Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

1. Construction of a ZIKV Infectious cDNA Clone in a BAC

NOTE: The strategy for the assembly of ZIKV in BACs is described for the RGN strain¹³ (accession number KU527068) (Figure 2).

1. Select unique restriction sites appropriately spaced in the viral genome that are absent in the plasmid pBeloBAC11 (Figure 2A).
   NOTE: For ZIKV-RGN, the restriction sites Pml I, Afe I, and BstB I (genomic positions 3,347, 5,969, and 9,127, respectively) were selected. In the case that no appropriate restriction sites are available in the viral genome, generate new restriction sites by introducing silent nucleotide mutations in the viral genome during the cDNA fragment design.
2. Generate by chemical synthesis four cDNA fragments spanning the full-length genome (Z1 to Z4), flanked by the CMV promoter at the 5'-end and the HDV RZ, the BGH termination, and polyadenylation sequences at the 3'-end ( Figure 2B). Each fragment must be flanked by the selected restriction sites (step 1.1).
   NOTE: Fragment Z1 is used as a backbone to clone the rest of the fragments in the pBeloBac11. For that end, it must contain the human CMV promoter and must be flanked at the 5'-end by ApaL I (to clone this fragment in pBeloBAC11) and Asc I (absent in the viral genome), and at the 3'-end by the restriction sites selected for the assembly of the infectious clone (Pml I, Afe I, and BstB I) followed by Mlu I (absent in the viral genome) and BamH I (to clone this fragment in pBeloBAC11). Fragment Z4 must contain the genomic region from the last restriction site selected (BstB I) to the end of the viral genome, followed by the HDV RZ, the BGH termination, and polyadenylation sequences, and the Mlu I restriction site (Figure 2B). Alternatively, the cDNA fragments (Z1-Z4) could be generated by a combination of standard reverse transcriptase polymerase chain reactions (RT-PCRs) and overlapping PCRs using specific oligonucleotides.
3. Assemble the infectious cDNA clone by sequential cloning of fragments Z1 to Z4 into pBeloBAC11 (Figure 2B).
   1. Digest the pBeloBAC11 plasmid and the Z1 fragment with ApaL I and BamH I. For that, mix 2 µg of pBeloBAC11 plasmid or 1 µg of Z1 fragment with 10 µL of 10x reaction buffer, 20 units of each enzyme, and water to reach a final volume of 100 µL. Incubate at 37°C for 2 h.
   2. Dephosphorylate the BAC vector using shrimp alkaline phosphatase (SAP). To that end, add 2.5 µL (2.5 units) of SAP to the digested BAC and incubate at 37°C for 1 h. Heat-inactivate the SAP by incubation at 75 °C for 15 min.
   3. Purify the dephosphorylated BAC vector and fragment Z1 by agarose gel electrophoresis using a commercial gel clean-up kit optimized for the purification of DNA fragments larger than 10 kb (see the Table of Materials).
   4. Perform the ligation reaction to generate the plasmid (p)BAC-Z1. To that end, mix 150 ng of purified digested BAC vector with the purified insert using a molar ratio of vector-to-insert of 1:3, 1.5 µL of 10x T4 DNA ligase buffer containing 10 mM ATP, one unit of T4 DNA ligase, and water to a final volume of 15 µL.
   5. Incubate the ligation mixture for 20 h at 16 °C. As control of the ligation reaction, perform in parallel a ligation reaction without insert. Heat-inactivate the ligase by incubation at 65 °C for 15 min.
      NOTE: In the case of blunt-ended DNAs, incubate the ligation reaction for 20 h at 14 °C. Due to the large size of the BAC vector (Figure 2A), it is essential to use larger amounts of vector and insert than ligations using conventional plasmids in order to increase the ligation efficiency.
   6. Transform 50 µL of E. coli DH10B electrocompetent cells with 2 µL of the ligation reaction (step 1.3.5) by electroporation (25 µF capacitance, 2.5 kV, and 100 Ω resistance), using electroporation cuvettes fitted with electrodes spaced at intervals of 0.2 cm, following standard protocols.
   7. Transfer the cells to a polypropylene tube with 1 mL of SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose [pH 7.0]) and incubate them at 37°C for 1 h with gentle shaking (200-250 rpm). Plate the cells onto Luria broth (LB) agar plates containing 12.5 µg/mL chloramphenicol and incubate them at 37°C for 16 h.
   8. Pick 8 to 12 bacterial colonies, make a replica on a fresh LB agar plate containing 12.5 µg/mL chloramphenicol, and test whether they contain the correct insert by direct PCR analysis using specific oligonucleotides.
   9. Pick a positive colony from the replica plate, grow it in 100 mL of LB containing 12.5 µg/mL of chloramphenicol, and isolate the BAC cDNA by the alkaline lysis method using a commercial plasmid midi kit, following the recommendation for the purification of large low-copy plasmids (see the Table of Materials).
      NOTE: BAC cDNA prepared by this method can be contaminated with up to 30% of bacterial genomic DNA, but it is suitable in quality to perform restriction analysis, sequencing, and cloning. Depending on the BAC size, yields of 4-6 µg of the BAC plasmid can be obtained.
   10. Verify the genetic integrity of the cloned cDNA by restriction analysis. Digest 500 ng of the pBAC-Z1 plasmid with Asc I and Pml I at 37 °C for 1 h and confirm the presence of the Z1 fragment by gel electrophoresis. To further confirm that unwanted mutations have not been introduced, sequence the insert with specific oligonucleotides.
   11. Starting from the plasmid pBAC-Z1 containing the selected restriction sites (Pml I, Afe I, BstB I, and Mlu I), sequentially clone fragments Z2 to Z4 to generate the full-length infectious cDNA clone pBAC-ZIKV (Figure 2B), following similar experimental approaches as described above for fragment Z1 (steps 1.3.1-1.3.10).

