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Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks
使用高或低含量防御病毒颗粒制备呼吸道合胞体病毒并从病毒原液中对其纯化   

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Abstract

Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). Here we discuss in detail how to prepare RSV stocks with a high or low content of DVGs, and how to purify defective viral particles containing DVGs from an RSV stock enriched in defective viral particles. These procedures are useful for the preparation of viral stocks and defective viral particles necessary for laboratory research. In brief, the different RSV stocks are produced in HEp2 cells, which are commonly used to amplify this virus in the laboratory. To generate an RSV stock with a high content of DVGs, HEp2 cells are sequentially infected with a high multiplicity of infection (MOI) multiple times followed by purification of the viral particles containing DVGs using gradient centrifugation. The procedure describe here has four parts: 1. Amplification of seed RSV stock with a low DVG content (RSV-LD), 2. Generation of a stock with a high DVG content (RSV-HD), 3. Purification of DVGs by gradient centrifugation, 4. Characterization of purified DVGs.

Keywords: Respiratory syncytial virus(呼吸道合胞病毒), Defective viral genomes(有缺陷的病毒基因组), Defective viral particles(有缺陷的病毒颗粒), DVGs(群), RSV preparation(呼吸道合胞病毒的制备)

Materials and Reagents

  1. Sterile polypropylene conical 15 and 50 ml tubes (BD, Falcon®, catalog number: 352070 , or equivalent)
    Note: Currently, it is “Corning, Falcon®, catalog number: 352070 ”.
  2. Disposable cell scraper (Thermo Fisher Scientific, catalog number: 08-100-241 )
  3. Sterile screw-cap microtubes, 2 ml (SARSTEDT AG & Co., catalog number: 15071353 )
  4. Sterile, aerosol-resistant micropipette tips (1-1,000 μl capacity) (Eppendorf AG or equivalent)
  5. Cotton-plugged, sterile serological pipettes (1-25 ml capacity) (Eppendorf AG or equivalent)
  6. Straight-neck polystyrene tissue culture flasks with vented caps, 75 cm2 (Bioexpress, catalog number: T-3001-2 ) and 225 cm2 (BD, FalconTM, catalog number: 353139 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353139”.
  7. Polystyrene tissue culture plates with lids 12 (Greiner Bio-One GmbH, catalog number: 665180 ) and 96 (Greiner Bio-One GmbH, catalog number: 655086 )
  8. 0.22 μm filter for pipette aid (VWR International, catalog number: 28145-481 )
  9. Vacuum-driven Stericup® 500 ml Millipore ExpressPLUS 0.22 μm PES (Merck Millipore Corporation, model: SCGPU05RE )
  10. Ultra-centrifuge tubes
    1. Ultra-ClearTM centrifuge tube, 14 x 89 mm (Beckman Coulter, catalog number: 344059 )
    2. Ultra-ClearTM centrifuge tube, 25 x 89 mm (Beckman Coulter, catalog number: 344058 )
  11. HEp2 cells (ATCC, catalog number: CCL-23 )
  12. Human RSV strain A2 (ATCC, catalog number: VR-1540 )
  13. Mycoplasma removal agent (MP BioMedical, catalog number: 093050044-5 ml )
  14. Dulbecco’s modified eagle medium (DMEM) (Life Technologies, catalog number: 11995073 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11995073”.
  15. UltraPureTM EDTA (0.5 M) (Gibco, catalog number: 15575 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575”.
  16. Trypsin-EDTA, 0.25% (wt/vol) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  17. Fetal bovine serum (FBS), heat-inactivated at 56 °C for 30 min (Thermo Fisher Scientific, catalog number: 10082-147 )
    Note: Aliquots should be stored at -20 °C and thawed before use.
  18. Gentamicin reagent solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15750-060 )
  19. Sodium pyruvate solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11360070 )
  20. L-glutamine (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25030-081 )
  21. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025-092 )
  22. Phosphate-buffered saline (PBS) (pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010056 )
  23. Ethanol (Thermo Fisher Scientific, DeconTM, catalog number: 64-17-5 )
  24. Methanol (HPLC) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A452 )
  25. Crystal violet (Thermo Fisher Scientific, catalog number: C581 )
  26. Dry ice
  27. Thermo ScientificTM GeneRulerTM 100 bp Plus DNA Ladder 100 to 3,000 bp (Thermo Fisher Scientific, catalog number: FERSM0322 )
  28. Electrophoresis, loading dyes, Thermo Scientific, 6x DNA loading dye (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )
  29. UltraPure agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500-500 )
  30. Tris-acetate-EDTA (TAE), 10x (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15558026 )
  31. D-sucrose (Sigma-Aldrich, catalog number: BP220 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: BP220”.
  32. Gelatin, from bovine skin (Sigma-Aldrich, catalog number: G9391 )
  33. Superscript® III first-strand synthesis system (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-051 )
  34. Platinum Taq DNA polymerase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10966018 )
  35. TRIzol reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596018 )
  36. dNTP set (100 mM) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297117 )
  37. PierceTM coomassie plus (Bradford) protein assay (Thermo Fisher Scientific, catalog number: 23236 )
  38. RT-PCR primers
    1. DI1 primer: 5’-CTTAGGTAAGGATATGTAGATTCTACC-3’
    2. gRSV/DI primer: 5’-CCTCCAAGATTAAAATGATAACTTTAGG-3’
  39. Regular tissue culture medium (TCM) (see Recipes)
  40. Infection medium (see Recipes)
  41. PNE buffer (see Recipes)
  42. 20% sucrose (see Recipes)
  43. 0.1% Gelatin in PBS (see Recipes)
  44. 1% crystal violet stock solution (see Recipes)
  45. Crystal violet working solution (see Recipes)

Equipment

  1. Class II biological safety cabinets
  2. Approved BSL-2 or enhanced BSL-2 laboratory facilities
  3. Personal protective equipment (PPE)
  4. Spray bottles for 70% (vol/vol) ethanol
  5. Micropipettes (1-1,000 μl capacity), multiple channel pipettes (1-200 μl capacity)

  6. Pipette controller (VWR International)
  7. Vortex mixer
(Thermo Fisher Scientific)
  8. Water bath, 37 °C
(Thermo Fisher Scientific)

  9. Refrigerated table-top centrifuge (for 15 ml and 50 ml conical tubes) (Eppendorf AG, model: 5810R )
  10. OptimaTM L-90K ultracentrifuge (Beckman Coulter, catalog number: 365670 )
  11. SW 32 Ti rotor package, swinging bucket (fit for 25 x 89 mm tube) (Beckman Coulter, catalog number: 369694 )
  12. SW 41 Ti rotor package, swinging bucket (fit for 14 x 89 mm tube) (Beckman Coulter, catalog number: 331362 )
  13. Gradient Master (BioComp Instruments Inc., catalog number: 107-201M )
  14. Freezers and refrigerators: -80 °C, -20 °C, and 4 °C

