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1 US Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944, USA
2 Lanzhou Veterinary Research Institute, Lanzhou, Gansu, PR China
3 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
P. W. Mason
pwmason{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AJ539136, AJ539137, AJ539138, AJ539139, AJ539140 and AJ539141.
Present address: Department of Pathology, Sealy Center for Vaccine Development, University of Texas Medical Branch, 3.206B MMN Pavilion, 301 University Boulevard, Galveston, TX 77555-0436, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Over the last few years, there has been an explosion in the use of molecular epidemiology to study the spread of FMD and other infectious diseases of plants, animals and man. In the case of foot-and-mouth disease virus (FMDV), its rapid rate of evolution, which results from a polymerase without proof-reading activity and the ability of the genome to accommodate considerable amounts of mutations, has made this pathogen particularly amenable to tracking outbreaks through comparisons of the nucleic acid sequences of the viral genome (Samuel & Knowles, 2001
). Shortly after the outbreak in the UK, analyses of a highly variable region of the virus genome encoding the capsid protein VP1 (also called 1D) revealed that the virus responsible for the UK outbreak was related to a virus that spread from India in the early 1990s to the Far East (Knowles et al., 2001b). These VP1 sequence data suggested that the closest relative of the UK outbreak virus was an isolate from an outbreak in the Republic of South Africa, which appeared to have resulted from the introduction of an Asian virus into the port of Durban in the province of KwaZulu-Natal (Knowles et al., 2001b).
Previous studies with a distantly related type O virus that caused an epizootic in pigs in Taiwan demonstrated that defined genetic changes outside the VP1-regions used for genotype and epidemiological analyses were responsible for its altered phenotype (Beard & Mason, 2000; Knowles et al., 2001a). Moreover, other authors have demonstrated that changes in different regions of the FMDV genome have correlated with altered viral properties (Escarmis et al., 1992; Martinez-Salas et al., 1993; Escarmis et al., 1995; Feigelstock et al., 1996; Núñez el al., 2001). Thus, we undertook analyses of the sequences of full-length genomes of PanAsia isolates to try to determine if specific changes could be responsible for its rapid spread throughout Asia, Africa and Europe. While these studies did not identify any obvious changes associated with the high fitness of these viruses in nature, they did confirm the close genetic relationships among the PanAsia viruses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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)]. O/SKR/2000gb is used to designate the data from a 2000 Republic of Korea isolate deposited in GenBank (accession no. AF377945).
RT-PCR and sequencing.
RNA isolated from the indicated source using TRIzol (Life Technologies) was reverse-transcribed with Superscript II polymerase (Life Technologies) and amplified by PCR with Herculase polymerase (Stratagene) and the indicated primers (see Table 1 of online supplementary data, available at http://vir.sgmjournals.org), using a modification of the long-distance PCR methodology of Tellier et al. (1996). Following amplification, the cDNA fragments were purified from acrylamide gels by elution (S-fragment amplicons) or agarose gels with Qiagen Resin and sequenced using selected primers (see Table 1 of online supplementary data, available at http://vir.sgmjournals.org) and asymmetric amplification with Big Dye terminators (ABI) followed by resolution on an ABI 3700 sequencer.
Genome scanning analyses.
A program written by one of the authors (N. J. K.) was used to compare the complete genome sequence of O/UKG/35/2001 to those of 15 FMDVs representing five of the seven serotypes. The percentage nucleotide identities were calculated for a sliding window of 300 nucleotides, stepping at 15 nucleotide intervals. The resultant values were plotted on a graph.
Phylogenetic analyses.
Distance matrices were calculated with the program CLUSTAL X (Thompson et al., 1997). The phylogenetic tree was constructed using a neighbour-joining algorithm (Saitou & Nei, 1987) implemented in the program CLUSTAL X and drawn using the program TREEVIEW 1.6 (Page, 1996). Confidence limits were calculated by the bootstrap re-sampling method (1000 replicates) (Efron et al., 1996) as implemented within CLUSTAL X.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In the case of FMDV, sequencing of its genome by RT-PCR methodology is further complicated by the presence of a long polypyrimidine tract (of variable length) near the 5' end of the genome, which contains predominantly cytosine (C) residues. Using oligonucleotide primers that border this region, it is possible to reverse-transcribe cDNAs and amplify them using PCR, although the poly(C) tracts are truncated by successive rounds of PCR. Using these methods, we developed full-length, authentic sequence data for a 1999 Taiwan isolate of the PanAsia topotype (O/TAW/2/99). This sequence reflects the authentic genome in all positions except the number of A residues in the poly(A) tract and the number of C residues in the poly(C) tract (displayed in Fig. 1 as 10 bases), as well as the possible small number of U and A residues that could be present in this poly(C) tract. Several nucleotides (positions 454, 460 and 461) are reported as C/U mixtures (see Fig. 1; denoted by Y), consistent with the expected quasispecies nature of the genome. All other positions could be assigned to a single nucleotide, but among these there were undoubtedly mixtures at multiple positions.