2. Preparation of High-purity pBAC-ZIKV for the Rescue of Infectious rZIKV

NOTE: The large-scale preparation of an ultrapure pBAC-ZIKV infectious clone, suitable for the transfection and rescue of infectious viruses, is performed by alkaline lysis with a commercial kit specifically developed for BAC purification (see the Table of Materials). The kit must include an ATP-dependent exonuclease digestion step that removes bacterial genomic DNA contamination, allowing the isolation of BAC cDNA with a grade of purity similar to that obtained with the cesium chloride method.

1. Grow a single colony of E. coli DH10B carrying the pBAC-ZIKV infectious clone in 5 mL of LB medium containing 12.5 µg/mL of chloramphenicol at 37°C for 8 h with gentle shaking (200-250 rpm).
2. Add 1 mL of bacterial culture (step 2.1) to 500 mL of LB with 12.5 µg/mL of chloramphenicol in a 2 L flask and grow the cells at 37°C for 14-16 h (till an OD₆₀₀ of 0.6-0.8).
   NOTE: Rich broths, such as Terrific Broth (TB), can produce extremely high cell densities, resulting in a lower yield and less purity of the BAC cDNA.
3. Purify the BAC infectious cDNA clone by alkaline lysis using a commercial kit specifically developed for BAC purification, following the manufacturer's specifications (see the Table of Materials). Keep the purified BAC cDNA at 4 °C. Depending on the BAC size, yields of 30 µg of ultrapure BAC cDNA clone can be obtained.
   NOTE: Do not dry the DNA pellet for more than 5 min, as overdrying will make it difficult to dissolve the BAC cDNA. Due to the large size of the BAC cDNA clone, avoid vortexing or pipetting to prevent plasmid shearing.

3. Rescue of Infectious rZIKV from the BAC cDNA Clone by Transfection of Vero Cells

NOTE: Infectious rZIKV is recovered by the transfection of Vero cells with the pBAC-ZIKV cDNA clone, using a commercial cationic lipid transfection reagent (see the Table of Materials; Figure 3).

