INTRODUCTION

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Rinderpest (RP) and peste-des-petits-ruminants (PPR) are two important diseases of domestic ruminants causing great economic losses due to their high morbidity and mortality. RP is a severe, acute disease of cattle, buffalo, and wild bovids, while PPR is a disease of sheep and goats that clinically resembles RP; both RP and PPR are regarded as List A diseases by the Office International des Epizooties due to their highly contagious nature and consequent capacity for rapid spread. RP virus (RPV) and PPR virus (PPRV) are both members of the Morbillivirus genus of the family Paramyxoviridae, a genus which also includes Measles virus (MV) (humans), Canine distemper virus (CDV) (canids and other wild carnivores), Phocid distemper virus (PDV) (seals), and the morbilliviruses of porpoises and dolphins (PMV/DMV). RP is currently the target of an international eradication campaign (20). Sheep and goats can be infected with RPV, but only the most virulent strains show any clinical disease in these species (7, 16). Because of the cross-reactive and cross-protective antibodies generated by either of these two related viruses, the normal tissue culture-adapted vaccine strain of RPV (RBOK) (37) is commonly used to vaccinate against PPR, as it is known to be both safe and clinically effective (47).

Recently, PPR has become a much more prominent disease because, besides causing disease in small ruminants, it influences diagnosis and vaccination carried out to prevent RP in large ruminants. PPRV can cause a subclinical infection in the latter, and it can be confused with RPV due to cross-reactive antibodies unless virus-specific reagents are used to analyze sera. This has important implications for the ongoing campaign for elimination of RP. In addition, areas which have been declared free of RP can no longer use the RPV vaccine strain to vaccinate against RP or PPR. A vaccine strain of PPRV [derived from PPRV(Nigeria 75/1)] has been isolated but has not yet been widely tested in the field. It would therefore be of great practical use to develop a vaccine against PPR which combines the known safety of the RBOK vaccine with usability in RP-free areas due to a unique serological signature.

Sequence comparisons show that RPV is most closely related to MV (3). Only about half of the sequence of PPRV is known; despite the similarity in the hosts of RPV and PPRV, such sequence data as are known show that PPRV is no more related to RPV than to CDV or PMV/DMV. The viruses all have a negative-strand RNA genome; in RPV it is 15,882 nucleotides in length (3). The morbillivirus genome is tightly encapsidated by the viral N protein and associated with the P and L proteins, which together make the viral polymerase. The nucleocapsid is in turn surrounded by a lipid envelope which contains two glycoproteins, F (fusion) and H (attachment). It is known that these glycoproteins are the major protective immunogens and are responsible for inducing neutralizing antibodies (6, 22, 49, 50). The high degree of sequence conservation between the F proteins of different morbilliviruses (9, 30) probably accounts for the extensive cross-protection observed between different viruses of this genus, enabling, for example, the RPV vaccine to be used to vaccinate against PPRV. The H proteins are more divergent (8) and may play a role in host cell specificity. By changing the F and/or H genes of the RPV vaccine strain for those of PPRV, the immune response can be further directed toward the latter virus.

The techniques by which single-segment, negative-strand RNA viruses can be rescued from cDNA copies of their genomes have already been applied to both MV (38) and RPV (4). Here we describe the rescue of a chimeric RPV in which both the F and H genes have been replaced by those of PPRV. The efficacy of this virus as a vaccine against PPRV infection has also been evaluated.

	MATERIALS AND METHODS

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Cells and viruses. Vero cells were grown in Dulbecco's minimal essential medium (MEM) containing 25 mM HEPES (pH 7.2) and 5% fetal calf serum (FCS). 293 cells were grown in Dulbecco's MEM without HEPES and with 10% FCS. B95a cells were grown in RPMI 1640 with 5% FCS. All media contained penicillin (100 IU/ml) and streptomycin (100 µg/ml).

Molecular biology techniques. Plasmids pKSMF, pMDBRPVII, pKSN1, pKS-P, and pGEM-L have been described in reference 4. Plasmid pKSF was derived from pKSMF by removing the SmaI-EcoRI fragment containing most of the F gene and ligating it into similarly cut pKS(+). Plasmid pKSL9, containing the whole of the RPV H gene plus parts of the flanking F and L genes, has been described in reference 3. Mutagenesis by elimination of unique sites was performed essentially as described in reference 18. Quick-Change mutagenesis (Stratagene) was performed as described in the instructions to the Quick-Change kit. All other DNA manipulations and cloning procedures were as previously described (4). RNA was purified from small-scale cultures (35-mm-diameter wells) using Trizol (Life Technologies, Inc.); large-scale preparations from virus-infected cells were performed according to the method of Chomczynski and Sacchi (15). For extraction of RNA from purified peripheral blood leukocytes (PBL), the cells were pelleted (1,280 × g, 10 min), and the pellet was resuspended in 100 µl of phosphate-buffered saline, which was then mixed with 1 ml of Trizol and processed according to the manufacturer's instructions. Ocular swabs were placed in 2-ml screw-cap tubes and extracted with 1 ml of Trizol by vigorous vortexing. The Trizol extract was then processed in the normal way. Reverse transcription-PCR (RT-PCR) was performed using Taq polymerase as described in reference 21. Preparative, gene-copying PCR using the proofreading Pfu polymerase was performed as described in reference 5.

Insertion of new restriction sites into the RPV genome sequence. A total of four new restriction sites were introduced into the RPV genome cDNA clone. Plasmid pKSL9 was mutated by unique site elimination (USE) mutagenesis to introduce either an AscI site at the position corresponding to 7195 to 7202 in the antigenome (pKSL9FAsc) or a PmlI site at the position corresponding to 9092 to 9097 in the antigenome (pKSL9HPml). The two mutations were combined by exchanging the EcoRI-SphI fragment containing the AscI site in pKSL9FAsc with the corresponding fragment in pKSL9HPml. The SunI-NcoI fragment from the resulting construct was then ligated to NcoI-AatII and AatII-SunI fragments from pRPVII to make pRPV2A. Plasmid pKSF was similarly mutated to introduce an SgfI site at the position corresponding to 5435 to 5442 in the antigenome. Plasmid pKSMF was mutated using the Quick-Change system (Stratagene) to introduce a SwaI site at the position corresponding to 4456 to 4463 in the antigenome. Because we observed mutations at secondary sites when using the Quick-Change system, we sequenced and removed the small AgeI-BsmBI fragment containing the SwaI site and ligated it into a nonmutated copy of pKSMF to give pKSMF-Swa. The Eco47III-NsiI fragment containing the SgfI site in pKSF-Sgf was then used to replace the corresponding section of pKSMF-Swa to give plasmid pKSMFSS. The SphI-SunI fragment from pKSMFSS, containing the P-M intergenic region, the M gene, and most of the F gene, was then ligated to the SunI-ClaI and ClaI-SphI fragments of pRPV2A to give pRPV2B.

RT-PCR and cloning of PPRV glycoprotein coding sequences. To obtain cDNA copies of the open reading frame (ORF) sequences of the H and F genes of PPRV with the appropriate restriction enzyme sites for cloning into pRPV2B, the two sequences were independently amplified by RT-PCR using RNA from Vero cells infected with the attenuated Nigeria 75/1 vaccine strain of PPRV. The H gene was amplified with primers PPRFAsc (5'-GGCGCGCCCTATTACACATTGGTCATC-3') and PPRHPml (5'-CACGTGTACTCAGACTGGATTACAT-3'), and the F gene was amplified with primers PPRFSgf (5'-CAATTAGCGATCGCCCATGTATAAACATCAT-3') and PPRFAscIR1 (5'-GGCGCGCCTTCTGGTCGGTGATCGGA-3'). The amplified DNAs were ligated into pGEM-T and sequenced.

