Establishing environmental DNA and RNA protocols for the simultaneous detection of fish viruses from seawater

Aquatic viruses are major threats to global aquacultural productivity. While conventional diagnostic methods for disease investigation are laborious, time-consuming, and often involve the sacrifice of animals, environmental DNA and RNA (eDNA/eRNA) tools have the potential in being non-invasive alternatives for the effective and early detection of various pathogens simultaneously. In this study, three seawater filtration methods-Sterivex syringe filtration, centrifugal ultrafiltration, and vacuum pump filtration with iron flocculation-were assessed for the recovery rates in co-detecting fish virus eDNA/eRNA from natural seawater that was spiked with fish red seabream iridovirus (RSIV, DNA virus) and nervous necrosis virus (NNV, RNA virus). The centrifugal ultrafiltration method was the most effective for the capture of small-sized viruses like NNV with a recovery rate of 63.23%, while the method of vacuum pump filtration with iron flocculation and chloroform disintegration of filter membranes had the highest RSIV recovery rate of 32.61%. We also optimized both automated and manual nucleic acid extraction methods and found comparable eDNA/eRNA extraction efficiencies. Our findings from the systematic comparison of seawater filtration and extraction methods suggest that each seawater filtration/nucleic acid extraction method can cater to different aquatic animal virus surveillance and disease investigation scenarios. These highlight the potential of virus eDNA/eRNA approaches for advancing the field of disease ecology and safeguarding aquatic animal health.