Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs

Virus Research 83 (2002) 131– 147 www.elsevier.com/locate/virusres

Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs Christopher L. Parks, Hai-Ping Wang, Gerald R. Kovacs, Nikos Vasilakis, Jacek Kowalski, Rebecca M. Nowak, Robert A. Lerch, Pramila Walpita, Mohinderjit S. Sidhu, Stephen A. Udem * Wyeth-Lederle Vaccines, Department of Viral Vaccine Research, 401 North Middletown Road, Pearl Ri6er, NY 10965, USA Received 14 August 2001; received in revised form 16 November 2001; accepted 16 November 2001

Abstract A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 °C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Recombinant canine distemper virus; cDNA rescue; Foreign gene expression

1. Introduction Canine distemper virus (CDV) is a member of the morbillivirus genus (Murphy, 1996). Like other members of this group, including measles virus, rinderpest virus, and peste des petits ruminants virus among others, CDV is an enveloped * Corresponding author. Tel.: + 1-845-602-5450; fax: +1845-602-5727. E-mail address: [email protected] (S.A. Udem).

RNA virus that contains a single-stranded, negative-sense genome of approximately 16 kb. The genome contains six non-overlapping gene regions, organized 3%-N –P–M–F–H–L-5%, that encode eight known viral polypeptides (Barrett, 1999; Harder and Osterhaus, 1997; Lamb and Kolakofsky, 1996). The viral polypeptides include: the nucleocapsid protein (N) that encapsidates viral genomic RNA; the matrix protein (M) that is a structural component of the virion; the fusion (F) and hemmagglutinin (H) envelope gly-

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coproteins; the catalytic polymerase subunit (L); and three proteins encoded by the P gene. In addition to encoding the phosphoprotein (P) polymerase subunit, the P gene encodes the C and V nonstructural proteins that are translated from a downstream translation initiation codon (C) or an edited version (V) of the P gene mRNA (Barrett, 1999; Harder and Osterhaus, 1997; Lamb and Kolakofsky, 1996). CDV is best known for causing disease in dogs (Barrett, 1999; Harder and Osterhaus, 1997). The virus is commonly spread by aerosol causing an initial infection in the upper respiratory tract epithelium. Replication at the site of infection leads to infection of lymphoid tissues causing immunosuppression and further dissemination to many organs and cell types. Some animals recover from the disease, but within a few days to weeks, a relatively high number will develop an active infection of the central nervous system that results in a progressive demyelinating disease that presents with neurological symptoms (Summers and Appel, 1994; Vandevelde and Zubriggen, 1995). Although classically associated with infection of dogs, recent investigations with improved detection techniques have demonstrated that CDV infects a wider range of hosts (Barrett, 1999; de Swart et al., 1995; Harder and Osterhaus, 1997; Summers and Appel, 1994). All Canidae are susceptible including domestic and wild dogs, foxes, wolves and coyotes. CDV infection has also been responsible for the deaths of large cats, including lions and tigers, in Africa and in zoos in the United States. Additionally, CDV outbreaks in seals have been reported, and the virus is known to cause disease in small carnivores like mink, ferrets, and raccoons. CDV has even been considered a suspect in some human diseases like Paget’s disease and multiple sclerosis (Fraser, 1997; Hodge and Wolfson, 1997; Mee and Sharpe, 1993). Live attenuated CDV vaccines have been effective controlling distemper in domesticated dog populations but there remains a need for additional vaccine research. The three commonly used CDV vaccines cannot be used in all susceptible animal populations, because ferrets, foxes, large cats, red pandas, and African wild dogs are vul-

nerable to disease caused by vaccine viruses (Barrett, 1999; Harder and Osterhaus, 1997; Summers and Appel, 1994). This is a particularly troubling problem for zoos and wildlife parks that need to protect their animals from CDV infection. Moreover, the ability of CDV to infect diverse hosts suggests that there may be additional potential for antigenic variation as well as further adaptation to other animal populations. Thus, vaccines that are safe for administration to a broader range of animals would be valuable, and it would be beneficial if these vaccines could be readily manipulated to take into account future antigenic variation. New CDV vaccines are being investigated. For example, vaccines based on recombinant vaccinia virus or canarypox vectors engineered to express CDV glycoproteins have been tested in dogs and ferrets (Pardo et al., 1997; Stephenson et al., 1997). These vaccines elicited a protective immune response; however, it has yet to be determined if the duration of this immune response is equivalent to responses induced by conventional live CDV vaccines (Barrett, 1999). DNA vaccines based on CDV glycoprotein genes have been tested in mice. Immunized mice survived intracerebral challenge with a neurovirulent strain of CDV, but some mice may not have been completely protected from the effects of infection (Sixt et al., 1998). In addition to testing these technologies, it also is worth considering whether live, attenuated CDV vaccines can be made safe for a broader range of hosts. The success of current CDV vaccines in controlling distemper in domesticated dog populations (Barrett, 1999; Harder and Osterhaus, 1997; Summers and Appel, 1994) suggests that a modified and improved live attenuated CDV strain may still be one of the most important options for further vaccine development. Viral cDNA rescue technology (Collins et al., 1999; Conzelmann, 1998; Marriot and Easton, 1999; Nagai, 1999; Radecke and Billeter, 1997; Roberts and Rose, 1998) has made it possible to generate recombinant negative-strand RNA viruses. Previously, genetic engineering of this class of viruses was impossible because introduction of viral genomic RNA or genomic cDNA

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into the cell by itself was insufficient to initiate the virus life cycle. Rescue technology has solved this problem (Pattnaik et al., 1992; Schnell et al., 1994), and it has since been used extensively to develop attenuated virus strains, perform genetic analyses, and insert foreign genes in a variety of negative strand viruses (Collins et al., 1999; Conzelmann, 1998; Marriot and Easton, 1999; Nagai, 1999; Radecke and Billeter, 1997; Roberts and Rose, 1998). To take advantage of this technology for studies of CDV, plasmid reagents have been generated enabling rescue of recombinant virus. Gassen et al. (Gassen et al., 2000) first reported successful CDV rescue and their findings are corroborated here. In addition, we have extended these findings to show that CDV can be used as a vector for expression of a foreign gene.

