Note: Figure 1 presents a strategy for the construction of a full-length infectious JEV cDNA as a BAC.²⁸ Table 1 provides a list of the oligonucleotides used in this protocol.²⁸

1. Extract Viral RNA from JEV Particles in Cell Culture Supernatants

1. Start with the cell culture medium containing JEV SA₁₄-14-2, a live JE vaccine virus that requires Biosafety Level 2 containment.
   Note: The viral titer is approximately 1-3 × 10⁶ plaque-forming units/ml.
2. Take the biosafety training necessary for all standard microbiological practices, safety equipment, and laboratory facilities prior to working with JEV SA₁₄-14-2.
3. Purify viral RNA from an aliquot of the virus-containing cell culture medium using a monophasic solution of phenol and guanidine isothiocyanate⁶⁶ (Figure 1A).
   1. Add 600 µl of the monophasic reagent to 200 µl of the culture supernatant in a 1.7 ml microtube. Homogenize the mixture by hand-shaking the tube vigorously for 30 sec and incubating for 5 min at RT.
   2. Add 160 µl of chloroform to the homogenized sample. Mix thoroughly by hand-shaking the tube vigorously for 15 sec and leaving it for 2-3 min at RT.
   3. Centrifuge the total lysate at 13,400 × g for 15 min at 4 °C, which results in a separation of two liquid phases, i.e., a lower organic phase and an upper aqueous phase. Transfer the upper aqueous phase (less than 200 µl) containing the RNA to a new microtube.
   4. Precipitate the RNA by adding 1 µl of 5 µg/µl glycogen and 400 µl of 100% isopropanol and incubating for 10 min at RT.
   5. Centrifuge the mixture at 13,400 × g for 10 min at 4 °C. Discard the supernatant and wash the RNA pellet with 1 mL of 75% ethanol by pulse-vortexing three to five times and centrifuging at 13,400 × g for 5 min at 4 °C.
   6. Air-dry the RNA pellet for 10 min and dissolve it in 40 µl of dH₂O. Store the extracted RNA at -80 °C until use.

2. Synthesize a Set of Four Overlapping cDNA Fragments (F1 to F4) Spanning the Entire Viral Genomic RNA by Reverse Transcription (RT)-PCR

1. Perform a 20 µl RT reaction with a 10 µl aliquot of the purified viral RNA as a template and a modified form of Moloney murine leukemia virus reverse transcriptase.⁶⁷
   1. Set up a 13 µl mixture containing 10 µl of the purified RNA, 1 µl 10 mM dNTP mix, 1 µl 4 pmol/µl primer, and 1 µl dH₂O. Use the fragment-specific primer for each RT reaction: 1RT for F1, 2RT for F2, 3RT for F3, and 4RT for F4.
   2. Incubate the mixture at 65 °C for 5 min, place on ice for 1 min, and then quick-spin to collect the contents at the bottom of the tube.
   3. Add 4 µl 5× RT buffer, 1 µl 0.1 M DTT, 1 µl 40 U/µl RNase inhibitor, and 1 µl 200 U/µl reverse transcriptase. Mix by pipetting up and down three to five times.
   4. Let the reaction proceed at 50 °C for 1 hr, then heat-inactivate the sample at 70 °C for 15 min. Store the synthesized first-strand cDNA at -20 °C until use.
2. Perform a 100 µl PCR reaction with a 5 µl aliquot of the heat-inactivated RT reaction as a template and a high-fidelity thermostable DNA polymerase⁶⁸ (Figure 1B).
   1. Set up a 100 µl PCR reaction on ice, containing 5 µl of the RT reaction, 20 µl 5× PCR buffer, 4 µl 10 mM dNTP mix, 5 µl 10 µM forward primer, 5 µl 10 µM reverse primer, 1 µl 2 U/µl DNA polymerase, and 60 µl dH₂O. Use the fragment-specific primer pair for each PCR reaction: 1F+1R for F1 (2573 bp), 2F+2R for F2 (4171 bp), 3F+3R for F3 (3922 bp), and 4F+4R for F4 (1798 bp).
   2. Mix gently by finger-flipping the tube three to five times and centrifuge briefly to collect its contents at the bottom.
   3. Begin thermocycling with an initial denaturation step of 30 sec at 98 °C, followed by 25-30 cycles with the following PCR profile: 10 sec at 98 °C, 30 sec at 60 °C, and 1-2 min (30 s/kb) at 72 °C. Store the PCR products at 4 °C until analyzed.
3. Run a 2-5 µl aliquot of each PCR reaction on a 0.8% agarose gel containing 0.5 µg/ml ethidium bromide (EtBr) (Figure 2).
   CAUTION: EtBr is a potent mutagen and requires lab coats, safety glasses, and gloves to be worn and extreme caution to be observed during its use, storage, and disposal.

