Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, AnHV and JEECV

The Japanese eel endothelial cell-infecting virus (JEECV) and Anguilla herpesvirus (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, respectively. Both viruses can result in severe mortality, either independently or in combination, necessitating rapid and early detection of their presence. In this study, we developed a highly efficient duplex quantitative PCR method (R² = 0.999) using TaqMan probes for the rapid and simultaneous detection 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 those of conventional PCR. Quantitative analysis of water and eel tissue samples from aquaculture facilities revealed that viruses could be detected in water 1–3 months prior to mortality, enabling early identification of infections through water testing alone. Moreover, 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. These findings underscore the critical role of advanced molecular diagnostic technologies in enhancing health management in aquaculture.