Drag Race: All Stars winner Alaska Thunderfuck did a parody song of “Despacito” called “Valentina of 4” in October 2017, valentinaof4 with lyrics about Valentina and her infamous elimination from the show. After Miller’s death in 2007, scientists examining sealed vials preserved from the original experiments were able to show that there were actually well over 20 different amino acids produced in Miller’s original experiments. There is a threshold in surface warming beyond which a partial or near-complete melting of the Greenland ice sheet occurs. The pair are both ice skaters, but compete separately; Ondrej, who was born in the Czech Republic, now skates for Italy alongside partner Valentina Marchei, while Anna skates with Luca Lanotte; both will be competing for Italy at Pyeongchang, although Ondrej and Valentina will be performing in figure skating pairs while Anna and Luca will compete in ice dancing. The trees are long-lived, as some specimens still fruit after 300 years. One day before the experiment, Vero E6 cells were seeded in 24-well plates at a density of 8 × 104 cells per well. One day before infection, Vero E6 cells were seeded in a 12-well removable chamber glass slide (Ibidi) at a density of 4 × 104 cells per well.

Cells were washed twice with PBS before permeabilization with 0.1% Triton X-100 and blocking with PBS supplemented with 50 mM NH4Cl, 0.1% (w/v) saponin and 2% (w/v) BSA (confocal buffer) for 60 min. T16 (Supplementary Table 1) using the HotStarTaq Master Mix (QIAGEN) according to the manufacturer’s instructions with a touchdown cycling protocol: 95 °C for 15 min; 15 cycles of 94 °C for 30 s, 65 °C touchdown to 50 °C for 1 min, 72 °C for 1 min; 25 cycles of 94 °C for 30 s, 50 °C for 1 min, Valentinaof4 72 °C for 1 min. Following reverse transcription, 1 μl RNase H (5 U μl−1, New England Biolabs) per 25 μl reaction was added, and the mixture was incubated at 37 °C for 20 min. Subsequently, 1 μl of this reaction was used for a nested re-amplification with the primer pair pWhSF-5utr-R17-273 and TagRACE (Supplementary Table 1) in a final volume of 50 μl following the same cycling protocol as described above. The PCR fragment was purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel) according to the manufacturer’s instructions, and the purified PCR fragment was sent to Microsynth for Sanger sequencing with the primer pWhSF-5utr-R17-273 (Supplementary Table 1). Sequencing raw data were assessed using the SeqManTM II sequence analysis software (DNASTAR).

The cDNA was immediately purified with the High Pure PCR product purification kit (Roche) according to the manufacturer’s instructions. The quantity and quality of the extracted RNA was assessed using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Q10211) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Agilent, DNF-471), respectively. Sequencing libraries were produced using an Illumina TruSeq Stranded mRNA Library Prep kit (Illumina, 20020595) in combination with TruSeq RNA UD Indexes (Illumina, 20022371) according to Illumina’s guidelines. The quality-control assessments, generation of libraries and sequencing run were all performed at the Next Generation Sequencing Platform, University of Bern, Switzerland. The TCID50 assay was performed for MERS-CoV and MERS-CoV-GFP in Vero B4 cells and SARS-CoV-2 and SARS-CoV-2-GFP in Vero E6 cells. In brief, cells were seeded 24 h before infection in a 96-well plate at a density of 2 × 106 cells per plate. Cell-culture supernatants were collected at the indicated time points after infection.

Cells were washed with PBS and inoculated with viruses serially diluted in cell-culture medium at 1:10 dilution. At the time of infection, cells were washed with PBS and inoculated with viruses serially diluted in cell-culture medium at 1:10 dilution. Viruses were serially diluted at 1:10 dilution from 10−1 to 10−8. After 72 h of incubation, the medium was removed and cells were fixed and stained with crystal violet. After 24 h of incubation, the overlay was removed and cells were fixed and stained with crystal violet. At 48 h after infection, cells were washed once with PBS and incubated in fresh PBS. In brief, 24 h before infection, Vero E6 cells were seeded at a density of 2 × 106 cells per plate. Cells were infected with rSARS-CoV-2 clone 3.1 (passage 2) or mock-infected as control. 0.01 or mock-infected as control. 0.01). Statistical significance was determined by two-sided unpaired Student’s t-test without adjustments for multiple comparisons. Mock, Vero B4 cells inoculated with the supernatant of BHK-21 cells that were electroporated without viral RNAs. The PFU ml−1 of SARS-CoV-2 and SARS-CoV-2-GFP was determined by plaque assay using Vero E6 cells in a 6-well format.