Transfection and virus rescue. Virus rescue in 293 cells was as previously described (5). For rescue in Vero cells, a new technique involving use of the recombinant fowlpox virus FP-T7 to express the T7 polymerase protein was used (17). Briefly, cells were plated at 2 × 10⁵ cells/well in six-well plates 1 day before use. Cells were approximately 70% confluent at the time of transfection. The cells were infected at a multiplicity of approximately 0.2 with FP-T7 for 1 h and then transfected with pKS-N, pKS-P, pGEM-L, and the appropriate genome cDNA construct, using 1 µg of each plasmid except pGEM-L (50 ng). Transfection was carried out using FuGENE6 (Roche Biologicals) according to the manufacturer's instructions at a ratio of 7.5 µl of FuGENE6 per µg of DNA. Transfected cells were incubated for 4 or 5 days, by which time either CPE was visible (positive controls) or the cells were trypsinized and transferred to 75-cm² flasks for further growth (chimeric constructs).

Virus characterization. RT-PCR to characterize recombinant viruses was carried out as described above, using RNA isolated from Vero cells that had been infected with RPV, PPRV, or chimeric virus, along with the following primer pairs: RPVF14 (5'-ACCAAATCCATCCGAGCATC-3') plus PPRF1 5'-ATCACAGTGTTAAAGCCTGTAGAGG-3'); PPRH1 (5'-TGGTCAGAGGGGAGAAT-3') plus RPVH6 (5'-GGAGGCCCTGGTTTATAA-3'); and PPRF1 (5'-ATCACAGTGTTAAAGCCTGTAGAGG-3') plus PPRH11 (5'-ATGTAGGGTCTTTCAATAGTT-3').

Animal studies. Indigenous British white goats between 3 and 12 months of age were used in these experiments, which were carried out under biosafety level 2 with regard to staff and at level 4 with regard to environmental release of pathogens. For vaccination, stocks of RPV2B, PPRV(Nigeria 75/1), and RPV-PPRFH were grown on Vero cells; 10⁴ TCID₅₀ of RPV2B or PPRV, or 10³ TCID₅₀ of RPV-PPRFH, was used to vaccinate each animal; 1 ml of virus was inoculated subcutaneously in the shoulder region. Challenge virus was 10⁴ TCID₅₀ of lamb kidney cell-grown PPRV(Ivory Coast 89/1), the kind gift of Adama Diallo, CIRAD/EMVT, Cedex, France. Rectal temperatures were recorded daily, and the animals were examined every second day for specific clinical signs such as oral lesions, salivation, or ocular or nasal discharges. Blood was collected at 2-day intervals for serum (nonheparinized) or PBL counting and purification (heparinized blood). PBL were purified as previously described (34). Swabs were taken at 2-day intervals from both eyes.

	RESULTS

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Plasmid construction. To construct RPV/PPRV chimeras, it was necessary first to insert suitable restriction sites at the beginning and end of the ORFs to be exchanged. We therefore inserted unique sites (i) just before the beginning of the F ORF (SgfI), (ii) just after the F ORF and before the F-H intergenic sequence (AscI), and (iii) at the end of the H ORF and before the H-L intergenic sequence (PmlI), as described in detail in Materials and Methods. These sites enable us to remove either or both of the sequences encoding the viral glycoproteins. RPVs rescued from the resulting clones of the RPV genome (pRPV2A and -B) were indistinguishable from the original virus in both ease of rescue and growth in tissue culture (not shown).

Rescue of chimeric virus. All three pRPV-based plasmids were used in rescue experiments as described previously (4, 5). Rescue of each plasmid was tried at least twice, using six 35-mm-diameter wells in each attempt. Although the parental plasmid pRPV2B was always rescued as live virus, no virus was recovered from cells transfected with pRPV-PPRF, pRPV-PPRH, or pRPV-PPRFH. Reasoning that the PPRV glycoproteins came from a virus that had been extensively adapted to Vero cells, and might therefore interact poorly with surface receptors in the initially transfected human cell line (293) or the marmoset lymphoblastoid cell line (B95a) used in the second stage of the rescue protocol, we attempted to perform the primary transfection in Vero cells. However, although the recombinant vaccinia virus MVA-T7 does not replicate in Vero cells, it causes sufficient CPE that most of the Vero cells had died 3 to 4 days after infection with this virus. Rescue of a slow-growing morbillivirus by this system was therefore not possible.

