On-site serological screening of Usutu virus in wild birds: Field evaluation of a lateral flow microarray immunoassay using the WHO REASSURED framework.

Arboviruses such as Usutu virus (USUV) and West Nile virus (WNV) cycle enzootically between competent mosquito vectors and avian reservoir hosts. With USUV established and WNV incidence rising in the Netherlands, detecting antibodies in wild birds captured at ringing sites is critical for surveillance. Currently, no multi-analyte, species-independent serological assays exist for on-site field analysis. Using a recently developed species-independent Lateral flow Microarray ImmunoAssay (LMIA) capable of detecting both USUV non-structural protein 1 (NS1)- and WNV NS1-specific antibodies, a total of 82 wild birds were captured and tested in the field. Of these, 8 birds were USUV LMIA-reactive, all of which were included in a subset of 18 birds that were additionally analysed using gold-standard USUV and WNV 90% Focus Reduction Neutralization Test (FRNT90). Compared to FRNT90 results, the LMIA showed a sensitivity and specificity for USUV of 100% and 89%, respectively. The LMIA also detected seroconversion to USUV in a Eurasian blackbird, which was confirmed by FRNT90. A scoring system based on the WHO's REASSURED criteria was also used as a framework to evaluate the LMIA field performance, receiving 81% of the highest possible score. While minor optimization and broader validation remain, this study demonstrates LMIA feasibility for on-site USUV serological screening. Its rapid, multi-analyte, and equipment-light format supports point-of-capture testing and targeted surveillance in Dutch bird-ringing sites and resource-limited settings.