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

Abstract

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.

Keywords

• iron flocculation
• oxalic acid
• ascorbic acid
• viral hemorrhagic septicemia virus
• viral genome recovery
• viral infective recovery