Phylogenetic analyses of the polyprotein coding sequences of serotype O foot-and-mouth disease viruses in East Africa: evidence for interserotypic recombination
© Balinda et al. 2010
Received: 22 April 2010
Accepted: 23 August 2010
Published: 23 August 2010
Foot-and-mouth disease (FMD) is endemic in East Africa with the majority of the reported outbreaks attributed to serotype O virus. In this study, phylogenetic analyses of the polyprotein coding region of serotype O FMD viruses from Kenya and Uganda has been undertaken to infer evolutionary relationships and processes responsible for the generation and maintenance of diversity within this serotype. FMD virus RNA was obtained from six samples following virus isolation in cell culture and in one case by direct extraction from an oropharyngeal sample. Following RT-PCR, the single long open reading frame, encoding the polyprotein, was sequenced.
Phylogenetic comparisons of the VP1 coding region showed that the recent East African viruses belong to one lineage within the EA-2 topotype while an older Kenyan strain, K/52/1992 is a representative of the topotype EA-1. Evolutionary relationships between the coding regions for the leader protease (L), the capsid region and almost the entire coding region are monophyletic except for the K/52/1992 which is distinct. Furthermore, phylogenetic relationships for the P2 and P3 regions suggest that the K/52/1992 is a probable recombinant between serotypes A and O. A bootscan analysis of K/52/1992 with East African FMD serotype A viruses (A21/KEN/1964 and A23/KEN/1965) and serotype O viral isolate (K/117/1999) revealed that the P2 region is probably derived from a serotype A strain while the P3 region appears to be a mosaic derived from both serotypes A and O.
Sequences of the VP1 coding region from recent serotype O FMDVs from Kenya and Uganda are all representatives of a specific East African lineage (topotype EA-2), a probable indication that hardly any FMD introductions of this serotype have occurred from outside the region in the recent past. Furthermore, evidence for interserotypic recombination, within the non-structural protein coding regions, between FMDVs of serotypes A and O has been obtained. In addition to characterization using the VP1 coding region, analyses involving the non-structural protein coding regions should be performed in order to identify evolutionary processes shaping FMD viral populations.
Elsewhere on the African continent, remarkable progress in FMD molecular epidemiology has been made particularly in Southern Africa, a region also characterized by SAT type endemicity. Molecular epidemiological studies have been able to reveal the origin and routes of FMD transmission in this region. In addition, the important epidemiological role of the African buffalo (Syncerus caffer) in maintenance of this virus and transmission to other cloven-hoofed animals has been highlighted [16–18].
In this study, we have analyzed the polyprotein coding sequence (6719 nt) of serotype O FMD viruses to get insights into relationships among these viruses to infer processes shaping their diversity in the East African region. Sequences analyzed in this study include isolates from Kenya and Uganda obtained between the years 1992 and 2006 and sequences already in the Genbank database.
Materials and methods
The viruses investigated in this study were collected during outbreaks in the indicated years in Kenya (K) and Uganda (U). Four samples; O/K/52/1992, O/K/117/1999, O/K/109/2000 and O/K/48/2005 were from Kiambu, Nakuru, Uasin Gishu and Kiambu districts, respectively. FMDV RNA was extracted from these samples following virus propagation in baby hamster kidney (BHK) cells. The O/U/25/2006 (Mpigi district) sequence was generated from RNA prepared directly from an oropharyngeal sample while the O/U/312/2006 virus was isolated from an orophyrangeal sample from Mbarara district and grown in primary bovine thyroid (BTY) cells.
RNA extraction, cDNA synthesis, PCR and Cycle sequencing
RNA was extracted from virus harvests and directly from orophyrangeal fluid using the QIAamp® Viral RNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The cDNA synthesis was carried out using Ready-To-Go™ You-Prime First - Strand Beads (GE Healthcare Life Sciences, Sweden) with random hexamer primers (pdN6).
Summary of primers used
8-A PN 2
Fragment 1: 8-A PN-35 & 8-A PN-2
8-A PN 3
8-A PN 4
Fragment 2: 8-A PN 3 & 8-A PN-4
8-A PN 6
8-A PN 14
Fragment 3: 8-A PN 34 & 8-A PN-83
8-A PN 22
8-A PN 23
Fragment 4: 8-A PN 51 & 8-A PN-6
8-A PN 34
8-A PN 35
Fragment 5: 8-A PN 84 & 8-A PN-85
8-A PN 45
8-A PN 46
Fragment 6: 8-A PN 98 & 8-A PN-64
8-A PN 51
8-A PN 64
Fragment 7: 8-A PN 86 & 8-A PN-45
8-A PN 68
8-A PN 80
Fragment 8: 8-A PN 22 & 8-A PN-23
8-A PN 83
8-A PN 84
Fragment 9: 8-A PN 46 & 8-A PN-87
8-A PN 85
8-A PN 86
Fragment 10: 8-A PN 99 & 8-A PN-68
8-A PN 87
8-A PN 98
Fragment 11: 8-A PN 113 & 8-A PN-14
8-A PN 99
8-A PN 101
Fragment 12: 8-A PN 80 & 8-A PN-101
8-A PN 113
Sequencher software 4.8 (Gene Code Corporation) was used to assemble the 12 overlapping fragment sequences generated per sample. Multiple alignments by log-expectation comparison were carried out using MUSCLE  incorporated within Geneious 4.7.6 software . Phylogenetic analyses involving the determination of models of evolution were performed using hierarchical likelihood-ratio test of 24 models using PAUP*(v. 4.0 beta 10)  and MrModeltest (v 2.2) . The GTR+I+G model was used and Bayesian inference analysis performed using MrBayes (v.3.1.2)  with the following settings: maximum likelihood model was six substitution types (nst = 6), with base frequencies set to variable values ("statefreqpr = dirichlet(1,1,1,1"). Rate variation across sites with a proportion of invariable sites was modeled using a gamma distribution (rates = invgamma). The Markov Chain Monte Carlo search was run with 4 chains for 500,000 generations with trees sampled every 100 generations; the first 1250 were discarded as burnin . Recombination between sequences was analysed using SimPlot method 2.5 software .
