Mouse actb mix

To inactivate virus in serum, TRIzol was added to the samples (1:3). For liver and spleen, PBS was added to each sample and pestles were used to crush the organs. Thereafter, the samples were centrifuged (5 min at 7,000 rpm) and 100 to 200 μL of each liver or spleen sample was added to TRIzol (1:3). RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. Viral RNA was measured by quantitative real-time PCR (qRT-PCR) using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) with primers and probe specific for the CCHFV L gene:
Forward, 5′- GCCAACTGTGACKGTKTTCTAYATGCT-3′,
Reverse 1, 5′- CGGAAAGCCTATAAAACCTACCTTC-3′,
Reverse 2, 5′-CGGAAAGCCTATAAAACCTGCCYTC-3′ and
Reverse 3, 5′-CGGAAAGCCTAAAAAATCTGCCTTC-3′, and
Probe, FAM-CTGACAAGYTCAGCAAC–MGB.
For liver and spleen samples, mouse ACTB mix (Thermo Fisher Scientific) was used as endogenous control. The cycling reactions was performed using a capillary Roche LightCycler 2.0 system.

Trizol (Sigma‐Aldrich) in a ratio of 1:3 was used to inactivate potential virus in nasal lavage samples (50 μl) from SARS‐CoV‐2‐infected K18‐hACE2 mice. For lung and spleen, PBS was added to each sample (1 g/ml) and pestles were used to crush the organs. Thereafter, the samples were centrifuged (5 min at 7,000 rpm) and 50 μl of each lung or spleen sample was added to Trizol (1:3). Total RNA was extracted using the Direct‐zol RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. Viral RNA were thereafter measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) using TagMan Fast Virus 1‐Step master mix (Thermo Fisher Scientific) with primers and probe for the SARS‐CoV‐2 E gene.
Forward: 5′‐ ACAGGTACGTTAATAGTTAATAGCGT ‐3′
Reverse: 5′‐ ATATTGCAGCAGTACGCACACA ‐3′
Probe: FAM‐ ACACTA GCC ATC CTT ACT GCG CTT CG MGB
For lung and spleen samples, mouse ACTB mix (Thermo Fisher Scientific) was used as endogenous control. The PCR reaction was performed using a capillary Roche LightCycler 2.0 system.