WSSV-infected gill tissues (1 g) from penaeid shrimp (Penaeus vannamei) were homogenized in 20 ml of TN buffer [20 mM tris-HCl and 400 mM NaCl (pH 7.5)]. After centrifugation at 3000g for 5 min, the supernatant was collected and further filtered by 0.45-μm filter. A 0.1-ml sample of supernatant was injected intramuscularly into healthy crayfish (Procambarus clarkii, 7 to 10 cm in length and 20 to 30 g in weight) between the third and fourth abdominal segments. After 4 to 7 days, dead and moribund crayfish were collected and kept at 4°C for virus isolation.
The tissues of 10 infected crayfish except for hepatopancreas were homogenized on ice for 5 min using a mechanical homogenizer (SCIENTZ, China) in 1 liter of TNE buffer [50 mM tris-HCl, 500 mM NaCl, and 5 mM EDTA (pH 8.0)] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 1 mM Na2S2O5) (34 (link)). After centrifugation at 3500g for 5 min at 4°C, the supernatant was collected and then subjected to ultracentrifugation at 30,000g for 30 min at 4°C (Beckman Optima XPN-100, USA). The supernatant was discarded, and the upper loose layer of the pellet was rinsed out carefully. The lower solid layer containing viral particles was resuspended in 50 ml of TNE buffer. The resuspension was subjected to another round of successive centrifugation steps at 3500g for 5 min and at 30,000g for 20 min at 4°C. The lower solid layer was resuspended in 1 ml of TM buffer [50 mM tris-HCl and 5 mM MgCl2 (pH 7.5)]. The final resuspension was centrifuged three to five times at 650g for 5 min to remove pink impurities. The milk-like fraction containing intact WSSV virions was stored at 4°C, and fresh samples were subjected to the following treatments.
Purified intact WSSV virions were mixed with an equal volume of 0.2% Triton X-100 in TM buffer and incubated at room temperature for 30 min to obtain oval-shaped WSSV capsids. The sediments were collected after centrifugation at 20,000g for 20 min at 4°C and resuspended in TM buffer. The treatment was repeated once to ensure that envelopes were removed completely. To obtain rod-shaped WSSV capsids, the Triton X-100–treated WSSV capsids were further mixed with an equal volume of 2 M NaCl in TM buffer and incubated at 4°C for 30 min. The sediments were collected after centrifugation at 20,000g for 20 min at 4°C and resuspended in TM buffer. Both rod-shaped and oval-shaped WSSV capsids were immediately subjected to high-resolution cryo-EM analysis.
In addition, the purified intact WSSV virions were directly treated with 2 M NaCl in TM buffer at 4°C for 30 min. The sediments were collected and suspended in TM buffer after centrifugation at 20,000g for 20 min at 4°C. The virions were still envelope-encapsulated, and we named them the capsid-detached WSSV virions. The capsid-detached WSSV virions were collected for the further infection assays.
The tissues of 10 infected crayfish except for hepatopancreas were homogenized on ice for 5 min using a mechanical homogenizer (SCIENTZ, China) in 1 liter of TNE buffer [50 mM tris-HCl, 500 mM NaCl, and 5 mM EDTA (pH 8.0)] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 1 mM Na2S2O5) (34 (link)). After centrifugation at 3500g for 5 min at 4°C, the supernatant was collected and then subjected to ultracentrifugation at 30,000g for 30 min at 4°C (Beckman Optima XPN-100, USA). The supernatant was discarded, and the upper loose layer of the pellet was rinsed out carefully. The lower solid layer containing viral particles was resuspended in 50 ml of TNE buffer. The resuspension was subjected to another round of successive centrifugation steps at 3500g for 5 min and at 30,000g for 20 min at 4°C. The lower solid layer was resuspended in 1 ml of TM buffer [50 mM tris-HCl and 5 mM MgCl2 (pH 7.5)]. The final resuspension was centrifuged three to five times at 650g for 5 min to remove pink impurities. The milk-like fraction containing intact WSSV virions was stored at 4°C, and fresh samples were subjected to the following treatments.
Purified intact WSSV virions were mixed with an equal volume of 0.2% Triton X-100 in TM buffer and incubated at room temperature for 30 min to obtain oval-shaped WSSV capsids. The sediments were collected after centrifugation at 20,000g for 20 min at 4°C and resuspended in TM buffer. The treatment was repeated once to ensure that envelopes were removed completely. To obtain rod-shaped WSSV capsids, the Triton X-100–treated WSSV capsids were further mixed with an equal volume of 2 M NaCl in TM buffer and incubated at 4°C for 30 min. The sediments were collected after centrifugation at 20,000g for 20 min at 4°C and resuspended in TM buffer. Both rod-shaped and oval-shaped WSSV capsids were immediately subjected to high-resolution cryo-EM analysis.
In addition, the purified intact WSSV virions were directly treated with 2 M NaCl in TM buffer at 4°C for 30 min. The sediments were collected and suspended in TM buffer after centrifugation at 20,000g for 20 min at 4°C. The virions were still envelope-encapsulated, and we named them the capsid-detached WSSV virions. The capsid-detached WSSV virions were collected for the further infection assays.
