Generation of recombinant Orfvirus using an enhanced green fluorescent protein reporter gene as a selectable marker

Generation of rORFVs using neo/gusA selection

Construction of recombinant cassettes with the novel transfer vector pSPV-EGFP

To shorten the time needed to generate rORFVs, the novel transfer vector pSPV-EGFP (Figure 1A) was constructed using the EGFP reporter gene to replace the neo/gusA cassette [7]. To compare the efficiency of plaque purification between pZIPPY-neo/gusA [7] and pSPV-EGFP, two ORFV proteins, ORFV113 and ORFV116, were used for this study. Two transfer vectors, pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF, were constructed using pSPV-EGFP. These cassettes encode EGFP under the control of the early-late VV 7.5 promoter, which allows for isolation and purification of recombinant viruses by using fluorescent signals under a fluorescent microscope. This novel experimental strategy streamlines the procedure. The experimental steps in this new strategy include: (i) generating the OV-IA82Δ113 and OV-IA82Δ116 after transfection of the pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF vectors into ovine fetal turbinate (OFTu) cells previously inoculated with OV-IA82; (ii) visual monitoring of infected cultures by fluorescent microscopy to assess the level of rORFV infection; (iii) determine the optimal time (usually 24 hours during the second round of limited dilution) to harvest cells for plaque assay (Figure 1B).

Generation of OV-IA82Δ113 and OV-IA82Δ116 using EGFP selection

When the pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF vectors were transfected into OFTu cells that had been previously exposed to OV-IA82 with 1.0 multiplicity of infection (MOI), spots of brightly EGFP-positive cells appeared within 6 h, and strong fluorescent signal was observed under fluorescent microscope after 24 h. Cytopathic effect (CPE) of OFTU cells infected with OV-IA82Δ113 or OV-IA82Δ116 (Figure 2A) was observed after 24 h during the second round of limited dilution, and abundant EGFP expression was observed in infected OFTu cells. EGFP-positive cells in 96-well plates with dilutions higher than 1:10, 000 were harvested for further plaque purification. After two rounds of limited dilutions, three different individual recombinant clones for each protein were selected to run 2 or 3 times plaque assays to remove parent virus contamination. Plaques with a fluorescent signal can be identified in OFTu cells after 12 h pi, and individual green plaques were picked under fluorescent microscope at 36 h pi (Figure 2B). This demonstrates that EGFP selection substantially shortens the time than conventional protocols such as neo/gusA selection.

Identification of OV-IA82Δ113 and OV-IA82Δ116

To determine the purity of OV-IA82Δ113 and OV-IA82Δ116, PCR was conducted using primers designed from internal coding regions of ORFV113 and ORFV116 or EGFP (Table 2). The results demonstrated that the EGFP gene was amplified from three different clones of OV-IA82Δ113 (Figure 3A) and OV-IA82Δ116 (Figure 3B), but not from OV-IA82 or mock infected cells. In contrast, the ORFV113 and ORFV116 genes were amplified from OV-IA82 infected cells only (Figure 3).

Primer	Sequence	Restriction enzyme*
GFPFw1	5'-AACTTAGAATTCGCCACCATGGTGAGCAAGGGCGA-3'	EcoR I
GFPRv1	5'-ATCAATGCGGCCGCTTACTTGTACAGCTCGTCCA-3'	Not I
GFPintrFw2	5'-GACGTAAACGGCCACAAGTT-3'
GFPintrRv2	5'-ACTGGGTGCTCAGGTAGTGG-3'
GFPseqFw3	5'-CGACCACTACCAGCAGAACA-3'
GFPseqRv3	5'-AAGTCGTGCTGCTTCATGTG-3'
113LFFw1	5'-AGGCCTCTAAGCTTCAGGTTCCGGCTTCAGATGCGCGT-3'	Hind III
113LFRv1	5'-ATTCGCGTCGACCACCAACACTTCCATTGTTGCGGC-3'	Sal I
113RFFw2	5'-TCTTATGCGGCCGCGAGCCGCCGATGCAGATCGAGGTA-3'	Not I
113RFRv2	5'-ATTCGCAGATCTTCGATCGCCAGTGCGCGGCGCATG-3'	Bgl II
113intrFw3	5'-CGCCGTAATATGCTTAACCGGAGC-3'
113intrRv3	5'-CGGACCGTGTTGGTCGTTGGGTCT-3'
113seqFw4	5'-TTAGCTTCCTTGTTTTTATC-3'
113seqRv4	5'-GTCCTTCGGGTCAGAGTCC-3'
116LFFw1	5'-GCCTCTACTAGTAGGAAGTGGCCTCGCCGACCACGA-3'	Spe I
116LFRv1	5'-ATTCGCGTCGACGTGGATGTCTCTAAGGTTCAATAC-3'	Sal I
116RFFw2	5'-TCTTATGCGGCCGCCTACCACTGGTACCAGCACCTCCT-3'	Not I
116RFRv2	5'-ATTCGCAGATCTGGCGCTACAGGCGTCCTGCAGGAA-3'	Bgl II
116intrFw3	5'-GAACAACACGTCAACCGATG-3'
116intrRv3	5'-AGGTGTGGGTTGACTTCCAG-3'
116seqFw4	5'-GTCGAGCAGATGTTCATGGA-3'
116seqRv4	5'-ATGCTGCACTTCCTGGAGAT-3'
001intrFw1	5'-CTCGGTGACCTGCCTGAC-3'
001intrRv1	5'-CTCGCGCACGTCGTAGAT-3'

