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1 Onderstepoort Veterinary Institute, Exotic Diseases Division, Private Bag X05, Onderstepoort 0110, South Africa
2 Plum Island Animal Disease Center, USDA, NAA, ARS, PO Box 848, Greenport, NY 11944, USA
Correspondence
H. G. van Rensburg
trudi{at}saturn.ovi.ac.za
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank accession number for the nucleotide sequence of the ZIM/7/83 genome reported in this paper is AF540910.
Present address: Department of Pathology, Sealy Center for Vaccine Development and WHO Collaborating Center for Tropical Diseases, University of Texas Medical Branch (UTMB), 301 University Boulevard, Galveston, TX 77555-0436, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Van Rensburg & Nel, 1999; Bastos et al., 2001, 2003) and antigenic variability (Esterhuysen, 1994). These types are further characterized by their ability to infect multiple wildlife hosts. African buffalo (Syncerus caffer) in southern Africa have been shown to be persistently infected with SAT type viruses (reviewed by Thomson et al., 2003) and are an important source of infection for domestic animals (Dawe et al., 1994; Bastos et al., 1999). The eradication of the disease from the region is therefore unlikely. Control in southern Africa consists mainly of vaccination as well as movement control of animals and animal products (Hunter, 1998). Due to the extensive variability of the SAT types, different lineages establish themselves in different geographical regions (Vosloo et al., 1995; Bastos et al., 2001, 2003). Current inactivated vaccines are often unable to control some lineages arising in these regions, arguing for the development of custom-made vaccines for use in specific geographical localities. However, development of useful cell-culture-adapted vaccine strains from field isolates is time-consuming and expensive, limiting the availability of custom-made vaccine strains.
The FMDV genome consists of a positive-strand RNA molecule of approximately 8·5 kb. This genome encodes a single translational ORF, and the product of the ORF is co- and post-translationally cleaved by viral proteinases (Sobrino et al., 2001). The fact that the viral RNA can be made infectious in the absence of other components of the virion opened the theoretical possibility of genetically engineering new viruses from in vitro-generated RNA molecules. Zibert et al. (1990) succeeded in the development of this technology, providing a powerful new tool for the study of FMD and the development of new methods to control the disease. Utilizing this technology, it is possible to engineer recombinant viruses containing manipulated and/or exchanged antigenic determinants (Rieder et al., 1994; Sa-Carvalho et al., 1997; Almeida et al., 1998; Baranowski et al., 1998; Beard & Mason, 2000). Thus, it is possible that chimeric SAT viruses could be designed to retain the desirable biological properties of good vaccine strains, whilst encoding the antigens required for vaccines in different geographical localities.
In a recent study, the usefulness of a type A genome-length clone in the construction of chimeras containing the structural proteins of SAT type 2 was reported (Van Rensburg & Mason, 2002). These studies showed that this chimera, created using a previously constructed type A12 genome-length backbone (Rieder et al., 1993), exhibited a slower growth rate than the parental SAT2 virus, suggesting that the A12 genome-length clone is not optimal for the expression of SAT2 capsid proteins. This paper describes how we have overcome this problem by constructing a genome-length cDNA copy of a SAT2 strain. Characterization of this strain (and several derivatives) together with our previously described chimera, demonstrated that the virus recovered from the genome-length SAT2 cDNA displayed the excellent growth properties of the parental ZIM/7/83 strain, performing considerably better than our previously reported chimera (Van Rensburg & Mason, 2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, and stained with 1 % (w/v) methylene blue in 10 % ethanol and 10 % formaldehyde prepared with PBS, pH 7·4. A SAT2 vaccine strain, originating from western Zimbabwe (ZIM/7/83; bovine passage 1, BHK passage 5, bovine passage 1), was selected for the construction of a genome-length backbone. This virus was previously obtained from the Botswana Vaccine Institute, Gabarone. To distinguish the parental virus from its cDNA derivative, we will refer to the natural isolate by its accession code (ZIM/7/83). The virus used for plaque assays and growth studies was further passaged three times in BHK cells. All genetically engineered viruses utilized for plaque assays and growth studies were passaged five times in BHK cells (see also ‘In vitro RNA synthesis, transfection and virus recovery’ below). A previously constructed genome-length cDNA copy of type A12 (pRMC35; Rieder et al., 1993) was used in the construction of the SAT2 genome-length cDNA copy. The derived virus, vRMC35, as well as another genetically engineered virus, vCRM4 (Sa-Carvalho et al., 1997), were included in this study as reference strains, using passage 5 on BHK cells. pCRM4 was previously constructed by exchanging the four capsid-coding proteins of type A12 in pRMC35 with the corresponding regions of type O1Campos.
RNA extraction and cDNA synthesis.
RNA was extracted from the tissue culture sample of ZIM/7/83 using TRIzol (Life Technologies) according to the manufacturer's specifications and used as a template for cDNA synthesis. Superscript Moloney murine leukaemia virus reverse transcriptase (Life Technologies) and random hexamers as primers were used for the reverse transcription reactions, which were carried out for 1 h at 42 °C.
PCR amplification, construction of genome-length cDNAs and sequencing.
A cassette-exchange strategy, based on an existing genome length cDNA of serotype A12 (pRMC35; Rieder et al., 1993) was used in the step-wise construction of an infectious SAT2 cDNA, similar to the strategy employed by Beard & Mason (2000) (Fig. 1). Regions of the SAT2 cDNA were obtained through PCR amplification from cDNA using AdvanTaq DNA Polymerase (Clontech). The reaction was carried out in the presence of oligonucleotides specifically designed to contain restriction enzyme sites that would facilitate insertion into the A12 genome (Fig. 1). The nucleotide sequence of pSAT2 was subsequently determined using selected oligonucleotides and the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Applied Biosystems), followed by resolution on an ABI PRISM 310 Genetic Analyser (Applied Biosystems). To confirm successful insertion into the A12 genome, the cloning sites of all the intermediate SAT2/A12 constructs were also verified through nucleotide sequencing.
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using an Electrocell Manipulator 600 (BTX). The electroporated cells were then diluted into cell growth medium and allowed to attach to 35 mm diameter tissue culture plates. Dead cells and medium were removed after 3–5 h and replaced with BME containing 1 % fetal bovine serum (Hyclone) and incubated at 37 °C for up to 24 h. Tissue culture plates containing the monolayers of electroporated cells were frozen at -70 °C. Following thawing, 10 % of the lysate was used to infect fresh BHK monolayers and incubated for up to 48 h at 37 °C prior to freezing. This process was repeated four times and aliquots of the lysate obtained during each step were frozen at -70 °C. Unless otherwise stated, passage 5 was used for analyses. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) with cDNAs encoding the corresponding regions of the genome of ZIM/7/83, several chimeric constructs were created, including a genome-length copy of ZIM/7/83 (pSAT2) (Fig. 1
). A single amino acid change [Val (GTC) to Leu (TTG)], was introduced in the 1B region of ZIM/7/83 to facilitate the insertion of the external capsid-coding region of the SAT2 strain into the A12 genetic backbone, resulting in pSAT2/A12 (step 1; Fig. 1). Substitution of the P2, P3 and 3' untranslated region into this cDNA clone was used to create pP1-AnSAT2 (step 2; Fig. 1). pCn-AnSAT2 was created by replacement of the poly(C)–1A region of A12 with the corresponding region of ZIM/7/83 in pP1-AnSAT2, producing a cDNA that contained almost the entire SAT2 genome, lacking only the S-fragment of this serotype (step 3; Fig. 1). To facilitate the assembly of a full-length SAT2 cDNA, the S-fragment was amplified using an oligonucleotide containing the first 18 nt of a different SAT2 strain, TAN/2/75 (Harris, 1980), and substituted for the A12 S-fragment in pCn-AnSAT2 (step 4; Fig. 1). An additional clone (pA12/SAT2) was created by substituting the external capsid-coding region of A12 into pSAT2, using the same restriction sites identified in Fig. 1.
Recovery of viable chimeras in BHK cells
These five genome-length cDNA clones, pSAT2/A12, pP1-AnSAT2, pCn-AnSAT2, pSAT2 and pA12/SAT2, together with pRMC35 and pCRM4 (A12 genetic background containing the four capsid proteins of a heparan-sulfate-binding O1Campos virus; Sa-Carvalho et al., 1997), were used as templates for the in vitro synthesis of RNAs which were used to transfect BHK cells. Viable viruses were recovered from RNAs obtained from all five cDNAs. To verify that the resulting viruses had the expected genetic composition, RNAs extracted from each recovered virus were sequenced utilizing RT-PCR. Analyses of the sequence data obtained from the junction sites of these viral genomes revealed that all of the recovered viruses had the expected genotype (results not shown). Despite our ability to recover viruses from all of these constructs, side-by-side transfections revealed that it was more difficult to recover viruses from some of the synthetic genomes. In particular, RNA synthesized from one construct, pP1-AnSAT2, failed to produce viable virus on several occasions, although when virus was recovered it had the expected sequence.
Comparisons of the ease with which viruses were recovered from all the constructs (except for pP1-AnSAT2) are shown in Table 1. These data showed that RNA derived from pSAT2 produced virus as early as that seen with the RNA derived from the highly tissue-culture-adapted virus (pCRM4; Sa-Carvalho et al., 1997). The CPE observed during the sequential passaging of viruses vSAT2/A12, vCn-AnSAT2, vA12/SAT2 and vRMC35 lagged behind vCRM4 and vSAT2, but by BHK passage 4, all viruses shown in Table 1 displayed similar ability to cause CPE.
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). These studies employed the five viruses described above along with ZIM/7/83 and the two viruses known to display different receptor specificity (vRMC35 and vCRM4). High titres were observed for all viruses on BHK and IB-RS-2 cells. All viruses containing ZIM/7/83 capsids yielded titres on CHO cells, similar to vCRM4, indicating that viruses with the ZIM/7/83 capsid could utilize heparan sulfate as a receptor.
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). In BHK cells the A12 virus (derived from pRMC35) and vA12/SAT2 displayed plaques with turbid morphology. All of the other viruses tested produced plaques in BHK cells that had a clear morphology, with vCRM4 producing plaques that were smaller than all the ZIM/7/83 external capsid-containing viruses (Fig. 3). Both viruses with the A12 capsid region failed to produce plaques on CHO cells (Fig. 3), consistent with studies showing that these cells do not make integrins suitable for FMDV infection (Mason et al., 1993). Among the remaining viruses the plaques produced by vCRM4 were extremely small (less than 0·5 mm diameter) on CHO cells (as reported by Sa-Carvalho et al., 1997), whereas all of the viruses with ZIM/7/83-derived capsids were small. Fewer differences in morphologies were readily distinguishable on IB-RS-2 cells (Fig. 3), suggesting that the turbid morphology produced by A12 capsid-containing viruses on BHK cells could be due to a limitation in the number of appropriate receptors for these viruses in BHK cells that were present in higher numbers on the IB-RS-2 cells.
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a). The chimeras in the SAT2 construction series performed poorly compared to vSAT2 and ZIM/7/83: vCn-AnSAT2>vSAT2/A12>vP1-AnSAT2. In the first experiment an approximately threefold lower titre for vCn-AnSAT2 (4·8x104 p.f.u. ml-1) was obtained at 10 h post-infection in comparison to vSAT2 (1·5x105 p.f.u. ml-1) and ZIM/7/83 (1·8x105 p.f.u. ml-1). In contrast, the titre obtained for vSAT2/A12 (2·8x104 p.f.u. ml-1) was five- to sixfold lower than that observed for vSAT2 and ZIM/7/83, whilst vP1-AnSAT2 produced a titre of only 7·3x103 p.f.u. ml-1 (20-fold lower than the titre produced by vSAT2) 10 h post-infection. The poorer performance of vSAT2/A12 in comparison to the parental virus has been observed previously (Van Rensburg & Mason, 2002), but the experiments were performed in the absence of vRMC35 (the genetic backbone used for this construct). However, in Fig. 4(b) similar growth was observed for vSAT2/A12 and vRMC35, reaching a titre of 5x104 p.f.u. ml-1 at 10 h post-infection, almost 10-fold lower than the titre obtained for vSAT2 (4·3x105 p.f.u. ml-1). vA12/SAT2 (A12 external capsids in SAT2 genetic backbone) exhibited a higher rate with which virus particles were released from infected cells compared to vRMC35, especially during initial stages of infection. The rate observed for vA12/SAT2 was comparable to vCRM4 10 h post-infection. Slightly lower titres of 1·1x105 p.f.u. ml-1 for vA12/SAT2 and 1x105 p.f.u. ml-1 for vCRM4 were observed compared to vSAT2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and O (Zibert et al., 1990; Beard & Mason, 2000) FMDV have been reported. Here we describe the first such clone for an SAT-type virus. Evaluation of the virus derived from our SAT2 genome-length clone revealed that it has maintained the high-titre growth of its parental virus, ZIM/7/83, in cell cultures. Furthermore, this virus, as well as a panel of chimeric viruses containing the capsid region of ZIM/7/83, all grew well in CHO cells, suggesting these viruses were able to use heparan sulfate as a receptor. Since ZIM/7/83 is a tissue-culture-adapted virus, this finding adds to those of others that tissue-culture-adapted viruses are capable of binding heparin and using heparan sulfate proteoglycans as cellular receptors (Sa-Carvalho et al., 1997; Neff et al., 1998; Fry et al., 1999; Jackson et al., 2003). These properties of vSAT2 suggest that the two viruses (vSAT2 and ZIM/7/83) are indistinguishable phenotypically. However, due to the fact that the complete genome sequence of the recovered vSAT2 virus was not compared to that of the parental strain, it is possible that the two viruses differ genotypically.
We previously reported that vSAT2/A12, a chimeric virus with the SAT2 capsid in an A12 genome, performed poorly in BHK cells when compared to the parental ZIM/7/83 (Van Rensburg & Mason, 2002). This finding, and studies showing that 3Cpro (which cleaves the capsid proteins of SAT2) differs significantly from type A 3Cpro (Van Rensburg et al., 2002), lead us to propose that the poor growth properties of vSAT2/A12 were due to suboptimal processing of the SAT2 external capsid proteins by the A12 3Cpro (Van Rensburg & Mason, 2002). However, side-by-side growth comparisons of this chimera with an infectious clone derived from a type A12 virus (vRMC35) and our genetically engineered SAT2 virus (vSAT2) indicated that the chimera grows just as well as the type A12 virus. This result suggests that, rather than being compromised by an incompatibility of various portions of its genome, the inferior growth of vSAT2/A12 relative to ZIM/7/83 may reflect the superior properties of the latter virus in cell culture.
Evaluations of the growth properties of other chimeras suggest that there is an incompatibility between different portions of the A12 and SAT2 genomes. The clearest case of this type of genetic incompatibility is revealed by the properties of the RNA derived from pP1-AnSAT2. The RNA derived from this chimeric genome did not reproducibly produce virus when transfected into cells. The resulting virus also produced plaques smaller than any of the other SAT2 capsid-expressing virus on all the cell lines investigated. This genome contains the 5' UTR, Lpro and protein 1A of type A12, with the remainder of the genome derived from ZIM/7/83. Since all of the fragments utilized in this clone produced a highly viable virus with excellent growth properties, we concluded that there were incompatibilities between the 5' UTR of A12 and the SAT2 polyprotein and 3' UTR. One of the components in this region, Lpro, was of particular interest since it was shown to differ substantially between A12 and SAT2 (Van Rensburg et al., 2002). Lpro is, however, followed by its own cleavage site and it therefore seems unlikely that Lpro incompatibility accounts for the poor growth properties observed for vP1-AnSAT2.
Investigation of the predicted RNA structural folding of the 5' UTR of SAT2, and that of the A12 genome (Mason et al., 2003) revealed a strong similarity in the structure, with only a few differences observed between the two genomes in the cre and previously mapped protein–RNA interaction sites for the FMDV IRES (results not shown), despite a low overall nucleotide identity (73 % for the pseudoknots and 77 % for the cre and IRES structures). However, it is unclear whether these differences could result in incompatible interactions between the 5' UTR and the non-structural proteins suggested by the poor growth of vP1-AnSAT2. Analyses of the predicted structure of the S fragment of SAT2 revealed a 37 nt deletion relative to that predicted for type A12 (results not shown). However, comparisons of vCn-AnSAT2 with vSAT2 (which differ only in their S fragments) failed to reveal any striking differences, providing further evidence of the difficulty in reaching a conclusion regarding functional interactions using predicted RNA structures.
Despite some slight differences observed with plaques on IB-RS-2 cells, reflected by small differences in growth kinetics in these cells (results not shown), the genetically derived virus, vSAT2, appears to grow as well as its parent, ZIM/7/83, in BHK cells. Since BHK is the cell line of choice for production of inactivated FMD vaccines, it appears that the cDNA used to derive vSAT2 is a good candidate for a genetic background upon which useful genetically engineered vaccine strains of FMDV can be built.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 July 2003;
accepted 23 September 2003.
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