Serological Cross-reactivity between Crimean-Congo Haemorrhagic Fever Virus (CCHFV) and Nairobi Sheep Disease Virus (NSDV) glycoprotein C (Gc)

Cross-reactivity to viruses of the Orthonairovirus genus can potentially interfere with serological assays when employed for serosurveillance in regions where two or more genus members overlap in their distribution. To investigate this, Crimean-Congo haemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV) were chosen due to both viruses cocirculating in small ruminant populations across South Asia and East Africa. In this study, sheep sera sampled from a region of confirmed CCHFV circulation and NSDV absence were utilized, thereby eliminating the possibility of co-exposure. Field sera were tested against in-house anti-NSDV ELISAs specific to the nucleoprotein (NSDV NP) and glycoprotein c (NSDV Gc) antigens as well as an in-house NSDV PRNT80. When comparing NSDV antigen-specific antibody responses against previously tested CCHFV antigen-specific antibody responses, a strong positive correlation was observed between the Gc-specific responses, while a weak positive correlation was observed between the NP-specific responses. Consequently, NPspecific ELISAs have a higher assay specificity compared to Gc-based ELISAs, making them more effective for serosurveillance in regions where multiple orthonairoviruses co-circulate.Crucially, only one seropositive sample to NSDV Gc-specific out of a set of 224 (0.4%) showed a neutralizing capacity at the lowest serum dilution (1:8), suggesting these field sera have not been exposed to NSDV. Hence, the in-house PRNT80 assay can be used as a confirmatory test in regions where both CCHFV and NSDV circulate. Finally, based on an analysis of known epitopes in NP targeted by antibodies in sheep serum, we propose that NP is less cross-reactive because dominant epitopes are highly dissimilar between CCHFV and NSDV.