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CR.pIX cells from the muscovy duck (Jordan et al. 2009b) and the virus isolate MVA-CR19 (Jordan et al. 2013a) have been described previously.
CR.pIX cells were maintained in adherent format in DMEM:F12 medium supplemented with 5% bovine serum (γ-irradiated, Gibco 26140-079), or in suspension cultures in CD-U4 medium (GE Healthcare #G3321 or Biochrom #F9185) supplemented with 10 ng/mL LONG-R3IGF (Sigma, USA). Both media were also supplemented with 2 mmol/L GlutaMAX I (Life Technology, USA). Infection and propagation of MVA was performed in 1:1 mixtures of CD-U4 and CD-VP4 (Merck-Millipore #F9127) as described previously, usually with 2 × 106 cells/mL, MOI of 0.01-0.1, and harvest 48 or 72 h post infection (p.i.) (Jordan et al. 2011). Suspension cultures were maintained in a shaking incubator (HT Multitron Cell, Infors AG, Switzerland) on a rotating platform with amplitude of 5 cm and rotation speed of 180 min-1. The CO2 atmosphere was set to 8% and temperature to 37 ℃. All culture vessels, shake tubes (Tubespin 50, TPP Techno Plastic Products AG, Switzerland) or baffled shake flasks (Corning, USA), were equipped with 0.2 µm filtered lids to allow gas exchange. Suspension culture volumes were maintained at 20%-40% of the vessel size.
Infectious titers of MVA were determined in PFU/mL (plaque forming units) or FFU/mL (fluorescence forming units) as described previously (Jordan et al. 2013a) on Vero cells. Viruses were visualized in the non-permissive indicator cells by immunostaining or, where applicable, with help of the fluorescing reporters in deletion site Ⅲ.
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Recombinant MVA was generated by homologous recombination in adherent CR.pIX cells by adaptation of published methods (Kremer et al. 2012). Here, 1 × 106 CR.pIX cells were seeded per well of a 6 well-plate. The culture monolayers were infected with receiving MVA with a MOI of 0.01 on the following day and transfected with 2.0 µg of shuttle plasmid for insertion into deletion site Ⅲ. Point mutations were introduced by homologous recombination with a synthetic fragment that additionally contained silent diagnostic sites for restriction enzymes to confirm successful insertion and maintenance (Table 1). The recombination flanks ranged 600-1000 bp on each side.
Gene Mutation Diagnostic site Wildtype Recombinant MVA082L (L3L) V110A ∆ Hph I (ggtga) ttg gTg aga
lys VAL argttg gCg aga
lys ALA argMVA113L Ava I* (ctcgag)
Psp XI* (vc/tcgagb)tgT tcT TCt
cys ser sertgC tcG AGt
cys ser serMVA114L (A3L) Nco I* (c/catgg) tca atg gat
ser met asptcc atg gat
ser met aspH639Y aga Cat att
arg HIS ileaga Tat att
arg TYR ileMVA120L (A9L) K75E aag Aag aat
lys LYS asnaag Gag aat
lys GLU asnEcoR I* (g/aattc)
∆ Xcm I (ccan9tgg)ccA aat tca ttt tgg
pro asn ser phe trpccG aat tca ttt tgg
pro asn ser phe trpMVA121L (A10L) K554K with
Sty I (ccwwgg)ccA aaG gtA
pro lys valccC aaG gtC
pro lys valMVA145R (A34R) D86Y Acc I* (gt/atac)
∆ BsaW I (a/ccgga)aga ccg Gat act
arg pro ASP thraga ccg Tat act
arg pro TYR thrThe silent mutations in MVA113L and MVA121L are markers to confirm that recombination includes the complete flanks. Note that GenBank sequence U94848 lists a mutation (cca aGA gta, R554K) in A10L at this site. However, this deviation is corrected in a subsequent analysis so that U94848 and AY603355 are considered identical ( Antoine et al. 2006 ).Table 1. Summary of mutations observed in MVA-CR19 and silent mutations used as markers in recombinant viruses.