* Restriction enzyme sites are underlined in prime sequences

To verify ORFV113 and ORFV116 genes were successfully deleted from OV-IA82 via the transfer vector pSPV-EGFP and the EGFP gene was incorporated into the OV-IA82 genome by homologous recombination; Southern blot analysis was performed on genomic DNA isolated from OV-IA82, OV-IA82Δ113 and OV-IA82Δ116. The ORFV113 probe detected the ORFV113 gene in OV-IA82 (Figure 4A lower panel, lane 1) but not in OV-IA82Δ113 (Figure 4A lower panel, lane 2). The ORFV001 gene is the only double copy located at the left and right ends of the viral genome. The ORFV001 probe detected the same pattern in both OV-IA82 and OV-IA82Δ113, indicating that the deletion of ORFV113 does not affect the left and right ends of the viral genome. The EGFP probe detected the EGFP gene in OV-IA82Δ113 but not in OV-IA82 (data not shown). Similar results were obtained when the ORFV116 probe was applied (Figure 4B). Sequencing of left and right flanking regions of the deleted gene, which were involved in the recombination, confirmed the integrity of parental virus sequences in the mutant virus (data not shown).

Deletion of ORFV113 and ORFV116 do not affect ORFV replication in vitro

To investigate the infectivity of the gene-deleted mutant viruses, a one-step growth curve was conducted. Replication properties of OV-IA82Δ113, OV-IA82Δ116, and OV-IA82 were compared after infection of OFTu cells. No significant differences in growth were detected between the mutant and wild type viruses (P > 0.05) in OFTu cells, indicating that ORFV113 and ORFV116 are not essential for ORFV replication in OFTu cells (Figure 5). The data also demonstrates that expression of EGFP in OV-IA82Δ113 and OV-IA82Δ116 does not affect ORFV growth and replication in vitro. Also shown in our previous report [23], the exogenous expression of EGFP did not affect the viral phenotype and the biological function in ORFV002 revertant virus (OV-IA82Rv002GFP) which was constructed by using the pSPV-EGFP system. Taken together, the pSPV-EGFP vector could be widely used for recombination and foreign gene expression studies.

Cells and viruses

Primary ovine fetal turbinate (OFTu) cells were kindly provided by Dr. Howard D. Lehmkuhl (USDA) and were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin,100 U/ml penicillin, 50 μg/ml gentamicin and 2 mM L-glutamine.

ORFV IA82 (OV-IA82) strain was isolated from lamb nasal secretion during an orf outbreak at the Iowa Ram Test station in 1982, and has been fully sequenced [5]. Low passage OV-IA82, which is fully virulent, was used to construct deletion mutants OV-IA82Δ113 (Figure 4A) and OV-IA-82Δ116 (Figure 4B) and was used here in all procedures involving infections with wild type virus, PCR amplification and cloning of viral genes. The virus was propagated in OFTu cells and was purified by sucrose gradient ultra-centrifugation before storage at -80°C for use.

