The authors would like to thank Kristen Bullard-Feibelman, Milena Veselinovic and Claudia R&#252;ckert for their assistance in characterizing the clone-derived virus. This work was supported in part by grants from the National Institute of Allergy and Infectious Diseases, NIH under grants AI114675 (BJG) and AI067380 (GDE).

1. Transform and Recovery of Infectious Clone Plasmids
<ol>
	<li>Transform both plasmids (separately) using a commercial transformation protocol (e.g., NEB 5 Minute Transformation Protocol) with some modifications. Both plasmids contain a gene encoding for ampicillin resistance, therefore use ampicillin or carbenicillin for selection. Carbenicillin is preferred, as it is more stable.
	<ol>
		<li>Remove cells (See Materials Table) from -80 &#176;C freezer and thaw on ice for 5-10 min. Prewarm lysogeny broth (LB) (containing 10 g/L NaCl and 25 &#181;g/mL carbenicillin; called LB-carb) plates and the broth with catabolite repression (SOC (without antibiotics) - coming with the cells) at 37 &#176;C.</li>
		<li>Add between 100 pg-10 ng in 1 &#181;L of plasmid DNA to a tube containing 50 &#181;L of competent cells; mix gently by flicking the tube.</li>
		<li>Incubate tubes on ice for 5 min.</li>
		<li>Heat shock tubes at 42 &#176;C for 30 s in a water bath. Return to ice for 2 min immediately following heat shock.</li>
		<li>Pipette 950 &#181;L of room temperature SOC into each tube and mix by pipetting. Spread 100 &#181;L of the bacterial mixture onto a LB-carb plate and incubate overnight at 37 &#176;C (roughly 12-14 h).</li>
	</ol>
	</li>
	<li>Remove plates from 37 &#176;C incubator after 12-14 h incubation. Place plates in a dark drawer at room temperature to allow colonies to continue to grow (roughly 8-9 h).</li>
	<li>Using 5 mL of Terrific broth (TB, containing 25 &#181;g/mL carbenicillin, called TB-carb), grow 5-10 small colonies for each plasmid overnight at 30 &#176;C in a bacterial shaker (roughly 14-16 h). 
	NOTE: Small colonies are an indicator that the plasmid is intact; colonies with plasmids containing deletions, rearrangements or mutations (herein called instability events) will grow faster, and thus be bigger. Therefore, one should attempt to pick the smallest colonies on the plate, specifically not selecting the largest colonies present. Some medium size colonies may also be selected for completeness. The lower temperature used reduces the probability that the cells will overgrow (OD600 of greater than 0.6-0.8). As most researchers perform overnight growth, it allows for more control and reduced chance of overgrowth.</li>
	<li>Check liquid cultures for turbidity (roughly OD600 of 0.6-0.8). Place any turbid tubes at 4 &#176;C and allow other tubes to become turbid by growing for longer. 
	NOTE: Do not grow bacteria to OD600 values greater than recommended, as plasmid instability events may occur.</li>
	<li>Extract plasmid DNA from bacteria with a commercial plasmid miniprep kit (See Materials Table). Elute in 15 &#181;L of pre-heated (to 70 &#176;C in a heating block) elution buffer. Allow the elution buffer to incubate on the column for 5 min before centrifugation. 
	NOTE: Preserve at least 1 mL of this starter culture at 4 &#176;C for further use in the next step.</li>
	<li>Digest 10 &#181;L of the recovered plasmids to assess whether plasmid instability events occurred during culture. 
	NOTE: Digest p1 with SalI/NheI and p2 with BamHI/HindIII at 37 &#176;C for 1 h. Confirm the presence of the correct bands following digestion (Figure 2a) by gel electrophoresis and proceed to step 2. If preparing plasmid DNA by rolling circle amplification (RCA), reserve some of the miniprep eluate to serve as a template.</li>
