Development of New Probe-Based Real-Time RT-qPCR Assays for the Detection of All Known Strains of Bovine Ephemeral Fever Viruses

Bovine ephemeral fever is an arthropod-borne viral disease that affects cattle and buffalo in many regions of the world; it causes heavy economic losses in the cattle industry. To date, all BEFV-specific diagnostic molecular assays have been based on the variable glycoprotein (G-protein)-coding genome region, potentially allowing the pathogen to escape detection. We developed two new assays, based on the less variable nucleoprotein genome region, and compared them with two G-protein-based assays. For this comparison, we used 245 samples comprising positive and negative field samples from Israeli outbreaks caused by different strains, belonging to lineage I and IIIa, as well as Australian and Japanese strains (lineages IV and IIIb). The new assays showed high agreement with the previous assay (Kappa = 0.92), detecting 144 out of 147 positive samples (sensitivity of 97.96%), and detected 6 more samples as positive out of 98 samples found negative by the G-protein-based assay. All nine non-agreeing results were validated as positive using a conventional RT-PCR assay. The new assays have higher analytical sensitivity than the previous assays, can be combined with internal controls, and enable the detection of all known BEFVs. The results indicate that these two nucleoprotein-based real-time RT-qPCRs can serve as fast, sensitive, and specific assays for the sustainable detection of BEFV strains.