96 well microtiter plates

An iIFA was performed as previously described [2 (link)]. Briefly, Vero cells persistently infected with BoDV-1 were mixed at a 1:2 ratio with uninfected Vero cells and cultured overnight in 96-well microtiter plates (Ibidi, Gräfelfing, Germany) to achieve confluent cell layers. Wells with uninfected Vero cells served as negative controls. After the removal of the supernatant, plates were dried for 2 h and then fixed at 80 °C for 2 h. Thereafter, heat-inactivated serum/plasma samples were added in a 2-fold dilution series in TRIS buffer (Sigma-Aldrich, St. Louis, MO, USA) (1:20, 1:40, 1:80). After incubation for 1 h, plates were washed three times with DPBS and incubated with a 1:200 dilution of Cy-3-conjugated polyclonal rabbit anti-human IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After a final washing step, the assays were analyzed using fluorescence microscopy. Samples with characteristic fluorescing spots in the nuclei of BoDV-1-infected Vero cells were considered positive. When similar fluorescence signals were detected in non-infected and BoDV-1-infected Vero cells, samples were considered unspecific. All samples were separately evaluated by two trained and experienced members of laboratory staff.

Samples with reactions against at least one ELISA antigen were re-tested by a BoDV-1 indirect immunofluorescence assay (iIFA), which was performed as previously described [10 (link), 13 (link)]. For the iIFA, a Vero cell culture persistently infected with a BoDV-1 isolate derived from human brain tissue (isolate Regensburg 2019) was used [11 (link)]. Infected Vero cells were mixed 1:2 with uninfected Vero cells and cultured in 96-well microtiter plates (Ibidi, Gräfelfing, Germany) overnight to achieve confluent cell layers. As negative controls, uninfected Vero cells were prepared in the same manner. The next day, supernatants were removed, plates were dried for 2 h and fixed at 80 °C for 2 h. Prepared plates were stored at − 20 °C before use. Heat-inactivated patient samples were added in a twofold dilution series (1:20, 1:40, 1:80). As secondary antibody, a Cy-3-conjugated polyclonal rabbit anti-human-IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) was used in a 1:200 dilution. Samples were analyzed by fluorescence microscopy. Samples with characteristic fluorescing spots in the nuclei of BoDV-1-infected Vero cells were considered positive. When similar fluorescence signals were detected in non-infected and BoDV-1-infected Vero cells, signals were considered unspecific.

An iIFA was performed following a recently published protocol [1 (link)]. Vero cells persistently infected with BoDV-1 were mixed 1:2 with uninfected Vero cells and cultured overnight in 96-well microtiter plates (Ibidi, Gräfelfing, Germany) to achieve confluent cell layers. Wells with uninfected Vero cells served as the negative control. After removal of the supernatant, plates were dried for 2 h and then fixed at 80 °C for 2 h. Thereafter, heat-inactivated serum/plasma samples were added in a 2-fold dilution series in TRIS buffer (Sigma-Aldrich, St. Louis, MO, USA) (1:20, 1:40, 1:80). After incubation for 1 h, plates were washed three times with DPBS and incubated with a 1:200 dilution of Cy-3-conjugated polyclonal rabbit anti-human-IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After a final washing step, the assays were analyzed using fluorescence microscopy. Samples with characteristic fluorescing spots in the nuclei of the BoDV-1-infected Vero cells were considered positive. When similar fluorescence signals were detected in non-infected and BoDV-1-infected Vero cells, samples were considered unspecific. All samples were evaluated separately by two trained and experienced members of the laboratory staff.