</ol>
2. Preparation of Sufficient Plasmid DNA for Ligation
NOTE: Ligation and subsequent electroporation requires a sufficient amount of plasmid DNA that must be generated via either maxiprep or RCA. While maxiprep is the traditionally used approach, RCA offers the advantage of not requiring bacteria, thus removing the potential for plasmid-induced toxicity in bacteria that can result in instability events.
<ol>
	<li>Perform the following maxiprep procedure.
	<ol>
		<li>For each plasmid that demonstrated the correct restriction enzyme digestion pattern in the previous section, add 500 &#181;L of starter culture to 1 L of TB-Carb (25 &#181;g/mL carbenicillin) broth and shake overnight at 30 &#176;C (roughly 14-16 h, roughly OD600 of 0.6-0.8). 
		NOTE: Do not allow the culture to grow over this OD600 value. This will potentially result in instability events within the plasmids.</li>
		<li>Harvest the bacteria and perform maxipreps per manufacturer&#39;s protocol (See Materials Table).</li>
	</ol>
	</li>
	<li>Using the saved miniprep from step 1.6, perform the following for the RCA procedure.
	<ol>
		<li>Transfer 1 &#181;L of plasmid DNA to a tube containing 50 &#181;L of sample buffer.</li>
		<li>Heat-denature the sample at 95 &#176;C for 3 min, and then incubate at 4 &#176;C until ready to use.</li>
		<li>Prepare the commercial amplification premix (See Materials Table). Combine 50 &#181;L of reaction buffer with 2 &#181;L of enzyme mix in a tube set on ice. 
		NOTE: The amplification premix should be kept on ice until ready for use. It is convenient to prepare a master mix by combining sufficient reagents for the required number of amplification reactions. Any unused premix must be discarded.</li>
		<li>Transfer 50 &#181;L of prepared amplification premix to the denatured sample.</li>
		<li>Incubate the reaction tubes at 30 &#176;C for 18 h.</li>
		<li>Incubate the tubes at 65 &#176;C for 10 min to inactivate the &#248;29 DNA polymerase.</li>
		<li>Store the reaction tubes at 4 &#176;C or -20 &#176;C until ready for use. 
		NOTE: At this point, plasmid DNA prepared via both methods should be quantified (using absorbance at 260 nm) and the ZIKV genome should be Sanger sequenced entirely to confirm no instability events (deletions and rearrangements) occurred during preparation.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer Name</td>
			<td>Sequence (5&#39; - 3&#39;)</td>
		</tr>
		<tr>
			<td>ZIKV PRVABC59 1For.</td>
			<td>AGTTGTTGATCTGTGTGAATCAGAC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 632For.</td>
			<td>GCCCTATGCTGGATGAGG</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 692Rev.</td>
			<td>GGTTCCGTACACAACCCAAGTTG</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 1313Rev.</td>
			<td>CTCCCTTTGCCAAAAAGTCCACA</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 1201For.</td>
			<td>CCAACACAAGGTGAAGCCTAC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 1663For.</td>
			<td>GCAGACACCGGAACTCCACACT</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 2008For.</td>
			<td>CAGATGGCGGTGGACATGC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 2350Rev.</td>
			<td>GTGAGAACCAGGACATTCCTCC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 2605For.</td>
			<td>TACAAGTACCATCCTGACTCCC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 3499Rev.</td>
			<td>GCCTTATCTCCATTCCATACCA</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 3961Rev.</td>
			<td>TTGCCAACCAGGCCAAAG</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 4132For.</td>