3. Subclone Each of the Four cDNA Fragments (F1 to F4) into a BAC Vector to Create pBAC/F1 to pBAC/F4 by Molecular Cloning Techniques

1. Digest the vector and insert DNAs with two appropriate restriction endonucleases, as follows:
   1. Perform a sequential digestion of pBAC/PRRSV/FL (vector),³⁰ a derivative of the pBeloBAC11 plasmid (7507 bp, GenBank accession number U51113), with Pme I and Not I in a total volume of 60 µl (containing ~500 ng DNA, 10 U enzyme, 1x digestion buffer, and 1× BSA) at 37 °C for 12-15 hr, which yields the 15426-bp vector fragment.
   2. Perform a sequential digestion of each of the four cDNA amplicons (insert) with Sma I and Not I in a total volume of 60 µl (containing ~1 µg DNA, 20 U enzyme, 1× digestion buffer, and 1× BSA) at 25 °C (Sma I) or 37 °C (Not I) for 12-15 hr, which yields the following insert fragments of the desired size: F1 (2559 bp), F2 (4157 bp), F3 (3908 bp), and F4 (1784 bp).
2. Purify the desired vector and insert DNA fragments by gel extraction.
   1. Separate the doubly digested products on a 1% low-melting point agarose gel containing 0.5 µg/ml EtBr. Cut out a band of the desired DNA fragment with a minimal amount of agarose (usually ~200 µl) under long-wave ultraviolet light.
   2. Add an equal volume of TEM buffer (10 mM Tris-Cl, 1 mM EDTA, and 10 mM MgCl₂) to the excised agarose in a 1.7 ml microtube. Incubate the sample at 72 °C for 10-15 min, with vortexing every 2-3 min until the agarose has completely melted.
   3. Add an equal volume of pre-warmed buffer-saturated phenol, vortex vigorously for 1 min, and centrifuge at 13,400 × g for 10 min at RT.
      Note: The mixture separates into a lower organic phase and an upper aqueous phase. Transfer the upper aqueous phase containing the DNA to a new microtube.
   4. Add an equal volume of chloroform:isoamyl alcohol (24:1), vortex for 1 min, and centrifuge at 13,400 × g for 10 min at RT. Transfer the upper aqueous phase to a new microtube.
   5. Add 0.5 µl of 10 µg/µl yeast tRNA, 1/10 volume of 3 M sodium acetate, and 2.5 volumes of 100% ethanol. Keep the mixture on ice for 20 min.
   6. Centrifuge the mixture at 13,400 × g for 10 min at RT. Remove the supernatant and wash the DNA pellet with 1 ml of 70% ethanol by pulse-vortexing three to five times and centrifuging at 13,400 × g for 10 min.
   7. Air-dry the DNA pellet for 10 min and dissolve it in 20 µl of TE buffer (10 mM Tris-Cl and 1 mM EDTA [pH 7.6]).
3. Ligate the desired vector and insert DNA fragments using T4 DNA ligase.
   1. Set up a 20 µl ligation reaction containing 50-100 ng of the vector DNA, a ~3-fold molar excess of the insert DNA, 400 U T4 DNA ligase, and 1× ligation buffer. Incubate the ligation reaction at 16 °C for 12-15 hr.
      Note: Include a negative control reaction, i.e., vector only without the insert, in parallel.
