The authors would like to thank Carla G&#243;mez for her technical assistance in the BAC cDNA clone generation and Snezhana Dimitrova for helping with the video preparation. This work was supported in part by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, grant number BFU2016-79127-R) to F.A.T. and the National Institutes of Health (NIH, grant number 1R21AI120500) to L.M.S. and F.A.T.

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).
<ol>
	<li>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.</li>
	<li>Generate by chemical synthesis four cDNA fragments spanning the full-length genome (Z1 to Z4), flanked by the CMV promoter at the 5&#39;-end and the HDV RZ, the BGH termination, and polyadenylation sequences at the 3&#39;-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&#39;-end by ApaL I (to clone this fragment in pBeloBAC11) and Asc I (absent in the viral genome), and at the 3&#39;-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.</li>
	<li>Assemble the infectious cDNA clone by sequential cloning of fragments Z1 to Z4 into pBeloBAC11 (Figure 2B).
	<ol>
		<li>Digest the pBeloBAC11 plasmid and the Z1 fragment with ApaL I and BamH I. For that, mix 2 &#181;g of pBeloBAC11 plasmid or 1 &#181;g of Z1 fragment with 10 &#181;L of 10x reaction buffer, 20 units of each enzyme, and water to reach a final volume of 100 &#181;L. Incubate at 37&#176;C for 2 h.</li>
		<li>Dephosphorylate the BAC vector using shrimp alkaline phosphatase (SAP). To that end, add 2.5 &#181;L (2.5 units) of SAP to the digested BAC and incubate at 37&#176;C for 1 h. Heat-inactivate the SAP by incubation at 75 &#176;C for 15 min.</li>
		<li>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).</li>
		<li>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 &#181;L of 10x T4 DNA ligase buffer containing 10 mM ATP, one unit of T4 DNA ligase, and water to a final volume of 15 &#181;L.</li>
		<li>Incubate the ligation mixture for 20 h at 16 &#176;C. As control of the ligation reaction, perform in parallel a ligation reaction without insert. Heat-inactivate the ligase by incubation at 65 &#176;C for 15 min. 
		NOTE: In the case of blunt-ended DNAs, incubate the ligation reaction for 20 h at 14 &#176;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.</li>
		<li>Transform 50 &#181;L of E. coli DH10B electrocompetent cells with 2 &#181;L of the ligation reaction (step 1.3.5) by electroporation (25 &#181;F capacitance, 2.5 kV, and 100 &#937; resistance), using electroporation cuvettes fitted with electrodes spaced at intervals of 0.2 cm, following standard protocols.</li>
		<li>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&#176;C for 1 h with gentle shaking (200-250 rpm). Plate the cells onto Luria broth (LB) agar plates containing 12.5 &#181;g/mL chloramphenicol and incubate them at 37&#176;C for 16 h.</li>
		<li>Pick 8 to 12 bacterial colonies, make a replica on a fresh LB agar plate containing 12.5 &#181;g/mL chloramphenicol, and test whether they contain the correct insert by direct PCR analysis using specific oligonucleotides.</li>
		<li>Pick a positive colony from the replica plate, grow it in 100 mL of LB containing 12.5 &#181;g/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 &#181;g of the BAC plasmid can be obtained.</li>
		<li>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 &#176;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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Grow a single colony of E. coli DH10B carrying the pBAC-ZIKV infectious clone in 5 mL of LB medium containing 12.5 &#181;g/mL of chloramphenicol at 37&#176;C for 8 h with gentle shaking (200-250 rpm).</li>
	<li>Add 1 mL of bacterial culture (step 2.1) to 500 mL of LB with 12.5 &#181;g/mL of chloramphenicol in a 2 L flask and grow the cells at 37&#176;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.</li>
	<li>Purify the BAC infectious cDNA clone by alkaline lysis using a commercial kit specifically developed for BAC purification, following the manufacturer&#39;s specifications (see the Table of Materials). Keep the purified BAC cDNA at 4 &#176;C. Depending on the BAC size, yields of 30 &#181;g 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.</li>
</ol>
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).
<ol>
	<li>One day before transfection, plate on 6-well plates 5 x 105 Vero cells/well in growth medium (Dulbecco&#39;s modified Eagle&#39;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.</li>
	<li>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.
	<ol>
		<li>Add 4 &#181;g of the BAC cDNA clone in 250 &#181;L&#160;of serum-reduced medium and mix carefully, avoiding vortexing to prevent plasmid shearing.</li>
		<li>In a separate tube, dilute 12 &#181;L of&#160;transfection reagent (1 mg/mL) (see the Table of Materials) in 250 &#181;L of serum-reduced medium, mix by vortexing, and incubate at room temperature for 5 min.</li>
