Dugbe orthonairovirus (DUGV) is a tick-borne arbovirus within the order Bunyavirales. DUGV was first isolated in Nigeria, but virus isolations in ten further African countries indicate that DUGV is widespread throughout Africa. Humans can suffer from a mild febrile illness, hence, DUGV is classified as a biosafety level (BSL) 3 agent. In contrast, no disease has been described in animals, albeit serological evidence exists that ruminants are common hosts and may play an important role in the transmission cycle of this neglected arbovirus. In this study, young sheep and calves were experimentally inoculated with DUGV in order to determine their susceptibility and to study the course of infection. Moreover, potential antibody cross-reactivities in currently available diagnostic assays for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) were assessed as DUGV is distantly related to CCHFV. Following subcutaneous inoculation, none of the animals developed clinical signs or viremia. However, all ruminants seroconverted, as demonstrated by two DUGV neutralization test formats (micro-virus neutralization test (mVNT), plaque reduction (PRNT)), by indirect immunofluorescence assays and in bovines by a newly developed DUGV recombinant N protein ELISA. Sera did not react in commercial CCHFV ELISAs, whereas cross-reactivities were observed by immunofluorescence and immunoblot assays.

Hazara orthonairovirus (HAZV) is a tick-borne arbovirus closely related to Crimean–Congo hemorrhagic fever orthonairovirus (CCHFV). Whereas CCHFV is a biosafety level (BSL) 4 agent, HAZV is classified as BSL 2, as it is not known to cause any disease in humans. Belonging to the same serogroup as CCHFV, HAZV might act as a model which can provide a better understanding of this important zoonosis. Furthermore, the serological relatedness may cause diagnostic problems if antibodies against HAZV interfere with current CCHFV serological assays. Therefore, sheep and cattle—important natural hosts for CCHFV—were experimentally infected with HAZV to prove their susceptibility and evaluate potential antibody cross-reactivities. According to this study, neither sheep nor cattle are susceptible to experimental HAZV infections. Consequently, the HAZV infection in ruminants is clearly distinct from CCHFV infections. Sera of immunized animals weakly cross-reacted between HAZV and CCHFV in immunofluorescence and immunoblot assays, but not in commercial CCHFV ELISAs commonly used for field studies.

Antibody cross-reactivities between related viruses are common diagnostic challenges, resulting in reduced diagnostic specificities and sensitivities. In this study, antibody cross-reactions between neglected members of the genus Orthonairovirus—Hazara (HAZV), Dugbe (DUGV), and Nairobi sheep disease orthonairovirus (NSDV)—were investigated. Mono-specific ovine and bovine sera following experimental infections as well immunization trials with HAZV, DUGV, and NSDV were tested in homologous and heterologous virus-specific assays, namely indirect ELISAs based on recombinant N protein, indirect immunofluorescence assays (iIFA), and two neutralization test formats (plaque reduction neutralization test (PRNT) and micro-virus neutralization test (mVNT)). The highest specificities were achieved with the ELISAs, followed by the mVNT, iIFA, and PRNT. Cross-reactivities were mainly observed within the Nairobi sheep disease serogroup–but surprisingly, HAZV antibodies in PRNT did also neutralize NSDV and DUGV. In conclusion, we recommend ELISAs and mVNTs for a discriminative diagnostic approach to differentiate between these antibodies. NSDV antisera were also used in serological assays for the detection of antibodies against the human pathogen Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). Interestingly, all CCHFV ELISAs (In-house and commercial) achieved high diagnostic specificities, whereas significant cross-reactivities were observed in a CCHFV iIFA. Previously, similar results were obtained when analyzing the HAZV and DUGV antisera.

Nairobi sheep disease orthonairovirus (NSDV) is a zoonotic tick-borne arbovirus, which causes severe gastroenteritis in small ruminants. To date, the virus is prevalent in East Africa and Asia. However, due to climate change, including the spread of transmitting tick vectors and increased animal movements, it is likely that the distribution range of NSDV is enlarging. In this project, sheep and cattle (hitherto classified as resistant to NSDV) were experimentally infected with NSDV for a comparative study of the species-specific pathogenesis. For this purpose, several new diagnostic assays (RT-qPCR, ELISA, iIFA, mVNT, PRNT) were developed, which will also be useful for future epidemiological investigations. All challenged sheep (three different doses groups) developed characteristic clinical signs, transient viremia and virus shedding—almost independent on the applied virus dose. Half of the sheep had to be euthanized due to severe clinical signs, including hemorrhagic diarrhea. In contrast, the course of infection in cattle was only subclinical. However, all ruminants showed seroconversion—implying that, indeed, both species are susceptible for NSDV. Hence, not only sheep but also cattle sera can be included in serological monitoring programs for the surveillance of NSDV occurrence and spread in the future.

Dugbe orthonairovirus (DUGV), a tick-borne zoonotic arbovirus, was first isolated in 1964 in Nigeria. For over four decades, no active surveillance was conducted to monitor the spread and genetic variation of DUGV. This study detected and genetically characterized DUGV circulating in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Kwara State, North-Central Nigeria. Blood and or ticks were collected from 1051 cattle at 31 sampling sites (abattoirs and farms) across 10 local government areas of the State. DUGV detection was carried out by RT-qPCR, and positive samples sequenced and phylogenetically analysed. A total of 11824 ticks, mostly A. variegatum (36.0%) and R. (B.) microplus (63.9%), were obtained with mean tick burden of 12 ticks/cattle. Thirty-four (32 A. variegatum and two R. (B.) microplus) of 4644 examined ticks were DUGV-positive, whereas all of the cattle sera tested negative for DUGV genome. Whole genome sequence (S, M and L segments) and phylogenetic analyses indicate that the positive samples shared up to 99.88% nucleotide identity with and clustered around the Nigerian DUGV prototype strain IbAr 1792. Hence, DUGV with high similarity to the previously characterised strain has been detected in Nigeria. To our knowledge, this is the first report of DUGV in North-Central Nigeria and the most recent information after its last surveillance in 1974.