   2. Perform four separate ligations, each joining the 15426-bp Pme I-Not I fragment of pBAC/PRRSV/FL with the 2559-bp (for F1), 4157-bp (for F2), 3908-bp (for F3), or 1784-bp (for F4) Sma I-Not I fragment of one of the four cDNA amplicons, to generate subclones pBAC/F1 to pBAC/F4.
4. Transform the ligated DNA into E. coli DH10B by the CaCl₂-heat shock method.
   1. Take 100 µl aliquots of the CaCl₂-treated competent DH10B cells⁶⁹ stored at -80 °C and thaw them on ice.
      Note: Use 100 µl of cells per transformation.
   2. Add a 10 µl aliquot of the DNA ligation reaction to 100 µl of the thawed cells in a 1.7 ml microtube, mix gently by tapping the tube, and keep on ice for 30 min.
   3. Heat-shock the DNA-cell mixture for 45 sec in a 42 °C water bath, place on ice for 2 min, and then add 900 µl of LB broth pre-warmed to RT.
   4. Incubate the heat-shocked cells at 35 °C for 1 hr, with shaking at 225-250 rpm.
   5. Spread 50 to 200 µl aliquots of the cultured cells on LB agar plates containing 10 µg/ml chloramphenicol (Cml). Keep the plates at RT, right side up, until they are dry.
   6. Turn the plates upside down and incubate at 35 °C for 15 hr.
5. Recover the cloned BAC DNA from the host cells by a column-based purification method (Figure 1C).
   1. Pick six to eight bacterial colonies from the LB-Cml agar plates and inoculate them into 3 ml of 2xYT broth containing 10 µg/ml Cml. Incubate the cultures at 35 °C for 10 hr with vigorous shaking (225-250 rpm).
   2. Isolate recombinant BAC DNAs from 1-1.5 ml of the bacterial cultures using spin columns, as directed by the manufacturer.⁷⁰ Elute the extracted DNA (typically 100-200 ng) in 20 µl of TE buffer.
   3. Perform two analytical restriction enzyme digestions of the isolated BACs for ~6 hr in a total volume of 10 µl, one to identify the presence of the vector with a correct insert using the same enzymes used for cloning (see Protocol 3.1), and the other to test the integrity of the cloned BACs with an appropriate enzyme (e.g., Bgl II, Nco I, or Pst I), generating a unique restriction fragment pattern.
   4. Propagate the correctly cloned BACs by inoculating 500 µl of the positive bacterial cultures (from Protocol 3.5.1) in 500 ml of 2xYT-Cml medium and cultivating the inoculum for 6 hr at 35 °C while shaking at 225-250 rpm. Purify the BAC DNA (typically 10-20 µg) from the 500 ml culture using filter columns, as recommended by the manufacturer.⁷¹
      Note: The initial four BAC subclones, each containing a cDNA fragment of JEV genomic RNA, may prove to have one or more unwanted mutation(s) when compared to the consensus sequence of the viral genome⁴² (which serves as a reference sequence). Any such mutation needs to be corrected by PCR-based site-directed mutagenesis prior to the assembly of a full-length JEV cDNA.²⁷

