Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

1. Transform and Recovery of Infectious Clone Plasmids

1. Transform both plasmids (separately) using a commercial transformation protocol (e.g., NEB 5 Minute Transformation Protocol) with some modifications. Both plasmids contain a gene encoding for ampicillin resistance, therefore use ampicillin or carbenicillin for selection. Carbenicillin is preferred, as it is more stable.
   1. Remove cells (See Materials Table) from -80 °C freezer and thaw on ice for 5-10 min. Prewarm lysogeny broth (LB) (containing 10 g/L NaCl and 25 µg/mL carbenicillin; called LB-carb) plates and the broth with catabolite repression (SOC (without antibiotics) – coming with the cells) at 37 °C.
   2. Add between 100 pg-10 ng in 1 µL of plasmid DNA to a tube containing 50 µL of competent cells; mix gently by flicking the tube.
   3. Incubate tubes on ice for 5 min.
   4. Heat shock tubes at 42 °C for 30 s in a water bath. Return to ice for 2 min immediately following heat shock.
   5. Pipette 950 µL of room temperature SOC into each tube and mix by pipetting. Spread 100 µL of the bacterial mixture onto a LB-carb plate and incubate overnight at 37 °C (roughly 12-14 h).
2. Remove plates from 37 °C incubator after 12-14 h incubation. Place plates in a dark drawer at room temperature to allow colonies to continue to grow (roughly 8-9 h).
3. Using 5 mL of Terrific broth (TB, containing 25 µg/mL carbenicillin, called TB-carb), grow 5-10 small colonies for each plasmid overnight at 30 °C in a bacterial shaker (roughly 14-16 h).
   NOTE: Small colonies are an indicator that the plasmid is intact; colonies with plasmids containing deletions, rearrangements or mutations (herein called instability events) will grow faster, and thus be bigger. Therefore, one should attempt to pick the smallest colonies on the plate, specifically not selecting the largest colonies present. Some medium size colonies may also be selected for completeness. The lower temperature used reduces the probability that the cells will overgrow (OD₆₀₀ of greater than 0.6-0.8). As most researchers perform overnight growth, it allows for more control and reduced chance of overgrowth.
4. Check liquid cultures for turbidity (roughly OD₆₀₀ of 0.6-0.8). Place any turbid tubes at 4 °C and allow other tubes to become turbid by growing for longer.
   NOTE: Do not grow bacteria to OD₆₀₀ values greater than recommended, as plasmid instability events may occur.
5. Extract plasmid DNA from bacteria with a commercial plasmid miniprep kit (See Materials Table). Elute in 15 µL of pre-heated (to 70 °C in a heating block) elution buffer. Allow the elution buffer to incubate on the column for 5 min before centrifugation.
   NOTE: Preserve at least 1 mL of this starter culture at 4 °C for further use in the next step.
6. Digest 10 µL of the recovered plasmids to assess whether plasmid instability events occurred during culture.
   NOTE: Digest p1 with SalI/NheI and p2 with BamHI/HindIII at 37 °C for 1 h. Confirm the presence of the correct bands following digestion (Figure 2a) by gel electrophoresis and proceed to step 2. If preparing plasmid DNA by rolling circle amplification (RCA), reserve some of the miniprep eluate to serve as a template.

2. Preparation of Sufficient Plasmid DNA for Ligation

NOTE: Ligation and subsequent electroporation requires a sufficient amount of plasmid DNA that must be generated via either maxiprep or RCA. While maxiprep is the traditionally used approach, RCA offers the advantage of not requiring bacteria, thus removing the potential for plasmid-induced toxicity in bacteria that can result in instability events.