  15. Tissue culture incubator (37 °C, 5-7% CO2) (Thermo Fisher Scientific)
  16. Cell counter
  17. Cell culture microscope (Nikon Corporation or equivalent)
  18. Microwave
  19. Glass Erlenmeyer flask (500 ml)
  20. Agarose gel casting tray (Thermo Fisher Scientific)
  21. Electrophoresis chamber (Thermo Fisher Scientific)
  22. Power supply (Thermo Fisher Scientific)
  23. UV light box (Thermo Fisher Scientific)
  24. Gel DocTM XR+ gel imaging system (Bio-Rad Laboratories)
  25. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
  26. VariokanTM Flash Multimode Reader (Thermo Fisher Scientific)

Procedure

  1. Preparation of an RSV seed stock with a low content of defective viral particles (RSV-LD)
    Note: This step aims to amplify the original virus purchased from ATCC. The goal is to have enough material to then generate an RSV-HD stock.
    1. For initial amplification of the virus, seed 5 x 105/well mycoplasma-free HEp2 cells in TCM in a 6-well plate one night before virus infection.
      Note: Cells and viral infections in this protocol are incubated in a tissue culture incubator at 37 °C, 7% CO2 (5% CO2 can also be used).
    2. Next day, wash the cells twice with sterile PBS and infect with RSV at an MOI of 0.01 tissue culture infectious dose (TCID50)/cell. Use the seeded number of cells for MOI calculation. Dilute the virus in infection medium and use a total volume of 200 μl per infection. Incubate at 37 °C in a tissue culture incubator for 2 h.
    3. Rock the plates once every 15-20 min to maintain an even virus distribution and avoid the cells drying out.
    4. After 2 h of incubation, add 2 ml infection medium (see Recipes) per well and incubate in a tissue culture incubator.
    5. Harvest the virus 5 days post-infection by scrapping the cells and collecting them together with the culture supernatants.
    6. Centrifuge the pooled cells and supernatants for 5 min at 280 x g in a table-top centrifuge at 4 °C and separate the supernatant leaving approx. 200 μl of supernatant in the tube with the pelleted cells.
    7. Resuspend the cell pellet with the leftover 200 μl supernatant and quick-freeze the mixture in dry ice/ethanol, followed by quick thaw in a 37 °C water bath. Repeat this quick freeze/thaw step at least 3 times and vortex after every time.
    8. Pool all the freeze-thawed cell debris with the saved supernatant, vortex to mix well, aliquot (500 μl/tube), and quick-freeze the virus in dry ice/ethanol. This is Passage 1 virus (P1).
    9. Amplify this P1 virus a second time (P2).
    10. For P2 amplification, seed 3 x 106 HEp2 cells in one T-75 flask and infect with 200 μl P1 virus diluted in 1 ml infection medium, following the same procedure as P1. After 2 h of incubation in a tissue culture incubator add 9 ml of infection medium to each flask.
    11. Harvest the virus 5 days after infection as described for P1. Aliquot (100 μl/vial) and quick-freeze the virus.
    12. Titrate the P2 (containing both supernatants and debris) before proceeding with the RSV-HD preparation (for titration method please see TCID50 section below).
      Note: Cell death should be noticed 4-5 days post infection (Figure 1). If earlier cell death is noticed, reduce the amount of P1 virus. If no cell death is noticed post 5 days infection, passage P2 another time following steps A10-12.


      Figure 1. Cell death during RSV infection. HEp2 cells were infected with RSV at MOI of 0.1. The images were taken 4 days post infection. Left: mock infection. Right: RSV-LD infection. Scale bar: 0.1 mm

  2. Preparation and concentration of an RSV stock with a high content of defective viral particles (RSV-HD).
    Note: This step aims to enrich DVGs in the RSV stock. The goal is to have enough DVG-containing defective viral particles to purify from the viral stock. The standard way to promote DVG production during viral infection is to sequentially passage the virus stock (generated from section A in vitro using a high MOI.
    1. To prepare an RSV-HD seed stock, infect HEp2 cells with RSV-LD P2 at a moi of 4 TCID50/cell for two passages to generate RSV-HD P1 and P2. Use the same procedure as for LD preparation. Specifically, T75 flasks are used for RSV-HD P1 and P2 amplification with an inoculum of 1 ml/flask. Additional 9 ml of infection medium should be added after 2 h of incubation. Calculate the viral titers using the TCID50 assay (see TCID50 section).
    2. Harvest viruses 2 days post-infection or when more than half of the cells are dead. Quick freeze/thaw the cell pellet at least 3 times as described in steps A6-7.
    3. Collect the supernatants and discard the cell debris. Aliquot (500 μl/vial) and quick-freeze the viruses.
    4. To generate an RSV-HD working stock, seed 107 HEp2 cells in one T-225 flask and infect with RSV-HD P2 at a moi of 10 TCID50/cell. Use an inoculum of 4 ml per T-225 flask and follow the infection procedure described above. Add 21 ml of infection medium after 2 h incubation of viruses and put it back in the tissue culture incubator.
    5. Two days later, or when more than half of the cells have detached from the plate, scrape the cells and collect them together with the culture supernatant to harvest the virus.
    6. Centrifuge for 5 min at 280 x g in a table-top centrifuge at 4 °C and collect the supernatant.
    7. Return 2-3 ml of the supernatant to each pellet, and put the rest of the supernatants on ice. Suspend the pellet, vortex, and quick freeze/thaw the mixtures at least 3-4 times as in steps A6-7.
    8.  Centrifuge for 5 min at 280 x g in a table-top centrifuge, the goal is to eliminate debris. Take the supernatants and combine them with the rest of the supernatants.
      Caution: Discard the debris at this step.
    9. Ultra-centrifuge the supernatants for 2.5 h at 59,000 x g at 4 °C using an SW 32 Ti Rotor and Ultra-ClearTM 25 x 89 mm centrifuge tube to concentrate the RSV-HD working stock.
    10. Lightly wash with 1 ml of HBSS twice without disturbing the pellet.
    11. Suspend the pellet in a total 200 μl of infection medium / T225 flask.
    12. Aliquot (200 μl /vial) and quick-freeze the viruses in dry ice/ethanol for storage.
    13. In order to obtain enough defective viral particles, we usually infect 48 T225-flasks, totaling 9 ml of ultra-centrifuged RSV-HD (less virus may be sufficient but the yield decreases).

