1 Mammal Research Institute, Department of Zoology & Entomology, University of Pretoria, Pretoria 0002, South Africa
2 ARC-Onderstepoort Veterinary Institute, Exotic Diseases Division, Private Bag X5, Onderstepoort 0110, South Africa
3 Centre for Tropical Veterinary Medicine, University of Edinburgh, Edinburgh EH25 9RG, UK
4 Laboratoire Central Veterinaire, BP 2295, Bamako, Mali
5 Organization of African Unity/Inter-African Bureau for Animal Resources (OAU-IBR), PO Box 30786, Nairobi, Kenya
Correspondence
A. Bastos (at University of Pretoria)
ADBastos{at}zoology.up.ac.za
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ABSTRACT |
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INTRODUCTION |
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African buffalo (Syncerus caffer) are particularly efficient maintenance hosts and play a central role in the epidemiology of the disease due to their ability to maintain and transmit FMD virus (reviewed by Thomson, 1996). Results indicate that SAT-type viruses can persist in an individual animal and in an isolated herd for at least 5 and 24 years, respectively (Condy et al., 1985
) and that numerous antigenic and genetic variants are generated in an individual animal during persistence (Vosloo et al., 1996
). Transmission of SAT-type virus from persistently infected African buffalo to cattle under experimental and natural conditions has been unequivocally demonstrated (Dawe et al., 1994a
, b
; Vosloo et al., 1996
; Bastos et al., 2000
). Separation of buffalo and livestock by fencing and vaccination of cattle with viruses that are antigenically closely related to those carried by nearby buffalo form an integral part of FMD control in southern African countries (Hunter, 1998
).
Overall antigenic diversity as measured by ELISA and virus neutralization test appears to be reflected genetically in the VP1 gene (Hunter et al., 1996; Araújo et al., 2002
), which is one of four structural proteins encoded within the P1 region of the genome. VP1 contains the major neutralizing sites of the virus (Crowther et al., 1993
) as well as the integrin cell-attachment site RGD, which is located on a highly flexible loop that protrudes from the surface of the virion (Acharya et al., 1990
). The multiplicity of roles that VP1 has in receptor-recognition, neutralization and antigenic diversity together with the valuable molecular epidemiological insights obtained from analysis of nucleotide sequences (reviewed by Sobrino et al., 2001
) has made this the most intensively studied gene of the FMD viral genome. However, most of these efforts have been directed at the European serotypes, O, A and C, with complete VP1 gene data being restricted to just three SAT 2-type viruses (van Rensburg & Nel, 1999
).
The benefits of extensive genetic characterization of the SAT-type viruses are perhaps best illustrated by reports from southern Africa where the central role played by buffalo in the epidemiological situation has been described (Dawe et al., 1994a; Vosloo et al., 1995
; Bastos et al., 2000
, 2001
). Here, the establishment of a partial VP1 gene sequence database for southern African SAT-type buffalo viruses was shown to be invaluable for accurately tracing the source of SAT 1-type outbreaks in this region (Bastos et al., 2001
) and for pinpointing the origin of illegally moved buffalo infected with SAT 3-type viruses (Bastos, 2001
; Vosloo et al., 2001
).
Of the six serotypes occurring in Africa, only serotype A has been studied at the continental scale. Analysis of partial VP1 gene nucleotide sequences revealed the presence of six regionally and genetically distinct virus lineages (Knowles et al., 1998) that conform to the topotype classification (Samuel & Knowles, 2001a
). The identification of regionally distinct viruses ensures that the origin of viruses imported trans-regionally and trans-continentally can be accurately determined (Knowles et al., 1998
; Sangaré et al., 2001
; Sangaré, 2002
). Studies similar to that of type A (Knowles et al., 1998
) applied to the remaining five African serotypes are beneficial not only for the African continent, but for all countries with FMD-free status, as the disease security of these countries is threatened by the importation of exotic viruses, as occurred in 2001 in the UK (Samuel & Knowles, 2001b
). Past records of multiple SAT-type virus incursions into the Middle East and known transmission routes from the latter region to Europe (Kitching, 1998
) make it imperative to characterize viruses occurring in FMD endemic areas.