Transfections were performed with Effectene Transfection Regent (Qiagen, Germany) according to the manufacturer's instructions 90 min after infection, and the medium was replaced 24 h post transfection. The infected/transfected culture was harvested after 48 h to 72 h, sonicated, and used to infect cell monolayers in a 6-well plate. Plaques of the appropriate fluorescent phenotype were picked usually after another 24 h to 48 h and total DNA was isolated from aliquots of individual plaques using QuickExtract DNA Extraction Solution 1.0 (Epicentre, USA). Another round of plaque purification was initiated with the candidate recombinant virus preparations that passed the PCR analysis. The material for infection was obtained by sonication of cell harvests using a Vial Tweeter (set to 20 s of 100% cycle and 90% amplitude) that allows handling of closed sample caps to avoid cross-contamination (Hielscher, Germany). Viruses with parental genotype or incomplete recombination were not detectable within 3-8 rounds of plaque purification.
Virus passages to assay genomic stability was performed in CR.pIX suspension cultures in a volume of 5 mL with 1:1 mixtures of CD-U4 and CD-VP4. Cell density was 2 × 106 cells/mL and MOI 0.01 (in blind passages a titer of 108 PFU/mL was assumed in the previous passage). The infected culture was sonicated 48 or 72 h post infection to harvest virus.
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80 µL of complete cell lysate was mixed with 20 µL of QuickExtract DNA Extraction Solution 1.0 (Epicentre, USA) and heated to 65 ℃ for 10 min and to 98 ℃ for 5 min. 4 µL of this preparation was subjected to PCR in a final volume of 25 µL with 0.15 µL Taq polymerase (Qiagen, Germany), 200 nmol/L each primer, and 125 µmol/L each nucleotide. The sequence of the primer pairs that span deletion sites Ⅰ-Ⅵ of the viral genome were obtained from the literature (Kremer et al. 2012). The expected sizes of the amplification products are 291, 354, 447, 502, 603, and 702 bp for wildtype virus deletion sites Ⅰ to Ⅵ (Kremer et al. 2012), and 1285 for deletion site Ⅲ in MVA-CR19.GFP. Thermocycling was initiated with 94 ℃ for 80 s, followed by 35 cycles of 94 ℃ for 20 s, 55 ℃ for 20 s and 72 ℃ for 90 s, and terminated with 72 ℃ for 5 min. Amplicons were separated by electrophoreses in 1.5% agarose gels.
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The shuttle plasmid for deletion site Ⅲ was cloned stepwise via insertion of the left and right flanks into pEGFP-N1 (Clontech, USA). The flanks were amplified from the genomic DNA of wildtype MVA with the primers shown in Supplementary Table S1. The left flank was cut with Nhe I (all restriction enzymes used in this study were obtained from New England Biolabs or Roche) and Pci I, the right flank with Dra Ⅲ and Not I for sequential insertion into the same sites of pEGFP-N1 while maintaining the EGFP open reading frame. The artificial EL promoter (Chakrabarti et al. 1997) was generated by annealing two complementary 72 bp-oligonucleotides (TIP MolBiol, Germany) with the sequence ATC TGC TAG CAC GTG GAC TAG TAA AAA TTG AAA TTT TAT TTT TTT TTT TTG GAA TAT AAA TAA GAT CTT ACC on the conding strand. The annealing was performed after denaturation at 95 ℃ for 2 min followed by a ramp down to 56 ℃ with - 0.1 ℃ per second. This fragment was cut with Bgl Ⅱ and Nhe I, precipitated with 300 mmol/L sodium acetate in two volumes of ethanol, purified by polyacrylamide gel electrophoresis, and inserted into the same sites of the pEGFP-N1 plasmid already containing the deletion site Ⅲ flanks. Sequencing confirmed integrity of the shuttle plasmid but revealed a transition from ttG aaa ttt to ttA aaa ttt in the EL promoter that was not corrected and maintained as GFP expression was strong in rMVAs. A viral transcription terminator signal (T5NT, Yuen and Moss 1987) is contained in the right flank. The DsRed1 derivative mCherry was synthesized with codon-optimization for duck (Eurofins Genomic, Germany) and inserted in antisense orientation to GFP and under control of the late P11 promoter (Bertholet et al. 1985). The resulting dual expression cassette spans 1615 bp from EL to P11 promoter, the amplification product for deletion site Ⅲ primers is 2087 bp long.
