Development of a Luciferase Immunosorbent Assay for Detecting Crimean-Congo Hemorrhagic Fever Virus IgG Antibodies Based on Nucleoprotein

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is a serious tick-borne disease with a wide geographical distribution. Classified as a level 4 biosecurity risk pathogen, CCHF can be transmitted cross-species due to its aerosol infectivity and ability to cause severe hemorrhagic fever outbreaks with high morbidity and mortality. However, current methods for detecting anti-CCHFV antibodies are limited. This study aimed to develop a novel luciferase immunosorbent assay (LISA) for the detection of CCHFV-specific IgG antibodies. We designed specific antigenic fragments of the nucleoprotein and evaluated their sensitivity and specificity in detecting IgG in serum samples from mice and horses. In addition, we compared the efficacy of our LISA to a commercial enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that the optimal antigen for detecting anti-CCHFV IgG was located within the stalk cut-off domain of the nucleoprotein. The LISA exhibited high specificity for serum samples from indicated species and significantly higher sensitivity (at least 128 times) compared with the commercial ELISA. The proposed CCHFV-LISA has the potential to facilitate serological diagnosis and epidemiological investigation of CCHFV in natural foci, providing valuable technical support for surveillance and early warning of this disease.

Keywords: Crimean–Congo hemorrhagic fever virus (CCHFV); IgG; detection; luciferase immunosorbent assay (LISA); nucleoprotein.

Figure 1

Schematic protocol for the luciferase immunosorbent assay (LISA).

Figure 2

Design and expression of the NLuc-nucleoprotein fusion antigen. (A) Schematic design of the antigen detection fragments. The NP sequence was fused to the end of the NanoLuc sequence and then cloned into pNLF1-N to construct the recombinant plasmid; (B) recombinant plasmids were validated by Western blotting. Anti-HA tag antibodies were used to detect the four fusion proteins, NP-full, NP-C1, NP-C2 and NP-C3. α-tubulin was used as an internal control. p: empty plasmid pNLF1-N.

Figure 3

Number of positive detected of four recombinant detection fragments. Serum samples from healthy mice (“normal serum”) and CCHFV antigen-immunized mice (“positive serum”) were assessed for the relative luminescence unit (RLU) of anti-CCHFV IgG antibodies by NP-full LISA (A), NP-C1 LISA (B), NP-C2 LISA (C) and NP-C3 LISA (D). The serum dilution ratio was 1:100 and ****, p < 0.0001.

Figure 4

Sensitivity analysis of four recombinant detection fragments after gradient dilutions. Two positive mouse serum samples were randomly selected for serial dilution and RLU was determined using NP-full (A), NP-C1 (B), NP-C2 (C) and NP-C3 (D) detection fragments.

Figure 5

Comparison of the novel LISA with the commercial ELISA for detecting anti-CCHFV IgG. (A) Correlation between LISA and ELISA based on 29 positive samples. The RLU of the NP-C2 LISA is plotted against the absorbance of the ELISA (p < 0.0001). The serum dilution ratio was 1:1000. (B) Sensitivity analysis of ELISA. Measurements were carried out using a gradient dilution of serum, and the positivity cut-off value for positive results is indicated by the dashed line.

Figure 6

Repeatability and cross-reactivity of CCHFV-LISA. (A) Repeatability of IgG antibody detection by CCHFV LISA. (B) Identification of reaction specificity of NLu-NP-C2 protein among horse sera after infection with CCHFV, RVFV, NIV and EBOV. (C) Cross-reactivity of the NP-C2-LISA using positive sera from patients with DENV, CHIKV and HCV, and sera from healthy individuals served as controls.