Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses

Abstract

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97); SEOV (China, n=5); DOBV (Romania, n=7); SNV (Canada, n=23); ANDV (Argentina and Chile, n=52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96%) and/or IgM (n=131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).

Conflict of interest statement

I have read the journal's policy and have the following conflicts: S. Lederer and E. Lattwein are employees of EUROIMMUN AG. K. Sonnenberg and M. Hanke were employees of EUROIMMUN AG at the time of conducting this study or writing the manuscript, respectively. W. Stoecker is a board member of EUROIMMUN AG.

Figures

Figure 1. Immunofluorescence microscope slides for the multiparametric detection of hantavirus-specific antibodies.

A microscope slide has ten reaction fields, each of which contains a biochip mosaic, allowing ten samples or sample dilutions to be incubated simultaneously with the same range of substrates. Due to identical incubation protocols, IgG and IgM testing can be performed on different reactions fields of the same slide using the respective secondary antibody conjugate. Hantavirus Mosaic 1 comprises mosaics of six biochips coated with Hantaan virus (HTNV)-, Puumala virus (PUUV)-, Seoul virus (SEOV)-, Sin Nombre virus (SNV)-, Dobrava virus (DOBV)- and Saaremaa virus (SAAV)-infected cells (-IC). Hantavirus Mosaic 3 consists of mosaics of two biochips coated with Sin Nombre virus (SNV)- and Andes virus (ANDV)-IC.

Figure 2. Multiparametric IFA-based reactivity patterns of hantavirus-specific serum antibodies.

Bars indicate reactivity rates or reciprocal geometric mean titers (rGMT) for IFA IgG positive sera (A and B; serum panel: I, n = 97; II, n = 5; III, n = 7; IV, n = 22; V, n = 52) and IFA IgM-positive sera (C and D; serum panel: I, n = 54; II, n = 5; III, n = 5; IV, n = 19; V, n = 48) for each substrate (cells infected with HTNV, PUUV, SEOV, SAAV, DOBV, SNV and ANDV). Dashed horizontal lines (right panels) indicate the reciprocal cut-off titer; black triangles indicate reactivity rates of 0% (left panels) or rGMTs of <10 (lower right panel). Results for panel IV with ANDV-infected cells were not available.

Figure 3. Hantavirus serotype determination by multiparametric indirect immunofluorescence analysis.

For sera which were IFA positive for both anti-hantavirus IgG and IgM (serum panel: I, n = 54; II, n = 5; III, n = 5; IV, n = 19; V, n = 48), percentages of hantavirus serotypes with the highest reciprocal IgG endpoint titers were calculated (left bars). Right bars represent results of serotyping by IgG in conjunction with IgM detection. For serum panels I and V, only those serotypes which accounted for more than 2% of the total number of sera are indicated. Results for panel IV with ANDV-infected cells were not available.