4. Create a Full-length JEV cDNA with the 5' SP6 Promoter and the 3' Run-off Site

1. Make three genetic modifications (see below Protocols 4.1.1-4.1.3) in the cloned cDNAs to allow in vitro run-off transcription of genome-length RNAs with the authentic 5' and 3' ends of the viral genome.
   1. Introduce an SP6 promoter directly upstream of the 5' end of the viral genome by overlap extension PCR (Figure 1D, pBAC/F1^SP6).
      1. Amplify two overlapping DNA fragments via the first standard PCR of pBAC/F1 with the two primer pairs SP6F+SP6R (product size, 173 bp) and F1F+F1R (product size, 676 bp), each in a 50 µl reaction containing 1 µl template DNA (~200 pg/µl), 10 µl 5× PCR buffer, 2 µl 10 mM dNTPs, 2.5 µl each of 10 µM forward and reverse primers, 0.5 µl 2 U/µl DNA polymerase,⁶⁸ and 31.5 µl dH₂O. Perform the PCR using the following cycling profile: 98 °C for 30 sec, and 25 cycles of 98 °C for 10 sec, 60 °C for 30 sec, and 72 °C for 20 sec.
      2. Gel-purify the two PCR-amplified DNA fragments after running each of the two PCR products on a 1.5% low-melting point agarose gel, as described in Protocol 3.2.
      3. Fuse the two gel-purified DNA fragments via the second fusion PCR using the outermost primers SP6F+F1R (product size, 821 bp) in a 100 µl reaction including 1 µl each of the two purified DNA fragments (~100 pg/µl), 20 µl 5× PCR buffer, 4 µl 10 mM dNTPs, 5 µl each of 10 µM forward and reverse primers, 1 µl 2 U/µL DNA polymerase,⁶⁸ and 63 µl dH₂O. Use the following thermal cycling profile: 98 °C for 30 sec, and 25 cycles of 98 °C for 10 sec, 60 °C for 30 sec, and 72 °C for 30 sec.
      4. Gel-purify the fused PCR amplicon from a 1% low-melting point agarose gel, as described in Protocol 3.2.
      5. Perform the five-step cloning procedure described in Protocols 3.1-3.5 to ligate the 760-bp Pac I-BsiW I fragment of the gel-purified fused PCR amplicon with the 9532-bp Pac I-BsiW I fragment of pBAC/F1 to generate pBAC/F1^SP6.
   2. Remove the pre-existing, internal Xba I site at nucleotide 9131 by introducing a silent point mutation (A⁹¹³⁴→T) via overlap extension PCR (Figure 1D, pBAC/F3^KO).
      1. Amplify two overlapping DNA fragments by the first standard PCR of pBAC/F3 with the two primer pairs X1F+X1R (product size, 746 bp) and X2F+X2R (product size, 316 bp) in a 50 µl reaction under the experimental conditions described in Protocol 4.1.1.1.
      2. Gel-purify the two PCR-amplified DNA fragments after electrophoretic separation on 1.5% low-melting point agarose gels, as detailed in Protocol 3.2.
      3. Fuse the two gel-purified DNA fragments via the second fusion PCR using the outermost primers X1F+X2R (product size, 1033 bp) in a 100 µl reaction under the experimental conditions described in Protocol 4.1.1.3.
      4. Gel-purify the fused PCR amplicon from a 1% low-melting point agarose gel, as detailed in Protocol 3.2.
      5. Perform the five-step cloning procedure described in Protocols 3.1-3.5 to ligate the 949-bp Avr II-Not I fragment of the gel-purified fused PCR amplicon with the 16245-bp Not I-BsiW I and 2141-bp BsiW I-Avr II fragments of pBAC/F3 to produce pBAC/F3^KO.
   3. Engineer a new artificial Xba I run-off site just downstream of the 3' end of the viral genome by PCR-based site-directed mutagenesis (Figure 1D, pBAC/F4^RO).
      1. Generate one DNA fragment by PCR of pBAC/F4 with primers ROF+ROR (product size, 324 bp) in a 100 µl reaction containing 1 µl template DNA (~200 pg/µl), 20 µl 5× PCR buffer, 4 µl 10 mM dNTPs, 5 µl each of 10 µM forward and reverse primers, 1 µl 2 U/µl DNA polymerase,⁶⁸ and 64 µl dH₂O. Perform the PCR using the following cycling profile: 98 °C for 30 sec, and 25 cycles of 98 °C for 10 sec, 60 °C for 30 sec, and 72 °C for 15 sec.
      2. Gel-purify the PCR-amplified DNA fragment from a 1% low-melting point agarose gel, as detailed in Protocol 3.2.
      3. Perform the five-step cloning procedure described in Protocols 3.1-3.5 to ligate the 283-bp Sfi I-Not I fragment of the gel-purified PCR amplicon with the 16933-bp Sfi I-Not I fragment of pBAC/F4 to create pBAC/F4^RO.
2. Assemble a set of the four modified, overlapping cDNAs into a single full-length SA₁₄-14-2 BAC (pBAC/SA₁₄-14-2) by joining at three natural restriction sites (BsrG I, BamH I, and Ava I) in a sequential manner using the five-step cloning procedures detailed in Protocols 3.1-3.5 (Figure 1E): F1^SP6 → F1^SP6F2 (by replacing the BsrG I-Not I fragment of pBAC/F1^SP6 with that of pBAC/F2) → F1^SP6F2F3^KO (by replacing the BamH I-Not I fragment of pBAC/F1^SP6F2 with that of pBAC/F3^KO) → F1^SP6F2F3^KOF4^RO (by replacing the Ava I-Not I fragment of pBAC/F1^SP6F2F3^KO with that of pBAC/F4^RO).

