1 Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, UK
2 Department of Research, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 7/9, 25124 Brescia, Italy
Correspondence
Donald P. King
donald.king{at}bbsrc.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences determined in this paper are AY875984AY876011.
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INTRODUCTION |
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SVD spreads rapidly through contact with infected pigs and following exposure to a contaminated environment (Dekker et al., 1995), and can cause up to 100 % morbidity in affected pens where animals are in direct contact. The disease is variable, but mild or subclinical infections now predominate in Italy, where there has been the greatest number of reported cases of the disease in the last 10 years. Pathogenic strains of SVDV can produce lesions on the snout and feet which are indistinguishable from those produced by the other vesicular disease viruses, namely foot-and-mouth disease (FMD) virus, vesicular stomatitis (VS) virus and vesiviruses [which include vesicular exanthema of swine virus (VESV)]. Due to the similarity of these clinical signs to FMD, SVD is a notifiable disease and therefore swift, accurate and sensitive diagnosis is necessary for effective disease control. Laboratory differentiation of SVDV from other vesicular disease viruses in clinical samples (typically faeces or vesicular epithelium) can be achieved by a combination of virus isolation (VI) in a permanent line of IB-RS-2 cells (De Castro, 1964
) and an antigen ELISA (Ferris & Dawson, 1988
). Whilst the ELISA is rapid, producing a result within a few hours, it has a limited sensitivity. VI is a sensitive method, but it can take up to 7 days to produce a definitive result, and other enteroviruses present in faeces can interfere with the isolation of SVDV. Recently, molecular methods, such as RT-PCR, have become more widely accepted for pathogen diagnosis (Belak & Thoren, 2001
). An RT-PCR preceded by immune-extraction of SVDV from faeces samples (Fallacara et al., 2000
) is routinely used in Italy for the virological surveillance programme, leading to the rapid and sensitive detection of pathogens. Real-time PCR may add further advantages, as it eliminates the need for post-PCR processing stages, such as gel electrophoresis. Two real-time RT-PCR assays have recently been developed for the detection of SVDV (Reid et al., 2004a
). Both of these assays target separate locations within the internal ribosomal entry site (IRES) located within the 5' untranslated region (5' UTR), which for SVDV is approximately 750 nt in length. This region was chosen as a diagnostic target because the IRES is critical for cap-independent translation and its structure is conserved in many other species of picornavirus (Belsham & Jackson, 2000
). Indeed, the primers and probes targeting this region have broad sensitivity against phylogenetically diverse SVDV isolates (Reid et al., 2004a
). However, nucleotide substitutions in the TaqMan probe regions resulted in the failure of both assays to detect a single (but different) SVDV isolate (Reid et al., 2004a
). Since the design of these real-time RT-PCR primers and probes was based upon just four IRES sequences available in GenBank (Seechurn et al., 1990
; Inoue et al., 1989
, 1993
; Rebel et al., 2000), the aim of this project was to generate additional sequence information for this region from a diverse selection of SVDV isolates, thereby facilitating the development of improved diagnostic assays. Interestingly, during the course of these analyses, large deletions located between the 3' end of the IRES and the initiation codon of the polyprotein were observed for the sequences of some isolates. In an attempt to investigate the significance of these deletions, in vitro growth studies were performed using these isolates.
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METHODS |
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Amplicons of the predicted size were excised from the gels, purified using the QIAquick gel-extraction kit (Qiagen) and quantified with the E-gel low range marker (Invitrogen). Approximately 50 ng of the purified PCR product was directly sequenced on both strands by the chain-termination method, employing the primers shown in Table 2, according to the manufacturer's instructions (Beckman Coulter, CEQ 8000 DNA analysis system). The resulting sequence of each isolate was assembled independently using SEQMAN (DNAStar, Lasergene) and aligned with other sequences using CLUSTAL X (Thompson et al., 1994
) and MegAlign (DNAStar, Lasergene). GCG (Wisconsin Package version 10.3, Accelrys Inc., San Diego) was used to determine nucleotide identity between isolates, and nucleotide variability was assessed using an algorithm described elsewhere (Proutski & Holmes, 1998
). RNA folding was performed using RNAStructure version 3.5 (Mathews et al., 1999
) and RNA DRAW version 1.1 (Mazura Multimedia, Sweden).
PCR analysis of 5' UTR spacer region.
Surprisingly, several isolates had deletions in the 5' UTR spacer region which prompted further investigation. SVDV isolates were screened by RT-PCR to identify those with 5' UTR spacer deletions. cDNA (2·5 µl) prepared as above was amplified using primers SASVD-3-IR-455-473F and SVD-5UTR REV3 (Table 2). Amplification conditions were as follows: 94 °C for 60 s, 63 °C for 45 s, 72 °C for 45 s for 35 cycles. Chain elongation at 72 °C was extended to 7 min for the final cycle. PCR products were analysed as described above and compared with the band size produced by viruses that had already been sequenced.
Virus plaque diameter.
Titrations of the stock virus were performed in initial plaquing experiments to determine the viral titre (plaque forming units ml1). Petri dishes confluent with IB-RS-2 cells were then inoculated with 0·5 ml of a dilution containing approximately 100 p.f.u. of stock virus. The virus was allowed to adsorb for 30 min and excess virus was removed by needle and syringe. Eight millilitres of overlay [MEM in 1·6 % Noble agar (Difco Laboratories) supplemented with 0·1 % fetal calf serum (Sigma)] was then added and allowed to set. Plates were incubated at 37 °C and plaques visualized at 72 h after methylene-blue staining. The diameters of the resulting plaques were measured.
