This project was funded by the National Institutes of Health (AI114851) to SR.

1. Generating the recombination vector
<ol>
	<li>Design primers to generate the selection cassette. Design each individual amplicon with overlapping sequences with neighboring amplicons and the vector to facilitate isothermal enzymatic assembly of DNA molecules, also called Gibson assembly, using any of several online primer design tools. 
	NOTE: This protocol can also be completed using traditional restriction endonuclease-based cloning methods. In that case, design primers with the appropriate restriction sites rather than with overlapping sequences.</li>
	<li>Using the primers designed in step 1.1, PCR amplify the following elements in order from 5&#8217; to 3&#8217; (Figure 1): ~500 nucleotides of the VACV genomic region 5&#8217; of E3L (5&#8217; arm), EGFP or the gene of interest, ~150 nucleotides from the VACV genomic region immediately 3&#8217; of E3L (short 3&#8217; arm), a synthetic early/late poxvirus promoter25, the mCherry-E3L fusion gene, and ~500 nucleotides from the VACV genomic region 3&#8217; of E3L including the short 3&#8217; arm (long 3&#8217; arm).
	<ol>
		<li>In a PCR tube, add the reagents in the following order for each amplicon: 17 &#956;L of DNase free water, 1.2 &#181;L of each primer (initial concentration = 10 &#956;M, final concentration = 0.5 &#956;M), 5 &#181;L of 5x PCR reaction buffer, template DNA (10 ng for amplicons amplified from plasmids: EGFP and E/L promoter-mCherry-E3L cassette; 100 ng for amplicons amplified from viral genomic DNA: 5&#8217; and 3&#8217; arms), and 0.5 &#181;L of DNA polymerase. Adjust the volume of water added for a final reaction volume of 50 &#956;L. 
		NOTE: The concentration of template DNA should be empirically determined, but we generally start with 10 ng/reaction.</li>
		<li>Place the tube(s) in a thermocycler, and melt the DNA at 98 &#176;C for 30 s, and then use 25 rounds of a three-step PCR protocol: 98 &#176;C for 5 s, 55 &#176;C for 10 s, and 72 &#176;C for 1 min. 
		NOTE: Determine the melting temperature based on the manufacturer&#8217;s suggested Tm for each primer set. Determine the appropriate extension time based on the length of each amplicon (1 minute/kb).</li>
	</ol>
	</li>
	<li>Visualize the amplification products on a 1% agarose gel. Add 10 &#956;L of each DNA product and 2 &#956;L of loading buffer to each well, and run at 8 V/cm for 1 h.</li>
	<li>Gel purify each amplicon using a DNA gel extraction kit and manufacturer&#8217;s protocol. Elute the amplicons from the column by adding 50 &#956;L of DNase free water and immediately centrifuging.</li>
	<li>Linearize the pUC19 cloning vector using EcoRI endonuclease digestion. To a tube, add 1 &#956;g of pUC19, water to a volume of 17 &#956;L, 2 L of reaction buffer, and 1 &#956;L (20 units) of EcoRI. Incubate at 37 &#176;C for 1 h.
	<ol>
		<li>Visualize the amplification products on a 1% agarose gel run at 8 V/cm for 1 h. Excise the band from the gel, and purify the product using the DNA gel extraction kit as described in step 1.4.</li>
	</ol>
	</li>
	<li>Ligate all of the individual, gel purified amplicons and the linearized vector using a master mix kit.
	<ol>
		<li>To a PCR tube, add 0.2 pmol of linearized pUC19 and each amplicon (5&#8217; arm, EGFP, short 3&#8217; arm, E/L promoter-mCherry-E3L cassette, 3&#8217;arm). Add DNase free water to a final volume of 10 &#956;L, and then add 10 &#956;L of DNA assembly master mix. Incubate samples at 50 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Transform chemically competent E. coli with 2 &#956;L of the assembled product from step 1.6 as previously described26,27. Plate 100 &#956;L of the transformed cells on LB agarose plates containing 100 &#956;g/mL ampicillin. Incubate the plates overnight at 37 &#176;C.</li>
	<li>Pick well-isolated colonies and transfer individual colonies to tubes containing Luria broth with 100 &#956;g/mL ampicillin. Incubate the tubes overnight at 37 &#176;C while shaking at 225 rpm.</li>
	<li>Isolate the plasmids from the overnight culture using a plasmid miniprep kit. Check the concentration and purity of the DNA using a spectrophotometer. An A260/A280 ratio between 1.8 and 2.0 is acceptable.</li>
	<li>Submit the plasmids for Sanger sequencing to determine whether the desired cloning product is correct. Store the DNA at -20 &#176;C.</li>