  3. Purification of RSV defective viral particles
    Note: This step aims to isolate the defective viral particles containing DVGs from the infectious viral particles with full-length genome. Since defective viral particles are much smaller and less dense compared to standard viral particles, the lighter (in our case is the top) fraction contains the most DVGs.
    1. Make 100 ml of PNE buffer fresh in the biosafety hood, shake and mix well.
    2. Prepare 20% and 60% sucrose solutions using PNE buffer, shake and mix well.
    3. Set out 6 tubes of Ultra-ClearTM 14 x 89 mm centrifuge tubes.
    4. Fill each tube with 5 ml of 20% sucrose solution. Carefully and slowly pipette 5 ml 60% sucrose solution starting at the bottom of the tube and slowly lift up the 20% sucrose layer.
      Caution: Slow action is critical. A sharp separation between 20% and 60% sucrose solutions should be observed.
    5. Carefully load the tubes containing sucrose solution on the Gradient Master to generate 20%-60% sucrose gradient. Do this in the biosafety hood to maintain sterility.
    6. Once finished, carefully layer 1.5 ml of ultra-centrifuged RSV-HD (prepared in step B) on top of each sucrose gradient. Use a P1000 micropipette to place the virus. The tubes should be filled up to 2-3 mm from the edge to avoid them collapsing during ultracentrifugation.
      Caution: Do not disturb the sucrose gradient when adding the virus.
    7. Ultra-centrifuge the gradients layered with virus for 2 h at 116,000 x g at 4 °C using an SW 41 Ti Rotor and Ultra-ClearTM 14 x 89 mm centrifuge tubes.
    8. Carefully remove the top pink layer once finished the centrifuge.
      Note: you should be able to observe several cloudy layers. Extract the cloudy layers from top to bottom using a P1000 micropipette. Transfer and combine the same fraction from all 6 tubes to a separate clean Ultra-Clear 14 x 89 mm centrifuge tube. In total, 3 layers are extracted from RSV-HD, named Fraction 1, 2, 3 from top to bottom (Figure 2).


      Figure 2. Schematic diagram of defective viral particles purified through sucrose gradient

    9. Fill up each tube to 2-3 mm from the top with PBS and ultra-centrifuge for 2 h at 4 °C at 106,000 x g using SW 41 Ti Rotor.
    10. Suspend the pellet from each fraction in 1 ml of 0.1% Gelatin in PBS, aliquot 100 μl per vial, snap freeze, and store at -80 °C.

  4. Virus Titration by TCID50 (infectious viral titer)
    Note: This step aims to quantify the infectious viral particles contained in each of the purified fractions based on their ability to replicate in permissive cells.
    1. The day before titration prepare 96 well plates with HEp2 cells. Seed the plate with 2 x 104 cells / well in TCM (100 μl /well of a solution of 2 x 106 cells/ 10 ml TCM). One plate will fit four test samples. Prepare as many plates as needed.
    2. The day of the titration the cells should be 80-90% confluent.
    3. In a separate 96 well plate, prepare 1/10 serial dilutions of fractions 1, 2, and 3 in triplicates in infection media. To do this add 90 μl of infection media to all wells in the plate. Add 10 μl of each fraction to each well on the first row. Prepare triplicates for each fraction. Leave three rows for media alone, and include a positive control with a virus of known titer. Use a multichannel pipette to mix the virus with the infection media in the first row and transfer 10 μl to the next row. Change tips, mix and transfer 10 μl to the next row, continue until the last row. Make sure to mix the virus well and change the tips for every dilution.
    4. Remove the TCM of the plate containing the cells and wash the monolayer twice with 100 μl of serum free media.
    5. Using a multichannel pipette, transfer 25 μl/well of the virus dilutions to the cells and incubate for 2 h at 37 °C in a tissue culture incubator. Start transferring from the bottom of the plate (higher dilution) to avoid carrying over virus from the higher concentrations.
    6. Add 75 μl of infection media per well and incubate for 4-5 days in a tissue culture incubator. Be careful not to contaminate the media with virus from the plate.
    7. To determine the viral titer, discard the media in a glass tray containing 10% bleach.
    8. Add 100 μl of crystal violet working solution to the wells. Be careful NOT to touch the bottom of the wells.
    9. Wait for 15-30 min.
    10. Wash the plate by submerging it upside down in water several times to eliminate the excess of crystal violet.
    11. Let it dry at RT.
    12. Score the titer by determining the last dilution with positive CPE (Figure 3). Score the number of positive wells for that last dilution (number of positive out of three). From this score calculate the TCID50/25 μl using the following formula, where “x” correspond to the dilution:
      “+ + +” 10x TCID50 = 10X+0.7/25 μl
      “+ + - ” 10x TCID50 = 10X+0.4/25 μl
      “+ - - ”  10x TCID50 = 10X-0.1/25 μl


      Figure 3. Crystal violet staining of TCID50. The titers from three different viruses were determined by TCID50 as illustrated above. 10-4 and 10-5 were the last dilutions with positive CPE for designated virus samples, which were the “x”. “+” stands for the positive CPE observed. “-” indicates no CPE observed. Based on the equation above, calculate the final titer of each virus as shown at the bottom of this figure.

  5. Estimation of total amount of viral particles (total virus)
    Note: This step aims to estimate the quantity of total viral particles present in each of the purified fractions. We use total amount of protein in each fraction measured by Bradford assay as an estimate of total viral particles.
    1. Dilute 5 μl of Fraction 1, 2, and 3 into 20 μl of dH2O separately → 1:5 dilution.
    2. Prepare the BSA standard (provided in the kit) using serial dilutions as shown in Table1.

      Table 1. BSA serial dilution for standard curve from 2 mg BSA stock
      Std number
      Volume of diluent
      (μl)
      Volume of stock or sample
      (μl)
      BSA conc.
      mg/ml
      1
      25
      75 (BSA: 2 mg/ml)
      1.500
      2
      65
      65 (BSA: 2 mg/ml)
      1.000
      3
      35
      35 of Std 1
      0.750
      4
      65
      65 of Std 2
      0.500
      5
      65
      65 of Std 4
      0.250
      6
      65
      65 of Std 5
      0.125
      7
      80
      20 of Std 6
      0.025

    3. Prepare 15 ml of Coomassie blue reagent (provided in the kit) per 96 wells plate and equilibrate it to room temperate before use.
    4. Add 150 μl of Coomassie blue reagent per well to the 96 well plate.
    5. Add 5 μl of the BSA standard and the diluted Fractions 1, 2, and 3.
    6. Mix by tapping the edge of the plate.
    7. Incubate the plate for 15 min at room temperature.
    8. Read O.D at 595 nm using a VariokanTM Flash Multimode Reader or equivalent.
    9. Based on the standard curve, calculate the total protein concentration of Fractions 1, 2, and 3.

  6. Infectivity/Total viral protein ratio calculation
    Note: This step aims to determine which fraction contains most of the defective viral particles with the least standard infectious viral particles.
    1. The ratio between Infectivity titer (I) and Total viral protein (T) equals to TCID50 per 25 μl / total viral protein per 25 μl.
    2. Select the Fraction with the lowest I/T to further validate the defective viral genome content.