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Confirmation of gene order in the chimeric virus by RT-PCR. (A) RNA isolated from virus-infected cells was analyzed by RT-PCR using the indicated primer pairs. The virus used was RPV-PPRFH (tracks 1, 4, and 7), RPV (tracks 2, 5, and 8), or PPRV (tracks 3, 6, and 9). (B) Line diagram the F-H gene region that should exist in the chimeric RPV-PPRFH virus. Positions of the primers used in the PCRs are shown, and lengths of the expected amplification products are indicated.

In vitro characterization of the chimeric virus. The nature of the recombinant virus was further confirmed by immunofluorescence microscopy on cells infected with RPV, PPRV, or RPV-PPRFH (Fig. 2). Rabbit polyclonal antibody MB18, which was raised against a fusion protein containing the C terminus of the RPV P protein and recognizes RPV P but not PPRV P (Fig. 2), was used to identify cells expressing RPV core proteins. Monoclonal antibodies specific for the H proteins of the parental viruses have been isolated and routinely used in virus-specific ELISAs (2). The chimeric virus produced the PPRV H protein, as seen by reaction with monoclonal antibody C77 (anti-PPRV H) but not with C1 (anti-RPV H). The same cells were also labeled by MB18, unlike cells infected with normal PPRV (Fig. 2), showing that the chimeric virus expresses a different combination of proteins than either parent.

View larger version (66K): [in this window] [in a new window]   FIG. 2.   Immunofluorescence microscopy of cells infected with parental (RPV or PPRV) or chimeric virus. All cells were incubated with rabbit anti-RPV P protein (MB18) combined with either mouse anti-RPV H protein (C1) or mouse anti-PPRV H protein (C77) and then with fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G combined with Texas red-labeled goat anti-mouse immunoglobulin G. (A to D) RPV2B; (E to H) RPV-PPRFH; (I to L) PPRV. (A, E, and I) Staining with MB18; (B, F, and J) same cells stained with C77; (C, G, and K) staining with MB18; (D, H, and L) same cells stained with C1. Bar in panel L = 10 µm.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Growth of RPV-PPRFH in tissue culture. Vero cells infected with PPRV (diamonds), RPV2B (stars), or RPV-PPRFH (squares) were frozen at different times postinfection, and the titer of virus present was determined as the TCID50.

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Plaque morphology of chimeric virus. Vero cells were infected with about 500 TCID50 of each virus and cultured under carboxymethyl cellulose. After staining, the cells were photographed at a magnification of ×100 (A to C) or the tissue culture dishes were photographed directly (D to F).

In vivo characterization of the chimeric virus. RPV-PPRFH was also tested for efficacy as a vaccine, comparing it to the two parental viruses, RPV and PPRV. We infected four goats with RPV2B, four with RPV-PPRFH, and two with PPRV(Nigeria-75/1); six were left unvaccinated (for clarity, data from only four of these control animals are shown in the following figures). All animals were challenged with a pathogenic strain of PPRV (Ivory Coast) 3 to 5 weeks postvaccination.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Rectal temperatures of animals subjected to vaccination and challenge. Animals were vaccinated with RPV-PPRFH (a to d), RPV2B (e to h), or PPRV (i and j) or were left unvaccinated (k to n). Rectal temperatures were recorded daily for 2 weeks following vaccination and challenge. The temperature (39.5°C) above which the animals were considered to be pyrexic is indicated by a dotted line.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   Leukocyte counts of animals subjected to vaccination and challenge. Animals were vaccinated as in Fig. 5, and blood samples were taken at 3- to 4-day intervals. Total leukocyte counts were determined as in Materials and Methods. Measurements which were less than 50% of the initial count for a particular animal are indicated by asterisks.

