Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus

Japanese eel endothelial cells-infecting virus (JEECV) and Anguillid herpesvirus 1 (AnHV) are major pathogens in farmed eels. JEECV causes eel viral endothelial cell necrosis (VECNE), while AnHV leads to symptoms such as head erythema and gill necrosis. Both viruses cause severe mortality alone or in combination, necessitating rapid and early detection of their presences. In this study, we developed a highly efficient duplex quantitative PCR method (r2 = 0.999) using hydrolysis probes for the rapid and simultaneous detections of AnHV and JEECV. This new diagnostic method demonstrated a 1.7-fold higher detection rate for AnHV and a 2.5-fold higher detection rate for JEECV than conventional PCR. Quantitative analysis of water and eel tissue samples from aquaculture facilities revealed that the two viruses could be detected in water 1–3 months prior to mortality, enabling their early identification of infections through water testing alone. Notably, the method reliably detected low viral loads (< 1 copy) in both water and tissue samples, facilitating preclinical detection and proactive disease management. This approach reduces the risk of mass mortality and economic losses in eel farming. This study underscores the critical role of advanced molecular diagnostic technologies in enhancing health management in aquaculture.