</ol>
2. Generating the recombinant virus
<ol>
	<li>Infect a confluent monolayer of suitable cells with the virus to be recombined at a multiplicity of infection of 1.0 (MOI = 1.0) in a 6-well plate. Incubate the infected cells at 37 &#176;C and 5% CO2 for 1 h. Then aspirate the medium and replace it with fresh DMEM. Incubate the infected cells at 37 &#176;C and 5% CO2. 
	NOTE: For replication competent viruses such as a vaccinia virus that lacks K3L22, a cell line such as European rabbit kidney cell line RK13 (ATCC #CCL-37) or BSC-40 is appropriate. However, for replication deficient viruses, such as the virus described in this paper lacking both PKR antagonists E3L and K3L, a complementing cell line expressing these two genes in trans or PKR knock-down or knock-out cells are required.</li>
	<li>Transfect the infected cells with 500 ng of the vector generated and validated in step 1.10 using a commercially available transfection reagent following the manufacturer&#8217;s protocol. Incubate the cells at 37 &#176;C and 5% CO2 for 48 h. 
	NOTE: If using a vaccinia virus lacking both E3L and K3L, PKR-mediated selective pressure will drive selection of recombined viruses and maintain expression of the mCherry-E3L fusion protein in these cells. If desired, it should also be possible to PCR amplify only the insert to use for transfection instead of the whole plasmid.</li>
	<li>48 hours post-infection, harvest the infected monolayer. In some cases, the cells can be harvested by pipetting, but if they are still tightly adhered, harvest them with a cell scraper. Freeze-thaw the cells three times, and then sonicate the lysates for 15 s at 50% amplitude. Store this lysate at -80 &#176;C until ready to use.</li>
	<li>Serially 10-fold dilute the lysate harvested in step 2.3 from 10-1 to 10-6 by adding 120 &#956;L of the lysate to 1080 &#956;L of DMEM (10-1), and then adding 120 &#956;L of this dilution to 1080 &#956;L of DMEM (10-2), and repeating this process four more times. Add 1 mL of each dilution to an individual, confluent well of a PKR competent cell line, in this case RK13 cells.
	<ol>
		<li>Incubate the infected cells at 37 &#176;C and 5% CO2 for 1 h. Then aspirate the medium and replace it with fresh DMEM Incubate the infected cells at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>24 to 48 hours post-infection, identify recombinant viruses by fluorescence microscopy. Plaques from recombinant viruses express red fluorescence due to integration the mCherry-E3L fusion gene (Figure 2). If a virus devoid of PKR inhibitors was used initially, all plaques will contain recombinant virus.</li>
	<li>Plaque purify recombinant viruses three times on RK13 cells. After the final round of plaque purification, all plaques should express red fluorescence.</li>
	<li>Infect a confluent 6-well plate of RK13 cells expressing the VACV PKR inhibitors E3L and K3L (RK13+E3L+K3L cells28) with the plaque-purified red fluorescing virus from step 2.6. Aim for approximately 50-100 plaques per well. 
	NOTE: These cells provide the VACV PKR antagonists in trans and alleviate the PKR-mediated selective pressure to maintain the mCherry-E3L fusion gene, thus promoting &#8220;scarless&#8221; generation of the recombinant virus.</li>
	<li>Identify collapsed viruses by fluorescence microscopy using an EVOS2 microscope, and a GFP filter cube (Excitation: 470/22, Emission: 525/50) and a RFP filter cube (Excitation: 531/40, Emission: 593/40). 
	NOTE: The frequency at which the mCherry-E3L fusion gene is lost is approximately 2.5% (Table 2). If EGFP is not included as a marker gene, plaques from mutant viruses that have lost the mCherry-E3L fusion gene will be colorless.</li>
	<li>Plaque purify green-only (VC-R4) or colorless plaques (E3L) three times on RK13+E3L+K3L cells. Ensure that no plaques fluoresce red.</li>
	<li>Confirm the loss of mCherry-E3L and the presence of the expected mutation by PCR and Sanger sequencing. 
	NOTE: If the gene or mutation of interest does not have PKR inhibitory activity, recombinant viruses must be grown on RK13+E3L+K3L cells or an equivalent PKR-inhibited or PKR deficient cell line (Figure 3).</li>
</ol>

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The authors declare no competing financial interests.

Here we present a variation of a transient marker selection strategy 32 to generate recombinant vaccinia viruses without retaining foreign DNA in the final recombinant virus. Our strategy uses selective pressure mediated by the host antiviral protein PKR rather than other forms of selective pressure such as antibiotics. The use of host antiviral genes eliminates the possibility of chemically induced phenotypic changes in the cells, or increased risk of mutation due to selection drugs. Furthermore, unlike with drug selection, there is no lag phase for our approach, because PKR is expressed constitutively in all cells. Secondary visual selection based on mCherry expression also improves the specificity of this method by ensuring that only plaques expressing the transgene are picked during the first phase, and is equally efficient as a negative selective marker while selecting mature recombinant viruses that have lost the mCherry-E3L gene.
The most critical steps for this recombination strategy are the generation of the appropriate recombination vector, and appropriate plaque purification to ensure that the selected virus is clonal. In this paper we suggest &#8220;Gibson assembly&#8221; to generate the recombination vector. This strategy is extremely efficient and allows assembly of all the fragments comprising the recombination vector in a single day. However, because the short 3&#8217; arm and the long 3&#8217; arm share identical sequences, these fragments have the potential to be joined together during the cloning reaction, and some vectors may not contain the mCherry-E3L cassette. In our experience this is rare but confirming the structure of the vector after cloning is necessary. We have also generated recombination vectors for this strategy using traditional endonuclease and ligase methods. This strategy avoids the problem described above but can be more labor intensive. Plaque purification is generally straightforward and is primarily reliant on using appropriate permissive cells for the initial recombination, PKR-competent cells for initial plaque purification to ensure that only recombinant viruses can replicate, and then permissive cells again to facilitate intramolecular recombination and loss of the selectable marker. Close attention to cell lines is therefore critical for the successful and efficient application of this strategy.
In this study, we demonstrate the use of this method to generate a VACV recombinant deleted for both PKR antagonists E3L and K3L and expressing EGFP under control of the E3L promoter. Going forward, this virus will serve as an efficient background for future recombinant viruses, as it is incapable of replicating in PKR competent cells. Therefore, there will be strong PKR-mediated selective pressure to drive the mCherry-E3L recombination cassette into progeny virions while at the same time essentially preventing replication of non-recombinant virus. Furthermore, the loss of EGFP by uptake of the recombination cassette is a useful secondary selection marker to ensure picked plaques are not co-infected with a non-recombinant virus. We observed rates of recombination consistent with previously reported rates for VACV, but the visual fluorescent markers increase the efficiency of generating recombinant viruses by ensuring that increasing the likelihood that the appropriate recombinant viruses are selected. Our observation of colorless plaques after two rounds of selection on PKR-competent cells, presumably due to the increased length of identical sequence between E3L and the mCherry-E3L marker gene, suggests that the rate of mCherry-E3L loss may be &#8220;tuned&#8221; by increasing or decreasing the length of the 3&#8217; short arm. The primary limitation of this technique is the use of PKR as the selective pressure for recombinants. The most efficient use of this recombination strategy is generating these viruses in a background lacking PKR antagonists. However, the colorimetric selection marker allows this recombination strategy to be used even without the selection mediated by PKR, simply by plaque purifying mCherry-expressing plaques. While the lack of PKR-mediated selective pressure will reduce the efficiency of the first screening step, the percentage of mCherry expressing plaques is still high enough that color-based selection is viable. Thus, this method can be used to insert nearly any gene into the poxvirus genome.
As demonstrated by the insertion of EGFP, with this approach, any gene can be rapidly inserted into the E3L locus under control of the native promoter, provided that PKR null cells or complimenting cell lines are used for downstream experiments if the transgene is not a PKR antagonist. This strategy, combined with the VC-R4 virus that we report here, adds a new and potent method to rapidly and reliably generate recombinant vaccinia viruses using host-mediated selective pressure and visual identification of recombinants early in the process.

Vaccinia virus (VACV) was instrumental for the first successful eradication of a human pathogen, variola virus (VARV), from nature. Ever since the extermination of variola virus, poxviruses including VACV have continued to be useful therapeutic viruses for both human and animal medicine. For example, a VACV-based rabies virus vaccine has been very effective in preventing transmission of sylvatic rabies in Europe1 and the United States2. More recently, recombinant poxviruses expressing a variety of anti-tumor molecules (e.g., single-chain antibodies or human erythropoietin) have seen encouraging success as oncolytic agents3,4,5. VACV is particularly attractive as a vector because it is readily amenable to genetic manipulation, possesses a broad host range, and it is stable under a variety of conditions, allowing easy transportation and vaccine viability in the field6,7. While multiple techniques have been developed to generate recombinant VACV for laboratory experiments and vaccine generation, current strategies to generate these viruses have notable limitations.
Because of the utility of VACV, multiple strategies to generate recombinant viruses have been developed. The first strategy employs homologous recombination to introduce a cassette including the transgene and a selectable marker gene such as an antibiotic resistance gene. The cassette is flanked by two ~500 nucleotides (nt) or larger arms directing the gene to a specific site in the viral genome, which is then stably integrated by double crossover events8,9,10. This strategy is rapid and efficient; however, it results in extra genetic material in the form of the marker gene that may produce unexpected effects. Furthermore, there is a practical upper limit to the number of transgenes that can be introduced limited by the number of unique selectable markers available. Transient dominant selection (TDS) strategies have addressed this issue by facilitating the generation of &#34;scarless&#34; recombinant viruses11. Using this strategy, a plasmid containing a mutant VACV gene and a selectable marker gene are integrated into the viral genome, but without additional flanking VACV DNA. This approach results in transient integration of the entire plasmid and duplication of the VACV gene as a result of integration by a single crossover event. This intermediate is stable as long as it is maintained under selection pressure, permitting enrichment of this construct. When selection is removed, the VACV duplication enables a second crossover event that results in the removal of the plasmid and subsequent formation of either the wild type (wt) or recombinant virus in an approximate 50:50 ratio. While TDS generates recombinant viruses without requiring the stable introduction of foreign DNA, multiple virus clones must be screened for the expected mutation by sequencing analysis, a potentially time consuming and costly step.
Here, we present an approach to generating recombinant poxviruses combining the best aspects of each of these approaches, similar to an approach that has been described for the replication incompetent modified vaccinia Ankara12,13,14. This strategy combines visual and host range selection to rapidly generate recombinant viruses by double crossover events, and subsequently eliminate the selectable marker gene by homologous recombination. This approach permits the rapid generation of mutants mediated by homologous recombination, with the &#34;scarless&#34; nature of TDS approaches, while not requiring a subsequent screening step to distinguish wild type and mutant viruses. Our method also uses host range selection in place of antibiotic selection, eliminating the risk of chemically induced phenotypic changes in the cell line. For this approach, we have chosen to use the host antiviral protein kinase R (PKR) as the selective agent to generate recombinant VACV. PKR is expressed as an inactive monomer in most cell types15. Upon binding double-stranded RNA (dsRNA) at the N-terminal dsRNA-binding domains, PKR dimerizes and is autophosphorylated16. This active form of PKR phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF2), ultimately inhibiting delivery of initiator methionyl-tRNA to the ribosome, thereby preventing intracellular translation and broadly inhibiting the replication of many virus families17,18.
In response to the broad and potent antiviral activity of PKR, many viruses have evolved at least one strategy to prevent PKR activation. Most poxviruses express two PKR antagonists, encoded by the E3L and K3L genes in VACV, which antagonize PKR through two distinct mechanisms19. E3 prevents PKR homodimerization by binding double-stranded RNA20,21, while K3 acts as a pseudosubstrate inhibitor by binding directly to activated PKR and thereby inhibiting interaction with its substrate eIF2&#945;22. Importantly, these two PKR antagonists do not necessarily inhibit PKR from all species. For example, the K3 homolog from the sheeppox virus strongly inhibited PKR from sheep, whereas the sheeppox E3 homolog did not show considerable PKR inhibition23,24. In this study, we present a method to use PKR-mediated selective pressure combined with fluorescence selection to generate a VACV recombinant deleted for E3L and K3L (VC-R4), which cannot replicate in PKR competent cells derived from diverse species. This recombinant virus provides an excellent background for rapid generation of recombinant viruses expressing genes under control of the native E3L promoter.

<table><tbody><tr><td>2X-Q5 Master Mix</td><td>NEB</td><td>M0492L</td><td>High-fidelity polymerase used in PCR</td></tr><tr><td>Ampicillin</td><td>ThermoFisher Scientific</td><td>11593027</td><td>Bacterial selective agent</td></tr><tr><td>Disposable Cell Scrapers</td><td>ThermoFisher Scientific</td><td>08-100-242</td><td>Cell scraper to harvest infected cells</td></tr><tr><td>EVOS FL Auto 2 Cell imaging system</td><td>ThermoFisher Scientific</td><td>AMAFD2000</td><td>Fluorescent microscope</td></tr><tr><td>EVOS Light Cube, GFP</td><td>ThermoFisher</td><td>AMEP4651</td><td>GFP Cube</td></tr><tr><td>EVOS Light Cube, RFP</td><td>ThermoFisher</td><td>AMEP4652</td><td>RFP Cube</td></tr><tr><td>GenJet</td><td>SignaGen Laboratories</td><td>SL100489</td><td>Transfection reagent</td></tr><tr><td>Luria Bertani (LB) Broth</td><td>Gibco</td><td>10855021</td><td>Bacterial growth medium</td></tr><tr><td>Monarch DNA gel extraction kit</td><td>NEB</td><td>T1020L</td><td>Gel purification kit used to purify amplicons and linearized vectors</td></tr><tr><td>Monarch Plasmid Miniprep kit</td><td>NEB</td><td>T1010L</td><td>Miniprep kit ussed to purify plasmids</td></tr><tr><td>NanoDrop One</td><td>ThermoFisher Scientific</td><td>ND-ONE-W</td><td>Spectrophotometer used to measure RNA and DNA concentration</td></tr><tr><td>NEBuilder Master Mix</td><td>NEB</td><td>E2621L</td><td>Isothermal enzymatic assembly kit used to generate the recombination vector</td></tr><tr><td>Q500 Sonicator</td><td>Qsonica</td><td>Q500-110</td><td>Sonicator for virus lysates</td></tr><tr><td>RK13 cells</td><td>ATCC</td><td>CCL-37</td><td>Rabbit kidney cells</td></tr><tr><td>VWR Multiwell Cell Culture plates</td><td>VWR</td><td>10062-892</td><td>Cell culture plates</td></tr></tbody></table>

rapid, seamless generation of recombinant poxviruses using host range and visual selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

We used the procedure diagrammed in Figure 1 to generate a VACV lacking both PKR antagonists E3L and K3L, by replacing E3L with EGFP in a virus already deleted for K3L (vP872). Figure 2 shows red fluorescent plaques in PKR competent RK13 cells indicative of viral expression of mCherry-E3L, as well as EGFP expressed in RK13+E3L+K3L cells confirming the loss of E3L and collapse of the mCherry-E3L selection marker. Figure 3 confirms that this recombinant virus, VC-R4, lacking both PKR antagonists cannot replicate in PKR competent RK13 cells, while the parent virus, vP872 expressing E3L, is replication competent. To confirm that this inability to replicate in RK13 cells was only due to the loss of E3L, we replaced EGFP in VC-R4 with E3L, to generate a revertant virus using the same selection protocol. Figure 3 also validates that this revertant virus replicates as efficiently as vP872 in RK13 cells. Interestingly, colorless plaques consistent with collapse of the mCherry-E3L selection marker were identified prior to selection in RK13+E3+K3 cells that are generally required to select &#8220;scarless&#8221; recombinants, likely because of the extended sequence identity between the mCherry-E3L recombination cassette and the E3L gene being inserted into VC-R4. Therefore, to determine the efficiency of recombination and the rate of collapse we elected to produce viruses expressing the poxvirus PKR antagonist K3L to avoid the problem of early collapse23. Figure 4 indicates the appearance of colorless plaques (arrowheads) after infection of RK13+E3L+K3L cells. Table 1 shows the results of three independent experiments, where on average 12.6% of progeny virions had undergone recombination with the transfected plasmid, similar to previously reported frequencies29,30,31. Table 2 details the frequency of colorless plaques relative to total plaques in RK13+E3L+K3L cells, demonstrating the rate of collapse and loss of the mCherry-E3L selection marker occurred at a frequency of approximately 1.8%.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61049/61049fig01.jpg" /> 
Figure 1: Diagram of p837-GOI-mCherry-E3L as well as the host-range and visual recombination strategy.&#160;(A) 5&#8217; arm (black) and 3&#8217; arm (grey) flank the E3L locus (brown) in VACV. (B) In p837-GOI-mCherry-E3L, these arms flank a cassette containing the gene of interest (GOI), in this case EGFP, (green) separated from an mCherry-E3L (red) fusion gene under control of the synthetic early/late poxvirus promoter25 blue) by a short 3&#8217; arm (grey). These external arms drive homologous recombination between VACV and the p837-GOI-mCherry-E3L. Black arrowheads indicate the sites of the overlapping primers used to generate this plasmid by Gibson cloning. (C) When PKR selective pressure is removed, viruses that have undergone intramolecular recombination between the short and long 3&#8217; arms can be selected. (D) Resulting in a virus (VC-R4) containing only the gene of interest in the E3L locus. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61049/61049fig01large.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/61049/61049fig02.jpg" /> 
Figure 2: Fluorescent micrographs of (top) a recombinant virus plaque 24 hours after recombination with p837-GOI-mCherry-E3L expressing both mCherry (left) and EGFP (right) in RK13 cells.&#160;(Bottom) Micrograph of a recombinant virus plaque 48 hours after PKR-mediated selective pressure has been removed in RK13++ cells, expressing EGFP (right) but not mCherry (left). The scale bar indicates 650 &#956;m for all panels. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61049/61049fig02large.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/61049/61049fig03.jpg" /> 
Figure 3: VC-R4 cannot replicate in PKR competent cells.&#160;The indicated cell lines were infected with vP872 (blue), VC-R4 (green), or VC-R4+E3L (magenta) at MOI = 0.1. 48 hours post-infection the infected cells were harvested and titered by serial dilution on RK13+E3L+K3L cells. Titers are reported in PFU/mL, errors bars represent the standard deviation of three replicate experiments. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61049/61049fig03large.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/61049/61049fig04.jpg" /> 
Figure 4: Loss of mCherry-E3L expression in RK13+E3L+K3L cells.&#160;Overlay of fluorescent and phase contrast micrographs of VC-R4+K3L-mCherry-E3L infected RK13+E3L+K3L cells. Three plaques no longer express mCherry (circles) due to collapse of the selection cassette yielding VC-R4+K3L. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61049/61049fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Experiment 1</td>
			<td>Experiment 2</td>
			<td>Experiment 3</td>
		</tr>
		<tr>
			<td>Red plaques (RK13)</td>
			<td>30</td>
			<td>11</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Total plaques (RK13+E3L+K3L)</td>
			<td>225</td>
			<td>64</td>
			<td>249</td>
		</tr>
		<tr>
			<td>Recombination Rate</td>
			<td>13.30%</td>
			<td>17.20%</td>
			<td>7.20%</td>
		</tr>
	</tbody>
</table>
Table 1: Recombination frequency of VACV with the p837-K3L-mCherry-E3L plasmid.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Experiment 1</td>
			<td>Experiment 2</td>
			<td>Experiment 3</td>
		</tr>
		<tr>
			<td>Total plaques (RK13+E3L+K3L)</td>
			<td>115</td>
			<td>44</td>
			<td>210</td>
		</tr>
		<tr>
			<td>Colorless plaques (RK13+E3L+K3L)</td>
			<td>3</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Recombination Rate</td>
			<td>2.60%</td>
			<td>2.30%</td>
			<td>0.50%</td>
		</tr>
	</tbody>
</table>
Table 2: Frequency of mCherry-E3L loss from VC-R4+K3L-mCherry-E3L in RK13+E3+K3 cells.

Watch this Scientific Journal Video about Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection at JoVE.com

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a ...

rapid-seamless-generation-recombinant-poxviruses-using-host-range

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.2K Views.  School of Medicine, University of California Davis. This is a method to generate “scarless” recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

Video: Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...