		<li>Combine the diluted BAC cDNA and the transfection reagent (with a 1:3 ratio of DNA:transfection reagent),&#160;mix carefully while avoiding vortexing, and incubate at room temperature for 20-30 min.</li>
	</ol>
	</li>
	<li>During the incubation period of the BAC&#160;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.</li>
	<li>Distribute dropwise&#160;the 500 &#181;L 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 &#176;C for 6 h.</li>
	<li>Remove the transfection medium, add 2 mL of fresh growth medium with antibiotics (100 U/mL penicillin and 100 &#181;g/mL streptomycin), incubate the cells in a 5% CO2 humidified incubator at 37 &#176;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.</li>
	<li>Collect aliquots (100 &#181;L) 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).</li>
	<li>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 &#176;C to remove cellular debris.</li>
	<li>Harvest the supernatants containing rZIKV and discard the cell pellets. Aliquot the supernatant in cryotubes and store them at -80 &#176;C to further confirm the presence of rescued rZIKV.</li>
</ol>
4. Titration of Recovered rZIKV
<ol>
	<li>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.</li>
	<li>Remove an aliquot of the supernatant from the freezer (step 3.8) and make serial 10-fold dilutions in growth medium without FBS.</li>
	<li>Wash Vero cells 2x with phosphate-buffered saline (PBS) and add 200 &#181;L/well of each virus dilution in triplicate. Place plates in a 5% CO2 humidified incubator at 37 &#176;C and rock every 15 min for a 90 min adsorption period.</li>
	<li>Remove the viral inoculum, overlay&#160;the cells with 2 mL of growth medium containing 2% FBS, 1% DEAE-dextran, and 0.6% Agar Noble, and incubate at 37 &#176;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.</li>
	<li>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.
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Alternatively, viral plaques can be visualized by immunostaining with the pan-flavivirus E protein mouse monoclonal antibody (mAb) 4G2 (Figure 4C, right panel).
	<ol>
		<li>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 &#181;L/well of 0.5% Triton-X100 in PBS for 15 min at room temperature.</li>
		<li>Remove the permeabilization solution, wash the cells 3x with PBS, and block them with 200 &#181;L/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.</li>
		<li>Remove the blocking solution and incubate the cells with 200 &#181;L/well of the pan-flavivirus E protein mAb 4G2 diluted in blocking solution (1 &#181;g/mL) for 1 h at 37 &#176;C. 
		NOTE: Other mAb or polyclonal antibodies (pAb) can be used instead of 4G2 for the immunostaining and detection of viral plaques.</li>
		<li>Wash the cells 3x with PBS, and incubate them with 200 &#181;L of biotinylated anti-mouse secondary antibody diluted (following the manufacturer&#39;s recommendation) in blocking solution for 1 h at 37 &#176;C.</li>
		<li>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&#39;s specifications (see the Table of Materials).</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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 &#181;L/well) in triplicate. Incubate the plates at 37 &#176;C for 90 min.</li>
	<li>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 &#176;C for 48 h.</li>
	<li>Remove the tissue culture supernatant, fix the cells with 150 &#181;L/well of 4% paraformaldehyde in PBS for 20 min at room temperature, and permeabilize the cells with 150 &#181;L/well of 0.5% Triton-X100 in PBS for 15 min at room temperature.</li>
	<li>After removing the permeabilization solution and washing the cells 3x with PBS, block the cells with 150 &#181;L/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).</li>
	<li>Remove the blocking solution and incubate the cells with 100 &#181;L/well of mAb 1176-56, specific for ZIKV E protein, diluted in blocking solution (1 &#181;g/mL) for 1 h at 37 &#176;C.</li>
	<li>Wash the cells 3x with PBS, incubate them at room temperature for 1 h with 100 &#181;L/well of Alexa Fluor 488-conjugated anti-mouse secondary antibody diluted (following the manufacturer&#39;s recommendation) in blocking solution, wash the cells extensively with PBS, and incubate them with 150 &#181;L/well of 4&#39;,6&#39;-diamidino-2-phenylindole (DAPI; 1 mg/mL) diluted 1:200 in PBS at room temperature for 10 min.</li>
	<li>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 &#176;C, protected from light.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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 &#176;C to remove the cellular debris.</li>
	<li>Harvest the supernatant containing rZIKV and discard the cell pellet. Aliquot the supernatant in cryotubes and store them at -80 &#176;C.</li>
	<li>Remove a virus aliquot from the freezer and determine the viral titer by plaque assay as described before (section 4).</li>
</ol>