5. Prepare a High-purity Maxi-prep of the Full-length SA₁₄-14-2 BAC

1. Grow a single colony of E. coli DH10B carrying pBAC/SA₁₄-14-2 in 3 ml of 2xYT broth containing 10 µg/ml Cml for 10 hr at 35 °C with shaking at 225-250 rpm, and then scale up by inoculating 500 µl of the 10 hr bacterial culture into 500 ml of 2xYT-Cml medium and cultivating the inoculum for 6 hr at 35 °C with vigorous shaking.
2. Centrifuge the bacterial culture in two 250 ml bottles at 3,107 × g for 15 min at 4 °C. Resuspend each pellet in 30 ml of GTE solution (50 mM glucose, 25 mM Tris-Cl, and 10 mM EDTA [pH 8.0]), and then add 500 µl of 60 mg/ml lysozyme. Incubate the two cell suspensions on ice for 10 min.
3. Add 60 ml of freshly made Lysis solution (0.2 N NaOH and 1% SDS) to each cell suspension, mix well until clear, and keep the lysates at RT for 10 min.
4. Add 45 ml of Neutralization solution (100 ml, consisting of 60 ml 5 M potassium acetate, 11.5 ml glacial acetic acid, and 28.5 ml dH₂O) to each bottle, mix thoroughly by inverting the bottles, and incubate the neutralized lysates on ice for 10 min.
5. Centrifuge the neutralized lysates at 18,566 × g for 20 min at 4 °C. Transfer the supernatant of both bottles into two new 250 ml bottles; add 0.6 volume of 100% isopropanol to each, and keep them on ice for 20 min.
6. Spin down the precipitates at 18,566 × g for 20 min at 4 °C. Dissolve each pellet in 5 ml of TE buffer, combine them in a 50 ml tube (10 ml total), and precipitate the RNA by adding an equal volume of 5 M lithium chloride. Incubate the mixture on ice for 10 min.
7. Centrifuge the RNA precipitate at 14,636 × g for 20 min at 4 °C. Transfer the supernatant to a new 250 ml tube and precipitate DNA by adding 2 volumes of 100% isopropanol. Incubate the mixture on ice for 20 min.
8. Spin down the DNA precipitate at 18,566 × g for 20 min at 4 °C. Aspirate the supernatant, resuspend the DNA pellet in 9.5 ml of TE buffer (pH 7.6), and add 10 g of cesium chloride (CsCl) and 390 µl of 10 mg/ml EtBr.
9. Load the DNA-CsCl-EtBr solution into a 16 × 76 mm sealable polypropylene tube using a syringe equipped with an 18 G needle. Spin the sealed CsCl gradient in an ultracentrifuge at 401,700 × g for 16 hr at 20 °C (Figure 3).
10. Collect a DNA band of BAC plasmid from the CsCl gradient using an 18 G needle to create an air vent at the top of the gradient and a 20 G needle-equipped syringe to retrieve the BAC DNA from the side of the gradient.
11. Add 2.5 volumes of dH₂O-saturated butanol to the EtBr-stained BAC DNA sample and mix by vortexing. Centrifuge the mixture at 13,400 × g for 1 min and transfer the lower aqueous phase to a new 1.7 ml microtube. Repeat this procedure six times.
12. Precipitate the EtBr-free BAC DNA by adding 1/10 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol to the butanol-extracted BAC DNA and incubating for 10 min on ice. Centrifuge the precipitate at 13,400 × g for 10 min, wash the DNA pellet with 1 ml of 70% ethanol, and re-pellet it by centrifugation.
13. Air-dry the DNA pellet for 10 min and dissolve it in 200 µl of TE buffer (pH 7.6).
    Note: Figure 4 shows an overview of the reverse genetics system for JEV SA₁₄-14-2.