1. One day before transfection, plate on 6-well plates 5 x 10⁵ Vero cells/well in growth medium (Dulbecco's modified Eagle's medium [DMEM] supplemented with 5% fetal bovine serum [FBS], 2 mM L-glutamine, and 1% nonessential amino acids) without antibiotics to raise 90% confluent cell monolayers by the time of transfection.
   NOTE: We recommend performing the virus rescues in triplicates.
2. Equilibrate serum-reduced medium (see the Table of Materials) at room temperature and prepare transfection mixtures in sterile microfuge tubes for each transfection sample as follows.
   1. Add 4 µg of the BAC cDNA clone in 250 µL of serum-reduced medium and mix carefully, avoiding vortexing to prevent plasmid shearing.
   2. In a separate tube, dilute 12 µL of transfection reagent (1 mg/mL) (see the Table of Materials) in 250 µL of serum-reduced medium, mix by vortexing, and incubate at room temperature for 5 min.
   3. Combine the diluted BAC cDNA and the transfection reagent (with a 1:3 ratio of DNA:transfection reagent), mix carefully while avoiding vortexing, and incubate at room temperature for 20-30 min.
3. During the incubation period of the BAC cDNA/transfection reagentmixture,wash Vero cells with growth medium without antibiotics and leave the cells in 1 mL of fresh medium without antibiotics.
   NOTE: The addition of antibiotics during the transfection process may decrease transfection efficiency.
4. Distribute dropwise the 500 µL of the BAC cDNA/transfection reagent mixture (of step 3.2.3) onto the Vero cells, mix them by rocking the plate back and forth, and incubate the cells in a 5% CO₂ humidified incubator at 37 °C for 6 h.
5. Remove the transfection medium, add 2 mL of fresh growth medium with antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), incubate the cells in a 5% CO₂ humidified incubator at 37 °C, and check every day for the induction of cytopathic effect (CPE).
   NOTE: ZIKV CPE is quite pronounced in Vero cells and it is characterized by the presence of rounded and birefringent cells that detach and float in the tissue culture supernatant.
6. Collect aliquots (100 µL) of the Vero cell tissue-culture supernatants (step 3.5) every 24 h to determine the efficiency of virus recovery by assessing the presence of ZIKV using a standard plaque assay on fresh Vero cells (Figure 4A).
7. After four to six days of transfection, when the CPE is around 50%-75% (Figure 4B), collect the tissue culture supernatants in 15 mL conical tubes and centrifuge at 2,000 x g for 10 min at 4 °C to remove cellular debris.
8. Harvest the supernatants containing rZIKV and discard the cell pellets. Aliquot the supernatant in cryotubes and store them at -80 °C to further confirm the presence of rescued rZIKV.

4. Titration of Recovered rZIKV

1. A day before titration, seed on 12-well plates 2.5 x 10⁵ Vero cells/well in growth medium to raise 90% confluent cell monolayers by the time of titration.
   NOTE: We recommend conducting the titration of the recovered rZIKV in triplicates.
2. Remove an aliquot of the supernatant from the freezer (step 3.8) and make serial 10-fold dilutions in growth medium without FBS.
3. Wash Vero cells 2x with phosphate-buffered saline (PBS) and add 200 µL/well of each virus dilution in triplicate. Place plates in a 5% CO₂ humidified incubator at 37 °C and rock every 15 min for a 90 min adsorption period.
4. Remove the viral inoculum, overlay the cells with 2 mL of growth medium containing 2% FBS, 1% DEAE-dextran, and 0.6% Agar Noble, and incubate at 37 °C under 5% CO₂ for 3-4 days.
   NOTE: It is possible to use agarose, methylcellulose, or other overlay media. The time for proper plaque formation will vary depending on the overlay used and the ZIKV strain.
5. Fix ZIKV-infected cells with 1 mL/well of 4% formaldehyde diluted in PBS at room temperature for 1 h, remove the overlay, and visualize the viral plaques by staining them with 0.1% crystal violet in 20% methanol at room temperature for 15 min.
   1. Discard the crystal violet, wash 3x with water, allow the plates to dry, and manually count the plaques (Figure 4C, left panel). Viral titer is calculated as plaque forming units per milliliter (PFU/ml).
6. Alternatively, viral plaques can be visualized by immunostaining with the pan-flavivirus E protein mouse monoclonal antibody (mAb) 4G2 (Figure 4C, right panel).
   1. To evaluate ZIKV plaques by immunostaining, after the fixing and removal of the overlay agar as described above (in step 4.5), wash the cells 2x with PBS and permeabilize them by incubation with 200 µL/well of 0.5% Triton-X100 in PBS for 15 min at room temperature.
   2. Remove the permeabilization solution, wash the cells 3x with PBS, and block them with 200 µL/well of blocking solution (10% FBS in PBS) for 1 h at room temperature.
      NOTE: Other standard blocking solutions (e.g., 2.5% BSA in PBS) can be used in this step.
   3. Remove the blocking solution and incubate the cells with 200 µL/well of the pan-flavivirus E protein mAb 4G2 diluted in blocking solution (1 µg/mL) for 1 h at 37 °C.
      NOTE: Other mAb or polyclonal antibodies (pAb) can be used instead of 4G2 for the immunostaining and detection of viral plaques.
   4. Wash the cells 3x with PBS, and incubate them with 200 µL of biotinylated anti-mouse secondary antibody diluted (following the manufacturer's recommendation) in blocking solution for 1 h at 37 °C.
   5. Remove the secondary antibody, wash the cells 4x with PBS, and visualize the viral plaques using an avidin/biotin-based peroxidase kit following the manufacturer's specifications (see the Table of Materials).