  7. Validation of purified RSV defective viral particles by PCR
    Note: This step aims to confirm that the fraction selected in section F is enriched in DVGs. For this purpose, permissive cells are incubated with each purified fraction and DVGs are identified using a specific RT-PCR assay (DVG-RT-PCR). For detailed illustration of the DVG RT-PCR assay please refer to Sun et al. (2015), Figure S1.
    1. HEp2 cells are supplemented with either PBS or 1, 10, 20 μl of the selected Fraction. In a separate well infect with RSV-HD working stock of MOI of 1 as a control.
    2. Harvest the infected cells 10 h post infection by adding TRIzol. Extract RNA from TRIzol following the manufacturer’s protocol. Quantify the RNA concentration via Nano-drop and use 1 μg of RNA for reverse transcription (RT).
    3. Mix the following reagents per sample in one tube: 1 μl of the DI1 primer (50 uM), 1 μl of dNTPs (10 mM), 1 μg of RNA, nuclease-free H2O (add up to 10 μl).
    4. Incubate 10 min at 65 °C.
    5. Prepare the RT reaction mix in tubes on ice using SuperScript III reverse transcriptase First-Strand System as follows (per sample): 5x RT buffer 2 μl, 25 mM MgCl2 4 μl, 0.1 M DTT 2 μl, RNase OUT 1 μl, SS III 1 μl.
    6. After incubation, add 10 μl of RT mix to each sample. Mix well.
    7. Incubate at 50 °C for 50 min.
    8. Heat at 85 °C for 5 min.
    9. Add 1 μl of RNase H per sample (from SuperScript III reverse transcriptase kit) and incubate at 37 °C for 20 min.
    10. cDNA can be stored at -20 °C or directly used for PCR.
    11. Spin down the cDNA and prepare the DI-PCR mix as below:

      Reagent
      Volume (μl)
      Water
      13.75
      Buffer (10x)
      2.5
      MgSO4 (50 mM)
      2.5
      dNTPs (10 mM)
      2.0
      RSV DI1 primer (10 μM)
      1.0
      DI gSeV Primer (10 μM)
      1.0
      Taq polymerase (5 units/μl)
      0.25
      cDNA
      2.0
      Total
      25

    12. Run PCR Program in a thermocycler.

      Program
      Temperature (°C)
      Time

      Denaturation
      95
      10 min
      Hold
      Denaturation
      95
      30 sec
      33-35 cycles
      Annealing
      55
      30 sec
      Extension
      72
      90 sec
      Final extension
      72
      5 min
      Hold

      4

      Hold

    13. Add 4 μl of the loading buffer to each sample.
    14. Load the samples and 100 bp Plus DNA Ladder in the 1.5% agarose gel.
    15. Observe the defective viral genome bands as shown in Figure 4.


      Figure 4. Content of defective viral genomes and standard genomes in Fraction 1. HEp2 cells were incubated with infection medium and supplemented with 0, 1, 10, or 20 μl of purified defective viral particles (pDP) (Fraction 1). Cells were infected with RSV-HD at an MOI of 1, serving as a positive control. Samples were harvested at 10 h post infection. RNA was extracted followed by DVG-RT-PCR to detect DVGs (A) and genome qPCR to quantify the full-length viral genome content (B). The bands observed during pDP supplementation mirrored the pattern in RSV-HD infection. Little viral replication of standard virus was detected during pDP supplementation, demonstrating that this fraction is enriched in DVGs (but not genome). The four major bands were further confirmed by sequencing [see Sun et al. (2015), Figure S2]. For details on the RSV genome qPCR, please refer to Tapia et al. (2013).

Recipes

  1. Regular tissue culture medium (TCM)
    500 ml DMEM supplemented with:
    10% fetal bovine serum
    1 mM sodium pyruvate (final concentration)
    2 mM L-Glutamine (final concentration)
    50 mg/ml gentamicin (final concentration)
    Pass the solution through a 0.22 μm filter
  2. Infection medium
    500 ml DMEM supplemented with:
    2% fetal bovine serum (final concentration)
    1 mM sodium pyruvate (final concentration)
    2 mM L-Glutamine (final concentration)
    50 mg/ml gentamicin (final concentration)
    Pass the solution through a 0.22 μm filter
  3. PNE buffer
    50 ml PBS supplemented with 200 μl of 0.5 M EDTA
  4. 20% sucrose
    10 g sucrose dissolved in a final volume of 50 ml of PNE buffer
    60% sucrose
    30 g sucrose dissolved in a final volume of 50 ml of PNE buffer
  5. 0.1% Gelatin in PBS
    Add 500 mg gelatin power to a total volume of in 500 ml dH2O, shake until fully dissolved.
    Pass the solution through a 0.22 μm filter
    Maintain sterile
  6. 1% crystal violet stock solution (100 ml)
    1 g crystal violet
    20 ml 100% ethanol
    80 ml dH2O
  7. Crystal violet working stock
    40 ml 1% crystal violet solution
    80 ml methanol
    180 ml dH2O

Acknowledgments

This work was supported by grant AI083284 from the National Institute of Health (CBL). The protocol described herein was based on the following paper: Sun et al. (2015).

References

  1. Lazzarini, R. A., Keene, J. D. and Schubert, M. (1981). The Origins of Defective Interfering Particles of the Negative-Strand Rna Viruses. Cell 26(2): 145-154.
  2. Lopez, C. B. (2014). Defective Viral Genomes: Critical Danger Signals of Viral Infections. Journal of Virology 88(16): 8720-8723.
  3. Prince, A. M., HuimaByron, T., Parker, T. S. and Levine, D. M. (1996). Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins. Journal of Viral Hepatitis 3(1): 11-17.
  4. Rao, D. D. and Huang, A. S. (1982). Interference among Defective Interfering Particles of Vesicular Stomatitis-Virus. Journal of Virology 41(1): 210-221.
  5. Sun, Y. and López, C. B. (2016). Respiratory syncytial virus infection in mice and detection of viral genomes in the lung using RT-qPCR. Bio-protocol 6(9): e1819.
  6. Sun, Y., Jain, D., Koziol-White, C. J., Genoyer, E., Gilbert, M., Tapia, K., Panettieri, R. A., Hodinka, R. L. and Lopez, C. B. (2015). Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans. Plos Pathogens 11(9).
  7. Tapia, K., Kim, W. K., Sun, Y., Mercado-Lopez, X., Dunay, E., Wise, M., Adu, M. and Lopez, C. B. (2013). Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity. Plos Pathogens 9(10).
  8. Yount, J. S., Kraus, T. A., Horvath, C. M., Moran, T. M. and Lopez, C. B. (2006). A novel role for viral-defective interfering particles in enhancing dendritic cell maturation. Journal of Immunology 177(7): 4503-4513.

