During June 2018–December 2019, we trapped 13 pools of Culicoides imicola, 8 pools of C. oxystoma, 5 pools of C. puncticollis, and 5 pools of C. newsteadii midges (each pool containing 50 midges) around livestock farms, and we tested CSF from 3 malformed 1-day-old lambs (born on July 3, 2019) and 1 malformed 11-day-old calf (born on November 1, 2019) (Figure; Appendix). We extracted RNA from Culicoides homogenates and CSF using Maxwell 16 Viral Total Nucleic Acid Purification Kit (Promega, https://www.promega.com) according to the manufacturer’s instructions. We used total viral nucleic acids (0.4 µg) for cDNA synthesis by UltraScript Reverse transcription (PCR Biosystems, https://www.promega.com) according to the manufacturer’s instructions. We performed reverse transcription (RT) nested qPCR targeting the L RNA segment of Simbu serogroup viruses according to Behar et al. (13). We further subjected samples suspected of being Simbu serogroup positive to RT-nested and seminested PCRs performed using S, M, and L segment-specific primer sets (Table 1; Appendix).

Of the 31 species-specific pools from the 4 Culicoides midge species that are known or suspected to be vectors of Simbu serogroup viruses (i.e., C. imicola, C. oxystoma, C. puncticollis, C. newsteadii) (2,12,15), we found that 11 contained RNA of Simbu serogroup viruses in 2018 and 2019 (35% of the total pools tested) (Table 2; Figure; Appendix Table). We identified partial nucleotide sequences of the S (370/830 bp) and L (370/6,882 bp) segments. Phylogenetic analysis of the samples showed that all positive samples were virtually identical to SBV (GenBank accession nos. MT816474–82, MT816485–95) (Appendix Figure, panels A, C). These samples were collected from several different geographic regions in Israel (Table 2, lines 1 and 3–12 in the Samples column; Figure; Appendix Table). In addition, we detected SBV RNA-specific fragments of the S (370/850 bp), M (430/4,373 bp), and L segments (370/6,882 bp) in a CSF sample from a malformed lamb born in July 2019 on a farm in southern Israel (Negev desert) and a malformed calf born in November 2019 on a farm in northern Israel (Galilee) (GenBank accession nos. MT816472, MT816473, MT816483, MT816484, MT816496, MT816497) (Table 2, lines 2 and 13 in the Samples column; Appendix Figure).

In general, the most susceptible period for induction of congenital malformations by Simbu serogroup viruses is 65–70 days of gestation in lambs and 150 days of gestation in calves (1,7). Thus, SBV detection in the respective ruminants fits with viral infection in March–April 2019, suggesting exposure to SBV in Israel in early spring 2019. Nevertheless, reports on severe decline in progeny prolificacy, stillbirths, and malformed lambs were reported by farmers to the Veterinary Field Services from autumn 2018 through December 2019 (Table 2). The detection of SBV in Culicoides pools collected from several of those farms (Table 2, lines 3–5, 7–9, and 11–12 in the Sample column; Figure; Appendix Table) suggests that SBV might have been clinically affecting ruminants in Israel as early as June 2018.

Our results demonstrate the circulation of SBV outside Europe. Future studies are needed to determine the seroprevalence of SBV in the Middle East, because this information is essential for understanding the risk of SBV spread into countries in Asia. Because SATV is found in the Middle East (12,13), virus neutralization tests will probably not be able to properly distinguish between antibodies against SBV and those against SATV. Therefore, developing of a competitive ELISA system using SBV-specific antibodies is crucial. Finally, the presence of both SATV and SBV in Israel provides a unique opportunity for comparative studies on possible cross-protection of SBV commercial vaccines between these viruses.