Genotyping field strains of African swine fever virus by partial p72 gene characterisation

Abstract

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.

Role of Wild Suids in the Epidemiology of African Swine Fever

“…The highly virulent Lil20/ 1 strain of East African origin was readily transmitted, but transmission failed when bushpigs were infected with VICT90/1, a genotype I virus that groups with West African, domestic pig cycle viruses (Bastos et al, 2003), that are generally associated with sub-clinical infection. Horizontal transmission between bushpigs did not occur, irrespective of the virus strain used , suggesting that this species may require higher doses of virus to become infected, or is only susceptible to particular virus variants that were not evaluated.…”

“…Variation in clinical disease and virus genome length and composition is known to occur, with infections in domestic swine ranging from sub-clinical to lethal and field strains having genomes up to 20 kbp longer than laboratoryadapted strains (Tulman et al, 2009). Recent molecular epidemiological studies have also uncovered substantial field heterogeneity with at least 22 genotypes being identified to date on the basis of C-terminus p72 gene sequencing (Bastos et al, 2003;Lubisi et al, 2005;Boshoff et al, 2007). The recent incursion of ASF into Georgia (Beltran Alcrudo et al, 2008;Rowlands et al, 2008) has highlighted the potential of the virus to expand its range towards Asia through the Caucasus and the Indian Ocean (Costard et al, 2009) as it encounters new populations of wild and domestic pigs, yet important gaps in our knowledge remain regarding the role of many wild Suidae in the maintenance of ASFV.…”

Respiratory syncytial virus(RSV)-induced allergy may be controlled by IL-4 and CX3C fractalkine antagonists and CpG ODN as adjuvant: hypothesis and implications for treatment

“…The usefulness of the C-terminal end of the p72 gene for genotyping ASF viruses and for inferring broad epidemiological relationships is well established [4,11,12], particularly in those areas where a sylvatic cycle occurs. 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.…”

“…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]. All reactions were performed in a final volume of 50 μl containing 200 μM dNTPs (Roche), 0.4 μM of each primer, 2.5 U of Taq DNA polymerase (Roche) and 3 μl of DNA extract.…”

“…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.…”