In Vitro Viral Recovery Yields under Different Re-Suspension Buffers in Iron Flocculation to Concentrate Viral Hemorrhagic Septicemia Virus Genotype IVa in Seawater

Simple Summary Environmental DNA (eDNA) has attracted attention as a monitoring tool in the aquaculture industry to detect aquatic viral diseases in seawater. To be able to use the low concentration of viruses in seawater, an additional concentration process needs to be employed. In this study, the iron flocculation method was applied to concentrate the viral hemorrhagic septicemia virus (VHSV), and the recovery rate was estimated to determine the suitability of two re-suspension buffers, oxalic and ascorbic acid. It was determined that, while both of these buffers could be efficient for recovering viral genome copy, the oxalic acid buffer was more suitable for preserving viral infectivity than the ascorbic acid buffer. Thus, the iron flocculation method using an oxalic acid buffer could be efficient for evaluating actual viral transmission and is expected to predict the occurrence of diseases in the environment. 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 x 10(1) to 1 x 10(5) 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 10(5) PFU/mL, the infective VHSVs were not sufficiently recovered at a low viral concentration (10(2) 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.

Automated summary

The iron flocculation method was used to concentrate viruses in water, and the suitability of oxalic and ascorbic acid re-suspension buffers was evaluated. It was found that the oxalic acid buffer was more effective in preserving viral infectivity than the ascorbic acid buffer. This study suggests that the iron flocculation method using an oxalic acid buffer can be used to evaluate viral transmission and predict disease occurrence in the environment.