The authors have nothing to disclose.

Infectious cDNA clones constitute essential molecular tools for basic research of RNA viruses and the development of vaccines and/or the identification of antiviral strategies. However, for many positive-stranded RNA viruses, including flaviviruses, the generation of infectious cDNA clones are difficult due to the instability of the cloned cDNAs when propagated in bacteria using standard high-copy-number plasmids. In the case of ZIKV and other flaviviruses, this instability is mainly due to the leaky expression of toxic viral proteins from cryptic bacterial promoters present in the viral genome14,15,16,17. Here, we describe an alternative and powerful protocol to generate a stable ZIKV full-length infectious cDNA clone as a single plasmid, based on the use of the BAC plasmid pBeloBAC1134 (Figure 2A) to overcome the toxicity problem, the use of the CMV promoter to allow the expression of the vRNA in the nucleus of transfected cells, and the HDV RZ to generate vRNAs with accurate 3&#39;-ends (Figure 2B). Using this method, we have successfully generated a fully stable infectious clone of the ZIKV strain RGN that allows the efficient and reliable recovery of infectious rZIKV after the direct transfection of susceptible Vero cells (Figure 3 and Figure 4).
A huge effort has been made in the last few years to overcome the instability problems associated with ZIKV infectious cDNA clones, and several approaches have been successfully implemented18, including the in vitro ligation of cDNA fragments24,25, low-copy plasmids19,20, the inactivation of cryptic bacterial promoters by the introduction of silent mutations26,27, intron insertion21,22,23, the Gibson assembly method30, the ISA method28,29, and the use of CPER31. Although these approaches overcome the toxicity problem and&#160;are useful to generate ZIKV infectious cDNA clones, some of them are laborious and present several disadvantages, including the need for in vitro ligation and transcription steps that reduce virus recovery efficiency or the introduction of a high number of silent mutations to inactivate cryptic bacterial promoter that could affect viral fitness, among others. The approach described in this protocol presents the following advantages. i) The BAC plasmid pBeloBAC1134 has a strictly controlled replication, keeping one or two copies of plasmid per cell, which minimizes toxicity and allows stable maintenance in bacteria of instable cDNAs. ii) The propagation and modification of BAC plasmids are almost similar to those of conventional plasmids, considering the slight modifications described in this protocol to manipulate large-size BAC-DNA fragments and low-copy plasmids. Notably, the BAC cDNA clone can also be modified into E. coli by homologous recombination using the Red recombination system42,43,44. iii) The use of CMV promoter allows the intracellular expression of capped ZIKV vRNA and the recovery of infectious viruses without requiring an in vitro transcription step. iv) Infectious rZIKV is generated after the direct transfection of susceptible cells (e.g., Vero) with the BAC cDNA clone. Since DNA transfection in mammalian cells is more efficient than RNA transfection, the virus recovery efficiency with the BAC approach is higher than that observed using RNA transcripts, reducing the number of passages in culture cells to generate a viral stock and, consequently, limiting the introduction of unwanted mutations by cell culture adaptation.
Finally, the potential of the BAC approach is supported by the successful use of this method (with slight modifications) to engineer infectious cDNA clones of other flaviviruses, including dengue virus36, and several coronaviruses of high impact in human and animal health, such as transmissible gastroenteritis coronavirus37 (TGEV), feline infectious peritonitis virus38 (FIPV), human coronavirus OC4339 (HCoV-OC43), severe acute respiratory syndrome coronavirus40 (SARS-CoV), and Middle East respiratory syndrome coronavirus41 (MERS-CoV), among others.
In the protocol described here, there are two critical steps that should be considered. One important consideration is identifying appropriate unique restriction sites in the viral genome that are absent in the BAC plasmid. If no adequate restriction sites are available, new restriction sites can be generated during the cloning design by the introduction of silent nucleotide mutations. Another important issue is that the BAC plasmids are present in only one or two copies per cell, and therefore, low yields of BAC plasmids with a high contamination of bacterial genomic DNA are obtained using standard protocols designed for high- and medium-copy-number plasmids. This potential problem is easily overcome using large culture volumes and purifying the BAC plasmid with a commercial kit specifically developed for BAC purification.
In summary, we have developed a powerful ZIKV reverse genetic approach based on the use of a BAC that could be adapted to generate stable and fully functional infectious cDNA clones of other positive-stranded RNA viruses to facilitate the study of the biology of these viruses and the development of vaccines and/or to facilitate the identification of antiviral drugs.

