A lateral flow microarray immunoassay to detect USUV NS1-specific antibodies in tissues of deceased blackbirds in the Netherlands.

Usutu virus (USUV) is an arbovirus that circulates between mosquitoes and birds with occasional spillover events into non-avian species. USUV continues to circulate in the Netherlands, particularly in Eurasian blackbirds (Turdus merula), although other USUV-susceptible avian species have also been found. Current serosurveillance systems make use of serological methods that can detect anti-USUV antibodies in sera collected from live birds. However, they require a dedicated laboratory and rarely include serological analysis of deceased avian specimens. As a proof of concept, this study evaluates the use of a previously developed lateral flow microarray immunoassay (LMIA) to detect USUV non-structural 1 (NS1)-specific antibodies in blood clots and lung and liver tissue extracts collected from blackbird carcasses found in the Netherlands. The likelihood of detection of USUV NS1 antibodies in USUV RT-PCR-positive blackbirds was found to trend higher in lung tissue extracts. Additionally, the LMIA exhibited only minimal cross-reactivity between USUV NS1-specific antibodies and USUV NS1 and West Nile virus (WNV) NS1 capture antigens on the LMIA. This initial study demonstrates the feasibility of the LMIA to specifically detect USUV NS1 antibodies in lung tissue extracts of deceased blackbirds. In future, a serological tool such as the one presented here, would allow for the rapid on-site analysis of both live and deceased blackbird specimens found in the field.