Beadblaster24r

Groups of male and female Roborovski dwarf hamsters were anesthetized using ketamine/dexmedetomidine and infected with 10⁴ pfu per animal (50 µl total volume, 25 µl per nare). Anesthesia was reversed with atipamezole. Hamsters were monitored daily (body weight, temperature, and clinical score). Treatment started 12 h after infection with nirmatrelvir (200 µl, 0.5% MC with 2% Tween80; 250 mg/kg or 125 mg/kg b.i.d.) and ritonavir (50 µl, 20% EtOH; 83.3 mg/kg, b.i.d.). Treatment continued twice daily for 5 and 7 days after infection for VOC δ and VOC o, respectively. Treatment was discontinued for VOC δ after 5 days due to treated hamsters developing pronounced diarrhea. Groups of hamsters were euthanized three days after infection to assess lower respiratory tract viral load. Lungs were harvested, washed with sterile phosphate buffered saline, and homogenized in PBS supplemented with antibiotic-antimycotic using a bead blaster (3 bursts of 30 s at 4 °C each, 30 s pause between bursts; Bead Blaster 24R, Benchmark). Lung homogenates were clarified by centrifugation (20,000 × g, 10 min, 4 °C), aliquoted, and stored at −80 °C until processed. For RNA quantification, lungs and tracheas were harvested and homogenized in RNeasy RNA lysis buffer (Qiagen) in accordance with the manufacturers protocol.

Frozen
mouse lung tissue (∼30 mg) was homogenized using a BeadBlaster
24R refrigerated homogenizer (Benchmark) at 3650 rpm for 30 s, with
a linear speed of 6.00 m/s at 4 °C. Two cycles were performed
at intervals of 30 s. Total RNA was extracted from the homogenized
tissue using the RNeasy fibrous tissue mini kit (Qiagen) following
the standard procedures. The EGFP mRNA was amplified by one-step RT-PCR
using the SuperScript IV one-step RT-PCR system (Invitrogen) following
the recommended protocol. In brief, 100 ng of isolated RNA was used
for the RT-PCR reaction, which proceeded at 55 °C for 10 min
and 98 °C for 2 min, followed by 40 cycles of amplification at
98 °C for 10 s, 68 °C for 10 s, and 72 °C for 15 s,
and final extension at 72 °C for 5 min. The resulting PCR products
were separated on a 4–20% gradient nondenaturing polyacrylamide
gel, and bands were visualized by staining with GelRed nucleic acid
gel stain in 0.1 M NaCl solution for 30 min (Biotium, Inc.) EGFP forward
primer: 5′-CGTAAACGGCCACAAGTTCAGCG-3′, reverse
primer: 5′-GTGGTGCAGATGAACTTCAGGGTC-3′.

For virus titration, the organs were weighed and homogenized in PBS supplemented with antibiotic-antimycotic solution (Gibco). Tissues were homogenized using a bead blaster (3 bursts of 30 s at 4 °C each, 30 s pause between bursts; Bead Blaster 24 R, Benchmark). Tissue homogenates were clarified by centrifugation (20,000 × g for 5 min at 4 °C). The clarified supernatants were harvested, frozen and used in subsequent TCID₅₀ or plaque assays. For detection of viral RNA, the harvested organs were homogenized in lysis buffer (Qiagen), and the total RNA was extracted using a RNeasy mini kit (Qiagen). For nasal lavages, RNA was extracted using a Quick-RNA Viral Kit (Zymo) in accordance with the manufacturer’s protocols.

Cultures of WT and Laccase⁺-riboF-Lysis⁺ were grown in liquid culture under 75 μmol photons m⁻² s⁻¹ with continuous shaking at 30 °C to an OD₇₅₀ of 0.5. 10 mL of cultures were pelleted by centrifugation at 4500 × g and aspirated. The pellets were then resuspended in 1 mL of cold lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) supplemented with 1 mM PMSF and Complete Mini Protease Inhibitor (Roche). The samples were then homogenized in a BeadBlaster 24 R (Benchmark Scientific) at 4 °C. The crude protein was then clarified by centrifugation at 4 °C for 20 min at 18,000 × g, after which the crude protein concentration was determined using a Pierce Coomassie Plus Protein Assay (Bradford), with bovine serum albumin used as a standard. After boiling the sample with 2× Laemmli Sample Buffer, 30 μg of sample was loaded into a Mini-PROTEAN® TGX Precast Gel (Bio Rad) and run at 150 V. The gel was then incubated in InstantBlue Coomassie Protein Stain (Abcam) overnight prior to imaging.