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con (Fig. 1A) was cloned into a plasmid vector previously used to construct a measles virus (MV) minireplicon (pMV107-CAT; Sidhu et al., 1995). Prior to cloning into this vector backbone, it was

2. Materials and methods

2.1. Cells and 6irus HEp2, A549, Vero, BHK, and chicken embyro fibroblasts (CEF) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HeLa suspension cells were grown in modified minimal essential medium (SMEM) containing 5% FBS. The laboratory-adapted Onderstepoort CDV strain (Haig, 1956) was propagated in HeLa cells as described previously (Sidhu et al., 1993a). A second laboratory-adapted Onderstepoort strain was provided by Dr Martin Billeter and was propagated in Vero cells. The recombinant attenuated vaccinia virus strains (MVA/T7, Wyatt et al., 1995 or MVAGKT7, Kovacs et al., 2002) that express the T7 RNA polymerase gene were propagated in CEF or BHK cells. The laboratoryadapted Edmonston strain of measles virus (MV) was grown in HeLa suspension cells (Udem, 1984).

2.2. Recombinant DNA Molecular cloning procedures were performed following standard protocols (Ausubel et al., 1987; Maniatis et al., 1982). The CDV minirepli-

Fig. 1. DNAs prepared for CDV rescue. The plasmid backbone used for constructing the CDV minireplicon (A) and full-length cDNA clone (B) has been described previously (Sidhu et al., 1995). Cis-acting signals that control T7 RNA polymerase transcription are drawn as grey boxes. The hepatitis delta virus ribozyme sequence (Been and Wickham, 1997) is drawn as a hatched box. Restriction enzyme digestion sites used for cloning are indicated in italics. The CDV minireplicon (A) is composed of the CAT reporter gene flanked by the 111-nucleotide CDV leader TCR and the 109-nucleotide trailer TCR (terminal open boxes). Transcription from the T7 RNA polymerase promoter generates a negative-sense minireplicon RNA. Part (B) shows a schematic diagram of the CDV full-length clone (pBS-rCDV). CDV cistrons are drawn as black boxes with white letters and gene boundaries. T7 RNA polymerase transcribes a positive-sense copy of the CDV genome. Part (C) shows the T7 RNA polymerase-dependent plasmid vectors (Moss et al., 1990; Radecke et al., 1995) that were prepared to direct expression of the N, P or L genes. The cDNA insert is cloned 3% of an IRES sequence to facilitate translation of the T7 RNA polymerase transcript. A stretch of approximately 50 adenosine residues is located at the 3% end followed by a T7 RNA polymerase terminator.

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modified by addition of a NotI site 3% of the T7 promoter. This modification was made by performing an end-to-end PCR amplification of the plasmid with primers that included an appropriately positioned NotI site. This PCR product was circularized by ligation and used for transformation. To generate pCDV-CAT, the region between the NotI and NarI sites (Fig. 1A) was synthesized by four sequential PCR reactions that copied the CAT gene from the MV-CAT plasmid while progressively adding CDV sequences to the 5% and 3% end of the amplified region with a series of nested primers containing 5% extensions. The series of nested PCR primers generated the CDV leader (CDV nucleotides 1– 111) and trailer (nucleotides 15 581– 15 690) TCRs, the T7 RNA polymerase promoter, part of the ribozyme sequence, and the NotI and NarI cleavage sites used for cloning. The full-length viral genomic cDNA also was cloned in the vector backbone described above (Fig. 1B). Six fragments of viral genomic cDNA were prepared from purified viral RNA by reverse transcription and PCR amplification (RT/PCR) (Sidhu et al., 1993a) using primers based on previously published sequences (McIlhatton et al., 1997; Sidhu et al., 1993a,b). The resulting cDNA fragments were cloned sequentially making use of existing restriction sites to clone the internal cDNA fragments (Fig. 1B). The terminal DNA fragment containing the CDV leader TCR, the N gene, and most of the P gene was amplified with an oligonucleotide primer that contained a 5% terminal extension that included a NotI site and T7 promoter. Similarly, the genomic 3% terminal fragment was prepared with a 3% oligonucleotide primer that included an extension containing a portion of the ribozyme sequence plus the NarI site. RT reactions were performed with Superscript RT (Invitrogen Life Technologies), and subsequent PCR amplification was performed with Taq DNA polymerase. Mutations introduced during cloning were repaired in plasmid subclones by oligonucleotide-directed mutagenesis using the Morph mutagtenesis kit (Eppendorf-5 prime, Inc.). A modified CDV cDNA clone suitable for foreign gene insertion was prepared by creating a small multiple cloning site (MCS) between the P