Sequence Characteristics and Phylogenetic Relationships
Almost the entire polyprotein coding sequence (6719 nt) of five FMDV isolates and a single Ugandan FMDV RNA sample were determined. Four of these samples had origins from the districts of Kiambu, Nakuru and Uasin Gishu in Kenya (from 1992, 1999, 2000 and 2005 respectively) while the two Ugandan isolates were from Mpigi and Mbarara districts (in 2006). All isolates belong to serotype O with percentage nucleotide sequence identities of the polyprotein coding region with representatives of six FMDV serotype as follows; O(89%), A(83%), C(84%), SAT1(73%), SAT2(77%) and SAT3(73%). The generated sequences have further been compared with sequences published in Genbank including the sequence of another recent Ugandan type O isolate from Kasese district (Accession no. EF611987), which was determined previously. Twenty one additional serotype O sequences previously isolated from Europe, Asia, South America and Africa were included in the analysis. Furthermore, sequences from serotypes A, C, SAT 1, SAT 2 and SAT 3 were included for comparative purposes.
Detection of Recombination
Sequencing of these recent viruses from Kenya and Uganda has allowed for the assessment of variation of the East African isolates across almost the entire coding region relative to viruses from Asia, Europe and to a smaller extent South America. These East African viruses belong to serotype O based on the sequence of the VP1 coding region (Figure 2) and are part of a single evolutionary lineage (topotype EA-2) except for K/52/1992 (topotype EA-1). The related virus strains U/312/2006 and O/UGA/2006 (accession no. EF611987)  have also been identified as serotype O by antigen ELISA (data not shown).
Within the East African lineage, two major divisions are observed corresponding to their geographical locations with the Ugandan isolates comprising one sub-lineage and the Kenyan strains another. In addition, the Ugandan viruses are grouped in a temporal manner with the Uganda (2006) isolates clustering together but separate from the Uganda (2002) isolates. The East African lineage appears to have evolved independently from other serotype O viruses which are circulating globally. From comparisons of published serotype O sequences, three other main lineages were observed (Figure 3). An Asian lineage includes O/Akesu/1958 and O/HKN/2002 while a second lineage, widely referred to as the PanAsia I lineage , includes recent European O and Asian strains, the PanAsia II lineage (Figure 2) is represented by a viral strain from Pakistan (EF494501/O/PAK/06/2006) collected in the year 2006. The last lineage is comprised of earlier European strains from 1966 and 1967 together with the Campos strain from South America, indicative of intercontinental transmission between the Asian continent and Europe as well as South America and Europe.
Phylogenetic relationships of the P2 and P3 coding regions placed the K/52/1992 strain in a close relationship with serotype A viruses (from Kenya in 1965 and Azerbaijan/USSR/1965) (Figures 4 and 5). This points to the possibility that this strain may have arisen by recombination between viruses of different serotypes, a process seen within many picornaviruses both in nature, e.g. for poliovirus, Human enterovirus B [6, 27–29], and in the laboratory including with FMD viruses . The mosaic pattern observed within the P3 coding region may suggest recombination although this could also indicate genetic convergence. Earlier studies have shown that inter serotypic genetic exchange occurs more frequently within the Euro-asiatic viruses in comparison to amongst SATs or even between SAT and non-SAT viruses .
It is noteworthy that the most prevalent serotypes in the East Africa region are O followed by A  strongly suggesting the possibility of co-infection with both or even more serotypes within some animals.
The findings reveal the complexity of FMDV evolution, consistent with earlier studies that have shown that recombination is mainly restricted to non-structural coding regions.
This study therefore supports recombination as an evolutionary force causing genetic diversity within FMDV and shows the need for full genome analyses to identify such events.
We thank Tina Frederiksen, Tina Rasmussen and Jani Christiansen for excellent technical assistance. We thank the Director of Veterinary Services, Kenya, for the Kenyan isolates. This work was supported by the Danish International Development Agency (DANIDA) under the Livestock Wildlife Diseases in East Africa project.
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