Prevalence and protein specificity of human antibodies to Inkoo virus infection

Abstract

Inkoo virus (INKV), a member of the California serogroup orthobunyaviruses, is circulating widely in northern Europe. Although the virus was discovered over 40 years ago, the disease associations and immune responses in human infection are poorly characterized. We first developed an immunofluorescence assay (IFA) for the detection of INKV antibodies in humans, and then we studied a panel of 1,292 sera in patients with a febrile illness in Finland. We found four acute (immunoglobulin M [IgM] positive) INKV infections and an IgG seroprevalence of 51.3%. The data indicate that the infection has become more common than it was in the 1960s, especially in southern Finland. Two distinct IgG IFA fluorescence patterns were observed: a granular pattern in sera from patients with acute INKV infection and a diffuse pattern in those with long-standing immunity. Further analysis with a panel of INKV-positive sera (n = 18; verified by neutralization assay) of protein-specific responses, using immunoprecipitation and IFA based on baculovirus-expressed INK N, Gn, and Gc proteins, demonstrated a strong IgG response predominantly towards N protein in the acute phase. In contrast, in patients with long-standing immunity, the Gc response was more prominent and the N response was weaker. In conclusion, a diagnostic IgG IFA pattern distinguishing between acute infection and long-standing immunity was observed. N protein seems to be the optimal antigen for the serodiagnosis of acute infection, and the Gc protein could be appropriate for the serosurveillance of INKV.

FIG. 1.

(A) Geographical distribution of INKV IgG seroprevalence in Finland. (B) Seroprevalence in different age groups.

FIG. 2.

IFA of INKV IgG- and IgM-positive acute and long-standing immunity serum samples using acetone-fixed INKV-infected Vero E6 cells as antigen. IgG IFA results for early (a) and late (b) immune response sera. IgM IFA results for early (c) and late (d) immune response sera.

FIG. 3.

Immunoprecipitation with MHAF of proteins from Sf-9 insect cells infected with baculoviruses expressing INKV N, Gc, or Gn, or from mock-infected cells. Asterisks indicate N, Gc, and Gn proteins.

FIG. 4.

IgG IFA using acetone-fixed Sf-9 cells expressing INKV N, Gn, and Gc proteins by recombinant baculovirus. IFA slides were stained with acute and old immunity human serum samples and hyperimmune mouse ascites fluid.

FIG. 5.

Immunoprecipitation of proteins from INKV-infected Vero E6 cells with (a) human sera (lanes 1 to 9), representing acute INKV infection (IgM positive and IgG positive) and an INKV IgG-negative serum (mock). (b) Human sera (lanes 10 to 18) from patients with long-standing INKV immunity (IgM negative and IgG positive) and a MHAF. Intensity of the N and Gc proteins bands are trace quantity values (intensity/millimeter). Statistical analysis for the Gc/N ratio between acute and long-standing immunity (Student's t test for independent samples) (the mean ± SD for long-standing-immunity cases was 0.6 ± 0.3, and that for acute cases was 0.3 ± 0.2) showed that the difference observed between the two groups was significant. P = 0.02; t = 2.6; df = 15.2 (equal variances not assumed).