Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions

Abstract

Variable regions of the African swine fever virus genome, which contain arrays of tandem repeats, were compared in the genomes of isolates obtained over a 40-year period. Comparison of the size of products generated by polymerase chain reaction (PCR) from four different genome regions, within the B602L and KP86R genes and intergenic regions J286L and BtSj, placed 43 closely related isolated from Europe, the Caribbean, West and Central Africa into 17 different virus sub-groups. Sequence analysis of the most variable fragment, within the B602L gene, from 81 different isolates distinguished 31 sub-groups of virus isolates which varied in sequence and number of a tandem repeat encoding 4 amino acids. Thus, each of these analysis methods enabled isolates, which were previously grouped together by sequencing of a more conserved genome region, to be separated into multiple sub-groups. This provided additional information about strains of viruses circulating in different countries. The methods could be used in future to study the epidemiology and evolution of virus isolates and to trace the sources of disease outbreaks.

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“…However, low levels of variation, principally within homogeneous pig and pig-tick associated genotypes, remains problematic and requires that an additional, more variable gene region be characterised in order to achieve optimal levels of resolution for epidemiological inferences. The value of a p72-CVR twostep approach has been demonstrated at a country, genotype and regional level [8,9,11] and its usefulness was confirmed in this study of genotype VIII viruses from East Africa, where seven CVR lineages were recovered from viruses that could previously only be resolved into two p72 genotype VIII lineages [4,12]. This is, however, the first time that the genes have been combined in a single analysis in order to improve resolution, which in the case of genotype VIII led to the recovery of eight discrete p72-CVR lineages, which clustered within three main monophyletic lineages (I-III in Fig.…”

“…Seven additional viruses identified as genotype VIII in previous studies [8,9,12] and for which CVR data were available were included, bringing the total number of viruses used in this study to 45 (Table 1). Viral DNA was extracted as described previously [12] and used as template for amplification with CVR with primers CVR-FL1 (5′TCG GCC TGA AGC TCA TTA G3′) and CVR-FL2 (5′CAG GAA ACT AAT GAT GTT CC3′) that bind in the regions flanking the repeat region [9].…”

“…Previous RFLP studies of 17 viruses recovered from outbreaks in Malawi between 1982 and 1989 confirmed the presence of a central variable region (CVR) within the central 125 kb conserved region of the genome [5]. This length variation was subsequently shown to result from differences openUP (December 2007) in a tetrameric repeat region located within the late viral gene, 9RL [6], also termed the B602L gene [7,8]. The protein, which varies in size in accordance with the number of repeats within the CVR is homogeneously distributed throughout the cytoplasm and is strongly recognised by convalescent swine serum [6].…”

“…The protein, which varies in size in accordance with the number of repeats within the CVR is homogeneously distributed throughout the cytoplasm and is strongly recognised by convalescent swine serum [6]. Recent PCR-based studies directed at the CVR have revealed both size and sequence variability [8,9,10] for the limited number of genotype VIII viruses included in these studies. Although the CVR is not suitable for resolution of inter-genotypic relationships [9,11], it has proved useful for resolving epidemiological complexities at the country [9], genotype [8] and regional level [11].…”

“…Recent PCR-based studies directed at the CVR have revealed both size and sequence variability [8,9,10] for the limited number of genotype VIII viruses included in these studies. Although the CVR is not suitable for resolution of inter-genotypic relationships [9,11], it has proved useful for resolving epidemiological complexities at the country [9], genotype [8] and regional level [11]. A two-step approach is advocated for epidemiological studies [9], whereby viruses are first assigned to their p72 genotype [12], prior to intra-genotypic resolution by CVR, and was followed in this study which represents the first comprehensive attempt to resolve the intra-genotypic relationships of 45 p72 genotype VIII viruses causing outbreaks in East Africa over a 40 year period.…”