A PCR-based sequencing method was developed which permits detection and characterization of African swine fever virus (ASFV) variants within 5 and 48 h, respectively, of receipt of a clinical specimen. Amplification of a 478 bp fragment corresponding to the C-terminal end of the p72 gene, confirms virus presence with genetic characterization being achieved by nucleotide sequence determination and phylogenetic analysis. The method was applied to 55 viruses including those representative of the major ASF lineages identified previously by restriction fragment length polymorphism (RFLP) analysis. Results confirmed that the p72 genotyping method identifies the same major viral groupings. Characterization of additional viruses of diverse geographical, species and temporal origin using the PCR-based method indicated the presence of ten major ASF genotypes on the African continent, the largest of which comprised a group of genetically homogeneous viruses recovered from outbreaks in Europe, South America, the Caribbean and West Africa (the ESAC-WA genotype). In contrast, viruses from southern and East African countries were heterogeneous, with multiple genotypes being present within individual countries. This study provides a rapid and accurate means of determining the genotype of field and outbreak strains of ASF and is therefore useful for molecular epidemiological clarification of ASF.

The virus isolate introduced to the Caucasus in 2007 is closely related to a group of viruses, genotype II, circulating in Mozambique, Madagascar, and Zambia.

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African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

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.

Virus diversity indicates a virus reservoir in this country.

Ten African swine fever virus isolates from the soft tick Ornithodoros erraticus collected on three farms in the province of Alentejo in Portugal were characterized by their ability to cause haemadsorption (HAD) of red blood cells to infected pig macrophages, using restriction enzyme site mapping of the virus genomes and by experimental infection of pigs. Six virus isolates induced haemadsorption and four were non-haemadsorbing (non-HAD) in pig macrophage cell cultures. The restriction enzyme site maps of two non-HAD viruses, when compared with a virulent HAD isolate, showed a deletion of 9?6 kbp in the fragment adjacent to the left terminal fragment and of 1?6 kbp in the right terminal fragment and an insertion of 0?2 kbp in the central region. The six HAD viruses isolated were pathogenic and produced typical acute African swine fever in pigs and the four non-HAD isolates were non-pathogenic. Pigs that were infected with non-HAD viruses were fully resistant or had a delay of up to 14 days in the onset of disease, after challenge with pathogenic Portuguese viruses. Non-HAD viruses could be transmitted by contact but with a lower efficiency (42-50 %) compared with HAD viruses (100 %). The clinical differences found between the virus isolates from the ticks could have implications for the long-term persistence of virus in the field because of the cross-protection produced by the non-pathogenic isolates. This may also explain the presence of seropositive pigs in herds in Alentejo where no clinical disease had been reported.

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