1. Perform the following maxiprep procedure.
   1. For each plasmid that demonstrated the correct restriction enzyme digestion pattern in the previous section, add 500 µL of starter culture to 1 L of TB-Carb (25 µg/mL carbenicillin) broth and shake overnight at 30 °C (roughly 14-16 h, roughly OD₆₀₀ of 0.6-0.8).
      NOTE: Do not allow the culture to grow over this OD₆₀₀ value. This will potentially result in instability events within the plasmids.
   2. Harvest the bacteria and perform maxipreps per manufacturer's protocol (See Materials Table).
2. Using the saved miniprep from step 1.6, perform the following for the RCA procedure.
   1. Transfer 1 µL of plasmid DNA to a tube containing 50 µL of sample buffer.
   2. Heat-denature the sample at 95 °C for 3 min, and then incubate at 4 °C until ready to use.
   3. Prepare the commercial amplification premix (See Materials Table). Combine 50 µL of reaction buffer with 2 µL of enzyme mix in a tube set on ice.
      NOTE: The amplification premix should be kept on ice until ready for use. It is convenient to prepare a master mix by combining sufficient reagents for the required number of amplification reactions. Any unused premix must be discarded.
   4. Transfer 50 µL of prepared amplification premix to the denatured sample.
   5. Incubate the reaction tubes at 30 °C for 18 h.
   6. Incubate the tubes at 65 °C for 10 min to inactivate the ø29 DNA polymerase.
   7. Store the reaction tubes at 4 °C or -20 °C until ready for use.
      NOTE: At this point, plasmid DNA prepared via both methods should be quantified (using absorbance at 260 nm) and the ZIKV genome should be Sanger sequenced entirely to confirm no instability events (deletions and rearrangements) occurred during preparation.

Table 1: Primers for sequencing cDNA clones. Plasmids can be sequenced entirely with the primers listed. Primers marked as "conserved" indicate that they are likely able to sequence/amplify all genotypes of ZIKV. Primers labelled as "PRVABC59" are specific to this strain but may also work for other Asian genotype strains and possibly other genotypes.

3. Preparation of In Vitro Transcribed RNA

1. Set up digestion reaction of either maxiprep or RCA prepared DNA.
   1. For p1 digestion, mix 10 µL of 10x reaction buffer, 3 µL of ApaLI, 3 µL of BamHI-HF, 3 µL of rSAP or CIP, and 3.3 µg of p1 DNA. Add ddH₂O to 100 µL.
   2. For p2 digestion, mix 10 µL of 10x reaction buffer, 3 µL of ApaLI, 3 µL of EcoRI-HF, and 13.3 µg of p2 DNA. Add ddH₂O to 100 µL.
   3. Incubate at 37 °C for 1-2 h.
   4. Purify using a commercial gel and PCR clean-up kit (See Materials Table). Elute in 30 µL of elution buffer pre-heated to 70 °C in a heating block (incubate the column for 5 min before spinning).
      NOTE: Keep 1 µL to run on a gel later.
2. Prepare ligation reaction.
   1. Combine 10 µL of 10x T4 DNA ligation reaction buffer, 3 µL of T4 DNA ligase (400 U/µL), 29 µL of purified p1, and 29 µL purified p2. Add ddH₂O to 100 µL.
   2. Incubate at room temperature for 2 h or 16 °C overnight.
   3. Purify using a commercial gel and PCR clean-up kit (See Materials Table). Elute in 10 µL of elution buffer pre-heated to 70 °C in a heating block (incubate column for 5 minutes before spinning).
      NOTE: Keep 1 µL to run on a gel later.
3. Run undigested plasmids, digested plasmids, and ligation on an agarose gel. The ligation product should have a higher molecular weight band (around 11 kb) than either digested plasmid alone, indicating the ligation was successful (Figure 2b).
4. Set up T7 in vitro transcription reaction.
   1. Combine 10 µL 2x ARCA/NTP Mix, 2 µL of T7 RNA polymerase mix, and 8 µL of purified ligation reaction for a final volume of 20 µL.
      NOTE: The ARCA included in the mix is a cap analog that is exclusively incorporated in the correct orientation. Standard cap analogs can result in only ~50% of the transcripts having a cap in the correct orientation.
   2. Incubate for 4 h at 37 °C.
   3. Measure RNA concentration via a UV spectrophotometer. Optionally, run a denaturing agarose gel to confirm the length of the RNA transcript.