			<td>CATTTGTCATGGCCCTGG</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 4561Rev.</td>
			<td>CTATTGGGTTCATGCCACAGAT</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 4665For.</td>
			<td>GACCACAGATGGAGTGTACAGAGT</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 5189Rev.</td>
			<td>AAGACAGTTAGCTGCTTCTTCTTCAG</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 5219For.</td>
			<td>GAGAGTTCTTCCTGAAATAGTCCGTGA</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 6086Rev.</td>
			<td>CTTGCTTCAAGCCAGTGTGC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 6119For.</td>
			<td>GCCTCATAGCCTCGCTCTATCG</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 6721Rev.</td>
			<td>CCATTCCAAAGCCCATCTTCCC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 6769For.</td>
			<td>CCAGCCAGAATTGCATGTGTCC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 7209Rev.</td>
			<td>ATCATTAGCAGCGGGACTCCAA</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 7343For.</td>
			<td>GTTGTGGATGGAATAGTGGT</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 8243For.</td>
			<td>TGCCCATACACCAGCACTATGA</td>
		</tr>
		<tr>
			<td>ZIKV PRVABC59 8893Rev.</td>
			<td>TGCATTGCTACGAACCTTGTTG</td>
		</tr>
		<tr>
			<td>ZIKV conserved 9133For.</td>
			<td>AGCCCTTGGATTCTTGAACGAGGA</td>
		</tr>
		<tr>
			<td>ZIKV PRVABC59 9673Rev.</td>
			<td>AACGCAATCATCTCCACTGACT</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 9686For.</td>
			<td>GATGATAGGTTTGCACATGCC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 10321Rev.</td>
			<td>GTCCATGTACTTTTCTTCATCACCTAT</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 10455For.</td>
			<td>CAGGAGAAGCTGGGAAACC</td>
		</tr>
		<tr>
			<td>ZIKV Conserved 10621Rev.</td>
			<td>CAGATTGAAGGGTGGGGAAGGTC</td>
		</tr>
	</tbody>
</table>
Table 1: Primers for sequencing cDNA clones. Plasmids can be sequenced entirely with the primers listed. Primers marked as &#34;conserved&#34; indicate that they are likely able to sequence/amplify all genotypes of ZIKV. Primers labelled as &#34;PRVABC59&#34; are specific to this strain but may also work for other Asian genotype strains and possibly other genotypes.
3. Preparation of In Vitro Transcribed RNA
<ol>
	<li>Set up digestion reaction of either maxiprep or RCA prepared DNA.
	<ol>
		<li>For p1 digestion, mix 10 &#181;L of 10x reaction buffer, 3 &#181;L of ApaLI, 3 &#181;L of BamHI-HF, 3 &#181;L of rSAP or CIP, and 3.3 &#181;g of p1 DNA. Add ddH2O to 100 &#181;L.</li>
		<li>For p2 digestion, mix 10 &#181;L of 10x reaction buffer, 3 &#181;L of ApaLI, 3 &#181;L of EcoRI-HF, and 13.3 &#181;g of p2 DNA. Add ddH2O to 100 &#181;L.</li>
		<li>Incubate at 37 &#176;C for 1-2 h.</li>
		<li>Purify using a commercial gel and PCR clean-up kit (See Materials Table). Elute in 30 &#181;L of elution buffer pre-heated to 70 &#176;C in a heating block (incubate the column for 5 min before spinning). 
		NOTE: Keep 1 &#181;L to run on a gel later.</li>
	</ol>
	</li>
	<li>Prepare ligation reaction.
	<ol>
		<li>Combine 10 &#181;L of 10x T4 DNA ligation reaction buffer, 3 &#181;L of T4 DNA ligase (400 U/&#181;L), 29 &#181;L of purified p1, and 29 &#181;L purified p2. Add ddH2O to 100 &#181;L.</li>
		<li>Incubate at room temperature for 2 h or 16 &#176;C overnight.</li>
		<li>Purify using a commercial gel and PCR clean-up kit (See Materials Table). Elute in 10 &#181;L of elution buffer pre-heated to 70 &#176;C in a heating block (incubate column for 5 minutes before spinning). 
		NOTE: Keep 1 &#181;L to run on a gel later.</li>
	</ol>
	</li>
