Application of iron flocculation to concentrate white spot syndrome virus in seawater

The iron flocculation method, which comprises the Fe-virus flocculate formation-filtration-resuspension steps, is extensively used to concentrate and precipitate viruses distributed in water. To apply this method to concentrate white spot syndrome virus (WSSV) in seawater, viral genomic and infective recovery yields were compared between polyethylene sulfone (PES) and polycarbonate (PC) membrane filters and two types of resuspension buffers (oxalate and ascorbate). Viral genome quantitation was determined above a 95 % limit of detection (11.48 viral DNA copies/mu L) using quantitative real-time PCR. From WSSV-spiked seawater (100-106 viral DNA copies/mL), the viral genomic recovery yields of the PES-Oxalate, PC-Oxalate, PES-Ascorbate, and PC-Ascorbate conditions were 78.67 % +/- 12.90 %, 84.53 % +/- 24.30 %, 85.59 % +/- 16.98 %, and 93.74 % +/- 7.44 %, respectively. The detectable Fe-virus flocculates collected by the PC membrane were approximately 101 WSSV DNA copies/mL of seawater, a value more than 10-fold higher than that compared to the PES membrane filter (102 WSSV DNA copies/mL), regardless of the resuspension buffer types. WSSV resuspended with oxalate buffer caused mass mortality among whiteleg shrimp (Litopenaeus vannamei), inducing the expression of the virus en-velope protein, VP28, similar to that of a native virus, suggesting stable viral activity during the resuspension process. Based on the PES-Ascorbate, WSSV particles could be successfully concentrated in seawater from shrimp farms with white spot disease outbreaks (approximately 10(2) WSSV DNA copies/mL). Collectively, these findings indicate that the simple and efficient method of iron flocculation is sufficient to concentrate WSSV in seawater and could be used as a non-invasive approach and one of the reasonable diagnostic processes for white spot disease surveillance.

Automated summary

The iron flocculation method is an effective approach for concentrating viruses in water and has shown promising results for white spot syndrome virus (WSSV). Comparisons were made between different membrane filters and resuspension buffers, and it was found that the combination of PC membrane and ascorbate buffer yielded the highest viral genomic recovery.