Genetic characterization of SAT 2 viruses from African buffalo maintenance host populations throughout Africa has not been adequately addressed, nor has any attempt been made to determine genetic variability at the continental scale. The former has implications for accurate determination of the source of SAT 2-type infections whilst the latter provides some measure of the complexity of disease control through vaccination. It is with these objectives in mind that SAT 2-type viruses from different regions and species, collected over a 52 year period have been selected for genetic characterization. Specifically, we examine these complete VP1 gene sequences for evidence of inter-lineage recombination, we relate the topological structure of the estimated phylogeny to the geographical location of the virus isolates, we examine the applicability of a molecular clock to describe the evolution of this gene both within and between lineages, and we report the distribution of substitutions along the gene.
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METHODS |
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Phylogenetic analysis.
Nucleotide sequences were aligned to published reference strains (Table 1) using DAPSA (Harley, 2001
) and trimmed so that a homologous region of 648 nt, corresponding to the complete VP1 gene, was obtained. All data were submitted to GenBank under the accession numbers provided in Table 1
and where grid references were available these were used to accurately plot geographical distribution.
Phylogenetic reconstruction was performed using a minimum evolution algorithm implemented in PAUPSTAR (v. 4.0b8; Swofford, 2001). Pairwise distances were computed using the HKY85 model of nucleotide substitution (Hasegawa et al., 1985
), with rate heterogeneity estimated using maximum-likelihood estimated over a topologically identical tree. 1000 bootstrap resamples were undertaken using the neighbor-joining method implemented in PAUPSTAR and assuming the same substitution model. Reconstruction was also conducted using maximum-parsimony, neighbor-joining and maximum-likelihood, in order to check for any substantial sensitivities to method of reconstruction. The program PLATO (Grassly & Holmes, 1997
) was used to examine whether or not there were areas of the alignment in which support for the reconstructed phylogeny was significantly reduced. This method works by examining the likelihood of the phylogeny within windows of different sizes that are moved sequentially along the alignment. The program TIPDATE (Rambaut, 2000
) was used to examine the average annual rate of nucleotide substitution over the phylogeny and to test the hypothesis that the phylogeny and various constitutive clades within it evolved in accordance with a molecular clock. This method uses a likelihood ratio test (LRT) to determine whether the reduction in likelihood of a tree with branch lengths estimated assuming a molecular clock is significantly reduced relative to the maximum-likelihood of a phylogeny assuming no clock. An LRT assumes that twice the difference in likelihood between two nested models is chi-squared distributed with degrees of freedom equal to the number of parameters by which the two models differ [see Rambaut (2000)
for further details of this test]. Several different plausible positions for the roots of the phylogeny were selected to test this hypothesis. As this program cannot handle identical sequences within the same phylogeny duplicate sequences were first removed.
The average number of synonymous and non-synonymous substitutions per synonymous and non-synonymous site along the gene was calculated in DNASP (Rozas & Rozas, 1999). The occurrence of synonymous and non-synonymous substitutions along the VP1 gene was examined using the average number of pairwise synonymous and non-synonymous substitutions per codon position. A permutation test was used to test for significant clustering in the distribution of both types of substitutions, using a sliding window of 5 codon positions in length.
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RESULTS |
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In West Africa two regionally distinct topotypes (E and F) having an overlapping distributional range in Sénégal were identified (Fig. 2). As sequence divergence values of viruses for topotypes E and F equalled that observed for viruses from different virus lineages, two West African virus lineages (II and III) were defined. All West African viruses comprising topotype F had a unique VP1 protein length of 215 instead of 214 due to an amino acid insertion at position 29.
The north-east African virus lineage (IV) comprised two viruses, ERI/12/98 and SAU/6/00, which were recovered from outbreaks occurring in Eritrea in 1998 and in Saudi Arabia in 2000, respectively. The 100 % bootstrap support for this node, together with the high level of sequence identity (>90 %), indicate that the north-eastern African region was the most likely source of the virus introduced to the Middle East.
In East and central Africa four topotypes were discerned, three of which (HJ) constitute lineage IV and are represented by a single virus each from Rwanda, the Democratic Republic of the Congo (DRC) and Kenya. The fourth topotype (B), represented by viruses from southern Kenya, Malawi and Burundi, was more closely related to topotypes occurring in the southern African region (topotypes A, C and D) than to those constituting the north-east African lineage (IV).
Molecular clock
The hypothesis that the rate of nucleotide substitution is constant over the whole phylogeny was strongly rejected (P<0·0001) irrespective of the position of the putative root. After removal of some of the more obvious temporally aberrant sequences (KEN/3/57, MAL/3/75 and RHO/1/48), the clock hypothesis was still strongly rejected (P<0·0001). Applying the test to each of the groups AF in Fig. 1 (each rooted on a sequence in a neighbouring topotype) yielded an average annual rate of nucleotide substitutions per site of 0·0023 (range 0·00020·0047). Statistics from the LRT applied to each of these six topotypes and the corresponding uncorrected P-values are reported in Table 2
. However, since six hypotheses are being tested, the critical P-value for rejection of the null hypothesis of constant rates within topotypes might be lowered from 0·05 to 0·05/6=0·0083, which would lead to the conclusion that a molecular clock could be applied to five of the six topotypes examined here.