and M genes. The SalI/NdeI subclone spanning the P and M intergenic region (Fig. 1B) was used as template for oligonucleotide-directed mutagenesis to introduce nine base substitutions between CDV nucleotide positions 3329 and 3377 (Fig. 4A). This modification created three unique restriction cleavage sites (AatII, FseI, MluI). The modified subclone fragment was inserted back into the full-length genomic clone positioning the MCS 3% of the P gene open reading frame and 5% of the P/M gene-end/gene-start (GE/GS) signal creating plasmid pBS-rCDVmcs. The luciferase gene from pGL2-Control (Promega) was then cloned between the FseI and MluI sites within the MCS of pBS-rCDVmcs. Primers used to amplify the luciferase coding sequence included the appropriate terminal restriction sites, and the 5% primer also contained a 5% extension equivalent to a synthetic copy of the GE/GS signal from the P– M intergenic region (Fig. 4A). The CDV genomic clone containing the luciferase gene (pBSrCDV-P/luc/M; Fig. 4) followed the rule-of-six (Kolakofsky et al., 1998). Expression vectors pT7CDV-N, pT7CDV-P, and pT7CDV-L (Fig. 1C) were prepared by inserting the appropriate coding region into a T7 expression vector (Radecke et al., 1995) based on pTM-1 (Moss et al., 1990). The CDV N and P genes were amplified from infected-cell RNA by RT/PCR. The L gene was PCR-amplified from the full-length CDV cDNA clone. Base substitutions introduced during PCR amplification and cloning were repaired by replacing sequences with fragments generated from an independent PCR amplification or by oligonucleotide-directed mutagenesis as described above. The MV N, P and L genes from the laboratory-adapted measles virus Edmonston strain (Udem, 1984) were cloned into the T7 vector after RT/PCR amplification from infected-cell RNA. An expression/recombination plasmid (pVVproN/P; Fig. 5A) was constructed containing both the CDV N and P genes under the control of two back-to-back vaccinia virus synthetic early/late promoters (Chakrabarti et al., 1997), and additional vaccinia virus DNA sequences (Fig. 5A) capable of directing homologous recombination (Sutter and Moss, 1992)

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to the region designated deletion III (Antoine et al., 1998) in the vaccinia virus Ankara (MVA) genome (Moss, 1996). Plasmid pVVproN/P was cloned by first modifying plasmid pGO8 (Sutter et al., 1995) through insertion of the b-glucuronidase (GUS) reporter gene. Subsequently, the CDV N and P gene fragments were amplified from CDV RNA by RT/PCR using gene-specific primers. The N gene was cloned using XhoI and NotI sites incorporated into the PCR primers. Similarly, the amplified P gene was cloned using PmeI and BssHII sites.

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2.3. DNA sequencing The nucleotide sequence of cloned DNAs was determined by cycle-sequencing (Griffin and Griffin, 1993; Kretz et al., 1994) using dye-terminator/Taq DNA polymerase kits (Perkin– Elmer). Sequencing reactions were purified using microspin G50 columns (Amersham-Pharmacia Biotech) and analyzed on an Applied Biosystems 377 automated sequencer. Data analysis was performed with MacVector software (Oxford Molecular). The genomic sequence of the CDV Onderstepoort strain was confirmed by generating a consensus sequence directly from amplified RT/ PCR products (Parks et al., 2001b).

2.4. Minireplicon and 6irus rescue Transfections were executed using several different procedures and transfection reagents during these studies. A calcium-phosphate protocol was eventually found to be most effective in our hands, and a detailed description of the procedure is provided below in the virus rescue protocol. Transfection with liposome reagents essentially followed the protocols recommended by the manufacturer.

Fig. 2.

Fig. 2. CDV-CAT minireplicon rescue. Part (A) illustrates the effect of cell culture incubation temperature on minireplicon activity. A549 cells in six-well plates were transfected then incubated at 32 or 37 °C. Plasmid minireplicon pCDV-CAT (50 ng) was co-transfected with expression plasmids (400 ng pT7CDV-N, 300 ng pT7CDV-P, 50 – 100 ng pT7CDV-L) using Lipofectace transfection reagent. Similarly, the measles virus minireplicon (100 ng pMV107-CAT) was co-transfected with measles virus protein expression vectors (400 ng pT7MV-N, 300 ng pT7MV-P, 100 ng pT7MV-L). Cells were infected with MVA/T7 when transfection was initiated. CAT activity was analyzed approximately 48 h after initiation of transfection. The CAT assay was performed with a fluorescent chloramphenicol substrate and reaction products were quantified using a fluorimager. CAT activity is expressed relative to the value given in lane 8. Part (B) illustrates the effect of heat shock on minireplicon expression. A549 cells in six-well plates were co-transfected with the pCDV-CAT minireplicon (10 ng) and expression vectors for N (400 ng), P (300 ng), and L (50 – 100 ng) using the calcium-phosphate procedure described in Section 2. At 3 h after transfection, appropriate cell cultures were heat shocked for 2 h then returned to 32 °C. CAT activity was analyzed as described above.

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The BES-buffered calcium-phosphate transfection procedure (Ausubel et al., 1987; Chen and Okayama, 1987; Tognon et al., 1996) was used for virus rescue. About 1– 2 h prior to transfection, 75– 90% confluent A549 or HEp2 cell monolayers cultured in six-well plates were fed with 4.5 ml of DMEM containing 10% FBS then shifted to an incubator set at 3% CO2 and 32 °C. This temperature (32 °C) was used because minireplicon experiments (Fig. 2A) indicated that this temperature improved rescue. Calcium-phosphate-DNA precipitates were prepared for transfection in 5 ml polypropylene tubes. Plasmid DNAs (5 mg full-length CDV plasmid, 400 ng pCDV-N, 300 ng pCDV-P, and 100 ng pCDV-L) were combined in a total volume of 225 ml followed by addition of 25 ml of 2.5 M calcium chloride. Next, 250 ml of 2× BES-buffered saline (50 mM BES [pH 6.95– 6.98], 1.5 mM Na2HPO4, 280 mM NaCl) was added drop-wise to each tube while gently vortexing the mixture. After completely adding the 2× BES-buffered saline solution, precipitates were allowed to form for an additional 20–30 min at room temperature before being added drop-wise to the culture medium. At this time, MVA/T7 was added to the culture medium at a multiplicity of infection (MOI) of approximately 1–2 and the plates were rocked gently to ensure uniform mixing of the medium, calcium-phosphate-DNA precipitate, and MVA/ T7 before returning the cells to the incubator set at 3% CO2 and 32 °C. Three hours after initiating transfection, the six-well plates were sealed in a zip-lock bag and submersed for 2 h in a water bath set at 43 °C. Following this heat shock step, the cells were returned to the 32 °C incubator set at 3% CO2. The following day, the transfection medium was removed and the cell monolayers were washed twice with a HEPES-buffered saline solution (10 mM hepes [pH 7.0], 150 mM NaCl, 1 mM MgCl2) and subsequently fed with 2– 3 ml of DMEM supplemented with 10% FBS. Cultures were incubated an additional 24– 48 h at 32 °C at which point the cells were scraped into the medium and transferred to a 10 cm2 plate containing a 70–80% confluent monolayer of Vero cells (Parks et al., 1999). Also 3– 5 h after incubating this coculture 3– 5 h, the medium was replaced

