Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

Abstract

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.

FIG. 1.

Structure of pKS336-CCHFV-NP. The gene, a cDNA of the NP gene of CCHFV strain 8402, was inserted into the BamHI restriction site within the multicloning site.

FIG. 2.

Positive IF staining of CCHFV rNP-expressing HeLa cells (a) and CCHFV-infected Vero E6 cells (b) with a CCHF antibody-positive serum sample.

FIG. 3.

Relationship between the titers of IgG antibody to CCHFV determined by IF using CCHFV slides and those determined by IF using CCHFV rNP slides. Serum from the monkey, which was immunized with the purified CCHFV rNP, was also tested by IF using authentic and recombinant antigens. The titers of the positive-control monkey serum sample (○) and the 13 CCHFV antibody-positive human serum samples (•) were plotted. Each data point represents one serum sample.