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DISCUSSION |
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Overlap in the distributional range of topotypes was observed in restricted areas in southern, eastern and western Africa. In southern Africa topotypes C and D display overlap in distributional range at the border region where Botswana, Angola, Namibia, Zambia and Zimbabwe converge. Within this area of convergence numerous disease outbreaks have been recorded which are attributed to intensive livestock trade (Mweene et al., 1996). Thus, the blurring of topotype boundaries arising from trans-boundary movements that occurs when a foreign topotype is introduced from a neighbouring country explains the presence of the predominantly Zambian topotype (topotype C) in buffalo from northern Botswana. Other countries containing multiple virus lineages (and topotypes) within their borders include Kenya (lineage I and IV) in East Africa, where cattle trading occurs across all borders (Ndiritu, 1984
), and Sénégal (lineage II and III) in West Africa, where the practice of transhumance is a contributing factor (Sangaré, 2002
). These three virus exchange hotspots' reveal that disease control would likely benefit through enforcing stricter border controls. The remaining seven topotypes show no evidence of overlap in geographical range.
From the amino acid sequence alignments it is clear that the VP1 gene of SAT 2-type viruses has features in common with serotype O. In both serotypes there is a conserved cysteine residue at the base of the GH loop that may account for the presence of conformational neutralizing epitopes in these serotypes (Xie et al., 1987; Crowther et al., 1993
). Complete conservation of this cysteine residue occurs in all SAT serotypes (Bastos, 2001
; Bastos et al., 2001
). However, the presence of the corresponding cysteine in VP2, with which Cys-134 of VP1 forms a heterodimer in type O viruses, has only been identified in two SAT 2-type viruses (van Rensburg & Nel, 1999
). A feature unique to SAT 2-type viruses is the presence of a highly conserved positively charged arginine residue within the GH loop flanking the carboxyl-end of the RGD. In all other serotypes, a non-polar leucine predominates at this position which together with a highly conserved leucine at the +4 position has been shown to be critical for modulating interactions with integrin receptors (Mateu et al., 1996
).
The steady rate of nucleotide substitution in the phenotypically plastic capsid genes of picornaviruses has been noted many times previously (e.g. Martinez et al., 1992; Vosloo et al., 1996
; Zhang et al., 1999
; Haydon et al., 2001b
; Jenkins et al., 2002
). Usually the rate of substitution can only be estimated within monophyletic groups rooted with a virus strain isolated close to the date of the root of the tree. The approach implemented in TIPDATE uses an LRT in the same spirit that Felsenstein (1993)
proposed a statistical test to distinguish between ultrametric clock-like trees and non-ultrametric trees. Felsenstein's original approach could not be validly applied to RNA virus phylogenies of strains isolated at different times as the rate of evolution is sufficiently high that even clock-like modes of substitution could not be expected to yield ultrametric phylogenies. It is not unusual to observe that the rates of estimated nucleotide substitution within the 1D genes become progressively less as they are measured over longer time-scales. For example, measured over just a few years, annual rates of between 0·5 and 1·5 % of sites incurring substitutions are not uncommon (Vosloo et al., 1996
; Haydon et al., 2001b
); however this rate cannot apply over longer time-periods and in the more clock-like clades of the phylogeny reconstructed here, rates were always estimated to be between 0·02 and 0·5 % per year. The determinants of longer term rates of evolution in RNA genomes remains a contentious subject (see for example Korber et al., 1998
). Interestingly, the highest rates of nucleotide substitution were observed in virus groups recovered from cattle (topotypes B, E and F) and the lowest rates observed in clades containing virus recovered from wildlife (topotypes A, C and D). The differences in rates between topotypes may reflect the different levels of epidemiological activity associated with different topotypes (with higher numbers of cases resulting in more virus replication and more opportunity for divergence). However, the sample sizes within each topotype do not permit these rates to be distinguished statistically.
