Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is an important virus responsible for die-off of farmed tilapia globally. Detection and quantification of the virus from environmental DNA/RNA (eDNA/eRNA) using pond water represents a potential, noninvasive routine approach for pathogen monitoring and early disease forecasting in aquaculture systems. Here, we report a simple iron flocculation method for viral concentration from water combined with a newly developed hydrolysis probe quantitative RT-qPCR method for detection and quantification of TiLV. The RT-qPCR method targeting a conserved region of TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of virus from water samples spiked with viral cultures. During disease outbreak cases from an open-caged system and a closed hatchery system, both tilapia and water samples were collected for detection and quantification of TiLV. The results revealed that TiLV was detected from both clinically sick fish and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms e.g. river water samples from affected cages (8.50 × 10² to 2.79 × 10⁴ copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 10² to 3.53 × 10³ copies/L) from affected and unaffected ponds of the hatchery. In summary, this study suggests that the eRNA detection system using iron flocculation coupled with probe based-RT-qPCR is feasible for concentration and quantification of TiLV from water. This approach might be useful for noninvasive monitoring of TiLV in tilapia aquaculture systems and facilitating appropriate decisions on biosecurity interventions needed.