with DMEM containing FBS. Plaques developed 4–6 days later. Rescued virus was harvested by scraping the cells into the medium and freezing at − 80 °C. CDV minireplicon DNA transfections were performed either with Lipofectace (InvitrogenLife Technologies) (Fig. 2A) or the calcium-phosphate procedure (Fig. 2B). About 10–50 ng of CDV minireplicon DNA was cotransfected with N (400 ng), P (300 ng) and L (50– 100 ng) support plasmids. In some experiments (Fig. 5), minireplicon RNA (0.5 mg) rather than DNA was transfected with DOTAP (Roche Molecular Biochemicals). Cells were infected with MVA/T7 at a MOI of 2 at the time transfection was initiated (Fig. 2) or 1 h prior to transfection (Fig. 5A). After overnight transfection, the cells were fed with fresh medium and incubated an additional day at which time cell extracts were prepared and CAT assays performed basically as described earlier (Parks et al., 1999). In most of these assays, 14C-chloramphenicol was replaced with a fluorescent 1-deoxychloramphenicol substrate (Hruby and Wilson, 1992; Young et al., 1991), and the assays were modified according to the substrate manufacturer’s recommendation (FAST CAT Yellow; Molecular Probes). Fluorescent products of the CAT assay were separated by thin-layer chromatography and quantified with a Fluorimager (Molecular Dynamics). Sequence polymorphism ‘tags’ within the genomes of rCDV isolates were analyzed by DNA sequencing or restriction enzyme digestion. Infected-cell RNA was isolated by the guanidinium–phenol–chloroform extraction method as described above, and the genomic region containing the appropriate sequence tag was amplified by RT/PCR using the Titan one-tube RT/PCR kit (Roche Molecular Biology). RNA subjected to PCR amplification without a prior RT step served as a negative control that tested for the presence of contaminating plasmid DNA. PCR fragments were sequenced as described above, or the amplified fragment was digested with an appropriate restriction enzyme (Fig. 4B). The rCDV genome containing the luciferase gene (rCDV-P/luc/M) was subjected to sequence analysis to verify that the luciferase gene was correctly inserted. Cells infected with rCDV-P/luc/

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M isolates were also analyzed for luciferase expression. Infected-cell extracts were prepared with Reporter Lysis Buffer (Promega) and analyzed for luciferase activity using assay reagents and a luminometer from Pharmingen.

3. Results

3.1. Preparation of cDNA rescue reagents Before cloning plasmid DNAs needed for CDV rescue, it was decided to first confirm our previously published sequence of the Onderstepoort strain (Sidhu et al., 1993a,b). This was done because sequencing methods with improved accuracy are now available for analysis of negative-strand RNA virus genomes, and because inaccuracies in earlier sequence determinations could lead to problems generating effective rescue system reagents. In addition, McIlhatton et al. (McIlhatton et al., 1997) accurately reported that the published sequence of the CDV L gene (Sidhu et al., 1993b) contained several errors. At this stage, it was also decided that it would be advantageous to continue working with the lab-adapted strain of the Ondestepoort vaccine virus (Haig, 1956), because it can be readily propagated in cultured cells and because it may be useful for studies of vaccine virus attenuation. The Onderstepoort strain genomic sequence was reanalyzed using RNA extracted from infected HeLa cells. Infected-cell RNA was then used for RT/PCR and the resulting DNA fragments were sequenced directly by cycle-sequencing (Parks et al., 2001b). Determination of a consensus sequence in this way avoided artifacts associated with earlier methods that relied on analysis of cloned RT/PCR products. An amended CDV genomic sequence was deposited in GenBank (accession number AF014953). The full-length CDV cDNA clone was assembled from six RT/PCR fragments taking advantage of unique restriction sites located in the genome (Fig. 1B). The viral cDNA was cloned with a T7 RNA polymerase promoter fused to the 5% end of the positive-sense cDNA sequence. The phage promoter was truncated at the 3% end to

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remove three G residues that normally serve as the preferred T7 polymerase initiation site so that a significant proportion of the transcripts would initiate at the first base in the positive genome strand. The hepatitis delta virus ribozyme sequence and two T7 transcriptional terminators were cloned downstream of the viral cDNA sequence. The ribozyme is responsible for cleaving the T7 RNA polymerase primary transcript at the precise 3% end of the CDV genomic RNA. The completed genomic cDNA clone contained nucleotide substitutions that were probably introduced during PCR amplification. Some base changes within protein coding regions were silent with respect to amino acid codon specificity, and these substitutions were not repaired; they served as useful ‘tags’ to identify recombinant virus. In addition, two nucleotide substitutions in the P gene were not repaired. These substitutions were silent with respect to P protein but did result in two amino acid changes in C protein. C protein amino acid codons 158 (histidine) and 159 (leucine) were changed to leucine and arginine, respectively. Finally, one noncoding region base change was not repaired in the intergenic region between the M and F genes (M/F intergenic region) at nucleotide 6837. The plasmid vector used to clone the full-length viral cDNA was also used to generate the pCDVCAT minireplicon plasmid (Fig. 1A). The CDVCAT minireplicon reporter gene was inserted between the T7 RNA polymerase promoter and ribozyme sequence in the opposite direction of the full-length clone (Fig. 1B). Thus, T7 RNA polymerase transcription generates the equivalent of a negative-sense minigenome RNA. In addition to a full-length CDV cDNA clone, virus rescue requires expression vectors that can direct synthesis of trans-acting factors needed for de novo initiation of virus replication in transfected cells. Accordingly, the N, P, and L genes were each cloned into a T7 expression vector (Fig. 1C). Messenger RNAs transcribed from these expression vectors contain the EMCV IRES at the 5% end and a template-encoded poly-A sequence at the 3% end.

