African swine fever virus (ASFV) was first reported in eastern Europe/Eurasia in 2007. Continued spread of ASFV has placed central European countries at risk, and in 2014, ASFV was detected in Lithuania and Poland. Sequencing showed the isolates are identical to a 2013 ASFV from Belarus but differ from ASFV isolated in Georgia in 2007.

Complete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach.

A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n = 260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous β-actin gene. Finally, the complete study offers a new validated real-time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF.

This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.

No abstract