The shuttle plasmids for introduction of the point mutations D86Y in A34R and V110A in L3L into wildtype MVA were cloned only with fragments amplified out of MVA-CR19 genomic DNA. These mutations already contain by chance diagnostic restriction enzyme sites to confirm successful recombination (Table 1). The A34R shuttle plasmid was cloned by amplification of 1393 bp with primers shown in Supplementary Table S1. The primers contained additional restriction sites for Nhe I and Xba I, respectively, at the 5′ termini for insertion into pEGFP-N1 [out of dam(-) bacteria] using these sites. The region of interest for the L3L shuttle plasmid was also obtained by PCR and inserted into pCR-Blunt Ⅱ-Topo ("pTopo") as described in the Zero Blunt TOPO PCR Cloning Kit (Invitrogen, USA).
The shuttle plasmids for the other point mutations, H639Y in A3L and K75E in A9L, were cloned by insertion of synthetic DNA (Eurofins Genomic) that contained the desired point mutation and silent diagnostic mutations (designed using http://resitefinder.appspot.com/).
For the generation of the shuttle plasmid for A3L a 2008 bp fragment of A3L was amplified (primer sequences shown in Supplementary Table S1) and inserted into pTopo. A 274-bp fragment therein from Sac I to Swa I was replaced with a synthetic DNA containing H639Y and the silent Nco I site. One flank of this shuttle plasmid had to be extended because first recombination attempts did not include the desired H639Y site: an additional synthetic DNA of 479 bp containing a new (but silent) Ava I site was appended to the Nco I-distal side using Swa I (in MVA) and Spe I (in pTopo).
Recombination of a 415 bp synthetic DNA that also contained a silent diagnostic mutation near to the desired K75E mutation transferred only the diagnostic mutation as well (revealed by sequencing of plaque-purified viruses). The flanks were therefore extended and additional diagnostic mutations were inserted so that K75E is framed by markers as follows: First, a 2905 bp fragment containing A9L and neighboring gene A10L was amplified out of wildtype MVA genomic DNA with primers shown in Supplementary Table S1. This 2905 bp fragment was cloned into pTopo to yield pTA10L. A synthetic DNA containing the diagnostic sites in A10L was then inserted via a three fragment ligation using Pme I (in the vector) to Nsi I (in the MVA insert) of pTA10L as new vector backbone, Spe I to Nsi I in the synthetic DNA to insert the silent Sty I diagnostic marker, and Nsi I to Pme I of the pTA10L to restore the initial amplification product. Three-fragment ligation to obtain pTLA10L-StyI was necessary to circumvent an additional Spe I site in the vector backbone. The A9L flank was inserted using a synthetic DNA fragment containing the K75E mutation framed by diagnostic silent mutations on both sides, EcoR I and BseR I. This fragment was inserted via a three-fragment ligation to circumvent a Tth111 I site in the vector, using Tth111 I in A9L to EcoR V in the multiple cloning site of the synthetic DNA vector, Tth111 I to Pme I in pTA10L and Tth111 I to Pme I in pTA10L to restore the vector. The resulting shuttle plasmid contains a MVA-derived fragment of 3620 bp.
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Genomic DNA of plaque-purified MVA-CR19.GFP was isolated by polyethylene glycol precipitation out of 100 mL of infected CR.pIX cells at 2 × 106 cells/mL as described previously (Jordan et al. 2013a). Sequences were obtained by GATC Biotech AG (Germany) with the PacBio RSII technology and assembled using an unforced (without guide sequence) algorithm.