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3.2. Analysis of CDV minireplicon reporter gene expression Analysis of transient reporter gene expression mediated by a minireplicon (Radecke and Billeter, 1997) is an invaluable tool for establishing a virus rescue system. Detection of minireplicon reporter gene expression confirms that newly-cloned N, P and L expression vectors direct synthesis of active proteins, and the assay also tests whether cis-acting sequences in the minireplicon plasmid are functional. Confirming that these cis-acting elements are functional was important because the minireplicon vector also served as the backbone for the full-length clone. Cis-acting sequences that must function properly include those in the vector backbone (T7 RNA polymerase promoter and ribozyme sequence) as well as CDV cis-acting sequence elements (leader and trailer TCRs). In addition to confirming that plasmid reagents are functional, the minireplicon assay also provides a convenient tool for optimizing transfection conditions in preparation for virus rescue. A variety of transfection conditions were examined using a typical minireplicon transient expression assay (Marriot and Easton, 1999; Radecke and Billeter, 1997). Cells were transfected with plasmids encoding CDV-CAT and the N, P and L support proteins. Synthesis of both negative-sense minigenome RNA and synthesis of N, P, and L mRNAs is controlled by T7 RNA polymerase promoters; therefore, transfected cell monolayers were infected with recombinant vaccinia virus (MVA/T7) that encodes T7 RNA polymerase (MVA/T7; Wyatt et al., 1995). In some minireplicon experiments, cells were transfected with a liposome reagent (Fig. 2A), but in later experiments a calcium-phosphate procedure was employed (Fig. 2B). This change was made because it has been observed that calcium-phosphate transfection improves recovery of recombinant MV (unpublished observation; Radecke et al., 1995; Schneider et al., 1997). Additional modifications were made to the virus rescue protocol based on the findings described below. The effect of maintaining transfected cells at reduced temperature was examined because earlier studies indicated that transient expression

from the MV minireplicon was elevated if transfected cells were maintained at 32 rather than 37 °C (data not shown). CDV minireplicon rescue at both 37 and 32 °C was analyzed in parallel (Fig. 2A), and at both temperatures it was clear that significant CAT activity was produced over background levels detected from negative control transfections that lacked the viral L polymerase expression vector (lanes 1 and 6). It also was noticeable that the background signal produced by the CDV minireplicon was greater than that produced by the MV minireplicon (lanes 5 and 10). This increased background signal probably was due to a sequence located within the CDV leader TCR that served as a functional vaccinia virus or RNA polymerase II promoter. This explanation is supported by the fact that the MV minireplicon is identical to the CDV minireplicon except for the differences in leader and trailer TCR sequences. To help minimize this background problem, less minireplicon plasmid DNA was used in many subsequent experiments (for example Fig. 2B). This improved the signal-tonoise ratio of the assay by dramatically diminishing the background signal, but it did also result in some reduction of CAT activity observed in the experimental samples. Evaluation of the results in Fig. 2A revealed that incubation at 32 rather than 37 °C produced up to two to three times more CDV minirepliconencoded CAT gene expression (compare lanes 2 and 3 to 7 and 8). Similarly, MV-CAT minireplicon-encoded CAT expression was increased by lower incubation temperature (compare lanes 4 and 9). Thus, 32 °C was used for virus rescue. Previous studies have indicated that induction of the cellular heat shock response increased transient expression from the MV minireplicon and enhanced rescue of recombinant MV (Parks et al., 1999). In addition, heat shock has been shown to increase CDV gene expression (Oglesbee et al., 1993). To assess whether heat shock would stimulate CDV rescue, CDV minireplicon-encoded reporter gene expression was examined in transfected cells that were subjected to heat shock (Fig. 2B). Three hours after initiating calciumphosphate transfection and MVA/T7 infection, the transfected cells were shifted to 43 °C for 2 h

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then returned to a 32 °C incubator. Analysis of CAT activity revealed that heat shock treatment increased reporter gene expression from 4- to 16-fold indicating that this treatment would likely stimulate CDV rescue.

3.3. Rescue of rCDV The transfection and culture conditions that produced the greatest levels of CDV minireplicon reporter gene expression were used for CDV rescue. A549 or Hep2 cells were cotransfected with full-length viral cDNA plasmid, pT7CDV-N, pT7CDV-P, and pT7CDV-L by the calcium-phosphate procedure (Section 2). Three hours after initiating transfection, the cells were heat shocked as described above. The following day, the culture medium was replaced and incubation continued for an additional day. At this stage, cytopathic effect caused by MVA/T7 infection reduced cell viability, and it was also unclear whether rCDV would form detectable plaques on HEp2 or A549 cells. Therefore, the transfected cells were then cocultured with an established Vero cell monolayer to promote replication of rescued rCDV and facilitate plaque detection (Section 2) (Parks et al., 1999). Syncytia were observed 4–6 days after initiating co-culture (Fig. 3A). In most experiments, these rescue conditions produced 4–6 rCDV-positive wells per transfected six-well plate. A limited comparison of rescue efficiency when using the calcium-phosphate procedure or a liposomal transfection reagent revealed that calcium-phosphate transfection resulted in CDV rescue about 2-fold more often than the liposome reagent (data not shown). rCDV isolates from several experiments were characterized to confirm that recombinant virus was indeed rescued. A number of nucleotide substitutions (sequence ‘tags’) distinguish rCDV from CDV strains propagated in the laboratory (labadapted Onderstepoort strains from our laboratory and Martin Billeter’s laboratory). For example, there were two closely-positioned base changes in the rCDV P gene (nucleotides 2295 and 2298) that distinguish it from the nonrecombinant Onderstepoort viruses. These base substitutions were silent with respect to the P open