简介

呼吸道合胞病毒(RSV)属于包括许多临床相关病毒(例如人偏肺病毒和麻疹)的副粘病毒家族。 RSV感染可以在婴儿,老年人和一些免疫受损的成人中引起严重的疾病。在RSV复制期间,产生一系列截短形式的病毒基因组。这些截短的病毒基因组被称为缺陷型病毒基因组(DVG),并且由许多病毒产生(Lazzarini等人,1981; Rao和Huang,1982; Prince等人, ,1996; Sun等人,2015; Tapia等人,2013)。 DVG可以限制全长病毒的复制,并且是针对RSV的先天免疫应答的主要天然触发因子(Sun等人,2015; Tapia等人 ,2013)。在这里,我们详细讨论如何准备具有高或低含量的DVG的RSV股票,以及如何从富含缺陷病毒颗粒的RSV原液中纯化含有DVG的缺陷病毒颗粒。这些程序可用于制备实验室研究所需的病毒原液和有缺陷的病毒颗粒。简言之,在HEp2细胞中产生不同的RSV种群,其通常用于在实验室中扩增该病毒。为了产生具有高含量DVG的RSV原液,HEp2细胞用高多重感染(MOI)多次感染,然后使用梯度离心纯化含有DVG的病毒颗粒。这里描述的方法具有四个部分:1.具有低DVG含量(RSV-LD)的种子RSV原种的扩增,2.具有高DVG含量(RSV-HD)的原种的产生,3.通过梯度离心,4.纯化DVG的表征。

关键字:呼吸道合胞病毒, 有缺陷的病毒基因组, 有缺陷的病毒颗粒, 群, 呼吸道合胞病毒的制备

材料和试剂

  1. 无菌聚丙烯锥形15和50ml管(BD,Falcon ,目录号:352070或等同物)
    注意:目前,"Corning,Falcon ® ,目录号:352070"。
  2. 一次性细胞刮刀(Thermo Fisher Scientific,目录号:08-100-241)
  3. 无菌螺旋盖微管,2ml(SARSTEDT AG& Co.,目录号:15071353)
  4. 无菌,抗气溶胶微量吸头(1-1000μl容量)(Eppendorf AG或同等品)
  5. 棉塞 - 无菌血清移液管(1-25ml容量)(Eppendorf AG或等同物)
  6. 带有通气盖的直颈聚苯乙烯组织培养瓶,75cm 2(Bioexpress,目录号:T-3001-2)和225cm 2(BD,Falcon TM ,目录号:353139)
    注意:目前,"Corning,Falcon ® ,目录号:353139"。
  7. 具有盖子12(Greiner Bio-One GmbH,目录号:665180)和96(Greiner Bio-One GmbH,目录号:655086)的聚苯乙烯组织培养板
  8. 用于移液管助剂的0.22μm过滤器(VWR International,目录号:28145-481)
  9. 真空驱动的Stericup 500ml Millipore ExpressPLUS0.22μmPES(Merck Millipore Corporation,型号:SCGPU05RE)
  10. 超离心管
    1. Ultra-Clear TM离心管,14×89mm(Beckman Coulter,目录号:344059)
    2. Ultra-Clear TM离心管,25×89mm(Beckman Coulter,目录号:344058)v
  11. HEp2细胞(ATCC,目录号:CCL-23)
  12. 人RSV株A2(ATCC,目录号:VR-1540)
  13. 支原体清除剂(MP BioMedical,目录号:093050044-5ml)
  14. Dulbecco改良的Eagle培养基(DMEM)(Life Techology,目录号:11995073)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11995073"。
  15. UltraPure TM EDTA(0.5M)(Gibco,目录号:15575) 注意:目前,"Thermo Fisher Scientific,Invitrogen TM ,目录号:15575"。
  16. 胰蛋白酶-EDTA,0.25%(重量/体积)(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  17. 胎牛血清(FBS),在56℃热灭活30分钟(Thermo Fisher Scientific,目录号:10082-147)
    注意:等分试样应储存在-20°C下,并在使用前解冻。
  18. 庆大霉素试剂溶液(Thermo Fisher Scientific,Gibco TM ,目录号:15750-060)
  19. 丙酮酸钠溶液(Thermo Fisher Scientific,Invitrogen TM,目录号:11360070)
  20. L-谷氨酰胺(Thermo Fisher Scientific,Invitrogen TM ,目录号:25030-081)
  21. Hanks平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM ,目录号:14025-092)
  22. 磷酸盐缓冲盐水(PBS)(pH 7.4)(Thermo Fisher Scientific,Gibco TM ,目录号:10010056)
  23. 乙醇(Thermo Fisher Scientific,Decon ,目录号:64-17-5)
  24. 甲醇(HPLC)(Thermo Fisher Scientific,Fisher Scientific,目录号:A452)
  25. 结晶紫(Thermo Fisher Scientific,目录号:C581)
  26. 干冰
  27. Thermo Scientific GeneRuler TM 100bp Plus DNA Ladder 100至3,000bp(Thermo Fisher Scientific,目录号:FERSM0322)
  28. 电泳,加样染料,Thermo Scientific,6x DNA加载染料(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:R0611)
  29. UltraPure琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500-500)
  30. Tris-acetate-EDTA(TAE),10x(Thermo Fisher Scientific,Invitrogen TM,目录号:15558026)
  31. D-蔗糖(Sigma-Aldrich,目录号:BP220) 注意:目前,它是"Thermo Fisher Scientific,目录号:BP220"。
  32. 明胶,来自牛皮肤(Sigma-Aldrich,目录号:G9391)
  33. Superscript III第一链合成系统(Thermo Fisher Scientific,Invitrogen TM ,目录号:18080-051)
  34. Platinum Taq DNA聚合酶(Thermo Fisher Scientific,Invitrogen TM ,目录号:10966018)
  35. TRIzol试剂(Thermo Fisher Scientific,Ambion TM ,目录号:15596018)
  36. dNTP集(100mM)(Thermo Fisher Scientific,Invitrogen TM ,目录号:10297117)
  37. Pierce 考马斯加(Bradford)蛋白质测定(Thermo Fisher Scientific,目录号:23236)
  38. RT-PCR引物
    1. DI1引物:5'-CTTAGGTAAGGATATGTAGATTCTACC-3'
    2. gRSV/DI引物:5'-CCTCCAAGATTAAAATGATAACTTTAGG-3'
  39. 常规组织培养基(TCM)(参见配方)
  40. 感染介质(见配方)
  41. PNE缓冲区(参见配方)
  42. 20%蔗糖(见配方)
  43. 0.1%PBS中的明胶(参见配方)
  44. 1%结晶紫储备溶液(见配方)
  45. 水晶紫工作液(见配方)