ZIKV is a mosquito-borne member of the Flavivirus genus within the Flaviviridae family that currently constitutes a global public health emergency1. Like other flaviviruses, ZIKV is an enveloped RNA virus with an icosahedral-like structure that contains a positive sense, single-stranded RNA molecule of about 10.8 kb (Figure 1)2. The viral genome encodes a large polyprotein of approximately 3,423 amino acids that is processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) that are involved in the formation of the viral particles and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that participate in genome replication, virus assembly, and evasion of the host immune response (Figure 1)3.
Historically, ZIKV infection has been associated with a mild febrile illness4,5. However, the explosive recent pandemic of ZIKV infections throughout South and Central America, the South Pacific, and the Caribbean6,7,8, and its association with the occurrence of Guillain-Barr&#233; syndrome and microcephaly9,10,11,12,13, have changed the historic perception and potentiated the relevance of ZIKV as an important human pathogen. In this sense, the development of molecular tools, such as infectious cDNA clones, will facilitate the study of viral pathogenesis and the development of genetically defined vaccines and the identification of antiviral drugs for the treatment of ZIKV infection. As described for other flaviviruses, the generation of ZIKV infectious clones is difficult due to the presence of cryptic bacterial promoters in the viral genome14 that allow the leaky expression of toxic viral proteins during the propagation of the cDNA clones in bacteria using standard high-copy-number plasmids15,16,17. To overcome this toxicity problem, several nontraditional approaches have been implemented successfully&#160;in the last two years18. These include the use of low-copy-number plasmids19,20, the insertion of introns to disrupt the toxic regions21,22,23, the in vitro ligation of cDNA fragments24,25, mutational silencing of cryptic bacterial promoters present in the viral genome26,27, infectious subgenomic amplicons (ISA)28,29, the Gibson assembly method30, and the use of circular polymerase extension reaction (CPER)31.
Herein, we describe the detailed protocol for the engineering of a full-length cDNA clone of the ZIKV strain ZIKV-RGN13, using a BAC to overcome the toxicity problem, and the rescue of infectious rZIKV by direct transfection of the BAC cDNA clone into Vero cells32, a cell line approved by the Food and Drug Administration (FDA) for the development of human vaccines33. In this system, the full-length cDNA copy of the viral genome is assembled in the BAC plasmid pBeloBAC1134 (Figure 2A), a low-copy-number plasmid (one to two copies per cell) derived from the Escherichia coli F-factor35, which minimizes the toxicity of flavivirus sequences during its propagation in bacteria. The cDNA of the ZIKV genome is assembled in pBeloBAC11 under the control of the human CMV immediate-early promoter, to allow the expression of the viral (v)RNA in the nucleus of transfected cells by cellular RNA polymerase II, and&#160;flanked at the 3&#39;-end by the hepatitis delta virus (HDV) ribozyme (RZ), followed by the sequences of the bovine growth hormone (BGH) termination and polyadenylation signals to produce synthetic RNAs bearing authentic 5&#39;- and 3&#39;-ends of the viral genome, respectively (Figure 2B). This cDNA-launched system results in the intracellular expression of capped viral RNA, allowing the recovery of infectious ZIKV without the need for an in vitro transcription step. The BAC approach provides a powerful methodology applicable to constructing stable and fully functional infectious cDNA clones for other flaviviruses, as well as other positive-stranded RNA viruses36,37,38,39,40,41.