The average ratio of synonymous to non-synonymous substitution across the whole VP1 gene was 5·0 : 1. Low ratios of synonymous to non-synonymous substitutions (less than one) measured across parts of the gene encoding viral epitopes has been interpreted as evidence of positive selection (Fares et al., 2001; Haydon et al., 2001a
), but this ratio is expected to rise rapidly as the more genetically constrained parts of the gene are included to estimate the average ratio. If virus infections induce a more vigorous immune response in cattle than in buffalo and impala (which may be the case given the difference in disease severity that infection produces), then we might expect to observe higher levels of positive selection and lower ratios of synonymous to non-synonymous substitutions in cattle than in wild hosts. However, we found that when we fitted a model that permitted different ratios of synonymous to non-synonymous substitutions to clades that contained only cattle (see Fig. 1
) no significant differences in selective pressure were indicated (results not shown). Synonymous substitutions occur evenly between codons along the gene, showing no sign of clustering, and thus do not indicate any evidence for patterns of larger scale constraints on synonymous substitutions. Surprisingly perhaps, non-synonymous substitutions also showed little evidence of spatial clustering along the gene, with just one weakly significant hotspot apparent around codon 140.
This study has provided some indication of the level of genetic diversity of SAT 2-type FMD in individual countries on the African continent. Of the 17 African countries included in this study, 13 have a single SAT 2 topotype within their borders. However within some countries, notably Botswana, Zimbabwe and Sénégal, there are at least two topotypes present. This translates to within-country genetic variation levels of between 24 and 27 % on nucleotide level and between 16·6 and 19·8 % on amino acid level, when pairwise comparisons are performed for two viruses from the same country but from different topotypes. By far the most heterogeneous is Kenya, which has viruses representative of two of the six major clusters (I and IV) and has the highest within-country levels of genetic diversity. Kenyan strains differed from each other by more than 30 % at nucleotide level and by more than 23·5 % at amino acid level. These within-country levels approximate the maximum observed for the continent where sequence divergence is 31·6 % and 24·1 % at nucleotide and amino acid levels, respectively.
The low level of nucleotide sequence conservation (only 38·4 % of sites are completely conserved across all SAT 2 viruses analysed in this study) has diagnostic implications, as the development of a single set of serotype-specific primers that will be suitable for detection of all SAT 2 field variants is unlikely. Through the generation of complete VP1 gene sequences detailed here, it may now be possible to design efficient serotype-specific primers, something that has eluded most researchers (Reid et al., 2001). However, given high levels of intratypic variation it is unlikely that a single primer set will permit unequivocal identification of all SAT 2 field strains and regional-specific primer sets may be more appropriate.
These high levels of genetic diversity within the SAT 2 serotype are likely to be reflected antigenically and have implications for control of the disease through vaccination. To be effective, vaccines should not only be stable and have a high antigen load, but should also be immunologically related to viruses circulating in the field at the time (Hunter, 1998). The r1 value provides a measure of antigenic similarity and is defined as the ratio of the serum titre of heterologous virus (e.g. an outbreak strain) to the serum titre of homologous virus (i.e. the vaccine strain) (Rweyemamu et al., 1977
). Serological comparisons of SAT 2 viruses from Kenya revealed that there is extensive antigenic variation in this country, where two of the six major SAT 2 virus clusters occur (Ndiritu et al., 1983
). In a comparative study of the antigenic and genetic diversity within SAT 1 (Hunter et al., 1996
), it was shown that viruses from different topotypes, i.e. those that differ from each other by more than 20 % on nucleotide sequence level (Bastos et al., 2001
), generally have r1 values below 0·4 and are therefore antigenically poorly related (Esterhuysen, 1994
; Hunter et al., 1996
). Similar results have been obtained for serotype A viruses where viruses differing from each other by
15 % and therefore of different topotypes (Knowles & Samuel, 2003
) were shown to be antigenically poorly related (Araújo et al., 2002
). Clearly, the identification of eleven discernable SAT 2 topotypes indicates that multiple region-specific vaccines may have to be developed to address this field heterogeneity. Given the regional diversity within the SAT 2 serotype and that documented for four other serotypes (Knowles et al., 1998
; Sangaré et al., 2001
; Bastos, 2001
; Bastos et al., 2001
), controlling the disease through vaccination will be extremely difficult. Furthermore, the costs arising from the administration of multivalent vaccines on a systematic basis places this means of disease control out of reach of most developing countries in sub-Saharan Africa. The twin problems presented by virus diversity and difficulty of establishing effective movement control in western, central and eastern Africa present an apparently insuperable difficulty for effective FMD control.
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ACKNOWLEDGEMENTS |
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Received 26 September 2002;
accepted 5 February 2003.