**4. Methods**

#### *4.1. Patients, Sample Collection and Handling, Biobanking, and Ethics Statement*

All COVID-19 patients were RT-PCR-confirmed and placed in negative pressure isolation rooms in our hospital. Nasopharyngeal or oropharyngeal throat swab specimens were collected on stated days post symptom onset for serial RT-PCR tracking. Pharyngeal specimens were also used to isolate and culture SARS-CoV-2 virus strains. One virus strain was used for a neutralization antibody test in our BSL-3 facility. Antibody tests were carried out using serum samples. Peripheral blood was collected for routine medical tests, and serum samples remaining after routine medical tests were used in our research.

Isolated virus strains are deposited in our institutional depository. Sequences of the virus strains are also deposited in the depository of the Taiwan Centers for Disease Control (Taiwan CDC).

This research was performed with informed consent from patients or their families. Specimen sampling and transportation were handled according to the criteria of the Taiwan CDC. This study was approved by the Institutional Review Board of Chang Gung Medical Foundation, Linkou Medical Center, Taoyuan City, Taiwan.

#### *4.2. SARS-CoV-2 Nucleic Acid Detection*

Nasopharyngeal or oropharyngeal throat swab specimens were collected from patients. Test for SARS-CoV-2 nucleic acid followed standard protocols. RNA was extracted from clinical samples with the LabTurbo system (Taigen, Taiwan). A 25 μL reaction contained 5 μL of RNA, 12.5 μL of 2 × reaction buffer provided with the Superscript III one-step RT-PCR system with Platinum Taq Polymerase (AgPath-ID One-step RT-PCR Kit), 1 μL of reverse transcriptase/Taq mixture from the kit, 0.4 μL of a 50 mM magnesium sulfate solution (Invitrogen), and 1 μg of nonacetylated bovine serum albumin (Roche). All oligonucleotides were synthesized and provided by Tib-Molbiol (Berlin, Germany). Thermal cycling was performed at 48 ◦C for 30 min for reverse transcription, followed by 95 ◦C for 10 min and then 45 cycles of 95 ◦C for 10 s, 65 ◦C for the 30 s [27].

## *4.3. COVID-19 Serum Antibody Detection*

To evaluate the antibody response, the levels of total IgG in patients' sera were semiquantified by ELISA (cat No. WS-1096, WANTAI SARS-CoV-2 Ab ELISA, China) through the use of a Triturus ELISA processor, following manufacturer's instructions. WANTAI SARS-CoV-2 Ab ELISA is a two-step incubation antigen "sandwich" enzyme immunoassay kit, which uses polystyrene microwell strips pre-coated with recombinant SARS-CoV-2 antigen. The results had previously been verified with another ELISA kit (Anti-SARS-CoV- 2 ELISA IgG, Euroimmun, Germany), and the ELISA was done in accordance with the manufacturer's instructions.

#### *4.4. Neutralization Antibody Test (NAT)*

The neutralizing antibody test of COVID-19 followed the standard protocol of a plaque reduction neutralization test. Vero cells were regularly maintained in minimal essential medium (MEM) supplemented with 10% (*v*/*v*) fetal bovine serum (FBS). COVID-19 virus was propagated in Vero cells in a maintenance medium consisting of MEM supplemented with 0% FBS. Serum samples were inactivated at 56 ◦C for 30 min before use. Serial two-fold dilutions of sera were mixed with an equal volume of COVID-19 virus suspension containing 100 × the median tissue culture infectious dose (TCID50). The mixture was incubated for 2 h at 37 ◦C, and then an equal volume of suspended VeroE6 cells (approximately 30,000 cells/well) was added to each well. Following incubation for 1 week at 37 ◦C, cells were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet. Serum neutralization titers were calculated and expressed as the reciprocals of the highest serum dilution that inhibits cytopathic effects.