设备

  1. II类生物安全柜
  2. 批准的BSL-2或增强的BSL-2实验室设施
  3. 个人防护装备(PPE)
  4. 用于70%(vol/vol)乙醇的喷雾瓶
  5. 微量移液器(1-1000μl容量),多通道移液器(1-200μl容量)
  6. 移液器控制器(VWR International)
  7. 涡旋混合器(Thermo Fisher Scientific)
  8. 水浴,37℃(Thermo Fisher Scientific)
  9. 冷冻台式离心机(15ml和50ml锥形管)(Eppendoff AG,型号:5810R)
  10. Optima TM sup- L-90K超速离心机(Beckman Coulter,目录号:365670)
  11. SW 32 Ti转子包,摆动斗(适用于25 x 89 mm管)(Beckman Coulter,目录号:369694)
  12. SW 41 Ti转子包,摆动斗(适合14 x 89 mm管)(Beckman Coulter,目录号:331362)
  13. Gradient Master(BioComp Instruments Inc.,目录号:107-201M)
  14. 冷冻箱和冰箱:-80°C,-20°C和4°C
  15. 组织培养孵育器(37℃,5-7%CO 2)(Thermo Fisher Scientific)
  16. 单元格计数器
  17. 细胞培养显微镜(尼康公司或同等产品)
  18. 微波
  19. 玻璃锥形烧瓶(500ml)
  20. 琼脂糖凝胶浇铸托盘(Thermo Fisher Scientific)
  21. 电泳室(Thermo Fisher Scientific)
  22. 电源(Thermo Fisher Scientific)
  23. 紫外灯箱(Thermo Fisher Scientific)
  24. Gel DocTM XR +凝胶成像系统(Bio-Rad Laboratories)
  25. NanoDrop 1000分光光度计(Thermo Fisher Scientific)
  26. Variokan TM Flash多模式读取器(Thermo Fisher Scientific)

程序

  1. 制备具有低含量病毒性粒子(RSV-LD)的RSV种子原料
    注意:此步骤旨在扩增从ATCC购买的原始病毒。 目标是有足够的材料,然后生成RSV-HD股票。
    1. 对于病毒的初始扩增,种子为5×10 5个/孔 无支原体HEp2细胞在TCM中在一个6孔板的前一天 病毒感染。
      注意:本协议中的细胞和病毒感染 在37℃,7%CO 2下的组织培养培养箱中孵育(5%CO > 2 可以   也可以使用)。
    2. 第二天,用无菌PBS洗涤细胞两次 并以0.01组织培养感染剂量的MOI感染RSV (TCID <50>)/小区。 使用种子数量的单元格进行MOI计算。 在感染培养基中稀释病毒,使用总体积200μl每次感染。 在组织培养箱中37℃孵育2小时。
    3. 每15-20分钟摇动平板一次,以保持病毒均匀分布,避免细胞干燥。
    4. 孵育2小时后,每孔加入2ml感染培养基(见Recipes),并在组织培养箱中孵育。
    5. 通过刮除细胞并与培养物上清液一起收集,在感染后5天收获病毒。
    6. 离心收集的细胞和上清液在280×g进行5分钟   在4℃的台式离心机和分离上清离开 约。 200μl上清液在具有沉淀的细胞的管中
    7. 用剩余的200μl上清液重悬细胞沉淀 在干冰/乙醇中快速冷冻混合物,然后快速解冻37℃水浴。 重复此快速冻/融步骤至少3次   并每次涡旋。
    8. 池所有冻融细胞 碎片与保存的上清液,涡旋混合好,等分(500 μl/管),并在干冰/乙醇中快速冷冻病毒。 这是Passage   1病毒(P1)。
    9. 第二次扩增该P1病毒(P2)。
    10. 对于P2扩增,在一个T-75烧瓶中种子3×10 6个HEp2细胞, 感染用1ml感染培养基稀释的200μlP1病毒 与P1相同。 在组织培养物中孵育2小时后 培养箱中向每个烧瓶中加入9ml感染培养基
    11. 如P1所述,感染后5天收获病毒。 等分试样(100μl/瓶)并快速冷冻病毒。
    12. 滴定P2(含有上清液和碎片)之前 继续进行RSV-HD制备(滴定方法参见 TCID <50> 部分)。
      注意:细胞死亡应注意4-5天 感染后(图1)。 如果注意到更早的细胞死亡,减少 P1病毒的量。 如果在感染5天后没有注意到细胞死亡, 在步骤A10-12之后的另一次通过P2。


      图1.细胞死亡   在RSV感染期间。 HEp2细胞用RSV感染,MOI为0.1。 感染后4天拍摄图像。 左:模拟感染。 右:RSV-LD感染。 比例尺:0.1mm

  2. 制备和浓缩具有高含量的缺陷病毒颗粒(RSV-HD)的RSV原液。
    注意:此步骤旨在丰富RSV股票中的DVG。目标是具有足够的含有DVG的缺陷病毒颗粒以从病毒原液中纯化。在病毒感染期间促进DVG产生的标准方法是使病毒原液(从A部分在体外使用高MOI产生)顺序通过。
    1. 为了制备RSV-HD种子原液,用RSV-LD P2在a。感染HEp2细胞 moi为4 TCID 50 /细胞,两次传代以产生RSV-HD P1和P2。使用 与LD制备相同的程序。具体来说,T75烧瓶是 用于用1ml /瓶的接种物进行RSV-HD P1和P2扩增。 在2小时后应加入额外的9ml感染培养基 孵化。使用TCID 50测定法计算病毒滴度(参见, TCID <50>部分)。
    2. 收获病毒2天后感染或何时 一半以上的细胞死亡。快速冻/融细胞沉淀 至少3次,如步骤A6-7中所述。
    3. 收集上清液并弃去细胞碎片。 等分试样(500μl/瓶)并快速冷冻病毒
    4. 为了产生RSV-HD工作原种,将10 7个HEp2细胞种在一个中 T-225瓶中,并以10TCID 50 /细胞的moi用RSV-HD P2感染。 使用   接种物,每个T-225瓶4ml,并遵循感染程序 如上所述。 加入21 ml感染培养基,孵育2 h后 病毒并将其放回组织培养箱中。
    5. 二 几天后,或当一半以上的细胞已从中分离时 板,刮细胞并与培养物一起收集 上清液收获病毒。
    6. 在4℃下在台式离心机中以280×g离心5分钟并收集上清液。
    7. 将2-3ml上清液返回到每个沉淀,并放置其余 的上清液。 悬浮颗粒,涡旋和快速 冷冻/融化混合物至少3-4次,如步骤A6-7。
    8.  在台式离心机中以280 x g离心5分钟,目标是 以消除碎片。 取上清液并将其与 剩余的上清液。
      警告:在此步骤丢弃碎片。
    9. 在4℃下以59,000×g超速离心上清液2.5小时 使用SW 32 Ti转子和Ultra-Clear TM 25×89mm离心管 集中RSV-HD工作库存
    10. 用1ml HBSS轻轻洗涤两次,不影响沉淀
    11. 将沉淀悬浮在总共200μl的感染培养基/T225烧瓶中。
    12. 等分试样(200μl/小瓶)并且在干冰/乙醇中快速冷冻病毒用于储存。
    13. 为了获得足够的有缺陷的病毒颗粒,我们通常 感染48个T225-烧瓶,共9ml的超离心RSV-HD(更少 病毒可能是足够的,但产量降低)。