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reading frame, but did generate a BstBI restriction enzyme digestion site. RT/PCR amplification of this region from infected-cell RNA, and subsequent digestion of the PCR product with BstBI, clearly showed that recombinant virus contained the BstBI tag while a nonrecombinant strain did not. Cleavage of the PCR product produced from recombinant virus generated a doublet that migrated faster than DNA that was resistant to digestion (Fig. 3B, compare lanes 2, 4, 6 to lanes 8, 9, 10). These results were confirmed by directly sequencing RT/PCR-amplified DNA fragments. Eleven additional sequence tags were analyzed similarly and the results conclusively showed that rCDV was rescued. The possibility that sequence tag analysis was compromised by contaminating plasmid carried over from transfected cells can be ruled out by two negative controls. First, RNA was prepared from cells that originated from a negative control transfection that received all plasmid DNAs except the pCDV-L expression vector. These cells were carried through all rescue steps including the coculture procedure. RNA prepared from this coculture did not yield detectable amounts of PCR product (Fig. 3B, lane 1). Second, no PCR product was evident if the reverse transcription step was omitted (Fig. 3B, lanes 3, 5, 7) indicating that there was no detectable plasmid DNA contamination. In addition to rescuing rCDV, it was of interest to determine whether rCDV could be used as an expression vector like a number of other negative strand viruses (Collins et al., 1999; Conzelmann, 1998; Marriot and Easton, 1999; Nagai, 1999; Radecke and Billeter, 1997; Roberts and Rose, 1998). Accordingly, the CDV genomic cDNA clone was modified to facilitate insertion of a foreign gene between the P and M genes. Earlier studies that utilized measles virus or rinderpest virus vectors indicated that this region of the CDV genome would likely support effective foreign gene expression (Radecke and Billeter, 1997; Walsh et al., 2000a,b). To simplify foreign gene insertion, nine nucleotide substitutions were introduced in the P/M intergenic region, between positions 3330 and 3373 (Fig. 4A), to create a small multiple cloning site (MCS) composed of three

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Fig. 3. Rescue of recombinant CDV. A549 or Hep2 cells in six-well plates were cotransfected with genomic clone pBS-rCDV and expression plasmids encoding CDV N, P, and L proteins using the calcium-phosphate method described in the Section 2. After 48 h, transfected cells were co-cultured with an established monolayer of Vero cells to allow amplification of rescued virus and better visualization of plaques. In part (A), representative plaques from two recombinant CDV strains are shown. The plaque labeled rCDV was rescued from the Onderstepoort strain genomic cDNA. The plaque labeled rCDV-P/Luc/M is a recombinant strain that contains the luciferase gene (Fig. 4A). Part (B) contains the results from an analysis of a Bst B1 sequence ‘tag’ found in the recombinant viruses. RNA from cells infected with two isolates of rCDV (rCDV1 and rCDV2) or the nonrecombinant Onderstepoort strain obtained from Martin Billeter (OND) was used as template for RT/PCR. A DNA fragment from the P gene containing a BstBI restriction site polymorphism was amplified by RT/PCR then digested with the restriction enzyme. The BstBI restriction enzyme digestion site tag is present in rCDV and not present in non-recombinant Onderstepoort (OND) strains. Lanes 1–7 contain the undigested products of the RT/PCR reactions. Lane 1 (-L) contains the result from a negative control performed with RNA originating from a rescue experiment that was performed without pCDV-L. Lanes 3, 5, and 7 were negative controls in which the RT step was omitted. Lanes 8 –10 contain the results of BstBI digestion of samples equivalent to the DNAs in lanes 2, 4 and 6. Cleaved PCR fragments yield a doublet of approximately 315 bp and undigested DNA is 630 bp. The DNAs were detected by scanning an ethidium bromide-stained gel with a Molecular Dynamics fluorimager.

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Fig. 4. Recombinant CDV expression vector. Part (A) shows the nucleotide substitutions that were introduced into pBS-rCDV to generate plasmid pBS-rCDVmcs creating unique AatII, FseI and MluI restriction sites. The MCS region was used to clone the luciferase gene after the reporter gene was amplified by PCR. The 5% luciferase primer included an FseI site and a synthetic GE/GS sequence equivalent to the P/M intergenic region. The 3% primer contained a MluI site. Part (B) shows results from a luciferase assay performed with extracts made from cells infected by five different isolates of rCDV-P/Luc/M virus. Each well of a six-well plate containing Vero cells was infected with different rCDV strains and cell extracts were prepared at approximately 48 h after infection when 75% or more of the monolayer displayed cell fusion. Extracts were diluted 104-fold to produce the results shown in the figure. The negative control samples were analyzed undiluted; these included a mock infection and infections performed with rCDV and rCDVmcs virus. When the rCDV-P/Luc/M viruses were rescued, a negative control transfection was performed in parallel that lacked L expression plasmid (no pCDV-L). Cell lysate from this parallel mock rescue also produced only background levels of luciferase activity.

unique restriction enzyme cleavage sites (AatII, FseI and MluI). Recombinant virus containing these base substitutions (rCDVmcs) was rescued demonstrating that these nucleotide substitutions did not noticeably impair the virus (data not shown). The MCS was then used to clone the luciferase reporter gene (Fig. 4A) and generate plasmid pBS-rCDV-P/luc/M. A P – M intergenic gene end/gene start (GE/GS) signal was added to the 5% end of the luciferase gene before insertion; thus, expression of the reporter gene from its location between the P and M genes should be controlled by the synthetic P– M GE/GS signal added to the 5% end of the reporter gene and the natural P–M GE/GS signal flanking the 3% end.

