White spot syndrome virus (WSSV) is the most problematic pathogen in crustaceans. In this study, we investigated the horizontal transmission model of WSSV based on the correlation between the disease severity grade and viral shedding rate and determined the minimum infective dose of WSSV via the waterborne route. Intramuscular injection challenges at different doses and water temperatures revealed that the thresholds of viral shedding and mortality were G1 (3.1 × 10³ copies/mg) and G2 (8.5 × 10⁴ copies/mg), respectively. Furthermore, a positive linear correlation was observed between viral copies of pleopods and viral shedding rate (y = 0.7076x + 1.414; p < 0.001). Minimum infective doses of WSSV were determined via an immersion challenge. Infection was observed within 1, 3, and 7 d in 10⁵-, 10³-, and 10¹ copies/mL of seawater, respectively. In the cohabitation challenge, infection was observed within six days with viral loads of 10¹ to 10² copies/mL of seawater, which further increased in the recipient group. Our results indicate a positive correlation between disease severity grade and viral shedding rate of infected shrimp and suggest that the waterborne transmission of WSSV depends on the viral load and exposure period.

Iron flocculation is widely used to concentrate viruses in water, followed by Fe-virus flocculate formation, collection, and elution. In the elution stage, an oxalic or ascorbic acid re-suspension buffer dissolved iron hydroxide. After the concentration of viral hemorrhagic septicemia virus (VHSV) in seawater (1 × 10¹ to 1 × 10⁵ viral genome copies or plaque-forming unit (PFU)/mL), the recovery yield of the viral genome using quantitative real-time PCR (qRT-PCR) and viral infectivity using the plaque assay were investigated to evaluate the validity of the two re-suspension buffers to concentrate VHSV. The mean viral genome recovery yield with oxalic and ascorbic acid was 71.2 ± 12.3% and 81.4 ± 9.5%, respectively. The mean viral infective recovery yields based on the PFU were significantly different between the two buffers at 23.8 ± 22.7% (oxalic acid) and 4.4 ± 2.7% (ascorbic acid). Notably, although oxalic acid maintains viral infectivity over 60% at a viral concentration above 10⁵ PFU/mL, the infective VHSVs were not sufficiently recovered at a low viral concentration (10² PFU/mL, <10%). To support this result, concentrated VHSV was inoculated in Epithelioma papulosum cyprini (EPC) cells to confirm cell viability, viral gene expression, and extracellular viral titer. All results demonstrated that oxalic acid buffer was superior to ascorbic acid buffer in preserving viral infectivity.