<table border="0" cellpadding="0" cellspacing="0" style="width:1048px;" width="1048">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">1. Molecular Biology Reagents</td>
			<td style="width:309px;"></td>
			<td style="width:123px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Afe I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0652S</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">AmpliTaq DNA Polymerase</td>
			<td style="width:309px;">ThermoFisher Scientific (Applied Biosystems)</td>
			<td>N8080161</td>
			<td>5,000 Units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">ApaL I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0507S</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Asc I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0558S</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">BamH I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0136S</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">BstB I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0519S</td>
			<td>20,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Chloramphenicol</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:123px;">C0378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">ElectroMAX DH10B Cells&#160;</td>
			<td style="width:309px;">ThermoFisher Scientific (Invitrogen)</td>
			<td style="width:123px;">18290015</td>
			<td>Electocompetent DH10B cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Electroporation Cuvettes, 0.2 cm</td>
			<td style="width:309px;">Bio-Rad</td>
			<td style="width:123px;">165-2086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Ethanol</td>
			<td style="width:309px;">Merck</td>
			<td style="width:123px;">100983</td>
			<td>Flamable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Isopropanol</td>
			<td style="width:309px;">Merck</td>
			<td style="width:123px;">109634</td>
			<td>Flamable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Large-Construct Kit (10)</td>
			<td style="width:309px;">QIAGEN</td>
			<td style="width:123px;">12462</td>
			<td>For high-purity BAC preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">LB Broth</td>
			<td style="width:309px;">ThermoFisher Scientific (Invitrogen)</td>
			<td style="width:123px;">12780029</td>
			<td>Can be homemade as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">LB with Agar</td>
			<td style="width:309px;">ThermoFisher Scientific (Invitrogen)</td>
			<td style="width:123px;">22700041</td>
			<td>Can be homemade as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Methanol</td>
			<td style="width:309px;">Merck</td>
			<td style="width:123px;">106009</td>
			<td>Flamable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Mlu I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0198S</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Oligonucleotides</td>
			<td style="width:309px;">IDT</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Plasmid pBeloBAC11</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">ER2420S (E4154S)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Plasmid Midi Kit (25)</td>
			<td style="width:309px;">QIAGEN</td>
			<td style="width:123px;">12143</td>
			<td>For midle-scale preparation of BAC plasmids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Pml I</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">R0532S</td>
			<td>20,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Polypropylene tubes (10 mL)</td>
			<td style="width:309px;">DeltaLab</td>
			<td style="width:123px;">175724</td>
			<td>Other commercial sources are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">QIAEX II Gel Extraction Kit (150)</td>
			<td style="width:309px;">QIAGEN</td>
			<td style="width:123px;">20021</td>
			<td>Gel-clean-up kit optimized for DNA fragments larger than 10 kb</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Shrimp AlKaline Phosphatase (rSAP)</td>
			<td style="width:309px;">New England BioLabs</td>
			<td style="width:123px;">M0371S</td>
			<td>1,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">SOC Medium</td>
			<td style="width:309px;">ThermoFisher Scientific (Invitrogen)</td>
			<td style="width:123px;">15544034</td>
			<td>Can be homemade as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Synthesis of cDNA fragments</td>
			<td style="width:309px;">Bio Basic</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T4 DNA Ligase</td>
			<td style="width:309px;">Sigma-Aldrich (Roche)</td>
			<td style="width:123px;">10481220001</td>
			<td>1,000 units/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">6-Well Plates</td>
			<td style="width:309px;">ThermoFisher Scientific (Nunc)</td>
			<td style="width:123px;">140675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">12-Well Plates</td>
			<td style="width:309px;">ThermoFisher Scientific (Nunc)</td>
			<td style="width:123px;">150628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">24-Well Plates</td>