Virus plaques were detected after using plasmid pBS-rCDV-P/luc/M in a rescue experiment (Fig. 4A, rCDV-P/Luc/M), and genomic sequence analysis revealed that the reporter gene was inserted as expected (data not shown). Vero cells infected with rCDV-P/Luc/M were also analyzed for luciferase expression. Infected cells exhibiting fusion over 70% or more of the cell monolayer were harvested for analysis of luciferase expression, and as shown in Fig. 4B, high levels of luciferase activity was observed in cells infected with several independent isolates of rCDV-P/Luc/M. Negative controls yielded very low background levels of luciferase (rCDV, rCDVmcs, no L plasmid).

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3.4. Rescue using a combined N and P expression 6ector The CDV rescue system was exploited further to examine the possibility that plasmid expression vectors based on vaccinia virus promoters would support virus rescue. A rescue system that relies primarily on vaccinia virus RNA polymerase for mRNA synthesis has a potentially important advantage; mRNA synthesized by vaccina virus RNA polymerase will be capped (Moss, 1990) and serve as a more effective substrate for translation.

In addition to being used for transfection, this type of expression plasmid could be made more versatile by including DNA sequences that would direct homologous recombination with the vaccina virus genome. This would provide the option for producing recombinant vaccinia virus strains that expresses CDV proteins if desired for use in a rescue system (Howley et al., 1999) or for other purposes that require high-level protein expression. To test the feasibility of using vaccinia virus promoter-based plasmid expression vectors, a

Fig. 5. Rescue with a modified vector for expression of N and P protein. (A) Plasmid pVVproN/P was designed to express both the CDV N and P. The CDV genes were cloned with synthetic early/late vaccinia virus promoters (Chakrabarti et al., 1997) at their 5% ends. This plasmid was designed primarily for use in rescue experiments, but a selectable marker (gpt) and reporter gene (b-GUS), and vaccinia virus DNA sequences (hatched bars) capable of mediating homologous recombination were added to create a vector that could be used also to generate recombinant vaccinia viruses (Sutter and Moss, 1992). In part (B), the pVVproN/P vector was used in a minireplicon experiment to determine if it would provide sufficient N and P protein to support minireplicon-encoded gene expression. Cells were infected with MVA/T7 and transfected with 0.5 mg CDV-CAT RNA, 0.5 mg pVVproN/V, and the amount of T7-L expression vector specified in the figure. The experiment was analyzed as described in Fig. 2A. In part (C), the pVVproN/P plasmid was used in four independent CDV rescue experiments that were performed as described in Fig. 4. HEp2 cells were infected with MVA/T7 (experiments A, B, D) or MVAGKT7 (Kovacs et al., 2002) (experiment C) and co-transfected with 5 mg pBS-rCDV, 100 ng of T7-L expression vector and either 500 ng of ppVVproN/P or 400 ng pT7CDV-N plus 300 ng pT7CDV-P. Plaques were detected as described in Fig. 3, and the experiment was scored 9 for the presence of plaques.

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plasmid was prepared that contained both the N and P genes (pVVproN/P; Fig. 5A). This plasmid was tested first in a minireplicon assay by using it in place of the typical T7-N and T7-P expression vectors (Fig. 5B). Minireplicon RNA was cotransfected into Vero cells with plasmid pVVproN/P and the T7-L expression plasmid, and the transfected cells were infected with MVA/ T7 1 h prior to transfection. The negative controls shown in Fig. 5B that lacked L (lane 3) or N/P (lane 2) expression vectors, or were not infected with MVA/T7 (lane 1), produced low levels of background CAT. Cells transfected with pVVproN/P plus the L expression plasmid did support minireplicon-encoded gene expression (lane 4 and 5), and as seen earlier (Fig. 2), the minireplicon system was sensitive to the amount of L protein expression (compare lanes 4, 5 and 6). Inhibition by higher amounts of pT7CDV-L expression vector was quite pronounced in this assay system when compared with the results shown in Fig. 2. The reason for this is unknown but may be related to the fact the amount or ratio of N and P expression may be substantially different when using the pVVproN/P plasmid rather than the conventional T7-N and T7-P plasmids. The positive results obtained in the minireplicon experiment led us to test the combined N/P expression vector in virus rescue experiments (Fig. 5C). Five wells of a six-well plate containing HEp2 cells were cotransfected with the CDV genomic clone, the T7-L expression vector, and the pVVproN/P expression vector. A positive control performed in the sixth well was transfected with the CDV genomic clone and T7 expression vectors for N, P and L as described earlier (Fig. 4). In independent experiments A, B and D (Fig. 5C) the cells were coinfected with MVA/T7 helper virus (Wyatt et al., 1995) and in experiment C the cells were infected with an MVA derivative (MVAGKT7) that expressed the T7 polymerase gene from a synthetic early/late promoter (Kovacs et al., 2002). In all four experiments, the positive control in well 6 produced virus detectable by plaque formation. Rescue transfections performed with pVVproN/P were also successful. The number of transfected wells generating virus ranged from one out of five in the first experiment to five

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out of five in the final two experiments. These results demonstrate that the vaccinia virus promoter-based expression system is a viable alternative.

