Analytical optimization of Al2(SO4)3 in the coagulation-flocculation-resuspension process for enhanced recovery of spring viraemia of carp virus in freshwater

Waterborne viruses are present at low concentrations in water and require a concentration process for environmental DNA (eDNA) analysis. The coagulation-flocculation–resuspension (C-F-R) process is an effective method for concentrating viral particles in water. However, its efficiency in freshwater environments is lower than that in seawater, making optimization of the process essential for improving performance. This study optimized the C-F-R process to recover spring viraemia of carp virus (SVCV) from freshwater by comparing two coagulants—Al₂(SO₄)₃ and FeCl₃. The optimal dosage for both coagulants was 2 mg/L, with Al₂(SO₄)₃ exhibiting a more stable performance at various dosages. Al₂(SO₄)₃ achieved effective flocculation within 15 min, whereas FeCl₃ required 60 min. Furthermore, Al-based flocs demonstrated a more efficient resuspension time of 2 h compared to Fe-based flocs. Using the optimized protocols, Al₂(SO₄)₃ presented superior recovery efficiency and consistency across different SVCV concentrations, with overall yields higher for Al than for Fe. To apply the optimized C-F-R process, viral dynamics were analyzed by injecting 10², 10⁴, and 10⁶ SVCV PFU/fish into common carp and monitoring the mortality rate, viral replication, and viral shedding. Viral shedding peaked earlier than viral replication in the liver, indicating that viral shedding was more active in the early to mid-stages of infection than in the later stage. Moreover, the optimized protocols could detect the virus as early as one-day post-infection. The C-F-R process with Al₂(SO₄)₃, therefore, could be applied to enhance the early detection of SVCV and provide a reliable, noninvasive approach for SVCV surveillance.