	<li>Run undigested plasmids, digested plasmids, and ligation on an agarose gel. The ligation product should have a higher molecular weight band (around 11 kb) than either digested plasmid alone, indicating the ligation was successful (Figure 2b).</li>
	<li>Set up T7 in vitro transcription reaction.
	<ol>
		<li>Combine 10 &#181;L 2x ARCA/NTP Mix, 2 &#181;L of T7 RNA polymerase mix, and 8 &#181;L of purified ligation reaction for a final volume of 20 &#181;L. 
		NOTE: The ARCA included in the mix is a cap analog that is exclusively incorporated in the correct orientation. Standard cap analogs can result in only ~50% of the transcripts having a cap in the correct orientation.</li>
		<li>Incubate for 4 h at 37 &#176;C.</li>
		<li>Measure RNA concentration via a UV spectrophotometer. Optionally, run a denaturing agarose gel to confirm the length of the RNA transcript.</li>
	</ol>
	</li>
</ol>
4. Rescue of Infectious Virus by Electroporation
<ol>
	<li>days prior to electroporation, prepare 1 T182 flask ofVero cells (between T150 and T182 is acceptable; grown in DMEM + 10% Fetal bovine serum (FBS)) per construct to be recovered. Include 1 T182 as a mock transfection control. Cells should be used at 70-80% confluency.</li>
	<li>When cells are ready, place 12 mL/reaction of DMEM + 15% FBS + 10 mM HEPES in a water bath heated to 37&#176;C.</li>
	<li>Detach cells by trypsinization with a standard protocol, and recover cells in media with 10% FBS to inactivate trypsin; centrifuge cells at 150 x g for 5 min at 4 &#176;C.</li>
	<li>Wash cells in 1x phosphate-buffered saline (PBS), pelleting cells at 150 x g for 5 min at 4 &#176;C. Repeat this step twice for a total of three washes.</li>
	<li>Resuspend cells in 200 &#181;L of RPMI 1640 + 10 mM HEPES (sterile) per # of samples. Transfer 200 &#181;L of cell suspension to a sterile tube. Cells should be ~0.5 x 107 cells/mL.</li>
	<li>Add 20 &#181;L of water (mock neg. control) or 20 &#181;L of RNA (preferably 5-10 &#181;g) produced in step 3.4 to each set of cells. Mix gently by flicking the tube and transfer the contents of the tube to a 2 mm gap electroporation cuvette.</li>
	<li>Set an electroporator to 170 V LV, omega (resistance) at 0 and 950 &#181;F (roughly to 625 V/cm and 20 ms time constant for other machines). Set resistance to the lowest available setting, either 0 or &#8734; if available.</li>
	<li>Pulse once and immediately add 600 &#181;L of DMEM +15 %FBS +10 mM HEPES that was warmed in step 4.2. Rinse inside of cuvette thoroughly to remove all cells. Add cells to a T75 tissue culture flask containing 10 mL of pre-warmed media. Remove additional media from cuvette by using the dropper that is included with the cuvette. Be careful to maintain sterility.</li>
	<li>Swirl flask to mix media and cells and place in 37 &#176;C incubator.</li>
	<li>Check the next day for cell viability, and do so every day until 50-75% cell death is observed. Cell death is observed by comparing to the mock electroporated control. 
	NOTE: ZIKV cytopathic effect (CPE) is quite pronounced in Vero cells, resulting in rounded up cells that detach and float in the culture medium. We have observed a range of 8-10 days as being ideal for harvest.</li>
	<li>Harvest supernatant in a conical tube and centrifuge to remove cellular debris at &#62;3,000 x g for 10 min at 4 &#176;C.</li>
	<li>Remove clarified supernatant to a new tube and supplement with a final concentration of 20% FBS (from 100% stock). Additionally, add sterile HEPES at a final concentration of 10 mM as a buffering agent to preserve virus infectivity while frozen (from a 1 M sterile stock).</li>
