Uvc 500

CHPV inactivation was carried out with a UV cross-linker (UVC 500, Hoefer scientific, USA) using short-wavelength UV radiation (UVC, 254 nm) at a distance of 5 cm for 25 minutes on ice as described earlier40 (link). Inactivation of virus was verified by plaque assay for all three sets of treated supernatant which showed the absence of viral plaque formation in the UV treated culture supernatant (Fig. 6C).

Cells were plated on 60- or 100-mm culture dishes. For ultraviolet (UV) irradiation, the culture medium was removed and the culture dishes were placed uncovered in a UV cross-linker (model UVC-500; Hoefer, Holliston, MA, USA). Cells were irradiated at 25–200 J/m2 UV-C in the initial dose response analysis, and then 50 J/m2 UV-C was used for experiments. Immediately after irradiation, medium was added to the cells, followed by culture for the appropriate time. For serum starvation, the culture medium was removed and DMEM without FBS was added to the cells, followed by further culture for 24 h. Cells were harvested and centrifuged at 1500 rpm for 5 min at room temperature. The medium was removed, and the cells were washed once in phosphate-buffered saline. Cells were then resuspended in cold binding buffer, followed by addition of annexin V-FITC and propidium iodide. The cells were incubated at room temperature for 15 min in the dark and then analyzed by flow cytometry.

Strains were cultured and treated with rifampicin as described for the chemical stability measurements. Samples (20 ml) were withdrawn and quenched with 4 ml of a solution of 5% phenol in ethanol. Cells were pelleted by centrifugation in an Eppendorf S‐4‐72 rotor at 4°C for 20 min. After suspension in 1.5 ml of Tri Reagent (MRC) using a MultiTherm agitator (37°C, 550 rpm, 10 min), cell debris was removed by centrifugation (20,000 g, 10 min, room temperature). An equal volume of ethanol was added to the supernatant and total RNA was prepared using a Direct‐zol^TM, RNA MiniPrep Plus kit (Zymo Research) following the manufacturer’s instructions. RNA was eluted in 80 µl of water. Concentration and purity were determined using a NanoDrop^TM spectrophotometer. RNA (2.5–4.0 µg) was heated at 65°C for 5 min in a 75 µl reaction containing 50% formamide, 2.5 M formaldehyde, 10 mM sodium MOPS, pH 7.0, 4 mM NaCl, 0.5 mM EDTA, 0.02% XC, 0.02% BPB and then vacuum blotted onto Hybond^TM‐XL filters (Amersham) using a P648 slot blot manifold (Amersham) following manufacturer’s instructions. After UV cross‐linking (Hoefer, UVC 500, 120,000 µJ/cm²), the blots were hybridized with 5′‐³²P‐DNA probes (Table S2). The rpsT, trxA and rpsO messages were hybridized with two probes to increase sensitivity. For normalization, the blots were stripped and then hybridized with a probe specific to 23S rRNA.

The self‐assembled structure consisting of nano‐spherical beads and a photonic organogel was synthesized following a previous report.^[43] PS beads with a diameter of 200 nm (Warrington, PA) and 3 wt% of aqueous solution were infiltrated to a 200‐µm gap. Then, the filtered precursor solution was slide‐casted at 1 mm min⁻¹. Thermal air blowing was employed on the sample to obtain a self‐assembled rigid nano‐bead lattice during water‐drying by evaporation.
To obtain the elastic host of the photonic gel, a monomer mixture comprising 4.68 m acrylamide (Sigma Aldrich, A8887) and 4.65 m 2‐hydroxyethyl methacrylate (Sigma, 128 635) was added to ethylene glycol (Sigma Aldrich, 324 558), followed by adding 0.57 wt% of ethylene glycol di‐methacrylate (Sigma, 335 681) and 0.17 wt% of lithium phenyl‐2,4,6‐trimethyl benzoyl phosphonate (with respect to acrylamide). The final host precursor mixture was introduced into the self‐assembled photonic crystal nano‐bead lattice and cured in an ultraviolet chamber (Hoefer, UVC 500) for 30 min, to induce stiffening. The final obtained elastic photonic organogel film exhibited red photonic reflection.

For Northern blot analysis, from 5 up to 100 μg of purified RNA from different plant tissues were examined in 5 % polyacrylamide gels containing 8 M urea and 1× TBE (89 mM Tris/89 mM boric acid/2.5 mM EDTA, pH 8.3). For double PAGEs, nucleic acids enriched in RNAs of the appropriate retrozyme size were obtained by cutting a section from nondenaturing 5 % polyacrylamide gels. These RNAs were examined in denaturing 5 % polyacrylamide gels containing 8 M urea and 0.25× TBE (22.5 mM Tris/22.5 mM boric acid/2.5 mM EDTA, pH 8.3). After ethidium bromide staining, RNAs were electroblotted to nylon membranes (Amersham Hybond-N, GE Healthcare) and UV-fixed with a crosslinker (UVC 500, Hoefer). Prehybridization, hybridization (at 68 °C in 50 % formamide for 16 h) and washing (twice with 0.1× SSC at 68 °C for 15 min) was done following the instructions of the manufacturer (GE Healthcare). The DIG-labelled probes were detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase (anti-digoxigenin-AP Fab fragments, 1:10⁴ dilution in blocking solution; Roche Diagnostics GmbH). The chemiluminiscence produced in the presence of the substrate CDP-Star (1:200 dilution in 0.1 M Tris-Cl, 0.1 M NaCl, pH 9.5; Roche Diagnostics GmbH) was finally visualized in a LAS-3000 Imaging System (Fujifilm).

The virus was inactivated as described by Groene and Shaw [52 (link)] with modifications. Purified RV [42 (link)] was distributed in aliquots of 50μl in 96-well multiwell plates and mixed with 40μg/ml of psoralen (AMT, 4’-aminomethyl trioxsalen hydrochloride). The plate was wrapped in aluminum foil and incubated on ice for 15min. Then, the plate was UV-crosslinked with 960 mJ/cm3 for 30 min (UVC500, Hoefer).