Culicoides biting midges, small hematophagous dipterans, are the demonstrated or putative vectors of multiple arboviruses of veterinary and public health importance. Despite its relevance in disease spread, the ceratopogonid genus Culicoides is still a largely neglected group of species, predominantly because the major human-affecting arboviruses are considered to be transmitted by mosquitoes. However, when a pathogen is detected in a certain vector species, a thorough search for further vectors often remains undone and, therefore, the relevant vector species may remain unknown. Furthermore, for many hematophagous arthropods, true vector competence is often merely suspected and not experimentally proven. Therefore, we aim to illuminate the general impact of Culicoides biting midges and to summarize the knowledge about biting midge-borne disease agents using the order Bunyavirales, the largest and most diverse group of RNA viruses, as an example. When considering only viruses evidentially transmitted by Culicoides midges, the Simbu serogroup (genus Orthobunyavirus) is presumably the most important group within the virus order. Its members are of great veterinary importance, as a variety of simbuviruses, e.g., the species Akabane orthobunyavirus or Schmallenberg orthobunyavirus, induces severe congenital infections in pregnant animals. The major zoonotic representative of this serogroup occurs in South and Central America and causes the so-called Oropouche fever, an acute febrile illness in humans.

The Simbu serogroup of orthobunyaviruses includes several pathogens of veterinary importance, among them Schmallenberg virus (SBV), Akabane virus (AKAV) and Shuni virus (SHUV). They infect predominantly ruminants and induce severe congenital malformation. In adult animals, the intra vitam diagnostics by direct virus detection is limited to only a few days due to a short-lived viremia. For surveillance purposes the testing for specific antibodies is a superior approach. However, the serological differentiation is hampered by a considerable extent of cross-reactivity, as viruses were assigned into this serogroup based on antigenic relatedness. Here, we established a glycoprotein Gc-based triplex enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of antibodies against SBV, AKAV, and SHUV. A total of 477 negative samples of various ruminant species, 238 samples positive for SBV-antibodies, 36 positive for AKAV-antibodies and 53 SHUV antibody-positive samples were tested in comparison to neutralization tests. For the newly developed ELISA, overall diagnostic specificities of 84.56%, 94.68% and 89.39% and sensitivities of 89.08%, 69.44% and 84.91% were calculated for SBV, AKAV and SHUV, respectively, with only slight effects of serological cross-reactivity on the diagnostic specificity. Thus, this test system could be used for serological screening in suspected populations or as additional tool during outbreak investigations.

Background: Despite its importance for Culicoides -borne disease risk assessment, the winter activity of species of this biting midge genus remains poorly characterized. Moreover, information on virus circulation in winter-active Culicoides vectors, both under endemic conditions and during outbreaks, is almost non-existent. Methods: UV-light traps to collect Culicoides biting midges were operated once a week for 24 h in cattle, sheep, goat and horse stables at 12 to 55 locations during four consecutive winter periods (November 1 to March 31, 2021 to 2025) throughout Germany. Collected females of the genus Culicoides were morphologically assigned to Obsoletus Group or Pulicaris Complex. Temperature data were recorded to relate biting midge activity to temperatures. Screening for bluetongue virus (BTV), Schmallenberg virus and epizootic hemorrhagic disease virus was done by real-time RT-PCR on 1,581 pools (46,358 females) of both groups collected before (winter 2022/2023) and during the BTV serotype 3 (BTV-3) outbreak (winters 2023/2024 and 2024/2025). Results: Culicoides collections displayed notable variation between sites and months. November and March accounted for 95% of captures, while mid-winter months (December to February) yielded only 5%, with January and February each contributing 1% of total captures. The March 2024 and 2025 activity peaks coincided with mild temperatures and the inclusion of new monitoring sites, suggesting that one or both factors contributed. Although blood-fed females were very rare during mid-winter, their abundance often exceeded the EU threshold for parous Culicoides to define a vector-free period of < 5 individuals per trap and night. However, only a single Culicoides pool from November 2024 tested positive for BTV-3-RNA. All other pools were negative for all three viruses. Conclusions: Potential Culicoides vectors are active and feed on blood in livestock housings during wintertime. The presence of blood-fed females, exceeding the EU threshold, challenges the concept of a ‘vector-free period’. However, despite extensive monitoring over four years including outbreak conditions, only one virus-positive Culicoides pool was detected during winter. Combined with the very low number of biting midges and the temperatures not allowing virus replication in arthropods, virus circulation during wintertime appears to be hardly existent even under endemic or outbreak conditions. Considering both Culicoides activity and virus circulation, the findings suggest that the risk of BTV infection in ruminants via biting midges during the central European winter (December to March) is negligible. BTV overwintering in central Europe likely depends on either rare infection events in ruminants and vectors during winter that sustain viremia until spring, or on persistence mechanisms that are currently unknown.

Introduction. Schmallenberg virus (SBV) is an arthropod-borne virus and belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus. Infection of naïve ruminants at critical stages of gestation can result in severe congenital malformations or abortion. Gap statement. Tools to measure virus infection parameters in cell culture such as replication efficiency, as well as neutralization assays, are mainly available in the form of assays based on the evaluation of cytopathic effects. The methods are labour-intensive and low-throughput, as they require long incubation periods of several days. Tools such as tagged SBV that allow for fast and automated readout are missing. Aim. We aimed to develop a tagged SBV that can be used for assays with fast and automated read-out. Methodology. We report the construction of a recombinant SBV stably expressing the nanoluciferase (nluc) enzyme (rSBV_nluc). Using reverse genetics, the nluc gene was integrated into the genome of an SBV variant naturally harbouring a large deletion within the Gc-head domain. The nluc gene was inserted into this locus. Results. The nluc-tagged virus showed in vitro no signs of attenuation and identical replication properties when compared to the parental virus in baby hamster kidney (BHK-21) cells. Our results demonstrate a new approach for rapid access to SBV replication. By performing nluc assays, we were able to track viral replication and also virus uptake in detail. We further evaluated neutralization properties of an SBV variant, which is lacking a major part of its antigenic domain (Gc-head) and developed a nanoluciferase activity-based serum neutralization assay. Conclusion. Overall, the nluc-tagged SBV is a suitable tool that further facilitates the study of viral infection dynamics and allows for high-throughput assays.