4. Rescue of Infectious Virus by Electroporation

1. days prior to electroporation, prepare 1 T182 flask ofVero cells (between T150 and T182 is acceptable; grown in DMEM + 10% Fetal bovine serum (FBS)) per construct to be recovered. Include 1 T182 as a mock transfection control. Cells should be used at 70-80% confluency.
2. When cells are ready, place 12 mL/reaction of DMEM + 15% FBS + 10 mM HEPES in a water bath heated to 37°C.
3. Detach cells by trypsinization with a standard protocol, and recover cells in media with 10% FBS to inactivate trypsin; centrifuge cells at 150 x g for 5 min at 4 °C.
4. Wash cells in 1x phosphate-buffered saline (PBS), pelleting cells at 150 x g for 5 min at 4 °C. Repeat this step twice for a total of three washes.
5. Resuspend cells in 200 µL of RPMI 1640 + 10 mM HEPES (sterile) per # of samples. Transfer 200 µL of cell suspension to a sterile tube. Cells should be ~0.5 x 10⁷ cells/mL.
6. Add 20 µL of water (mock neg. control) or 20 µL of RNA (preferably 5-10 µg) produced in step 3.4 to each set of cells. Mix gently by flicking the tube and transfer the contents of the tube to a 2 mm gap electroporation cuvette.
7. Set an electroporator to 170 V LV, omega (resistance) at 0 and 950 µF (roughly to 625 V/cm and 20 ms time constant for other machines). Set resistance to the lowest available setting, either 0 or ∞ if available.
8. Pulse once and immediately add 600 µL of DMEM +15 %FBS +10 mM HEPES that was warmed in step 4.2. Rinse inside of cuvette thoroughly to remove all cells. Add cells to a T75 tissue culture flask containing 10 mL of pre-warmed media. Remove additional media from cuvette by using the dropper that is included with the cuvette. Be careful to maintain sterility.
9. Swirl flask to mix media and cells and place in 37 °C incubator.
10. Check the next day for cell viability, and do so every day until 50-75% cell death is observed. Cell death is observed by comparing to the mock electroporated control.
    NOTE: ZIKV cytopathic effect (CPE) is quite pronounced in Vero cells, resulting in rounded up cells that detach and float in the culture medium. We have observed a range of 8-10 days as being ideal for harvest.
11. Harvest supernatant in a conical tube and centrifuge to remove cellular debris at >3,000 x g for 10 min at 4 °C.
12. Remove clarified supernatant to a new tube and supplement with a final concentration of 20% FBS (from 100% stock). Additionally, add sterile HEPES at a final concentration of 10 mM as a buffering agent to preserve virus infectivity while frozen (from a 1 M sterile stock).
13. Mix virus by vortexing and aliquot into screw cap vials. Store at -80 °C for future use.

5. Titration of Recovered Virus

1. Seed the appropriate number of 6- or 12-well plates of Vero cells the day before titration.
2. Remove virus from the freezer and make serial 10-fold dilutions in DMEM+ 2%FBS in tubes or 96-well plates.
   NOTE: The expected titer from recovered clones is between 1 x 10⁶ and 5 x 10⁷ PFU/mL.
3. Add 200 or 400 µL of each dilution in duplicate to a well of a 12- or 6-well plate, respectively.
4. Place plates in 37 °C incubator and rock every 15 min, for a total of ~1-1.5 h adsorption period.
5. Remove viral inoculum and add 1 mL of (12-well) or 2 mL (6-well) DMEM-Tragacanth overlay to each well.
   NOTE: It is possible to use agarose, agar, methylcellulose or other overlay media here.
6. Incubate cells in 37 °C incubator for 5 days. The time for proper plaque formation will vary depending on the overlay used.
7. To fix cells, remove plates from incubator and decant overlay gently into disinfectant.
8. Add sufficient 20% ethanol/0.1% crystal violet (CV) to each well to cover and swirl to mix.
   NOTE: Many other fixatives exist and can be used here. Additionally, if using an agarose or agar overlay, a second overlay can be used with in conjunction with neutral red to visualize plaques without fixation. 20% ethanol has shown to be effective in inactivating enveloped viruses in previous studies; do not use this amount for non-enveloped viruses as they will not be inactivated¹⁹.
9. Incubate plates for 30-60 min at room temperature (in a class II biosafety cabinet); discard CV (per local regulations) and wash plates with tap water.
10. Allow plates to dry, and manually count plaques.
    NOTE: Reference 20 can be used as an additional reference for performing plaque assays.