Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA
© Rasmussen et al.; licensee BioMed Central Ltd. 2013
Received: 31 July 2013
Accepted: 14 November 2013
Published: 22 November 2013
Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria.
A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence.
These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.
Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3–6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus  and Dengue virus ) have been inserted into BACs.
BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11]. Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13]. The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins Erns, E1 and E2) and eight non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15–17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope  within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.
Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see ). The use of homologous recombination allows site-directed mutagenesis of BACs  and, by employing a counter-selection scheme, specific modifications can be obtained without leaving residual “foreign” sequences . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25].
Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain “C-strain Riems”. The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain “Gifhorn”) and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.
Cells and viruses
Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO2) in Dulbecco’s minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV “C-strain Riems” (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain “Gifhorn”  was used for amplification of the Gifhorn E2-coding sequence.
Oligonucleotide primers used are listed in Additional file 1: Table S1.
The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described  using RNA derived from the “C-strain Riems”. Briefly, full-length viral cDNAs flanked by Not I sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a Not I site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a Not I site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the C-strain). The product (ca. 12.3 kbp) was digested with Not I and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad). BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).
Modification of the CSFV cDNA by Red/ET recombination
Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, StrepR) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany).
The Red/ET recombination involved three steps (i-iii). Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporation-competent E.coli DH10B cells containing the parental BAC (phenotype CamR, StrepR). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring TetR) was verified by visual inspection of BAC-DNA preparations from the CamR/TetR colonies using agarose gel electrophoresis. Step ii) counter-selection marker cassettes with an extra Not I site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (StrepS) onto the resistant DH10B strain and the neo confers Kanamycin resistance (KanR). The correct phenotype (CamR, KanR, TetR, StrepS) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be StrepS indicating incorporation of a functional rpsL gene. The structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts. Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counter-selection against the rpsL-neo cassette (phenotype CamR, KanR, TetR, StrepS) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype CamR and StrepR).
For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsL-neo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below).
Rescue of viruses and virus growth curves
BAC DNA (1 μg) was linearized with Not I or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat anti-mouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days. At 2, 8, 24, 48 and 72 hours post infection (PK15) or at 3, 12, 24, 48 and 72 hours post infection (SFT-R), cell samples were harvested for virus titration. Cell samples containing virus from each time point were assayed on PK15 or SFT-R cells by limiting dilutions and grown for three days to determine the virus titre (as TCID50/ml).
BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm  were used for mapping the reads to the expected sequence. A combination of Samtools  and LoFreq SNV-caller  was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).
Generation of a BAC containing full-length cDNA corresponding to the modified live vaccine “C-strain Riems”
Nucleotide and amino acid differences between the consensus cDNA sequences of the parental vaccine virus (vRiemser), the cloned BAC cDNA (pBeloR26) and cDNA of the rescued vR26/P-12
pBeloR26 (BAC cDNA)
aa change (in pBeloR26)
Homologous recombination to obtain CSFV E2 chimeric constructs
To determine the utility of the targeted recombination-mediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain “Gifhorn” as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC structure as determined by Not I digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV “Gifhorn”. Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).
Rescue of modified virus from recombined BACs
After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein (Figure 2). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins (Figure 2) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the “Gifhorn” E2 protein using staining with an anti-BDV mAb (Figure 2). The presence of the BDV epitope TTVSTSTLA in vR26_TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.
Genetic stability of the BACs in the bacterial host
The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.
Genetic stability of viruses rescued from the BACs
In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions (Table 1). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV  and for insertions of heterologous reporter sequences into CSFV replicons . Using Red/ET recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses.
Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope  and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain “Gifhorn” is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents C-strain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).
Genetic stability in the bacterial host
Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for “C-strain Riems”. Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2-chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing full-length pestivirus cDNAs . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli[8, 10].
Genetic stability of rescued viruses
Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa change D3431G) within the NS5B coding region. The same reversion to the consensus sequence for the “C-strain Riems” virus was observed in two independent virus populations, derived from independent BACs, and indicates that this substitution is important for efficient growth of the “C-strain Riems” virus in SFT-R cells. In contrast, all other nucleotide positions remained unchanged during the extensive virus propagation including the heterologous E2 sequences (Figure 5B). The glycine (G) residue at position 3431 in the polyprotein is highly conserved amongst CSFV strains; indeed, alignment of CSFV sequences retrieved from GenBank did not reveal any variation at that position (data not shown). Whether this mutation was derived from the cloning procedure or reflects a low-level variant present in the parental vaccine stock is unclear.
