African swine fever virus (ASFV) is a complex DNA virus causing severe hemorrhagic disease in domestic pigs and wild boar. The disease has spread worldwide, with important socio-economic consequences. Early virus detection and control measures are crucial as there are no effective vaccines nor antivirals on the market. While the diagnosis of ASFV is fast and based primarily on qPCR, the detection of infectious ASFV is a labor-intensive process requiring susceptible macrophages and subsequent antibody-based staining or hemadsorption. The latter cannot detect ASFV isolates devoid of functional CD2v (EP402R) expression. Here, we report the development of a plasmid-based reporter assay (RA) for the sensitive detection and titration of infectious ASFV. To this end, we constructed a plasmid for secreted NanoLuc luciferase (secNluc) expression driven by the ASFV DNA polymerase gene G1211R promoter. Infection of plasmid-transfected immortalized porcine kidney macrophages (IPKM) followed by measurement of secNluc from cell culture supernatants allowed reliable automated quantification of infectious ASFV. The RA-based titers matched the titers determined by conventional p72-staining or hemadsorption protocols. The novel assay is specific for ASFV as it does not detect classical swine fever virus nor porcine reproductive and respiratory syndrome virus. It is applicable to ASFV of different genotypes, virulence, and sources, including ASFV from sera and whole blood from infected pigs as well as non-hemadsorbing ASFV.
Keywords: ASFV; African swine fever virus; High-throughput screening; Live virus detection; Reporter assay; Virus titration.
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