  3. RSV缺陷病毒颗粒的纯化
    注意:此步骤旨在将具有DVG的缺陷病毒颗粒与具有全长基因组的感染性病毒颗粒分离。 由于缺陷病毒颗粒比标准病毒颗粒小得多且密度较小,所以较轻(在我们的情况下是顶部)级分含有最多的DVG。
    1. 在生物安全罩中新鲜制备100 ml PNE缓冲液,摇匀并混匀
    2. 使用PNE缓冲液制备20%和60%蔗糖溶液,摇动并混匀
    3. 设置6支超透明 TM 14 x 89mm离心管。
    4. 用5ml 20%蔗糖溶液填充每个管。 小心和 缓慢吸取5 ml 60%蔗糖溶液,从底部开始 并缓慢提起20%蔗糖层 注意:慢动作至关重要。 应观察到20%至60%蔗糖溶液之间的明显分离。
    5. 小心地装载含有蔗糖溶液的管 梯度Master产生20%-60%蔗糖梯度。 这样做生物安全罩保持无菌
    6. 一旦完成,仔细 层上1.5ml的超离心RSV-HD(在步骤B中制备) 每个蔗糖梯度。 使用P1000微量移液管放置病毒。 的 管应填充距离边缘2-3mm以避免它们 在超速离心期间崩解。
      警告:在添加病毒时,请勿打扰蔗糖梯度。
    7. 超离心机梯度与病毒分层2小时 116,000xg在4℃下使用SW 41 Ti转子和Ultra-Clear TM sup 14x89mm 离心管
    8. 一旦离心机完成,小心删除顶部粉红色层。
      注意:你应该能够观察到几个云层。 提取 使用P1000微量移液管从顶部到底部的多云层。 转让并将来自所有6个管的相同级分合并到单独的清洁 超透明14 x 89毫米离心管。 总共提取3层   从RSV-HD,从上到下命名为部分1,2,3(图2)。


      图2.通过蔗糖渐变纯化的缺陷病毒颗粒示意图

    9. 使用PBS从顶部填充每个管2-3mm,并使用SW 41 Ti Rotor在4℃下以106,000×g超速离心2小时。
    10. 将来自每个级分的沉淀物悬浮在1ml的0.1%明胶中 PBS,等分试样每个小瓶100μl,快速冷冻,并储存在-80℃。

  4. 通过TCID <50> (感染性病毒滴度)的病毒滴定
    注意:此步骤旨在根据其在允许细胞中复制的能力来量化每种纯化级分中含有的感染性病毒颗粒。
    1. 滴定前一天用HEp2细胞制备96孔板。种子 该板在TCM中以2×10 4个细胞/孔(100μl/孔的溶液 2×10 6个细胞/10ml TCM)。一个板将适合四个测试样品。 根据需要准备多个板。
    2. 滴定当天,细胞应该是80-90%汇合
    3. 在单独的96孔板中,制备1/10系列稀释的 级分1,2和3在感染培养基中一式三份。要做到这一点 向板的所有孔中加入90μl感染培养基。加入10μl每个 分数到第一行上的每个孔。为每个准备三次 分数。为媒体单独留出三行,并包括一个正值 用已知滴度的病毒对照。使用多通道移液器混合病毒与感染培养基在第一行转移10微升 到下一行。 更改提示,混合和传输10μl到下一行, 继续直到最后一行。 确保混合病毒和改变 每次稀释的提示。
    4. 取出含有细胞的板的TCM,并用100μl无血清培养基洗涤单层两次。
    5. 使用多通道移液器,转移25微升/孔的病毒 稀释至细胞并在37℃下在组织培养物中孵育2小时   孵化器。 开始从板的底部(更高 稀释),以避免从较高浓度携带病毒
    6. 每孔加入75μl的感染培养基,在a中孵育4-5天   组织培养孵化器。 小心不要弄脏介质 病毒从板。
    7. 为了确定病毒滴度,将培养基丢弃在含有10%漂白剂的玻璃托盘中。
    8. 向孔中加入100μl结晶紫工作溶液。 小心不要触摸井底。
    9. 等待15-30分钟。
    10. 通过将其倒置在水中几次洗涤板,以消除过量的结晶紫
    11. 让它在室温下干燥。
    12. 通过确定具有阳性CPE的最后稀释度来评价滴度 (图3)。 对最后一次稀释的阳性孔数进行分数 (三的正数)。 从这个分数计算TCID <50> /25μl,其中"x"对应于 稀释:
      "+ + +"10 x TCID 50 = 10 0.7 /25微升
      "+ + - "10 x TCID 50 = 10 0.4 "+ - - "  10 x TCID 50 = 10 X-0.1 /25μl


      图3. TCID 50的结晶紫染色。 三分之一的效价 如上所述通过TCID 50测定不同的病毒。 10 -4 和10 -5 是指定病毒的阳性CPE的最后一次稀释 样品,其为"x"。 "+"代表观察到的阳性CPE。 " - "表示未观察到CPE。基于上述方程,计算 每种病毒的最终滴度如图所示。

  5. 估计病毒颗粒总量(总病毒)
    注意:此步骤旨在估计每个纯化级分中存在的总病毒颗粒的量。我们使用通过Bradford测定法测量的每个级分中的蛋白质总量作为总病毒的估计
    1. 将5μl级分1,2和3稀释到20μldH 2 O单独→1:5稀释物中。
    2. 使用如表1所示的系列稀释液制备BSA标准品(试剂盒中提供)。

      表1.来自2mg BSA储液的标准曲线的BSA系列稀释
      标准号
      稀释剂体积
      (μl)
      库存或样品的数量
      (μl)
      BSA浓度。
      mg/ml
      1
      25
      75(BSA:2mg/ml) 1.500
      2
      65
      65(BSA:2mg/ml) 1.000
      3
      35
      35 of std 1
      0.750
      4
      65
      65 of std 2
      0.500
      5
      65
      第4段的第65条
      0.250
      6
      65
      第5条第65条
      0.125
      7
      80
      20 of std 6
      0.025

    3. 准备15ml的考马斯蓝试剂(试剂盒中提供) 96孔板,并在使用前平衡至室温。
    4. 每孔加入150μl考马斯蓝试剂到96孔板中
    5. 加入5μlBSA标准品和稀释的级分1,2和3.
    6. 点击板的边缘进行混合。
    7. 在室温下孵育平板15分钟。
    8. 使用Variokan TM Flash多模读取器或等同物读取595 nm的O.D。
    9. 基于标准曲线,计算级分1,2和3的总蛋白浓度。

  6. 感染性/总病毒蛋白比例计算
    注意:此步骤旨在确定哪个部分包含大多数具有最低标准感染性病毒颗粒的缺陷病毒颗粒。
    1. 感染滴度(I)和总病毒蛋白(T)之间的比率等于每25μl/总25μg/总病毒蛋白的TCID 50次。
    2. 选择具有最低I/T的级分以进一步验证有缺陷的病毒基因组内容。

