Tecnai g2

Powder X-ray diffraction (XRD) patterns were collected using a Rigaku Smartlab diffractometer with Cu Kα radiation (λ = 1.5418 Å) in the scan range of 10° to 90° at a step of 0.01°. Scanning electron microscopy (SEM) measurements were performed on an XL 30 ESEMFEG field emission scanning electron microscopy. Transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and elemental mapping analysis were performed on a Philips TECNAI G2 electron microscope operating at 200 kV. High-annular dark-field scanning transmission electron microscopy (HAADF-STEM) images were performed on a Titan 80-300 scanning/transmission electron microscopy operated at 300 kV. The surface electronic states were determined by X-ray photoelectron spectroscopy (XPS) using a Thermo Fisher Scientific ESCALAB 250Xi unit with Al-Kα (1486.6 eV) as the X-ray source. Inductively coupled plasma optical emission spectroscopy (ICP-OES; X Series 2, Thermo Scientific USA) was used to determine the ratio of M/Ru in the as-prepared samples and the dissolution of M and Ru after the stability tests.

Virus purification was conducted as described previously [30 ] with some modifications. Briefly, 100 g of mustard leaves were homogenized in 200 mL of 0.067 M sodium phosphate buffer (pH 7.2) and 50 mL of 0.1 M ascorbic acid. The homogenate was centrifuged at 9700× g for 10 min at 4 °C. The separated supernatant was mixed with 10% chloroform and shaken for 10 min at 4 °C, followed by a pH adjustment to 5.3. The mixture was centrifuged at 200,000× g for 2 h in a fixed-angle (Beckman Ti 35) rotor. The pellets were suspended in a cold 0.01 M sodium phosphate buffer (pH 7.0). Transmission electron microscopy (TEM) was used to identify the purified viral particles. For TEM visualization, a purified virion sample (3.5 μL) was applied onto 300 mesh carbon-coated copper TEM grids for 30 s. Excess fluids were blotted, and after a wash with distilled water, the grids were stained with 2% uranyl acetate and visualized using a Tecnai G², FEI-Philips (Philips, Eindhoven, The Netherlands).

Viruses not identified by PCR were also viewed by electron microscopy using the negative staining technique. In the grids after glow discharge, supernatant of the positive samples (20–40 μl) was added for 10 min to achieve adherence to the grids. The samples were washed, fixed and contrasted using a 5% solution of ammonium molybdate, again washed and dried before electron microscopic analysis. The samples were observed using a Morgagni 268 D (Philips) operating at 100 60 keV and a Tecnai G2 operating at 200 keV (Supplementary Figure S3).
The environmental giant virus samples are summarized in Table 1 and results are expressed in Figure 2.