	<li>Mix virus by vortexing and aliquot into screw cap vials. Store at -80 &#176;C for future use.</li>
</ol>
5. Titration of Recovered Virus
<ol>
	<li>Seed the appropriate number of 6- or 12-well plates of Vero cells the day before titration.</li>
	<li>Remove virus from the freezer and make serial 10-fold dilutions in DMEM+ 2%FBS in tubes or 96-well plates. 
	NOTE: The expected titer from recovered clones is between 1 x 106 and 5 x 107 PFU/mL.</li>
	<li>Add 200 or 400 &#181;L of each dilution in duplicate to a well of a 12- or 6-well plate, respectively.</li>
	<li>Place plates in 37 &#176;C incubator and rock every 15 min, for a total of ~1-1.5 h adsorption period.</li>
	<li>Remove viral inoculum and add 1 mL of (12-well) or 2 mL (6-well) DMEM-Tragacanth overlay to each well. 
	NOTE: It is possible to use agarose, agar, methylcellulose or other overlay media here.</li>
	<li>Incubate cells in 37 &#176;C incubator for 5 days. The time for proper plaque formation will vary depending on the overlay used.</li>
	<li>To fix cells, remove plates from incubator and decant overlay gently into disinfectant.</li>
	<li>Add sufficient 20% ethanol/0.1% crystal violet (CV) to each well to cover and swirl to mix. 
	NOTE: Many other fixatives exist and can be used here. Additionally, if using an agarose or agar overlay, a second overlay can be used with in conjunction with neutral red to visualize plaques without fixation. 20% ethanol has shown to be effective in inactivating enveloped viruses in previous studies; do not use this amount for non-enveloped viruses as they will not be inactivated19.</li>
	<li>Incubate plates for 30-60 min at room temperature (in a class II biosafety cabinet); discard CV (per local regulations) and wash plates with tap water.</li>
	<li>Allow plates to dry, and manually count plaques. 
	NOTE: Reference 20 can be used as an additional reference for performing plaque assays.</li>
</ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+amplifiable+multicopy+DNA+cloning+vehicles+derived+from+the+P15A+cryptic+miniplasmid.">Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.</a> J Bacteriol. 134 (3), 1141-1156 (1978).</li><li>Weger-Lucarelli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+Recombinant+Virus+Generated+from+a+New+World+Zika+Virus+Infectious+Clone.">Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.</a> J Virol. 91 (1), (2017).</li><li>Roberts, P. L., Lloyd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+inactivation+by+protein+denaturants+used+in+affinity+chromatography.">Virus inactivation by protein denaturants used in affinity chromatography.</a> Biologicals. 35 (4), 343-347 (2007).</li><li>Baer, A., Kehn-Hall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+concentration+determination+through+plaque+assays:+using+traditional+and+novel+overlay+systems.">Viral concentration determination through plaque assays: using traditional and novel overlay systems.</a> J Vis Exp. (93), e52065 (2014).</li><li>Weger-Lucarelli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+Recombinant+Virus+Generated+from+a+New+World+Zika+Virus+Infectious+Clone.">Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.</a> J Virol. , (2016).</li><li>Grubaugh, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Drift+during+Systemic+Arbovirus+Infection+of+Mosquito+Vectors+Leads+to+Decreased+Relative+Fitness+during+Host+Switching.">Genetic Drift during Systemic Arbovirus Infection of Mosquito Vectors Leads to Decreased Relative Fitness during Host Switching.</a> Cell Host Microbe. 19 (4), 481-492 (2016).</li></ol>

