Development and validation of a universal S-segment-based real-time RT-PCR assay for the detection of Simbu serogroup viruses

Simbu serogroup viruses induce acute clinical diseases and abnormal courses of pregnancies in livestock. In Israel, two members of this serogroup, namely Akabane virus (AKAV) and Shuni virus (SHUV), were recently detected and, in Europe, Schmallenberg virus (SBV) poses a threat to ruminants. To address this emerging problem, a universal S-segment-based real-time RT-PCR assay (Uni-S) for the detection of Simbu serogroup viruses was established, which, additionally, enabled species identification of the detected viruses by subsequent sequencing. The newly developed probe-based PCR system enabled reliable detection of a comprehensive panel of Simbu viruses. Furthermore, several SBV isolates and German field samples were tested by the new Uni-S system in comparison to a SBV-specific real-time RT-PCR and both assays exhibited equally high levels of sensitivities. Finally, co-circulation of AKAV and SHUV in Israel was confirmed by analyzing field samples using the Uni-S assay followed by sequence analysis of the positive samples. To validate the test specificity, blood and tissue samples from animals negative for Simbu viruses, preparations of genetically related viruses and additional ruminant pathogens were examined and all were found to be negative.In conclusion, the new assay enabled sensitive and rapid universal molecular detection of Simbu viruses and is expected to serve as a valuable method for infection diagnosis, especially in regions where several Simbu serogroup members circulate.