4. Discussion Gassen et al. (Gassen et al., 2000) first demonstrated that CDV could be recovered from cloned DNAs adding it to the growing list of nonsegmented negative-strand RNA viruses that have been successfully rescued (Collins et al., 1999; Conzelmann, 1998; Marriot and Easton, 1999; Nagai, 1999; Radecke and Billeter, 1997; Roberts and Rose, 1998). Our results confirm their conclusions and also demonstrate that CDV, like other negative-strand RNA viruses (Collins et al., 1999; Conzelmann, 1998; Marriot and Easton, 1999; Nagai, 1999; Radecke and Billeter, 1997; Roberts and Rose, 1998), can effectively function as a viral expression vector. Taken together, these studies indicate that molecular genetic manipulation of CDV is feasible, and that rescue technology can facilitate rational design of future attenuated CDV strains and viral expression vectors. Rescue of CDV relied on established techniques (Conzelmann, 1998; Marriot and Easton, 1999; Nagai, 1999; Radecke and Billeter, 1997; Roberts and Rose, 1998), but several technical modifications were added during our studies. These modifications were introduced after identifying conditions that enhanced transient expression from a CDV minireplicon. For example, it was observed that incubating cells at 32 rather than 37 °C increased minireplicon-encoded gene expression (Fig. 2A). The mechanism responsible for this observation is unclear, but it could be related to a number of explanations. For example, maybe one of the CDV trans-acting proteins is slightly temperature sensitive. Alternatively, it could be possible that the lower temperature simply slows the rate of MVA/T7 infection and increases cell viability during the transfection period. The effect of lower temperature also may be related to the observation that infected cells produce increased levels of MV at reduced temperature (Coulter-Mackie et al., 1980; Udem,

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1984; Wechsler and Fields, 1978). Regardless of the mechanism, the practical value of this finding led us to routinely perform rescue experiments at the lower temperature. We also found that transient heat shock treatment improved CDV minireplicon reporter gene expression (Fig. 2B). The effect of heat shock was examined because earlier studies showed that induction of the cellular stress response stimulated CDV gene expression (Oglesbee et al., 1993) and also improved measles virus rescue efficiency (Parks et al., 1999). The positive effect of heat shock on CDV minireplicon-encoded gene expression led us to incorporate a heat shock step into the CDV rescue protocol. Each of these optimization steps produced modest increases in minireplicon-encoded gene expression, but taken together they may have had a significant effect on virus rescue efficiency. We regularly obtained 4– 6 CDV-positive wells from a transfected six-well plate when using the modified protocol. This relatively efficient rescue scheme may be helpful in the future if new rCDV strains under study prove to be difficult to rescue. The protocol modifications mentioned above were used in combination with a calcium-phosphate transfection technique. It was found that calcium-phosphate transfection generally increased the percentage of transfected cultures that produced virus by about 2-fold over a liposome transfection method that was tested (data not shown). Again, this may not be a large effect, but it could be important when isolating a highly attenuated strain. The reason that calcium-phosphate was more effective is not known, but has also been observed by others (Radecke et al., 1995; Schneider et al., 1997). Maybe calciumphosphate is less damaging to cell membranes than liposomal reagents, and healthier cell membranes promote maturation and budding of relatively rare rescued viruses. It could also be true that calcium-phosphate precipitates are somewhat more effective at introducing multiple different plasmids into the same cell, and actually generate a greater number of cells that contain the complete set of N, P, and L expression plasmids together with the genomic cDNA.

The CDV rescue system was also used to test an alternative expression vector system. A plasmid was constructed containing both the N and P genes under control of vaccinia virus transcription regulatory signals (Fig. 5). This plasmid effectively complemented both minireplicon and virus rescue. At this stage, the rescue system based on plasmids controlled by vaccinia virus promoters requires further development and analysis before the effectiveness of this type of system can be compared with the more traditional T7 expression vector systems. As an alternative to T7 expression plasmids, the vaccinia virus promoter vectors may be attractive in some cases because these plasmids also can be designed to serve as recombination plasmids for making recombinant vaccinia virus strains. The rescue of rCDV provides one avenue to pursue development of safer live attenuated CDV vaccines. It would be a valuable improvement if a new live attenuated CDV vaccine was developed that would remain highly effective for immunization of dogs while being safe and effective for use in other animals such as large cats, small carnivores and seals. One approach to achieving this goal may be to use a rational vaccine design strategy. There have been results from a number of studies that may help identify potential attenuating amino acid substitutions and cis-acting signal changes that could be introduced into rCDV for further examination. For example, analysis of recombinant strains of human parainfluenza virus type 3 and respiratory syncytial virus have identified attenuating mutations that may have good correlates in CDV. These include amino acid substitutions in the L protein (Collins et al., 1999; Skiadopoulos et al., 1998, 1999; Whitehead et al., 1999), and mutations in cis-acting sequences in the leader and in GE/GS signals (Collins et al., 1999; Juhasz et al., 1999; Skiadopoulos et al., 1999; Whitehead et al., 1998). In addition, the complete genomic sequence of pathogenic and nonpathogenic strains of measles virus and rinderpest virus been examined (Baron et al., 1996; Parks et al., 2001a,b; Takeda et al., 1998). Coding and noncoding genome changes have been identified in the less pathogenic strains and some of these changes may be attenuating if introduced

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into rCDV. Furthermore, a gene inactivation approach may be useful. There are examples of viruses defective for C or V protein expression that exhibit some degree of attenuation (Baron and Barrett, 2000; Durbin et al., 1999; Escoffier et al., 1999; Garcin et al., 1997; Kato et al., 1997; Mrkic et al., 2000; Nagai, 1999; Patterson et al., 2000; Tober et al., 1998; Valsamakis et al., 1998). Finally, it may be possible to utilize the novel gene shuffling approach (Ball et al., 1999; Wertz et al., 1998) to develop a safer and more attenuated CDV vaccine strain. Our studies have also demonstrated that CDV can be used as an expression vector. This raises the possibility of using a live attenuated CDV vaccine to immunize dogs against distemper plus one or more infectious agents. Antigen genes from a variety of viruses (Appel, 1999; Carmichael, 1999), including rabies virus, canine adenovirus types 1 and 2 (infectious canine hepatitis, respiratory illness), or canine parvovirus (severe gastrointestinal illness), could prove interesting for further analysis of a CDV vaccine vector.

Acknowledgements The authors thank Martin Billeter for his gift of CDV, and thank T. Zamb, C. Pachuk, C. Satishchandran, and M. Billeter for helpful discussion.

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