The School of Biology and Biochemistry, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK1
Department of Veterinary Science, The Queen's University of Belfast, Stormont, Belfast BT4 3SD, UK2
Author for correspondence: Elizabeth Hoey.Fax +44 28 90 236505. e-mail e.hoey{at}qub.ac.uk
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Abstract |
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Introduction |
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Although some resulting infections from various PEVs are asymptomatic, virulent strains can give rise to syndromes which are of economic importance. Firstly, infection may be associated with abortion, stillbirth and infertility, designated the SMEDI syndrome (swine mummification, embryonic death and infertility). Certain virus strains of serotypes 1, 3, 6 and 8 have been isolated from such cases. Secondly, infection with some virus strains, particularly of serotype 1, can give rise to porcine polioencephalomyelitis. Infection by highly virulent strains can result in extensive neuronal destruction, paralysis and death. Epidemics of this type have occurred in central and eastern Europe and the disease was given the name Teschen disease, derived from the village name where the outbreak was first reported (Trefny, 1930 ). Milder forms of the disease caused by less virulent serotype 1 strains and some from other group I serotypes are common elsewhere with younger animals being generally infected. Animals may develop paralysis; however, mortality rates are low and those infected often recover. An outbreak of this type occurred in Britain in 1956 and was called Talfan disease (Harding et al., 1957
), with the isolated PEV-1 strain being designated the Talfan virus isolate.
To date, few molecular studies have been performed with virus strains from the majority of serotypes, thus their classification has depended on basic biological and biophysical properties. Recently assignment of viruses to specific genera within the picornavirus family has relied not only on these facts but on the genomic sequence and organization of the polyprotein. The complete sequence of a PEV-9 isolate has been determined and partial sequences are available for isolates from serotypes 8 and 10. The generated data have confirmed that viruses of these serotypes belong within the Enterovirus genus (Hyypiä et al., 1997 ). Recently, doubt has been expressed concerning the validity of assigning the PEVs that cause Teschen-like disease (group I) within the Enterovirus genus on the basis of subtle differences in their capsid properties (N. J. Knowles, personal communication).
In this study we have determined the majority of the genomic sequence from a serotype 1 virus, designated F65. This was originally isolated from a pig which showed no disease symptoms but when inoculated into specific-pathogen free pigs it produced clinical signs resulting from neuroinvasiveness (Alexander & Betts, 1967 ). Following cloning and sequencing of the genome from this isolate, the data generated suggest that viruses of this serotype do not belong within the Enterovirus genus. The level of sequence identity observed with representatives of other picornavirus genera has suggested that PEV-1 should be assigned to a new genus within the picornavirus family. This has been agreed with the members of the Picornaviridae Study Group of the International Committee on Taxonomy of Viruses.
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Methods |
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cDNA synthesis and cloning.
RNA was obtained from purified virus by phenolacetate extraction (Todd & Martin, 1979 ). The aqueous layer was washed three times with ether and RNA was ethanol-precipitated. Double-stranded (ds) cDNA was prepared using a Riboclone cDNA synthesis kit (Promega), following the instructions supplied by the manufacturer. Oligo(dT)20 was used as primer. EcoRI adapters were ligated to the ds cDNA prior to ligation to pGEM3z plasmid molecules. The resulting products were used to transform competent E. coli DH5
cells. Ampicillin-resistant colonies were initially screened by hybridization with a [32P]dCTP-labelled virus genomic cDNA probe. The size of inserts in positive clones was estimated by PCR using primers at either side of the multiple cloning site of the plasmid. Large scale preparations of plasmids with large inserts (>600 bp) were carried out. Intervening sequences and that from the 5' UTR were obtained by analysis of PCR fragments. The latter procedure used an oligo(dC)20 primer and one from inside the coding region. Routinely the Expand Long Template PCR system (Boerhinger Mannheim) was used and the instructions of the manufacturer were followed.
DNA sequencing and analyses.
