VHSV and IHNV in the Environment: Assessment and Comparison of eRNA‐Based Methods for Detection in Aquaculture

ABSTRACT Infectious hematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) are major pathogens in aquaculture. Detection of both viruses requires lethal sampling, as it is typically performed by testing of internal organs. However, this approach only identifies the pathogens once viral transmission has already occurred and infection is underway. As both viruses can spread through water, an efficient environmental detection method could significantly improve disease control and prevention of transmission, and enhance welfare by reducing lethal sampling. In this study, we assessed the feasibility of detecting IHNV and VHSV environmental RNA (eRNA) in water during in vivo infection trials in Oncorhynchus mykiss , using RT‐qPCR. By sampling at multiple time points post‐exposure, we could evaluate the efficacy of detection at decreasing viral concentration in water over time. Viral eRNA was recovered using different methods: viral concentration with polyethylene glycol (PEG) precipitation, filter membranes, filtered water, or unprocessed water samples. RT‐qPCR values were compared to the quantification of infectious particles using viral titration, with RT‐qPCR consistently detecting higher eRNA copies/mL than the cell‐based assay to detect infectious particles (TCID 50 /mL). eRNA detection from the filter membranes significantly outperformed the other tested methods, enhancing eRNA recovery particularly at lower viral concentrations. Notably, eRNA detection in water was still possible after the peak of mortality for both viruses. Additionally, IHNV eRNA was successfully detected in farm water samples, even up to 50 days post initial fish tissue diagnosis, confirming the feasibility under real conditions. This study provides the first quantification of IHNV eRNA from aquaculture water and demonstrates the effectiveness of a filtration‐based viral concentration method for environmental surveillance. These findings suggest that eRNA‐based RT‐qPCR detection of IHNV and VHSV from water could be a valuable addition to current diagnostic tools, potentially enabling earlier detection and improved containment in aquaculture and the surrounding environment.