Construction of pSPV-EGFP

To obtain the EGFP gene sequence, two primers were designed, sense-primer GFPFw1 and antisense-primer GFPRv1. An EcoR I site was added to the sense-primer and Not I to the antisense-primer (Table 2). Thermostable high fidelity DNA polymerase and dNTPs were obtained from Invitrogen (Carlsbad, CA, USA). Polymerase chain reaction (PCR) was performed in 50 μl thin-wall polypropylene tubes (Denville Scientific, NJ, USA) using a PTC-200 DNA Engine thermal cycler (Bio-Rad, Shelton, CA, USA) under the following conditions: denaturation at 96°C for 2 min followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 54°C for 45 s, elongation at 72°C for 1 min, and a final extension at 72°C for 7 min. PCR products were visualized in 1% agarose gel with ethidium bromide staining, cut out, and purified using a Qiagen gel extraction kit (Qiagen, Valencia, CA, USA). The products were then cloned into vector pZIPPY-neo/gus [7], which had been linearized with EcoRI and NotI (New England Biolabs, Ipswich, MA, USA) to generate the novel transfer vector pSPV-EGFP (Figure 1A). The plasmid was propagated in Escherichia coli strain Top10 (Invitrogen, Carlsbad, CA, USA). DNA sequencing of the pSPV-EGFP was done to ensure that the correct construct had been obtained. The complete DNA sequence of the pSPV-EGFP was deposited in GenBank under the accession number: GU062789.

Construction of recombinant cassettes

DNA preparations and manipulations were performed using standard methods as described by Sambrook et al. [27] or followed instructions from manufactures. Two recombination cassettes were constructed by PCR amplifying ORFV113 and ORFV116 left and right flanking regions from OV-IA82 genome using primers listed in Table 2. The PCR products were cloned into the pSPV-EGFP MCSs to generate pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF. In addition, to compare the efficiency between the pSVP-EGFP and pZIPPY-neo/gus, seven recombinant cassettes were also constructed using pZIPPY-neo/gus vector. The ORFV002, ORFV012, ORFV024, ORFV113, ORFV116, ORFV120 and ORFV121 left and right flanking regions from OV-IA82 genome were amplified and cloned into the pZIPPY-neo/gus MCSs, flanking the neomycin resistance (neo) and β-glucuronidase reporter (gus) genes under control of the VACV VV7.5 and modified H5 promoters, respectively [7]. The resulting recombinant vectors were named pZNG-ORFV002 [23], pZNG-ORFV012, pZNG-ORFV024 [24], pZNG-ORFV113, pZNG-ORFV116, pZNG-ORFV120, pZNG-ORFV121 [25]. All sub-cloning procedures were confirmed by using restriction enzymes and DNA sequence analysis.

Generation of recombinant viruses

OV-IA82Δ113 and OV-IA82Δ116 viruses were constructed by infecting OFTu cells (in T25 flasks) at a multiplicity of infection (MOI) of 1.0 with wild type OV-IA 82 for 3 hours and subsequently transfecting the cells with 10 μg of pSVP-113LF-EGFP-113RF and pSVP-116LF-EGFP-116RF transfer vectors by standard in vivo recombination protocols [28, 29]. Transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Viruses were harvested 48 h pi by scraping infected/transfected OFTu cells into sterile 15 ml conical tubes. The cell suspensions were vortexed, frozen/thawed 3 times, and then centrifuged at 1000 rpm, for 10 min at 4°C (Eppendorf Centrifuge 5810R, 15 amp version, Hamburg, Germany). The supernatant (viruses) were transferred into 2 ml cryogenic vials (Corning, NY, USA) and stored at -80°C for future use.

In order to select and purify recombinant viruses, limited dilution and plaque assays were performed. 3 ×10⁴ of OFTu cells per well were seeded into 96-well plates one day before infection. On the second day, the cells were infected with serial 10-fold dilutions of viruses from 10^-1 to 10^-11 (900 μl 1 × MEM + 100 μl of virus). The cells were examined under a fluorescent microscope (Leica DMI4000B inverted microscope) 12 h after inoculation. The cells exhibiting fluorescent signal with dilutions higher than 1:10, 000 were collected for the second round of limited dilution. Typically, after 24 h pi, strong green fluorescent signal was observed in 96-well plates. The cell suspensions were harvested and frozen/thawed as above. The supernatant (viruses) was stored at -80°C for use.

After 2 or 3 rounds of limited dilution, plaque assays were carried out for further purification. 7 × 10⁵ of OFTu cells per well were passed into 6-well plates one day before infection. On the next day, cells were infected with serial dilutions of viruses from 10^-1 to 10^-6. The viruses were allowed to absorb to the cells for 1 h at 37°C, in 5% CO₂ incubator. The medium was removed and then the cells were overlaid with 3 ml of MEM containing 5% of FBS and 0.5% low melting point agarose (Sea Kem^® GTG^®, Lonza, Rockland, ME, USA). Plaques with a GFP signal were visualized and picked 24 or 36 h pi. A minimum of 2 or 3 plaques were picked for further plaque purification. The last plaque purified recombinant viruses were expanded in 35 × 10 mm dishes; genomic DNA was extracted using QIAamp DNA blood kit (QIAGEN, Germany). PCR was performed to screen for the wild type virus contamination using internal primers from ORFV113 and ORFV116 coding regions (Table 2). PCR conditions were the same as described above except that the annealing temperature was based on the Tm values of each primer. Southern blotting was further carried out to confirm that double homologous recombination had occurred.