Inserts contained in identified cDNA plasmids were used to determine nucleotide sequences using a Taq DyeDeoxy Terminator Cycle sequencing kit and a 373A DNA sequencer (Perkin Elmer). The generated data were used to predict sequence-specific primers for the production of PCR products across the virus genome using the Expand Long Template PCR system (Boehringer Mannheim). The PCR fragments were also sequenced. The data were compiled and analysed using the Microgenie suite of programs (Queen & Korn, 1984 ) and the polyprotein sequence was predicted. The P1 and 3D protein sequences were compared with those from other picornaviruses using the multiple alignment program CLUSTAL W (Thompson et al., 1994
) and the subsequent phylogenetic analyses were performed using maximum likelihood mapping (PUZZLE v4.0;Strimmer & von Haesler, 1996
, 1997
) with the JTT substitution model (Jones et al., 1992
) to produce unrooted neighbour joining trees. In some cases the
2 test for homogeneity of sequence composition showed significant deviations, such sequences were excluded from the analysis. In several instances statistical tests (Templeton, 1983
) were conducted to assess the likelihood of the occurrence of constrained topologies relative to the most parsimonious trees: these were implemented using the program PROTPARS of the PHYLIP package (Felsenstein, 1989
) to test rejection of the null hypothesis at P<0·05. Possible secondary structure formation in the 3' UTR was examined using the program MFOLD (Jaeger et al., 1989
) and plots were generated using the program PlotFold (Zuker, 1989
). All analyses were carried out using the SEQNET facility (Daresbury, UK). The accession numbers of picornavirus sequences used in the phylogenetic analysis are as follows: Aichi virus AB010145 (Yamashita et al., 1998
); bovine enterovirus type 1 (VG-527; BEV-1a) D00214 (Earle et al., 1988
); bovine enterovirus type 1 (M4; BEV-1b) not submitted (McIlhatton et al., 1993
); bovine enterovirus type 2 (PSU 87; BEV-2a) X79368 (McNally et al., 1994
); bovine enterovirus type 2 (RM2; BEV-2b) X79369 (McNally et al., 1994
); coxsackievirus B1 (CBV-1) M16560 (Iizuka et al., 1987
); enterovirus type 70 (EV-70) D00820 (Ryan et al., 1990
); encephalomyocarditis virus (EMCV) M81861 (Duke et al., 1992
); equine rhinitis A virus (ERAV) X96870 (formerly equine rhinovirus serotype 1; Wutz et al., 1996
); equine rhinovirus serotype 2 (ERV-2) X96871 (Wutz et al., 1996
); foot-and-mouth disease virus O (FMDV-O1) X00871 (Forss et al., 1984
); human hepatitis A virus (HHAV) M14707 (Cohen et al., 1987
); human parechovirus 1 (HPeV-1) L02971 (Hyypiä et al., 1992
); human rhinovirus 14 (HRV-14) K02121 (Stanway et al., 1984
); poliovirus type 1 (Sabin strain; PV-1) V01150 (Nomoto et al., 1982
); porcine enterovirus type 9 (PEV-9) Y14459 (J. H. Peng, J. W. McCauley, R. P. Kitching & N. J. Knowles, unpublished); swine vesicular disease virus (SVDV) D00435 (Inoue et al., 1989
); Theiler's murine encephalomyelitis virus (TMEV) M20562 (Pevear et al., 1988
).
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Results and Discussion |
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Genomic cloning and sequencing
RNA was extracted from purified virus. Denaturing gel electrophoresis suggested that this RNA was substantially larger than BEV RNA, which is 7414 nt long prior to the poly(A) tract (Earle et al., 1988 ), and was of the order of 8 kb (data not shown). Purified PEV-1 RNA was used initially for the generation of a cDNA library. Fifty-one strongly positive clones were detected using a virus genomic cDNA probe and those with the largest inserts were sequenced. Sequence identity with other picornaviruses, determined by database interrogation, allowed the position of these on the virus genome to be determined. This permitted the prediction of primer sequences, which were used to generate PCR fragments from which data were obtained across areas of the genome which had not previously been assessed. The sequence generated is presented. Over 7100 nt of the virus genome were obtained (Fig. 1
), which falls short of the estimated size obtained from gel electrophoresis studies. Approximately 90% of this partial genomic sequence was determined in both directions. The predicted genomic organization of this PEV-1 strain is characteristic of picornaviruses and contains a large open reading frame flanked by 5' and 3' UTRs.
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The 3' untranslated region (3' UTR)
None of the preliminary 51 clones contained a poly(A) tract. Thus cDNA synthesized using oligo(dT) was used in a PCR reaction using an oligo(dT)30 primer and one within the 3D polymerase coding region. This generated a fragment which suggested that the sequence of the 3' UTR is 68 nt to the poly(A) tract. The predicted secondary structure of this region indicated that it was composed of a single hairpin loop. The two folds obtained with MFOLD (Jaeger et al., 1989 ) did not differ significantly in energy (-9·8 and -9·4 kcal/mol) and had similar structures (Fig. 2
). This is a similar situation to that encountered with HPeV-1 (Auvinen & Hyypiä, 1990
) and HRV-2 (Pöyry et al., 1996
), which also is predicted to have a single hairpin loop, whereas viruses of the enterovirus genus have either two or three such structures (Pöyry et al., 1996
) and EMCV has two hairpin loops (Cui & Porter, 1995
) in this region of the genome.