  7. 通过PCR验证纯化的RSV缺陷病毒颗粒
    注意:此步骤旨在确认在章节F中选择的部分富含DVG。 为此,将允许细胞与每种纯化的级分一起温育,并使用特异性RT-PCR测定(DVG-RT-PCR)鉴定DVG。 有关DVG RT-PCR测定的详细说明,请参阅Sun et al。 (2015),图S1。
    1. HEp2细胞补充有PBS或1,10,20μl的 选择分数。 在单独的井感染RSV-HD工作股票 作为对照的1的MOI
    2. 收获感染细胞10 h后 感染加入TRIzol。 从TRIzol提取RNA 制造商的协议。 通过纳米滴定量RNA浓度 并使用1μgRNA进行逆转录(RT)。
    3. 混合 每个样品在一个管中的以下试剂:1μl的DI1引物(50uM),1μldNTP(10mM),1μgRNA,无核酸酶的H 2 O 2(加起来为10μM)   μl)。
    4. 在65℃孵育10分钟。
    5. 准备RT反应   使用SuperScript III逆转录酶在冰上管中混合 第一链系统如下(每个样品):5x RT缓冲液2μl,25mM MgCl 22μl,0.1M DTT2μl,RNase OUT1μl,SS III1μl。
    6. 孵育后,向每个样品中加入10μlRT混合物。 混合良好。
    7. 在50℃孵育50分钟。
    8. 在85℃加热5分钟
    9. 每个样品加入1μlRNase H(从SuperScript III逆转录酶试剂盒),并在37℃孵育20分钟。
    10. cDNA可以储存在-20℃或直接用于PCR
    11. 旋转cDNA并制备如下的DI-PCR混合物:

      试剂
      体积(μl)

      13.75
      缓冲区(10x)
      2.5
      MgSO 4(50mM) 2.5
      dNTPs(10mM)
      2.0
      RSV DI1引物(10μM)
      1.0
      DI gSeV引物(10μM)
      1.0
      Taq聚合酶(5单位/μl)
      0.25
      cDNA
      2.0
      总计
      25

    12. 在热循环仪中运行PCR程序
      程序
      温度(℃)
      时间

      变性
      95
      10分钟
      暂停
      变性
      95
      30秒
      33-35个周期
      退火
      55
      30秒
      扩展
      72
      90秒
      最后延长
      72
      5分钟
      暂停

      4

      暂停

    13. 向每个样品中加入4μl加样缓冲液
    14. 将样品和100 bp Plus DNA梯子装入1.5%琼脂糖凝胶中
    15. 观察缺陷病毒基因组条带,如图4所示。


      图4.缺陷病毒基因组和标准基因组的内容部分1.将HEp2细胞与感染培养基一起温育 补充有0,1,10或20μl纯化的缺陷型病毒 颗粒(pDP)(级分1)。 细胞用RSV-HD以MOI感染 为1,作为阳性对照。 在10小时后收获样品   感染。 提取RNA,然后进行DVG-RT-PCR以检测DVG(A) 和基因组qPCR以定量全长病毒基因组内容(B)。 在pDP补充期间观察到的条带反映了模式 RSV-HD感染。 标准病毒的小病毒复制 在pDP补充期间检测到,证明该级分是   富集DVG(但不是基因组)。 四个主要乐队进一步 通过测序确认[参见Sun等人(2015),图S2]。 详情 关于RSV基因组qPCR,请参见Tapia等人(2013)。

食谱

  1. 常规组织培养基(TCM)
    500ml DMEM补充:
    10%胎牛血清 1mM丙酮酸钠(终浓度)
    2mM L-谷氨酰胺(终浓度)
    50mg/ml庆大霉素(最终浓度)
    将溶液通过0.22μm过滤器
  2. 感染介质
    500ml DMEM补充:
    2%胎牛血清(终浓度)
    1mM丙酮酸钠(终浓度)
    2mM L-谷氨酰胺(终浓度)
    50mg/ml庆大霉素(最终浓度)
    将溶液通过0.22μm过滤器
  3. PNE缓冲区
    补充有200μl0.5M EDTA的50ml PBS
  4. 20%蔗糖 10g蔗糖溶于最终体积为50ml的PNE缓冲液中 60%蔗糖 30g蔗糖溶解在最终体积为50ml的PNE缓冲液中
  5. 0.1%明胶在PBS中
    向500ml dH 2 O中的总体积中加入500mg明胶粉,摇动直至完全溶解。
    将溶液通过0.22μm过滤器
    保持无菌
  6. 1%结晶紫储备溶液(100ml) 1克结晶紫
    20ml 100%乙醇 80毫升dH 2 O 2 /
  7. 水晶紫工作股
    40ml 1%结晶紫溶液
    80 ml甲醇
    180ml dH 2 O

致谢

这项工作是由国家卫生研究所(CBL)授权AI083284支持。 本文所述的方案基于以下论文:Sun等人(2015)。

参考文献

  1. Lazzarini,RA,Keene,JD和Schubert,M。(1981)。  阴性干扰RNA病毒的有缺陷干扰颗粒的起源。 细胞 26(2):145-154。
  2. Lopez,CB(2014)。  有缺陷的病毒基因组:关键病毒感染的危险信号。 病毒学杂志 88(16):8720-8723。
  3. Prince,AM,HuimaByron,T.,Parker,TS和Levine,DM(1996)。  丙型肝炎病毒粒子和从低密度脂蛋白分离的推定的有缺陷的干扰颗粒的可视化。病毒性肝炎杂志 3(1):11-17。 br />
  4. Rao,DD和Huang,AS(1982)。  水泡缺陷性干扰颗粒之间的干扰病毒 - 病毒。病毒学杂志 41(1):210-221。
  5. Sun,Y.和López,C. B.(2016)。 小鼠中的呼吸道合胞病毒感染和使用RT-qPCR检测肺中的病毒基因组。 Bio-协议 6(9):e1819
  6. Sun,Y.,Jain,D.,Koziol-White,CJ,Genoyer,E.,Gilbert,M.,Tapia,K.,Panettieri,RA,Hodinka,RLand Lopez,CB(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26336095"target ="_ blank">来自呼吸道合胞病毒的免疫刺激性有缺陷的病毒基因组在感染期间促进强烈的先天抗病毒反应在小鼠和人类中。

    Plos病原体 11(9)。

  7. Tapia,K.,Kim,WK,Sun,Y.,Mercado-Lopez,X.,Dunay,E.,Wise,M.,Adu,M.and Lopez,CB(2013)。  在体内产生的有缺陷的病毒基因组提供关键的危险信号触发肺抗病毒免疫。 Plos病原体 9(10)。
  8. Yount,JS,Kraus,TA,Horvath,CM,Moran,TM和Lopez,CB(2006)。  病毒缺陷性干扰颗粒在增强树突状细胞成熟中的新颖作用。 177(7):4503-4513。 br />
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sun, Y. and López, C. B. (2016). Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks. Bio-protocol 6(10): e1820. DOI: 10.21769/BioProtoc.1820.
  2. Sun, Y., Jain, D., Koziol-White, C. J., Genoyer, E., Gilbert, M., Tapia, K., Panettieri, R. A., Hodinka, R. L. and Lopez, C. B. (2015). Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans. Plos Pathogens 11(9).
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