			<td style="width:309px;">ThermoFisher Scientific (Nunc)</td>
			<td style="width:123px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Agar Noble</td>
			<td style="width:309px;">VWR</td>
			<td style="width:123px;">214230</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Alexa Fluor 488 Conjugate ant-mouse secondary antibody</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Biotinylated Anti-Mouse Secondary Antibody</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Cell Culture Dishes (100x21 mm)</td>
			<td style="width:309px;">ThermoFisher Scientific (Nunc)</td>
			<td style="width:123px;">172931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Conical Tubes (15 mL)</td>
			<td style="width:309px;">VWR</td>
			<td style="width:123px;">525-0150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Conical Tubes (50 mL)</td>
			<td style="width:309px;">VWR</td>
			<td style="width:123px;">525-0155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Crystal Violet</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:123px;">C6158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">DAPI</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:123px;">D9542</td>
			<td>Toxic and carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">DEAE-Dextran</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:123px;">D9885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:309px;">ThermoFisher Scientific (Gibco)</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:309px;">ThermoFisher Scientific (HyClone))</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Formaldehyde</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:123px;">F8775</td>
			<td>Toxic and carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine</td>
			<td style="width:309px;">ThermoFisher Scientific (Gibco)</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td style="width:309px;">ThermoFisher Scientific (Invitrogen)</td>
			<td>11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonessential amino acids</td>
			<td style="width:309px;">ThermoFisher Scientific (Gibco)</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:309px;">ThermoFisher Scientific (Gibco)</td>
			<td>31985070</td>
			<td>Transfection medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Pan-flavivirus E protein mAb 4G2</td>
			<td style="width:309px;">BEI Resources</td>
			<td style="width:123px;">NR-50327</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Paraformaldehyde</td>
			<td style="width:309px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">15710-S</td>
			<td>Toxic and carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">PBS</td>
			<td style="width:309px;">ThermoFisher Scientific (Gibco)</td>
			<td style="width:123px;">14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td style="width:309px;">ThermoFisher Scientific (Gibco)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">ProLong Gold Antifade Reagent</td>
			<td style="width:309px;">ThermoFisher Scientific (Invitrogen)</td>
			<td style="width:123px;">P10144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Triton-X-100</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:123px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Vectastain ABC Kit</td>
			<td style="width:309px;">Vector Laboratories Inc</td>
			<td style="width:123px;">PK-4010</td>
			<td>Avidin/biotin-based peroxidase kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Vero Cells</td>
			<td style="width:309px;">ATCC</td>
			<td style="width:123px;">CCL-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">ZIKV E Protein mAb 1176-56</td>
			<td style="width:309px;">BioFront Technologies</td>
			<td style="width:123px;">BF-1176-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">3. Equipment</td>
			<td style="width:309px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Agarose Gel Electrophoresis System</td>
			<td style="width:309px;">Bio-Rad</td>
			<td style="width:123px;">1704468</td>
			<td>Other commercial sources are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Class II Biosafety CO2 Cabinet</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td>Other commercial sources are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Desktop Refrigrated Centrifuge</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Fluorescence Microscope</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">High-Speed Refrigrated Centrifuge</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">MicroPulser Electroporator</td>
			<td style="width:309px;">Bio-Rad</td>
			<td style="width:123px;">1652100</td>
			<td>Other machines are acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">SimpliAmp Thermal Cycler</td>
			<td style="width:309px;">ThermoFisher Scientific (Applied Biosystems)</td>
			<td style="width:123px;">A24811</td>
			<td>Other machines are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Vortexer</td>
			<td style="width:309px;">Varies</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