The authors have nothing to disclose.

Here we describe a method for the recovery of a bipartite infectious cDNA clone system for ZIKV. Previously described clones for ZIKV suffer from either attenuation or require the addition of introns, making plasmids larger and preventing rescue in insect cells. Infectious virus can be recovered using the two-plasmid clone system in either mammalian or insect cells (data not shown). In addition, virus recovered from this system behaves similarly to wild-type virus in several cell lines, in an immunocompromised mouse mode...

Zika virus (ZIKV; Family Flaviviridae: Genus Flavivirus) is a mosquito-borne flavivirus that arrived in Brazil in 2013-14 and was subsequently associated with a massive outbreak of febrile illness that spread throughout the Americas1. In addition, ZIKV has been linked with severe disease outcomes, such as Guillain-Barr&#233; syndrome in adults and microcephaly in fetuses and neonates2. Little was known about ZIKV before its rapid spread in the western hemisphere. This included a lack of molecular tools, thus hindering mechanistic research. Molecular tools for viruses, such as infectious cDNA clones, facilitate vaccine and antiviral therapeutic development, and allow for the assessment of viral genetic factors related to differential viral pathogenesis, immune response and viral evolution.
Flavivirus infectious clones are known to be highly unstable in bacteria due to cryptic prokaryotic promoters present in their genomes3. Several approaches have been used to ameliorate this problem; including insertion of tandem repeats upstream of viral sequences4, mutation of putative prokaryotic promoter sequences5, splitting the genome into multiple plasmids6, low-copy number vectors (including bacterial artificial chromosomes)7,8 and insertion of introns in the viral genome9. One full-length system without modifications has been described for ZIKV; however, this clone appeared to be attenuated in cell culture and in mice10. Other groups have engineered introns into the ZIKV genome, allowing for disruption of unstable sequences in bacteria that can be spliced out in mammalian cells in vitro for the production of infectious virus11,12. Additionally, a PCR-based system entitled Infectious-Subgenomic-Amplicons has been used successfully to rescue the prototype MR766 strain of ZIKV13. The approach described here requires no foreign sequences, but rather disrupts the genome at the region of high instability by using multiple plasmids, which has previously been used successfully with yellow fever6, dengue14,15 and West Nile viruses16. Furthermore, the addition of the hepatitis D ribozyme sequence at the viral genomic termini facilitates the creation of an authentic 3&#39; end without the need for the addition of a linearization site. Additionally, both plasmids are constructed in a low-copy number vector (pACYC177, ~15 copies per cell) to mitigate any residual toxicity17. The virus recovered displays a growth profile comparable to parental virus in in vitro growth curves in 8 cell lines comprising a variety of cell-types derived from both mammals and insects, and has exhibited identical pathogenic profiles in mice and infection, dissemination and transmission rates in mosquitoes18.
Herein, we detail a protocol describing how to grow the infectious clone plasmids, generate full-length viral RNA (the viral genome) in vitro, and recover infectious virus in cell culture. First, we describe the propagation of plasmids in bacteria or bacteria-free amplification using rolling circle amplification (RCA). Next, we show how the two plasmids are digested and then subsequently ligated together to generate full-length virus. Finally, we describe the production of transcribed RNA and its subsequent electroporation into Vero cells, followed by titration of recovered virus (Figure 1). The approach described is rapid, allowing for recovery of infectious virus stocks in 1-2 weeks.