Because large gaps at the left side of the genome remained after sequence assembly, and because PCR against the deletion sites indicated a loss of deletion site Ⅰ (that is located near the left terminus of MVA) 5′-end RACE was performed. Primer D2 RII (Supplementary Table S1) against a 5′ terminal region still covered by the genomic sequence assembly was designed using the Clone Manager Professional suite version 9 (Sci-Ed Software, USA). This primer was extended on 500 ng of viral genomic DNA in 100 µL of 1 × PCR buffer, 1 × Q solution, and 5 U Taq and 0.2 U ProofStart Taq polymerase (all Qiagen, Germany), 0.4 µmol/L primer D2 RII and 0.05 mmol/L each dNTP. The thermocycler was programmed for 35 cylces of 94 ℃ for 10 s, 57 ℃ for 60 s, and 68 ℃ for 3 min (with 95 ℃ for 2 min at the start and 72 ℃ for 10 min at the end of the program). This PCR reaction was purified with the QIAquick PCR Purification Kit, 25 µL thereof were incubated with terminal transferase (TdT, New England Biolabs #M0315S) in a final volume of 50 µL of 1 × Tailing Buffer, 0.25 mmol/L CoCl2 and 0.1 mmol/L dCTP. The tailing reaction was preceded by denaturation at 94 ℃ for 3 min, followed by addition of 0.5 µL of the TdT and incubation at 37 ℃ for 30 min, and termination at 70 ℃ for 10 min.
A nested PCR was next performed to recover the 5′ extended and dC-tailed product using primers D2 and the universal anchored primer AAP in a final volume of 100 µL as described above for D2 RII primer extension but with an extension temperature of 59 ℃ for 60 s (instead of 57 ℃).
This first nested PCR was diluted 1:50 and subjected to a second nested PCR in a final volume of 50 µL, without Q solution, primers GSPD2-R and AAP, with the same thermocycler program as in the first nested PCR. A fragment of approx. 700 bp was isolated and purified by agarose gel electrophoresis with the Qiagen Gel Extraction kit and sequenced with primers AAP and GSPD2-R. Primers were designed on this sequence as a diagnostic pair for amplification of 469 bp spanning the newly discovered recombination site (RS469).
The long-PCR for amplification of the presumed left ITR of MVA-CR19 was performed with primers D2 RII and ITR-M to obtain 21, 312 bp on MVA-CR19 and 9360 bp on wildtype MVA (Fig. 3). The ITR-M primer binds in forward orientation from 533 to 553 and in reverse orientation from 165, 956 to 165, 976 in GenBank sequence AY603355 whereas primer D2 RII binds only once, in reverse orientation from 9869 to 9892. The possible amplicons are therefore 9360 bp and 165, 444 bp (ITR-M single-primer amplification) with wildtype MVA as template, but not 21, 312 bp. LongRange PCR (Qiagen) was performed in 50 µL final volume with 200 ng of viral genomic DNA according to the manual. The thermocycler program was initiated with 93 ℃ for 3 min; followed by 10 cycles of 93 ℃ for 10 s, 57 ℃ for 30 s and 68 ℃ for 15 min; followed by 25 cycles of 93 ℃ for 15 s, 57 ℃ for 30 s and 68 ℃ for 21 min with extension by 20 s per cycle. Restriction enzyme analysis with Bcl I, Nru I or ApaL I was performed with 3 µL of PCR product in 20 µL final volume and 0.5 µL of enzyme according to the manufacturer's instructions.
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Single-cell cultures (not suspended aggregates) of 20 mL × 2 × 106 CR.pIX cells/mL were infected with MOI of 8 and cultivated for 30 h. The infected cells were collected by centrifugation for 5 min with 200 ×g and resuspended in 2.5% glutaraldehyde in 100 mmol/L phosphate buffer, pH 7.3, and stored at 4 ℃ until further processing. The fixed cells were rinsed in 100 mmol/L phosphate buffer, pH 7.4 and then treated with 2% osmium tetroxide in 100 mmol/L phosphate buffer, pH 7.4 at 4 ℃ for 2 h. They were next dehydrated in ethanol and acetone prior to embedding in LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were prepared using a Leica ultracut UCT (Leica, Wien, Austria) and contrasted with uranyl acetate followed by lead citrate. The ultrathin sections were examined in a Tecnai G2 Spirit BioTWIN transmission electron microscope (FEI Company, Eindhoven, The Netherlands) and digital images were acquired using a 2 k × 2 k side-mounted Veleta CCD camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).