In summary, the present study shows that targeted recombination-mediated mutagenesis of BACs containing full-length CSFV cDNA facilitate manipulation and rescue of chimeric pestiviruses. The system shows high genetic stability of the BAC constructs (within E. coli) and the chimeric viruses rescued from the BACs can be efficiently and stably propagated in SFT-R cells and this represents a suitable system for the production of virus stocks for future marked vaccine experiments. The strategies employed in this study have applicability not only for CSFV (and other pestiviruses) but should be adaptable to the study of other RNA viruses.
We are grateful to Gabriela Adam and Lone Nielsen for excellent technical assistance. This work was supported by the Danish Research Council for Technology and Production Sciences [DRCTPS grant 274-07-0198 (to TBR) and 274-08-0305 (to GJB)].
- Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH: Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA. 1997, 94 (26): 14759-14763. 10.1073/pnas.94.26.14759.PubMed CentralView ArticlePubMedGoogle Scholar
- Tischer BK, Kaufer BB: Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences. J Biomed Biotechnol. 2012, 2012: 472537-PubMed CentralView ArticlePubMedGoogle Scholar
- Almazan F, Gonzalez JM, Penzes Z, Izeta A, Calvo E, Plana-Duran J, Enjuanes L: Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA. 2000, 97 (10): 5516-5521. 10.1073/pnas.97.10.5516.PubMed CentralView ArticlePubMedGoogle Scholar
- Balint A, Farsang A, Zadori Z, Hornyak A, Dencso L, Almazan F, Enjuanes L, Belak S: Molecular characterization of feline infectious peritonitis virus strain DF-2 and studies of the role of ORF3abc in viral cell tropism. J Virol. 2012, 86 (11): 6258-6267. 10.1128/JVI.00189-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Almazan F, Dediego ML, Galan C, Escors D, Alvarez E, Ortego J, Sola I, Zuniga S, Alonso S, Moreno JL, Nogales A, Capiscol C, Enjuanes L: Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J Virol. 2006, 80 (21): 10900-10906. 10.1128/JVI.00385-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Almazan F, Galan C, Enjuanes L: Engineering infectious cDNAs of coronavirus as bacterial artificial chromosomes. Methods Mol Biol. 2008, 454: 275-291. 10.1007/978-1-59745-181-9_20.View ArticlePubMedGoogle Scholar
- Hotard AL, Shaikh FY, Lee S, Yan D, Teng MN, Plemper RK, Crowe JE, Moore ML: A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis. Virology. 2012, 434 (1): 129-136. 10.1016/j.virol.2012.09.022.PubMed CentralView ArticlePubMedGoogle Scholar
- Yun SI, Kim SY, Rice CM, Lee YM: Development and application of a reverse genetics system for Japanese encephalitis virus. J Virol. 2003, 77 (11): 6450-6465. 10.1128/JVI.77.11.6450-6465.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Pierro DJ, Salazar MI, Beaty BJ, Olson KE: Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation. J Gen Virol. 2006, 87 (Pt 8): 2263-2268.View ArticlePubMedGoogle Scholar
- Fan ZC, Bird RC: An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA. J Virol Methods. 2008, 149 (2): 309-315. 10.1016/j.jviromet.2008.01.011.View ArticlePubMedGoogle Scholar
- Rasmussen TB, Reimann I, Uttenthal A, Leifer I, Depner K, Schirrmeier H, Beer M: Generation of recombinant pestiviruses using a full-genome amplification strategy. Vet Microbiol. 2010, 142 (1–2): 13-17.View ArticlePubMedGoogle Scholar
- Collett MS, Anderson DK, Retzel E: Comparisons of the pestivirus bovine viral diarrhoea virus with members of the flaviviridae. J Gen Virol. 1988, 69 (Pt 10): 2637-2643.View ArticlePubMedGoogle Scholar
- Meyers G, Rumenapf T, Thiel HJ: Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology. 1989, 171 (2): 555-567. 10.1016/0042-6822(89)90625-9.View ArticlePubMedGoogle Scholar
- Beer M, Reimann I, Hoffmann B, Depner K: Novel marker vaccines against classical swine fever. Vaccine. 2007, 25 (30): 5665-5670. 10.1016/j.vaccine.2006.12.036.View ArticlePubMedGoogle Scholar
