Cross-Reaction or Co-Infection? Serological Discrimination of Antibodies Directed against Dugbe and Crimean-Congo Hemorrhagic Fever Orthonairovirus in Nigerian Cattle

Abstract

Dugbe orthonairovirus (DUGV) and Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are tick-borne arboviruses within the order Bunyavirales. Both viruses are endemic in several African countries and can induce mild (DUGV, BSL 3) or fatal (CCHFV, BSL 4) disease in humans. Ruminants play a major role in their natural transmission cycle. Therefore, they are considered as suitable indicator animals for serological monitoring studies to assess the risk for human infections. Although both viruses do not actually belong to the same serogroup, cross-reactivities have already been reported earlier-hence, the correct serological discrimination of DUGV and CCHFV antibodies is crucial. In this study, 300 Nigerian cattle sera (150 CCHFV seropositive and seronegative samples, respectively) were screened for DUGV antibodies via N protein-based ELISA, indirect immunofluorescence (iIFA) and neutralization assays. Whereas no correlation between the CCHFV antibody status and DUGV seroprevalence data could be demonstrated with a newly established DUGV ELISA, significant cross-reactivities were observed in an immunofluorescence assay. Moreover, DUGV seropositive samples did also cross-react in a species-adapted commercial CCHFV iIFA. Therefore, ELISAs seem to be able to reliably differentiate between DUGV and CCHFV antibodies and should preferentially be used for monitoring studies. Positive iIFA results should always be confirmed by ELISAs.

Keywords: CCHFV; Crimean-Congo hemorrhagic fever orthonairovirus; DUGV; Dugbe orthonairovirus; Nigeria; cattle; cross-reactivity; sensitivity; serology; specificity.

Figure A1

Comparative ROC analyses: Graph (a) shows the ROC curve for all 300 serum samples, graph (b) the 150 CCHFV seronegative samples and graph (c) the 150 CCHFV seropositive samples only.

Figure 1

ROC analysis employing 300 Nigerian cattle sera: Diagnostic sensitivity of the DUGV ELISA is 95.7% (95% CI 91.7–98.1) and diagnostic specificity is 88.6% (95% CI 81.3–93.8) with AUC being 0.951 (p-value < 0.001).

Figure 2

Correlation of mVNT titers and corrected OD values: only 8 sera were false-negative (green triangles) and 12 sera false-positive (red dots) out of 300 tested cattle sera. Six false-negative sera and two false-positive sera actually yielded CCHFV antibodies (marked with *).

Figure 3

Corrected OD values for 300 Nigerian cattle sera: The distribution patterns between CCHFV seropositive sera (red dots) and CCHFV seronegative sera (green dots) do not apparently vary between each other.

Figure 4

Cross-reactivity N protein-based ELISAs: The percentage of DUGV antibody-positive sera is slightly lower for the CCHFV seropositive samples, indicative of good serological discrimination of DUGV and CCHFV antibodies via ELISA.

Figure 5

DUGV IFA: Cells infected with DUGV (I), as well as non-infected control wells (II), are depicted. Sera A and B represent DUGV seropositive samples, with serum A being higher seropositive than serum B. Sera C, D and E yielded CCHFV antibodies only. Whereas serum C is an example of a true-negative serum, sera D and E show specific staining of DUGV-infected cells. Serum F (CCHFV−/DUGV−) served as a negative control.

Figure 6

CCHFV IFA (Euroimmun, Lübeck): Cells expressing CCHFV GPC (I) and non-transfected control cells (II) are depicted. Serum A (CCHFV+/DUGV−) led to an undistinguishable fluorescent signal compared to sera B and C (CCHFV−/DUGV+). Serum D (CCHFV−/DUGV−) served as the negative control.