Time-course study of virus growth.
The growth of selected SVDV isolates in confluent IB-RS-2 cells was monitored in 25 cm3 flasks (Falcon). After washing off the maintenance media, the cell sheet was washed twice with PBS, pH 7·4. Cultures were inoculated with 500 µl of virus dilution containing 200 p.f.u. Virus was allowed to adsorb for 30 min at 37 °C before adding 5 ml serum-free modified Eagles maintenance medium. Flasks of each virus were incubated at 37 °C or 32 °C (four replicates at each temperature). A sample from each flask was collected at 0, 12, 24, 48 and 72 h post-inoculation; 0·2 ml of the cell-culture supernatant fluid was removed, added to 1 ml Trizol reagent (Invitrogen) and stored at 80 °C until tested by real-time RT-PCR.
Real-time RT-PCR.
RNA was extracted using the QIAamp extraction kit on a QIAamp BioRobot 9604 (Qiagen). A QIAamp BioRobot 3000 was then used for the liquid-handling steps of the reverse transcription and PCR procedures with pipetting volumes similar to those described before (Reid et al., 2004b) and the 2B-IR primers/probe set (Reid et al., 2004a
). A dilution series of an RNA standard transcribed in vitro (MEGAscript, Ambion) from a plasmid clone containing the SVDV IRES (pGEM3Z/J1 IRES; Sakoda et al., 2001
) enabled viral genome (RNA copies in each sample) to be quantified, as described elsewhere (Inoue et al., 2005
). Each RNA standard dilution (6·0 µl) was added manually to reverse transcription reaction mix (9·0 µl) immediately before the reverse transcription incubation reaction, as previously described (Reid et al., 2004b
).
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RESULTS |
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In common with the 5' UTRs of other picornaviruses, a polypyrimidine tract approximately 20 nt upstream of a conserved cryptic AUG was present in all 33 SVDV sequences. Both these features have been shown to play a role in translation initiation (Pelletier et al., 1988; Pilipenko et al., 1995
). As previously reported (Seechurn et al., 1990
), SVDV secondary-structure predictions reveal a picornaviral IRES structure similar to that proposed for poliovirus 3 (Skinner et al., 1989
; Le & Maizel, 1998
). Fig. 1
shows the nucleotide base-pairing for four of these proposed stemloop structures (designated stemloops II to V; Beales et al., 2003
). It was difficult to reliably model the final stemloop (VI) containing the cryptic AUG proposed for other enteroviruses (Semler, 2004
) for all SVDV sequences, and it is therefore not shown in Fig. 1
. A GNRA (GYRA) tetraloop (Jucker et al., 1996
) present in other picornavirus IRES elements (Semler, 2004
) is generated (positions 345348) at the apex of stemloop IV. Interestingly, there was evidence for compensatory nucleotide substitutions at 25 positions in these four stemloops in order to maintain RNA base-pairing (Fig. 1
). These compensatory substitutions were located particularly at the apex and base of the stemloops. In contrast, there was a degree of plasticity in the predicted structures generated in the middle of stemloops II, IV and V.
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DISCUSSION |
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In contrast to the relative conservation of the IRES element, there was considerable nucleotide variability in the spacer region located after the cryptic AUG. Interestingly, this study also highlighted the presence of block deletions of between 6 and 125 nt in this region for a number of SVDV isolates. A close ancestral relationship was shared among nine of the 11 isolates shown to have a deletion. It is possible to speculate that the largest deletion (POR 3/95) is a product of stepwise (2885125) deletions observed in other SVDV isolates. Some of these sequences with deleted regions (HKN 1/80 and POR 1/2003) were recovered directly from clinical material from infected animals, suggesting that these unexpected findings did not necessarily arise through tissue-culture adaptation.
In vitro growth studies showed that selected isolates with these deletions had a significantly reduced plaque diameter, a finding similar to observations with artificially generated mutants of poliovirus 1 (Gmyl et al., 1993; Slobodskaya et al., 1996
). Furthermore, POR 3/95 grows to a significantly lower titre than the reference strain UKG 27/72. It is attractive to speculate that these effects relating to the in vitro viral growth of these selected SVDV isolates are related to the remarkable deletions evident in their 5' UTR. However, since the region sequenced in this study only contributes a minor proportion of the total SVDV genome length, it is impossible to rule out the influence of changes elsewhere in these viruses. The mechanism of this effect on viral growth is not clear. One possible explanation is that these effects could be related to the ability of these viruses to form the final stemloop VI of the IRES (Beales et al., 2003
) containing the cryptic AUG, or may simply be the result of a shorter distance between critical motifs in this region. There was a striking similarity between the length of the spacer for POR 3/95 and that of rhinovirus 14 (Callahan et al., 1985
). However, there was no evidence for a differential effect of temperature, as might be expected for a respiratory phenotype.
In summary, this paper describes sequences for the 5' UTR of SVDV which unexpectedly reveal the presence of block deletions in the genome of some isolates. Of further interest is the finding that viruses with these deletions grow less well in cell culture. It is possible that these in vitro observations may reflect altered in vivo characteristics for these viruses. Indeed, previous studies have shown that plaque size can be correlated to the pathogenicity of SVDV, although in an earlier study these effects were mapped to the 2A region of the genome (Kanno et al., 1999). Further work is required to define the significance of the deletions highlighted by this study and to assess whether they impact upon SVD.
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ACKNOWLEDGEMENTS |
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Received 23 February 2005;
accepted 29 June 2005.
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