WSSV virion purification

Description

WSSV is a large enveloped virus of approximately 275 by 120 nm in size with an olivaceous-to-bacilliform shape. The WSSV virion consists of four components: a ~300 kbp double-stranded DNA genome, a nucleocapsid, a tegument, and an outer envelope. Purified WSSV virions can be prepared from the hemolymph and other tissues of heavily infected shrimp, crayfish or crabs, but the crayfish hemolymph is the recommened resource.

Brief protocol

The viral stock described previously injected intramuscularly into healthy crayfish Procambarus clarkii (1:2.5 dilution in PBS; 5 μl/g of body weight) between the second and third abdominal segments. Beginning 4 to 6 days later, hemolymph was extracted from the infected moribund crayfish and then centrifuged at 1,500 ×g for 10 min at 4 °C. The supernatant was layered on top of a 35% (wt/vol in TNE buffer: 50 mM Tris-HCl, 400 mM NaCl, 5 mM EDTA, pH 8.5) sucrose solution and centrifuged at 89,000 ×g using a 28 SA rotor in a Hitachi ultracentrifuge (SCP85H2) for 1 h at 4 °C. The virus pellet was resuspended with TNE and then subjected to a linear 35 to 65% sucrose gradient centrifugation at 89,000 ×g for 1 h at 4°C. The collected virus band was then mixed with TNE buffer and repelleted at 89,000 ×g for 1 h at 4 °C. The resulting pellet was again dissolved in TNE. To check for quality and quantity, virus samples were negatively stained with 2% sodium phosphotungstate and examined under a transmission electron microscope (TEM).

Purification of intact WSSV viral particles from tissues of infected crayfish

The tissues of infected crayfish excluding hepatopancreas were collected in an ice-bathed beaker. Ten grams of infected tissues were homogenized in 500 ml TNE buffer containing a combination of protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM benzamidine, and 1 mM Na₂S₂O₅), and then centrifuged at 3,500 ×g for 5 min at 4 °C. After filtering by nylon net (400 mesh), the supernatant was centrifuged at 30,000 ×g for 30 min at 4 °C. Then the upper loose pellet was rinsed out carefully, and the lower white pellet was suspended in 10 ml TM buffer (50 mM Tris-HCl, 10 mM MgCl₂, pH 7.5). After centrifugation at 3,500 ×g for 5 min, the virus particles were sedimented by centrifugation at 30,000 ×g for 20 min at 4 °C, and then resuspended and kept in 1 ml TM buffer containing 0.1% NaN₃. The degree of purity of virus isolated was evaluated by negative-staining TEM.

References

1. Xie XX, Li HY, Xu LM,Yang F. (2005) A simple and efficient method for purification of intact white spot syndrome virus (WSSV) viral particles. Virus Res 108: 63–67.

Some of our publications that have used this platform

1. Wang, C. H., C. F. Lo, J. H. Leu, C. M. Chou, P. Y. Yeh, H. Y. Chou, M. C. Tung, C. F. Chang, M. S. Su, and G. H. Kou. (1995) Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Panaeus monodon. Dis Aquat Org 23: 239-242.
2. Tsai, J.-M., H.-C. Wang, J.-H. Leu, H.-H. Hsiao, A. H.-J. Wang, G.-H. Kou, and C.-F. Lo. (2004) Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol 78:11360-11370.