rescue of recombinant zika virus from a bacterial artificial chromosome cdna clone

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial&#160;propagation.

The protocol described here allows for the generation of stable ZIKV full-length cDNA infectious clones using a BAC to minimize the toxicity problems associated with several flaviviral sequences. Efficient recovery of infectious rZIKV from the BAC cDNA clone can be easily achieved after the transfection of susceptible Vero cells (Figure 2). Using this protocol, we have generated a stable full-length cDNA clone of the ZIKV strain RGN32 by sequentially cloning four overlapping cDNA fragments into the BAC plasmid pBeloBAC1134 using conventional cloning methods and unique restriction sites present in the viral genome (Figure 2). The full-length cDNA clone was flanked at the 5&#39;-end by the human CMV promoter to allow the expression of vRNA in the nucleus of transfected cells, and at the 3&#39;-end by the HDV RZ followed by the BGH polyadenylation and termination sequences, to produce RNAs containing the exact 3&#39;-end of the viral genome (Figure 2). The stability in bacteria of the generated BAC cDNA clone, together with its easy manipulation using standard recombinant DNA technologies, highlights the potential of the BAC cDNA approach for the rapid and reliable generation of stable full-length cDNA clones of ZIKV and other flaviviruses or positive-stranded RNA viruses with unstable viral genomes.
Once the BAC cDNA clone was assembled (Figure 2), infectious virus could be easily recovered after the direct transfection of susceptible Vero cells with the BAC cDNA clone using cationic liposomes(Figure 3). This cDNA-launched system allowed the intracellular expression of the capped vRNA, allowing the recovery of infectious viruses without the need for an in vitro transcription step. Using this approach, we were able to rescue rZIKV-RGN with titers higher than 106 PFU/mL at 5 days posttransfection (Figure 4A). In addition, the rescued virus induced a clear CPE (Figure 4B), generated homogeneous plaques of about 2 mm in size (Figure 4C), and its identity was confirmed by sequencing (data not shown) and immunofluorescence analysis using the mAb specific for ZIKV E protein, 1176-56 (Figure 4D). In vitro data indicate that the recovered rZIKV-RGN replicated efficiently in Vero cells and to levels compared to a natural ZIKV isolate32 (data not shown). Overall, these results demonstrate that infectious rZIKV can be rescued from full-length cDNA clones assembled in a BAC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59537/59537fig1.jpg" /> 
Figure 1: ZIKV genome organization and virion structure. (A) Genome organization: ZIKV contains a positive single-stranded RNA that is translated as a single polyprotein. The translated polyprotein was cleaved by viral (arrows) and host (diamonds) proteases to produce the structural proteins capsid (C, blue), matrix (M, brown), and envelop (E, green), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The 5&#39; and 3&#39; untranslated regions (UTRs) at the end of the viral genome are indicated with black lines. (B) Virion structure: ZIKV virions were decorated with the E and M proteins, anchored in a lipid bilayer with an icosahedral-like structure. Under the viral envelop was the viral nucleocapsid composed of the C protein associated with the viral genomic RNA. This figure has been adapted from &#193;vila-P&#233;rez et al.18. <a href="https://www.jove.com/files/ftp_upload/59537/59537fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59537/59537fig2.jpg" /> 