5. Confirmation of Successful rZIKV Rescue

NOTE: To further confirm the identity of the rescued virus, ZIKV E protein expression is analyzed by immunofluorescence using the mouse mAb 1176-56 specific for ZIKV E protein (Figure 4D). This mAb is specific for ZIKV E protein, contrary to the situation of the pan-flavivirus E protein mAb 4G2 (step 4.6.3). Alternatively, the virus identity can be confirmed by sequencing.

1. A day before immunofluorescence analysis, seed coverslips in 24-well plates containing 1 x 10⁵ Vero cells/well in growth medium to raise 90% confluent cell monolayers by the time of infection.
2. Wash the cells 2x with PBS and infect them with a multiplicity of infection (MOI) of 0.5 PFU/cell of the rescued virus in growth medium without FBS (100 µL/well) in triplicate. Incubate the plates at 37 °C for 90 min.
3. After viral adsorption, remove the viral inoculum, add 0.5 mL of fresh growth medium with 2% FBS, and incubate the cells in a 5% CO₂ humidified incubator at 37 °C for 48 h.
4. Remove the tissue culture supernatant, fix the cells with 150 µL/well of 4% paraformaldehyde in PBS for 20 min at room temperature, and permeabilize the cells with 150 µL/well of 0.5% Triton-X100 in PBS for 15 min at room temperature.
5. After removing the permeabilization solution and washing the cells 3x with PBS, block the cells with 150 µL/well of blocking solution during 1 h at room temperature.
   NOTE: Cell scan be blocked with alternative blocking solutions (e.g., 2.5% BSA in PBS).
6. Remove the blocking solution and incubate the cells with 100 µL/well of mAb 1176-56, specific for ZIKV E protein, diluted in blocking solution (1 µg/mL) for 1 h at 37 °C.
7. Wash the cells 3x with PBS, incubate them at room temperature for 1 h with 100 µL/well of Alexa Fluor 488-conjugated anti-mouse secondary antibody diluted (following the manufacturer's recommendation) in blocking solution, wash the cells extensively with PBS, and incubate them with 150 µL/well of 4',6'-diamidino-2-phenylindole (DAPI; 1 mg/mL) diluted 1:200 in PBS at room temperature for 10 min.
8. Wash the cells 3x with PBS, mount the coverslips on an antifade mounting medium (see the Table of Materials), and analyze the samples under a fluorescence microscope.
   NOTE: For long-term storage, store samples at 4 °C, protected from light.

6. Amplification and Generation of Viral Stocks

NOTE: Once the identity of the rescued virus is confirmed (section 5), amplify the virus on Vero cells and generate viral stocks for further studies.

1. Grow Vero cells in 100 mm x 21 mm plates at 90% confluence and infect them with an MOI of 0.1 PFU/cell as described before.
2. When the CPE is around 75% (approximately 48-72 h postinfection), collect the tissue culture supernatant in a 50 mL conical tube and centrifuge at 2,000 x g for 10 min at 4 °C to remove the cellular debris.
3. Harvest the supernatant containing rZIKV and discard the cell pellet. Aliquot the supernatant in cryotubes and store them at -80 °C.
4. Remove a virus aliquot from the freezer and determine the viral titer by plaque assay as described before (section 4).