For comparison studies, OV-IAΔ002, ORFVΔ012, OV-IA-82Δ024, OV-IA82Δ113, OV-IA82Δ116, OV-IA82Δ120 and OV-IA82Δ121 were also constructed using neo/gus selection following the procedures as described above. The only difference is that those recombinant viruses produced blue plaques (gusA activity) in the presence of X-gluc (Clontech, Palo Alto, CA, USA). Blue plaques were picked at day 4 or day 5 pi.

Southern blots

Genomic DNA was extracted from OV-IA82, OV-IA82Δ113 and OV-IA82Δ116 viruses, respectively. One microgram of viral DNA was digested with AflII or EcoRI (New England Biolabs, Inc.), electrophoresed via a 1.0% agarose gel and transferred via capillary action to a nylon membrane (Bio-Rad, CA) using standard protocols [30]. The membranes were hybridized with specific digoxin (Roche, Mannheim, Germany) labeled probes, which were generated specifically for the ORFV113, ORFV116, ORFV001 and EGFP genes. The probes for ORFV113, ORFV116, EGFP and ORFV001 were amplified by using internal primers (Table 2), same primers applied for PCR detection as described above. Amplicons were purified and labeled with digoxin followed the manufacture's instruction (Roche, Mannheim, Germany). To determine the ends of recombinant viruses, the same blots were stripped with N, N-dimethylformamide (Sigma, St. Louis, MO, USA) at 50-60°C for 1 h, until color had been removed completely. During the stripping process, the solution needed to be changed every 20 min. The stripped membrane was incubated in stripping buffer (0.2 M NaOH and 0.1% sodium dodecyl sulfate) for 30 min at 37°C, and re-hybridized with a specific digoxin labeled probe that were generated toward the ORFV001 gene cassette. The NBT/BCIP Detection kit (Roche, Mannheim, Germany) for nonradioactive color development was used for hybridization analysis of Southern blots according to the manufacture's instructions.

Sequencing analysis

To determine homologous recombination, three plaques, which were confirmed by PCR detection using ORFV113 and ORFV116 internal primers (113intrFw3 and 113intrRv3 or 116intrFw3 and 116intrRv3, table 2) were applied for sequencing analysis. Two pairs of primers (Table 2), 113seqFw4 and GFPseqRv3; GFPseqFw3 and 113seqRv4 or 116seqFw4 and GFPseqRv3; GFPFw3 and 116seqRv4, were utilized to amplify the regions involved in recombination. PCR conditions were the same as described above except that the annealing temperature was employed based on the Tm values of each primer. The PCR products were cloned into pCR2.1 TA cloning plasmid (Invitrogen, CA) for sequencing analysis using an Applied Biosystems PRISM 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Virus expansion, titration, and growth curve

Based on the Southern blot and sequence analysis, one clone of OV-IA82Δ113 and OV-IA82Δ116 were used for expansion. Confluent monolayers of OFTu cells in four T150 cm² flasks were infected at MOI of 1. The viruses were harvested when all cells were rounded but still attached (about 3 or 4 days pi). The cell suspensions were prepared as above. The supernatants were dispensed into 2 ml cryogenic vials (1 ml/vial) and stored at -80°C for future use.

96-well plates of 90% confluent monolayers OFTu cells were prepared as above. Rows of cells were infected with serial 10-fold dilutions of wild type and mutant viruses of 10^-1 to 10^-9, with one row for control (no virus). One column was used for one dilution. CPE was read at 2 d pi, and the final read was carried out at 7 d pi. The median tissue culture infected dose (TCID₅₀) per ml was calculated using a spreadsheet.

One step growth curves were conducted as our pervious reports [23–25]. OFTu cells were prepared in 35 × 10 mm dishes (7 × 10⁵ cells/dish) one day before the experiment. The cells were counted on the second day and were infected at a MOI of 10 with wild type and mutant virus. Virus was harvested at 0, 6, 12, 24, 36, 48 h pi and titrated. The growth curves were plotted as titer (log₁₀ TCID₅₀/ml) versus time course (h) pi. Data are presented as the mean ± standard deviation from three independent experiments. P values were determined by using the unpaired two-tailed Student's t-test. Statistical significance was set at the 95% confidence level.