RT-qPCR analysis.

Vero E6 cells were seeded into 6-well plates at a density of 2 × 10⁵/well, and after culture for 24 h, SARS-CoV-2 was infected at an MOI of 0.008 and incubated for the specified time. Viral RNA in the supernatant was isolated with the QIAamp viral RNA kit (Qiagen; 52906) following the manufacturer’s instructions. Viral copy numbers were then detected by absolute quantitative real-time PCR (RT-qPCR) methodology using the HiScript II one-step RT-qPCR SYBR green kit (Vazyme Biotech, Nanjing, China) and an ABI 7500 real-time PCR system (Applied Biosystems, CA). The protocol for the RT-qPCR was as follows: 50°C for 15 min and 95°C for 30 s, followed by 45 cycles at 95°C for 10 s and 63°C for 35 s. The specific primers used to detect the SARS-CoV-2 N and E genes were as follows: for the N gene, 5′-GGGGAACTTCTCCTGCTAGAAT-3′ (forward) and 5′-CAGACATTTTGCTCTCAAGCT-3′ (reverse), and for the E gene, 5′-CGATCTCTTGTAGATCTGTTCTC-3′ (forward) and 5′-ATATTGCATTGCAGCAGTACGCACA-3′ (reverse). The sequences of the TaqMan probe were as follows: for the N gene, 5′-FAM-TTGCTGCTGCTTGACAGATT-6-carboxytetramethylrhodamine (TAMRA)-3′, and for the E gene, 5′-6-carboxyfluorescein (FAM)-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3′. For lung tissues or xenografted human lung tissues isolated from mice, samples were collected and homogenized in PBS. Viral RNA in the homogenate was isolated and processed as described above.