View larger version (52K): [in this window] [in a new window]   FIG. 7.   Antibody responses in animals subjected to vaccination and challenge. Serum was isolated at weekly intervals from the animals described in the legends to Fig. 5 and 6, and levels of specific anti-RPV H (open bars) and anti-PPRV H (filled bars) were determined by cELISA. The average of duplicate readings is plotted. Values above 50% inhibition (indicated by the dotted lines) were taken to be positive, and the day of challenge is indicated by an arrow for the vaccinated animals (A to J).

	DISCUSSION

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All paramyxoviruses have two envelope glycoproteins, one (F) which appears to mediate fusion of the virus envelope with the host cell plasma membrane, and a second (variously H or HN, or G in the pneumoviruses) which is the attachment or receptor-binding protein by which the virus associates with the target cell. In infected cells, or when expressed from cDNA clones, these proteins cause fusion of the plasma membranes of adjacent cells to create syncytia, a characteristic of paramyxovirus cytopathology. Although the F protein of simian virus 5 appears to be able to cause syncytium formation by itself (25, 36), as does that of respiratory syncytial virus (35) and MV (1), other studies with a number of paramyxoviruses have found that both glycoproteins are required for cell-cell fusion (19, 23, 31, 32, 38, 46, 48). This discrepancy has been ascribed to differences in the expression systems used (23). Although it was originally thought that the role of the H/HN protein in syncytium formation was simply to bring cell surfaces close enough together for fusion to take place, a number of studies showed that functional interaction of the two glycoproteins required that they be derived from the same virus (10, 23, 25, 26) and that sometimes even the glycoproteins of different strains of the same virus could not bring about syncytium formation (14). If syncytium formation by a pair of viral glycoproteins can be taken as a measure of the ability of a virus expressing those proteins to enter its host cell, these observations would suggest that any chimera between two paramyxoviruses should contain F and H/HN genes from the same virus. However, two other studies showed that combinations of glycoproteins from two morbilliviruses (CDV and MV) could function together to cause cell fusion (33, 43). The rate of appearance was lower and the syncytia were smaller when a heterologous pair of proteins was used than when both proteins were from the same virus (43), and MV H together with CDV F was more effective than the reverse pairing (33). It was therefore possible that heterologous combinations of RPV and PPRV F and H proteins might function adequately to allow recovery of viable virus.

Despite many attempts, in all of which control RPV genome plasmids were rescued into viable virus, we were unable to rescue virus in which only one of the two glycoprotein genes of RPV was swapped with the corresponding PPRV gene. That the clones of the PPRV F and H genes were functional was demonstrated by the reproducible rescue of RPV-PPRFH. These observations suggest that type-specific interactions between F and H/HN are important not only in syncytium formation but also in virus entry, or possibly in virus assembly and budding. Some combinations of viral proteins, expressed in certain ways, may allow low levels of cell-cell fusion, but this does not appear to mean that these proteins could function in vivo to fuse the viral envelope with the host cell. The only published report of a similar paramyxovirus chimera published, between human parainfluenza virus types 1 and 3 (hPIV1 and hPIV3) (45), also involved a change of both glycoproteins simultaneously.