6. Transcribe Synthetic RNAs In Vitro from a Linearized Full-length JEV BAC DNA

1. Perform a large-scale restriction enzyme digestion of pBAC/SA₁₄-14-2 with Xba I in a total volume of 100 µl (containing 3 µg DNA, 60 U enzyme, 1× digestion buffer, and 1× BSA) at 37 °C for 12-15 hr. Examine a 3 µl aliquot of the digestion reaction on a 0.8% agarose gel containing 0.5 µg/ml EtBr.
2. Incubate the digestion reaction further with 25 U of mung bean nuclease (MBN) at 30 °C for 2 hr (Figure 4A).
3. Bring the volume of the Xba I-digested, MBN-treated sample up to 300 µl with dH₂O. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the diluted sample, vortex vigorously for 1 min, spin at 13,400 × g for 10 min, and then transfer the upper aqueous phase to a new 1.7 ml microtube. Add an equal volume of chloroform and repeat the extraction procedure.
4. Recover the phenol/chloroform-extracted, linearized BAC by ethanol precipitation: Add 1/10 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol, and incubate on ice for 20 min. Centrifuge the precipitate at 13,400 × g for 10 min, wash the DNA pellet with 1 ml of 70% ethanol, and then spin it down by re-centrifugation.
5. Air-dry the DNA pellet for 10 min and dissolve it in 30 µl of dH₂O. Examine a 1 µl aliquot of the recovered BAC on a 0.8% agarose gel with 0.5 µg/ml EtBr (Figure 5A).
6. Perform a run-off transcription of the linearized BAC DNA in a total volume of 25 µl (containing ~200 ng template DNA, 0.8 mM cap analog [m⁷G(5')ppp(5')A], 1 mM rNTPs, 40 U RNase inhibitor, 20 U SP6 RNA polymerase,⁷² and 1× transcription buffer) at 37 °C for 1 hr (Figure 4B). Include 0.5 µM [³H]UTP for RNA quantification on the basis of [³H]UTP incorporation, as monitored by adsorption to DE-81 filter paper.⁶⁹
7. Run a 1-2 µl aliquot of the run-off transcription reaction on a 0.6% agarose gel containing 0.5 µg/ml EtBr (Figure 5B).

7. Determine RNA Infectivity and Virus Yield

1. Cultivate BHK-21 cells in 150 mm culture dishes at a density of 3 × 10⁶ cells/dish for 24 hr at 37 °C with 5% CO₂.
   Note: Maintain the BHK-21 cells in alpha minimal essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, vitamins, and penicillin/streptomycin.
2. Rinse the cell monolayer with 10 ml of cold Solution A (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, and 1.5 mM KH₂PO₄). Detach the cells from the dishes by treatment with 4 ml of trypsin-EDTA (0.25%), and collect them by centrifugation at 270 × g in a desktop centrifuge for 2 min.
3. Resuspend the cell pellet with 50 ml of cold Solution A in a 50 ml conical tube and centrifuge the cell suspension at 270 × g for 2 min. Repeat this wash procedure three times; after the last wash, resuspend the cell pellet at a density of 2 × 10⁷ cells/ml in Sol A.
4. Mix a 400 µl aliquot of cell suspension with 2 µg of synthetic RNA in a 2-mm gap cuvette, and promptly electroporate the mixture with an electroporator under optimal electroporation conditions: 980 V, 99 µsec pulse length, and 5 pulses (Figure 4C).
   Note: Use the ³H-labeled RNA synthesized in Protocol 6.5 directly for electroporation without further purification.
5. Leave the electroporated cells at RT for 10 min and transfer to a 1.7 ml microtube containing 600 µl of complete culture medium.
6. Prepare a 10-fold serial dilution of the electroporated cells in 1 ml of complete culture medium and plate a 100 µl aliquot of each dilution on the monolayers of unelectroporated BHK-21 cells (5 × 10⁵) in a 6-well plate.
7. After 4-6 hr of incubation, overlay the cells with 0.5% agarose in minimal essential medium containing 10% fetal bovine serum. Incubate the plates for 4 days at 37 °C with 5% CO₂.
8. Visualize the infectious centers (plaques) by fixation with 7% formaldehyde and staining with 1% crystal violet in 5% ethanol²⁷ (Figure 6A).
   1. Optional: Examine RNA-electroporated cells at 18-20 hr post-transfection for JEV protein expression by immunofluorescence assays^27,73 (Figure 6B), and harvest the supernatants from the RNA-electroporated cells at 22 and 40 hr post-transfection for virus titration by plaque assays^27,63 (Figure 6C).