<table><tbody><tr><td>NEB Stable CompetentE. coli</td><td>New England BioLabs</td><td>C3040H</td><td></td></tr><tr><td>Carbenicillin, Disodium Salt</td><td>various</td><td></td><td></td></tr><tr><td>Zyppy Plasmid Miniprep Kit</td><td>Zymo Research</td><td>D4036</td><td></td></tr><tr><td>ZymoPURE Plasmid Maxiprep Kit</td><td>Zymo Research</td><td>D4202</td><td></td></tr><tr><td>SalI-HF</td><td>New England BioLabs</td><td>R3138S</td><td>20,000 units/ml</td></tr><tr><td>NheI-HF</td><td>New England BioLabs</td><td>R3131S</td><td>20,000 units/ml</td></tr><tr><td>ApaLI</td><td>New England BioLabs</td><td>R0507S</td><td>10,000 units/ml</td></tr><tr><td>EcoRI-HF</td><td>New England BioLabs</td><td>R3101S</td><td>20,000 units/ml</td></tr><tr><td>BamHI-HF</td><td>New England BioLabs</td><td>R3136S</td><td>20,000 units/ml</td></tr><tr><td>HindIII-HF</td><td>New England BioLabs</td><td>R3104S</td><td>20,000 units/ml</td></tr><tr><td>illustra TempliPhi 100 Amplification Kit</td><td>GE Healthcare Life Sciences</td><td>25640010</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-up</td><td>Macherey-Nagel</td><td>740609.5</td><td></td></tr><tr><td>Shrimp Alkaline Phosphatase (rSAP)</td><td>New England BioLabs</td><td>M0371S</td><td>1,000 units/ml</td></tr><tr><td>Alkaline Phosphatase, Calf Intestinal (CIP)</td><td>New England BioLabs</td><td>M0290S</td><td>10,000 units/ml</td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202S</td><td>400,000units/mL</td></tr><tr><td>HiScribe T7 ARCA mRNA Kit</td><td>New England BioLabs</td><td>E2065S</td><td></td></tr><tr><td>Vero cells</td><td>ATCC</td><td>CCL-81</td><td></td></tr><tr><td>ECM 630 High Throughput Electroporation System</td><td>BTX</td><td>45-0423</td><td>Other machines are acceptable.</td></tr><tr><td>LB Broth with agar (Miller)</td><td>Sigma</td><td>L3147</td><td>Can be homemade as well.</td></tr><tr><td>Terrific Broth</td><td>Sigma</td><td>T0918</td><td>Can be homemade as well.</td></tr><tr><td>Petri Dish</td><td>Celltreat</td><td>229693</td><td></td></tr><tr><td>Culture Tubes</td><td>VWR International</td><td>60818-576</td><td></td></tr><tr><td>T75 flasks</td><td>Celltreat</td><td>229340</td><td></td></tr><tr><td>T182 flasks</td><td>Celltreat</td><td>229350</td><td></td></tr><tr><td>1x PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>DMEM with L-glutamine and 4.5 g/L glucose</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlas Biologicals</td><td>FP-0500-A</td><td></td></tr><tr><td>Tragacanth Powder</td><td>MP Bio</td><td>MP 104792</td><td></td></tr><tr><td>Crystal Violet</td><td>Amresco</td><td>0528-1006</td><td></td></tr><tr><td>Ethanol Denatured</td><td>VWR International</td><td>BDH1156-1LP</td><td></td></tr><tr><td>6 well plate</td><td>Celltreat</td><td>229106</td><td></td></tr><tr><td>12 well plate</td><td>Celltreat</td><td>229111</td><td></td></tr><tr><td>Sequencing Oligos</td><td>IDT</td><td>see table 1</td><td></td></tr><tr><td>Qubit 3.0</td><td>ThermoFisher</td><td>Qubit 3.0</td><td>other methods are acceptable.</td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>ThermoFisher</td><td>Q32850</td><td>other methods are acceptable.</td></tr><tr><td>Qubit RNA HS Assay Kit</td><td>ThermoFisher</td><td>Q32852</td><td>other methods are acceptable.</td></tr><tr><td>Class II Biosafety Cabinet</td><td>Varies</td><td>N/A</td><td>This is necessary for live-virus work.</td></tr></tbody></table>

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.

The protocol described here allows for the recovery of infectious clone-derived Zika virus. Manipulating the two-plasmid infectious clone system is straightforward when performed with care, as compared to full-length versions which are highly unstable (data not shown). After digestion and ligation of the two distinct pieces, capped RNA is produced using in vitro transcription with T7 polymerase which is then electroporated into Vero cells (Figure 1)....

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

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 ...

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JoVE Journal

Immunology and Infection

9.6K Views.  Colorado State University. This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Video: Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious 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 ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

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 ...

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 ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Neuroscience

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 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 ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

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

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Generation of Recombinant Influenza Virus from Plasmid DNA

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

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

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

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

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

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

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication