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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

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 ...

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

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

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Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses