Development of a PCR-based method for the detection and characterization of foot-and-mouth disease virus

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Primer-pair recognition of FMDV serotypes

In order to assess the applicability of published primers for FMDV diagnosis in southern Africa, three different primer pairs were initially evaluated. These include the primer pair described by Höfner et al. (1993), which targets the P1 structural protein region and the primer pairs of Vosloo et al. (1996) and Laor et al. (1992) which are designed to amplify the 1D (VP1) and 3D (replicase) genes respectively. Comparison of the three primer pairs revealed that the amplification efficiency was in the order of P1<1D<3D, for the SAT-types tested (results not shown). The difference in amplification efficiency between structural (1D) and non-structural (3D) protein is demonstrated in Figure 2.2. Only two of the seven SAT-2 type field stains from various southern African localities amplified with the 1D primers, whilst all viruses tested with the 3D primers amplified the expected 978 bp band. Although the 3D primers were shown to be suitable for detecting a variety of FMDV types, this conserved, non-structural protein is serotypically non-informative and therefore of no use for genetic differentiation of viruses by nucleotide sequencing.
Five new primers, which amplify a product of under 1000 bp and which specifically target the VP1 gene were subsequently designed, on the basis of consensus sequences of data available in Genbank (Table 2.1). The different primer pair combinations and their expected product sizes are summarized in Fig. 2.1. Each of these primer pairs were tested against one representative of each of the European and Asian serotypes and two of each of the SAT-types in order to establish general FMDV and SAT-type recognition capabilities of different combinations by PCR. Primer pairs 2B+VP3U and P1+W-US scored equally well in terms of SAT-type amplification (Table 2.2), with pair 2B+VP3U having a higher overall recognition for all FMDV types.
The P1+W-US pair was, however, selected for optimization as amplification of all three SATtypes was obtained (2B+VP3U did not amplify the SAT-2 strains) and the level of amplification was generally higher. In addition, the product size of the P1+W-US primer pair is closer to the 200-400 bp amplification efficiency range (Rychlik 1993) and the 500 bp product can potentially be sequenced in its entirety with the external PCR primers alone.

Primer optimization and testing

It was noted that the 3′ terminal nucleotide of the W-US primer corresponds to a third base amino acid position which is known to be highly variable and have a high mutation frequency (Vosloo et al. 1996). By comparing the only complete VP1 gene sequence of a SAT-type (Brown et al. 1989) with the sequences of European serotypes A, O and C (see Table 2.1), a 22mer primer (termed VP1Ua) based on the consensus sequence of these aligned sequences was identified and synthesized. The primer was designed to end at a 2nd base position of the corresponding amino acid in order to stabilize the terminal end of the oligonucleotide. Although recognition of SATtypes increased to 86 %, the P1+VP1Ua pair was not capable of amplifying all SAT-type field strains tested. This was in all likelihood due to variability in the first base position, one nucleotide upstream of the terminal 3′ base. Mismatches in this position are known to affect polymerization, a characteristic exploited for diagnostic purposes in the amplification refractory mutation system (ARMS) PCR (Wenham et al. 1991). A second VP1U primer, termed VP1Ub was therefore synthesized. This primer was based on the consensus sequence of Asia-1, in addition to SAT-3, A, O and C and was extended by 3 nucleotides on the 3′ end so that the final oligonucleotide length was 25. Alignment of various representatives of the five serotypes on which the primer sequence was based revealed that the terminal three nucleotides are highly conserved amongst the different FMDV serotypes. In addition, internal stability plots of these primers (results not shown) indicate that VP1Ub and the P1 primer conform to the 3′ terminal pentameric requirements of successful PCR and sequencing oligonucleotides (Breslauer et al. 1986; Rychlik 1993). Testing of these primers not only revealed significantly improved SAT-type recognition, but also enabled amplification of various subtypes of the European strains in addition to amplification of an Asia-1 isolate. Product sizes varied due to inter- and intratypic differences in VP1 gene amino acid sequence length (Fig. 2.3). The relative amplification efficiencies of the upstream VP1 primers combined with the P1 primer are summarized in Table 2.3. With the exception of KEN 1/91 all 30 isolates tested with the VP1Ub + P1 primer pair amplified the expected band of approximately 500 bp. KEN 1/91 was, however, amplified with the 2B+VP1Ub primer pair, thereby permitting sequencing of the VP1 gene. Testing of the optimized VP1Ub primer in combination with 2B in PCR revealed that this primer pair successfully amplifies all European and Asian types tested but has limited success in amplifying SAT-types (results not shown). These results indicate that of the two potential cDNA and/or antisense PCR primers, P1 (Beck & Strohmaier, 1987) is the more conserved of the two, across all seven serotypes.

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Confirmation of specificity 

In order to assess the specificity of the VP1 gene primer pair for FMDV alone, cDNA was prepared from genetically and/or symptomatically related Picornaviridae such as swine vesicular disease virus (SVDV). The strains tested were representative of the enterovirus [coxsackie B2 and – B4; echo 11; polio-1, -2 and -3; bovine enterovirus (BEV); SVDV] and cardiovirus (encephalomyocarditis) genera. Integrity of the cDNA was confirmed by amplification with enterovirus-specific primers (Rotbart 1990). Interestingly, all enterovirus strains with the exception of BEV isolates amplified the expected 154 bp fragment (results not shown). This is perhaps not surprising in view of the grouping of BEV outside the enterovirus and rhinovirus cluster on the basis of VP1 gene phylogenetic analysis (Palmenberg 1989). Encephalomyocarditis was negative with the enterovirus primers. All enterovirus and cardiovirus virus strains tested were negative for PCR with the VP1Ub and P1 primer pair.

CHAPTER 1: Literature Review
1.1 Introduction
1.2 Picornavirus Taxonomy
1.3 Foot-and-mouth disease virus
1.4 Genetic variation
1.5 Antigenic variation
1.6 Geographical distribution of FMDV
1.7 FMD in wildlife in southern Africa
1.8 FMD as a zoonosis
1.9 The role of carriers in the epidemiology of FMD
1.10 Characterization of field strains of FMDV
1.11 Objectives of this study
CHAPTER 2: Development of a PCR-based method for the detection and characterization of foot-and-mouth disease virus in southern Africa 
2.1 Introduction
2.2 Materials and Methods
2.3 Results
2.4 Discussion
CHAPTER 3: Investigating the possibility of sexual transmission of foot-and-mouth disease in African buffalo (Syncerus caffer) 
3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.4 Discussion
CHAPTER 4: Evidence of natural transmission of foot-and-mouth disease virus between African buffalo (Syncerus caffer) and impala antelope (Aepyceros melampus)
4.1 Introduction
4.2 Materials and Methods
4.3 Results
4.4 Discussion
CHAPTER 5: Phylogeographic distribution of SAT-type foot-and-mouth disease virus in African buffalo populations in southern Africa 
5.1 Introduction
5.2 Materials and Methods
5.3 Results
5.4 Discussion
CHAPTER 6: Tracing the origin and course of foot-and-mouth disease epizootics in southern Africa
6.1 Introduction
6.2 Materials and Methods
6.3 Results
6.4 Discussion
CHAPTER 7: Concluding remarks and future prospects 
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