In tissue culture, RPV-PPRFH grew much more slowly than either of the parental viruses. In this respect, the RPV/PPRV chimera resembled recombinant MVs in which the F and H genes were replaced with the single attachment/fusion glycoprotein G from vesicular stomatitis virus (42). In the same study, it was shown that the cytoplasmic domain of the MV F protein was required for incorporation of the viral M protein into the budding virion. There is also direct evidence for interactions of the M protein of the related Sendai virus with the cytoplasmic domains of both viral glycoproteins (40, 41). It is possible, therefore, that the defective growth of RPV-PPRFH is due to defective interaction of the cytoplasmic domains of one or both of the PPRV glycoproteins with the RPV M protein. However, a number of other observations provide evidence against this hypothesis. The cytoplasmic domains of morbillivirus F proteins are highly conserved, being identical over the last 14 amino acids in all morbilliviruses studied (30), and so the change introduced to this domain in exchanging the F proteins of the two viruses is small. In addition, another study of recombinant MVs (13) showed that changes to the cytoplasmic domains of F and H glycoproteins, including deletion of 14 amino acids from the cytoplasmic domain of H and replacement of the cytoplasmic domain of F with that of Sendai virus F (no sequence similarity to the cytoplasmic domain of MV F), led to viruses which grew to titers similar to those for the parental MV strain, albeit with a highly fusogenic phenotype, with large syncytia, similar to the type of CPE caused by RPV-PPRFH. These viruses with highly altered cytoplasmic domains on their glycoproteins showed reduced incorporation of the M protein into virions, and it was suggested that M protein binding to the one or other of the glycoproteins suppresses fusogenic potential (13) as a means of reducing active fusion of newly synthesized virus with membranes of the host cell. Further studies will be required to determine if our chimera is defective in incorporation of the M protein into virions and if the growth defect can be rectified by including the PPRV M protein as well as the F and H proteins. Since the morbillivirus M protein also interacts specifically with the viral nucleocapsid (24, 44), such a substitution may introduce other defects, and it may prove necessary to identify and swap specific M protein domains which interact with the viral envelope glycoproteins. An important result from these studies has been that the exchange of glycoproteins between two morbilliviruses with relatively closely related sequences led to a defective virus. By contrast, a chimera of hPIV3 containing the F and HN of hPIV1 was fully viable (45). Despite being in the same genus, hPIV3 and hPIV1 show almost no sequence similarity between the corresponding cytoplasmic domains of their glycoproteins. There may therefore be a significant difference in the assembly of viruses of these two groups.

An unexpected finding in this study was that the chimera could not be rescued by transfecting 293 cells. These cells have been used for the rescue of both MV and RPV (4, 38), even though these viruses were not specifically adapted to growth in these cells. It was subsequently observed that the (Vero-adapted) Nigeria strain of PPRV did not appear to grow in this cell line. It may be that these cells lack or are deficient in a specific attachment protein or an intracellular host protein necessary for efficient PPRV replication. The change of glycoproteins appeared to be a strong determinant of host range for the chimera since it readily infected and grew in Vero cells but not in the B95a lymphoblastoid cell line; these cells are a good host for RPV (27) but did not support replication of our PPRV strain unless the virus was adapted through five or six blind passages.

Despite its apparent attenuation in growth relative to its parents, the chimera successfully protected vaccinated animals from subsequent challenge. The effect of PPRV challenge on unvaccinated animals seen here was much less severe than seen in earlier studies on American mixed-breed animals (12). This may be due to differences in the susceptibility of different strains of goat or to the fact that we used PPRV grown in tissue culture rather than whole blood from an infected goat. However, the clinical responses in unvaccinated animals upon challenge in the present study were clear and readily distinguishable from those of vaccinated animals. Although two of the animals vaccinated with RPV-PPRFH showed no serum antibody to PPRV H protein before challenge, all four experimental animals were protected, possibly due to cell-mediated immunity, priming of the antibody response, or a combination of the two. As yet, very little is known about the role of cell-mediated immune responses in protection from RPV or PPRV infection. All animals vaccinated with RPV-PPRFH showed a clear anamnestic response in serum anti-PPRV H after challenge, which is not seen in animals vaccinated with RPV, showing that the antibody response is more PPRV specific in this system. In addition, vaccinated animals should generate a serological signature distinct from that generated by exposure to either RPV or PPRV, with antibodies recognizing unique epitopes on RPV N (28, 29) and on PPRV H (2). This may allow the chimera to be used as a genetically marked vaccine, which will be useful both in the RPV eradication campaign and for the control of PPRV, requiring epidemiological seromonitoring of PPRV prevalence and spread in the presence of vaccination. However, further clinical trials involving animals of different genetic backgrounds, and tests of longevity of the protection afforded by the vaccine, are required to establish its clinical safety and effectiveness.