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The cleavage sites of the PEV-1 open reading frame were predicted by alignment with those of other picornaviruses (Table 1). The junction sites appeared to be mainly at Q/G residues, indicating that these proteins may be cleaved by the virus protease 3C (3Cpro). Exceptions were observed at the VP4/VP2 and the VP2/VP3 cleavage sites, which are predicted to occur between A/E and Q/S residues respectively. In addition, the 2A/2B cleavage site is predicted to occur at an NPG/P junction. Although the organization of the polyprotein is typical of other picornaviruses, particularly those of the cardio- and aphthovirus genera, the level of sequence identity of individual PEV-1 proteins with representative members of the picornavirus genera is low (Table 2
) and, with the exception of the 2A protein, never exceeds 40%.
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Examination of the three-dimensional structural analyses of all picornavirus capsid regions analysed to date has revealed that each of the major capsid proteins has an eight-stranded ß-barrel structure and it is assumed that this is the case for those of PEV-1. Loop regions located between each sheet confer specific biological properties shown by the various viruses. When the sequence of the P1 capsid region of PEV-1 was aligned with those of other picornaviruses the greatest sequence identity was observed in residues forming the conserved structural core, with the loop regions being the most variable both in sequence and size. In particular, the ßEB `puff' region of VP2 was found to be considerably larger than comparable regions of viruses of the cardio- and aphthovirus genera (Fig. 3
). VP1 showed the greatest variation when compared with equivalent proteins of other picornaviruses. In addition an RGD sequence was not observed in VP1 of PEV-1 (or in any of its capsid proteins). This RGD sequence is located in the main antigenic determinant of FMDV VP1 and is part of the receptor binding site. The absence of this sequence is of importance as it implies binding of PEV-1 to the cell surface through a receptor different from the integrin
vß3 used by FMDV (Neff et al., 1998
).
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The 2C protein sequence aligns well with those of other picornaviruses. The sequence GAPGQGKS, which occurs at position 117, conforms with the conserved nucleic acid binding motif which is present in viruses of other genera.
The 3B (VPg) protein has the conserved tyrosine residue located at position 3; however, only one copy of the gene is present. In comparison, three copies of the VPg gene are reported in FMDV (Forss et al., 1984 ), and in ERV-2 there is an additional VPg pseudo-coding region in addition to that encoding the normal product (Wutz et al., 1996
).
The 3D polymerase protein showed the highest level of sequence identity when proteins were compared across genera, although in no instance did this exceed 40%. However, four highly conserved motifs were present. These are KDELR, PSG, YGDD and FKLR, which are located at positions 159, 289, 324 and 371 respectively in the polymerase sequence.
Phylogenetic analyses
Phylogenetic analysis was performed on two regions of the polyprotein (P1 and the 3D polymerase). Using either the P1 or the 3D polymerase sequences, the enteroviruses including PEV-9 and rhinoviruses formed a well resolved clade (Figs 5 and 6
), which was strongly supported by the quartet puzzling values. In both these phylogenies PEV-1 was found to form a separate branch away from PEV-9 and adjacent to ERV-2 and the cardio/aphthovirus group. The best tree that could be found supporting monophyly for PEV-1 and PEV-9 using the 3D polymerase sequences added 160 substitutions to the most parsimonious tree (most parsimonious score was 3619) and the corresponding value for the P1 sequences was 250 (most parsimonious score was 9979). These phylogenies were judged to be significantly worse than the phylogenies which place PEV-9 among the entero- and rhinoviruses and PEV-1 in the cardio/aphthovirus group. This again supports our contention that from a molecular evolutionary standpoint PEV-1 is significantly different from PEV-9 and should not be grouped with it among the entero- and rhinoviruses, regardless of similarities in its physiological effects or shared gross morphological properties.
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Conclusions
The sequence data presented in this paper indicate that PEV-1 is distinct from viruses of the enterovirus genus to which it was previously assigned on the basis of biophysical and physiological properties. The low level of sequence identity with other enteroviruses in most regions of the PEV-1 genome, the fact that it contains a leader protein, the features of the 2A protease sequence and the phylogenetic analysis are all supportive of the fact that PEV-1 should be assigned to a new genus. Interestingly the polymerase gene of the Talfan strain of PEV-1 has recently been determined (Kaku et al., 1998 ) and has been found to have a similarly low level of sequence identity with the enteroviruses.
To date molecular analysis has been performed with viruses from only four of the 13 different PEV serotypes, three of which have been confirmed as members of the enterovirus genus. The re-classification of PEV-1 highlights the importance of having sequence data prior to determining the taxonomic position of viruses. We are currently studying virus strains from other serotypes in order to assess to which genus they belong.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 January 1999;
accepted 6 May 1999.