- van Gennip HG, van Rijn PA, Widjojoatmodjo MN, de Smit AJ, Moormann RJ: Chimeric classical swine fever viruses containing envelope protein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response. Vaccine. 2000, 19 (4–5): 447-459.View ArticlePubMedGoogle Scholar
- Reimann I, Depner K, Trapp S, Beer M: An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus. Virology. 2004, 322 (1): 143-157. 10.1016/j.virol.2004.01.028.View ArticlePubMedGoogle Scholar
- Rasmussen TB, Uttenthal A, Reimann I, Nielsen J, Depner K, Beer M: Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus. J Gen Virol. 2007, 88 (Pt 2): 481-486.View ArticlePubMedGoogle Scholar
- Lin M, Lin F, Mallory M, Clavijo A: Deletions of structural glycoprotein E2 of classical swine fever virus strain alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J Virol. 2000, 74 (24): 11619-11625. 10.1128/JVI.74.24.11619-11625.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Reimann I, Depner K, Utke K, Leifer I, Lange E, Beer M: Characterization of a new chimeric marker vaccine candidate with a mutated antigenic E2-epitope. Vet Microbiol. 2010, 142 (1–2): 45-50.View ArticlePubMedGoogle Scholar
- Holinka LG, Fernandez-Sainz I, O’Donnell V, Prarat MV, Gladue DP, Lu Z, Risatti GR, Borca MV: Development of a live attenuated antigenic marker classical swine fever vaccine. Virology. 2009, 384 (1): 106-113. 10.1016/j.virol.2008.10.039.View ArticlePubMedGoogle Scholar
- Hall RN, Meers J, Fowler E, Mahony T: Back to BAC: the use of infectious clone technologies for viral mutagenesis. Viruses. 2012, 4 (2): 211-235.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Y, Buchholz F, Muyrers JP, Stewart AF: A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet. 1998, 20 (2): 123-128. 10.1038/2417.View ArticlePubMedGoogle Scholar
- Muyrers JP, Zhang Y, Testa G, Stewart AF: Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 1999, 27 (6): 1555-1557. 10.1093/nar/27.6.1555.PubMed CentralView ArticlePubMedGoogle Scholar
- Friis MB, Rasmussen TB, Belsham GJ: Modulation of translation initiation efficiency in classical swine fever virus. J Virol. 2012, 86 (16): 8681-8692. 10.1128/JVI.00346-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Risager PC, Fahnoe U, Gullberg M, Rasmussen TB, Belsham GJ: Analysis of classical swine fever virus RNA replication determinants using replicons. J Gen Virol. 2013, 94 (8): 1739-1748.View ArticlePubMedGoogle Scholar
- Rasmussen TB, Reimann I, Hoffmann B, Depner K, Uttenthal A, Beer M: Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon. J Virol Methods. 2008, 149 (2): 330-333. 10.1016/j.jviromet.2008.01.029.View ArticlePubMedGoogle Scholar
- Uttenthal A, Storgaard T, Oleksiewicz MB, de Stricker K: Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA. Vet Microbiol. 2003, 92 (3): 197-212. 10.1016/S0378-1135(02)00364-4.View ArticlePubMedGoogle Scholar
- Li H, Durbin R: Fast and accurate long-read alignment with burrows-wheeler transform. Bioinformatics. 2010, 26 (5): 589-595. 10.1093/bioinformatics/btp698.PubMed CentralView ArticlePubMedGoogle Scholar
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R: 1000 Genome project data processing subgroup: the sequence alignment/Map format and SAMtools. Bioinformatics. 2009, 25 (16): 2078-2079. 10.1093/bioinformatics/btp352.PubMed CentralView ArticlePubMedGoogle Scholar
- Wilm A, Aw PP, Bertrand D, Yeo GH, Ong SH, Wong CH, Khor CC, Petric R, Hibberd ML, Nagarajan N: LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 2012, 40 (22): 11189-11201. 10.1093/nar/gks918.PubMed CentralView ArticlePubMedGoogle Scholar
- Ruggli N, Rice CM: Functional cDNA clones of the Flaviviridae: strategies and applications. Adv Virus Res. 1999, 53: 183-207.View ArticlePubMedGoogle Scholar
- Quinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010, 26 (6): 841-842. 10.1093/bioinformatics/btq033.PubMed CentralView ArticlePubMedGoogle Scholar
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