Figure 2: Assembly of the ZIKV full-length infectious cDNA clone in a BAC. (A) Schematic representation of the pBeloBAC11 BAC:The regulatory genes parA, parB, parC, and repE,the F-factor replication origin (OriS), the chloramphenicol resistant gene (Cmr), and the lacZ gene are indicated. The relevant restriction sites used to assemble the infectious ZIKV cDNA clone are underlined. (B) Assembly of ZIKV full-length infectious cDNA clone into the pBeloBAC11 BAC:Four overlapping DNA fragments (Z1-Z4), covering the entire ZIKV genome (Figure 1) and flanked by the indicated restriction sites, were generated by chemical synthesis and sequentially cloned into pBeloBAC11 to generate the infectious ZIKV cDNA clone pBAC-ZIKV. The full-length ZIKV infectious cDNA was assembled under the control of the human cytomegalovirus immediate-early promoter (CMV) and flanked at the 3&#39;-end by the HDV ribozyme (RZ) and the bovine growth hormone (BGH) termination and polyadenylation sequences. The acronyms for the viral genes and regulatory elements are as described in Figure 1. <a href="https://www.jove.com/files/ftp_upload/59537/59537fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59537/59537fig3.jpg" /> 
Figure 3: Workflow to generate rZIKV from the BAC cDNA clone. Vero cells at 90% of confluence were transfected in monolayer with the ZIKV full-length infectious cDNA clone pBAC-ZIKV (Figure 2)&#160;using cationic liposomes. At 4-6 days posttransfection, when CPE was evident, tissue culture supernatants containing rZIKV were collected and evaluated for the presence of virus (Figure 4) and used for viral amplification in Vero cells. <a href="https://www.jove.com/files/ftp_upload/59537/59537fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59537/59537fig4.jpg" /> 
Figure 4: Recovery and in vitro&#160;characterization of rZIKV. (A) Rescue of infectious rZIKV from the BAC cDNA clone:&#160;Vero cells at 90% of confluence (6-well plate format, triplicates) were mock-transfected or transfected with 4 &#181;g/well of pBAC-ZIKV (Figure 3), and at the indicated days posttransfection, virus titers in tissue culture supernatants were determined by plaque assay (PFU/mL). The error bars indicate standard deviations from three different transfection experiments.The dotted black line indicates the limit of detection (50 PFU/mL). (B) Viral CPE:Vero cells at 90% confluence (6-well plate format, triplicates) were mock-infected (top) or infected (MOI of 0.5 PFU/cell) with rZIKV, and at 48 h postinfection, the presence of CPE was evaluated by light microscopy. Scale bars = 100 &#181;m. (C) Viral plaque assay and immunostaining: Vero cells at 90% confluence (12-well plate format) were infected with rZIKV, and at 72 h postinfection, viral plaques were visualized by crystal violet staining (left) or by immunostaining (right) using the pan-flavivirus E protein mAb 4G2. Scale bars = 5 mm. (D) Immunofluorescence:Vero cells at 90% confluence (24-well plate format, triplicates) were infected (MOI of 0.5 PFU/cell) with rZIKV and, at 48 h postinfection, analyzed by immunofluorescence using the mAb 1176-56, specific for ZIKV E protein. Cell nuclei were stained with DAPI. A representative merged image of ZIKV-infected Vero cells is shown. The white square in the top right represents an enlarged image of ZIKV-infected Vero cells. Scale bars = 100 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/59537/59537fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone at JoVE.com

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

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines ...

rescue-recombinant-zika-virus-from-bacterial-artificial-chromosome

Research

JoVE Journal

Immunology and Infection

26.5K Views.  University of Rochester Medical Center. The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

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

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...