定细胞可以改善这种感染率。此外,当我们将MOI增加到50时,我们经常在Huh7细胞中达到~100%感染该菌株。在这些条件下,在Huh7细胞中没有观察到明显的细胞病变效应的迹象。

    图4.在Huh7细胞中的寨卡病毒感染的成像。用MOI为1的ZIKV MR766或H / PF / 2013株感染Huh7细胞或未感染。感染后48小时,将细胞固定,透化并用抗dsRNA和抗ZIKV NS4B抗体标记。用DAPI染色观察细胞核。用Zeiss LSM780共聚焦显微镜观察细胞。对于ZIKV H / PF / 2013,将显示受感染的群集。比例尺:20μm。

  3. 相关评论
    1. 10厘米培养皿:1,000,000 Huh7细胞。感染5毫升
    2. 6孔板:200,000 Huh7细胞。感染在1毫升
    3. 12孔板:100,000 Huh7细胞。感染500μl
    4. 24孔板:50,000 Huh7细胞。感染250-300μl
    5. 96孔板:10,000 Huh7细胞。感染50μl
    &NBSP;当进行功能测定,例如评估药物或宿主基因表达调节对ZIKV滴度的影响时,应选择低MOI(对于Huh7细胞通常在0.01和0.1之间)。实际上,如果使用高MOI,滴度饱和超过10 6 PFU / ml的事实可能掩盖了一些表型。使用低MOI可确保在收集样本时病毒复制处于其对数动态窗口中。
    &NBSP;最后,理论上任何细胞系或原代细胞都可以允许ZIKV感染。为了测试这一点,我们通常进行复制动力学实验,其中我们用MOI为1的ZIKV感染细胞。收集细胞上清液并在感染后1,2,3和4天过滤,类似于ZIKV原种收获。然后使用噬斑测定评估推定的上清液相关感染性。可以使用本手册中描述的方案确定感染百分比。值得注意的是,应始终牢记使用噬斑测定法测定的感染性滴度与VIK E6细胞对ZIKV的放射性相关。由于给定的测试细胞系(例如,病毒受体可用性/表达,抗病毒应答的高诱导...)和/或病毒株所固有的许多可能原因,感染率可能低,即使用MOI为1.为了规避这一限制,MOI可能会增加到10-50,并且感染程序在未来的实验中得到优化(例如,感染时间,接种物中血清的存在......)最终达到接近100%的价值。


  1. 完成DMEM
  2. Plaquing medium(MEM-CMC)
    1. 将7.5 g CMC放入装有磁棒的可高压灭菌的玻璃瓶中
    2. 高压灭菌瓶子对粉末和搅拌器进行灭菌(121°C,30分钟,20巴;干燥25分钟)
      C。在细胞培养箱的无菌环境中加入500ml MEM瓶中。用手接触瓶子而不接触瓶颈。通过倒置混合。将原始MEM瓶保持在4°C以备进一步使用(见下文)
      d。将MEM / CMC瓶在4℃下在磁力搅拌板上孵育直至CMC完全溶解。这通常需要两到三天。
      即将重构的培养基从玻璃瓶转移到细胞培养箱的无菌环境内的原始MEM塑料瓶中。这将避免将任何玻璃制材料引入BSL2区域 F。储存在4°C。 CMC的最终浓度为1.5%
  3. 甲醛固定剂
  4. 多聚甲醛固定剂
    1 L 1x PBS
    在15 ml管中制备12 ml等分试样并储存在-20°C
  5. 结晶紫染色液
    溶于52.6毫升95%乙醇中 加入445毫升双蒸水
    结晶紫和乙醇的最终浓度分别为1%和10% 在室温下储存
  6. Triton X-100透化液
    1毫升Triton X-100(终浓度0.2%)
  7. BSA封锁解决方案
    1. 将10 g BSA溶于200 ml PBS(5%[w / v]终浓度)
    2. 加入1 ml 10%(w / v)叠氮化钠溶液(NaN 3 ;终浓度:0.05%)
    3. 储存在4°C
    1. 可选:溶液可以通过过滤灭菌,以避免污染,并在几个月内保持溶液的质量。
    2. 免疫荧光染色当天,用正常山羊血清补充溶液至终浓度为10%(v / v)。


LCC正在接受魁北克省圣保罗基金会(FRQS)的研究学者(初级2)工资支持。 LCC的研究得到了加拿大自然科学和工程研究委员会(NSERC; RGPIN-2016-05584),加拿大卫生研究院(CIHR; PJT153020; ICS154142),魁北克省自然科学基金会(CEDT de la Recherche duQuébec-Nature et Technologies)的资助( FRQNT; 2018-NC-205593),Armand-Frappier Foundation和Institut National de la Recherche Scientifique。该协议主要改编自我们之前的工作(Chatel-Chaix et al。,2016)中使用的实验程序,其中我们优化了ZIKV的生产和检测。我们感谢欧洲病毒档案全球(EVAg)和Xavier de Lamballerie博士(Aix-Marseille大学的病理学Virales),提供ZIKV MR766和H / PF / 2013原始库存。我们感谢PatrickLabonté(国立科学研究院),Tom Hobman(阿尔伯塔大学)和Anil Kumar(阿尔伯塔大学)慷慨地提供Huh7和Vero E6细胞。




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引用:Freppel, W., Mazeaud, C. and Chatel-Chaix, L. (2018). Production, Titration and Imaging of Zika Virus in Mammalian Cells. Bio-protocol 8(24): e3115. DOI: 10.21769/BioProtoc.3115.