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), so the 19 bases reported in Fig. 1(a)
are highly likely to reflect the authentic genome sequences. In addition, primer-determined sequences account for the last base of the S-fragment for O/TAW/2/99bov, O/Tibet/CHA/99, O/SKR/2000 and O/SAR/19/2000, and the last 10 bases of the S-fragment of O/UKG/35/2001. In addition, for all viruses except O/TAW/2/99 and O/Tibet/CHA/99 the first 22 bases of the L-fragment of the genome are from primer-encoded sequences, although, once again, due to conservation among viruses, these are also likely to be identical to the authentic genome given the high degree of conservation observed elsewhere. Among these five genomes, unambiguous assignments (with the caveats listed above) could be made for all but one base in the O/SAR/19/2000 sequence (an A/G mix at position 2182) and one base in the O/SKR/2000 sequence (a C/G mix at position 4009). Interestingly, our O/SKR/2000 sequence data showed numerous differences to a recently deposited sequence (GenBank accession no. AF377945) of the L-fragment of O/SKR/2000 (2·76 % different).
5' and 3' untranslated regions (UTR)
The 5' and 3' UTRs of the FMDV genome contain a number of distinct elements that have been identified on the basis of their predicted secondary structure and in some cases, biological functions. Fig. 2(a–c) shows the predicted secondary structures of these elements for O/TAW/2/99. These same panels show the position of differences with the other genomes (derived from data shown in Fig. 1a, b). As expected, the vast majority of the sequence differences are in regions between the elements, or do not affect the predicted secondary structure. Of interest is the finding that the 3' UTR of the Republic of Korea sequence deposited in GenBank has five deletions that were not present in the genomes of the seven other PanAsia isolates (Fig. 1b; see also Concluding comments).
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). In all of the coding regions, the conservation at the protein level is greater than at the nucleotide level, indicating that silent codon changes were greatly preferred (data not shown). Of interest is the fact that there is not a single deletion or insertion within the polyprotein, although the sequence of O/SKR/2000gb contains a deletion closely followed by an insertion in the coding region for VP2 (1B), which is most likely explained by a sequencing error (producing two new codons, shown as CY at positions 66 to 67 of VP2; see Fig. 3).
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shows all the type O viruses compared to O/UKG/35/2001. All the PanAsia virus sequences are very closely related across the whole genome, with O/SAR/19/2000 being the most closely related and O/SKR/2000gb the most different. The other two type O viruses are clearly distinct. A similar comparison of O/UKG/35/2001 to viruses of other serotypes revealed clear differences among all the coding regions, with the greatest differences detected in the region encoding the outer capsid polypeptides (see Fig. 1 of online supplementary data, available at http://vir.sgmjournals.org).
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), one nucleotide in the pseudoknot (PKN) region, one nucleotide in the internal ribosome entry site (IRES) region of the 5' UTR (Figs 1 and 2b) and no changes detected in the S-fragment or 3' UTR (Figs 2a, c and 3).
Using the complete genome sequence data, the relationships between the eight PanAsia viruses were determined using phylogenetic algorithms (Fig. 5). In general, the year of isolation correlates well with genetic relationship, and these analyses reveal that O/UKG/35/2001 is most closely related to O/SAR/19/2000, consistent with earlier analyses with data from the VP1-coding region of the genome (Knowles et al., 2001b). However, phylogenetic analysis of the VP1-coding region alone from the same set of viruses shown in Fig. 5 revealed that O/Yunlin/TAW/97 fell slightly closer to the PanAsia group (see Fig. 2 of online supplementary data, available at http://vir.sgmjournals.org). It is not clear why this was the case; however, the bootstrap value of the branch leading to the O1/Kaufbeuren-O1/Campos and PanAsia groups in the complete-genome-tree was quite low (423) in contrast to that leading to the O/Yunlin/TAW/97 and PanAsia groups in the VP1 tree (916) (see Fig. 2 of online supplementary data, available at http://vir.sgmjournals.org).
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Concluding comments
Taken together, analyses of the complete genome sequence data reveal a remarkable conservation among the PanAsia virus isolates, which appear to be much more stable than other type O viruses circulating in Asia during the same time period (Knowles et al., 2001a; Samuel & Knowles, 2001). Not only are the PanAsia genomes devoid of specific hot-spots for change, the scanning analyses reveal that there has not been any recent intra- or intertypic recombination of the PanAsia viruses.
These findings, together with the information cited above on how slight changes in the polyprotein correlate with profound differences in virulence, demonstrate that with our current level of understanding of FMDV genetics we are unable to identify changes in sequences that are diagnostic for the new properties. Thus, we have failed to identify specific genetic changes that can help explain why the PanAsia viruses have been so effective in their spread across Asia and appear to have, in some areas, replaced enzootic strains of FMDV type O (N. J. Knowles & A. R. Samuel, unpublished observations). If the reasons for the fitness of the PanAsia virus can be ascribed to some marker in laboratory or animal tests, then reverse genetics technology might be suitable for divining the cause of the apparently high fitness of the PanAsia viruses in nature.
Comparisons of the sequence data of O/SKR/2000, an isolate from early May 2000 in the Kyunggi province of the Republic of Korea, revealed a large number of differences (2·76 %) to a recently deposited sequence of the L-fragment of another O/SKR/2000 sequence (GenBank accession no. AF377945). This large number of differences strongly suggests that multiple genetic lineages of FMDV were responsible for the outbreaks reported in the Republic of Korea in 2000, a particularly intriguing hypothesis given the reported re-introduction of FMD in 2002.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 June 2002;
accepted 4 February 2003.
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