Introduction of ORF3a-Q57H SARS-CoV-2 Variant Causing Fourth Epidemic Wave of COVID-19, Hong Kong, China

We describe an introduction of clade GH severe acute respiratory syndrome coronavirus 2 causing a fourth wave of coronavirus disease in Hong Kong. The virus has an ORF3a-Q57H mutation, causing truncation of ORF3b. This virus evades induction of cytokine, chemokine, and interferon-stimulated gene expression in primary human respiratory cells.

length >29,700 nt and sequence coverage >100 by using an Illumina (https://www.illumina.com) sequencing protocol we described previously (1). In brief, virus genome was reverse transcribed with multiple gene-specific primers targeting different regions of the viral genome. The synthesized cDNA was then subjected to multiple overlapping 2-kb PCRs for full-genome amplification. PCR amplicons obtained from the same specimen were pooled and sequenced by using Nova sequencing platform (Illumina). Sequencing library was prepared by using Nextera XT (Illumina).

Phylogenetic Analysis
Generated sequencing reads were mapped to a reference virus genome by BWA (http://bio-bwa.sourceforge.net), and genome consensus was generated by Geneious version 11.1.4 (https://www.geneious.com). Sequences from each phylogenetic clade (G, GH, GR, L, O, S, and V) of SARS-CoV-2 were selected from GISAID for phylogenetic analysis (Appendix Table 2). Viral sequences were aligned by using BioEdit (https://www.bioedit.com) and phylogenetically analyzed by using MEGA-X (https://www.megasoftware.net). We constructed the phylogenetic tree by using the neighbor-joining method with 500 bootstraps.

Virus Isolates Used in Characterization
Virus isolated from the wave 1 (clade L), wave 3 (clade GR), and wave 4 (clade GH) of COVID-19 in Hong Kong were used for phenotypic characterization. The wave 1 virus was isolated from an imported case identified in a 39-year-old symptomatic man, as previously described (2). The wave 3 virus was isolated from a locally transmitted case in a 57-year-old symptomatic woman. The wave 4 virus was isolated from patient A2 in this study, a 43-year-old asymptomatic man. These patients recovered and were discharged from hospital.

Ex Vivo Cultures and Infection of Human Respiratory Tract
Infection procedures were performed as previously described (2). In brief, nontumor bronchus and lung tissues were obtained from patients undergoing elective surgery, as detailed previously (3,4). Fragments of human tissues were infected with each virus at 5 × 10 5 TCID50/mL for 1 h at 37°C. The explants were washed 3 times with phosphate buffered saline (PBS) and placed in culture medium (F-12K nutrient mixture with L-glutamine and antibiotics) and incubated at 37°C with 5% CO2. Infectious viral titers in culture supernatants were assessed at 1 h, 24 h, 48 h, 72 h, and 96 h post-infection (hpi) by titration in Vero E6 cells. One set of bronchus and lung tissues were fixed at 96 hpi in 10% formalin and processed for immunohistochemistry staining.

2-Dimensional Differentiated Human Airway Organoid Culture and Infection
The human airway organoids were established from human lung tissues, as previously described (5). The 2-dimensional differentiated airway organoid model was further built on the airway organoids, as previously described (6), with a few modifications. In brief, airway organoids were dissociated into single cells using TrypLE select (GIBCO, https://www.thermofisher.com) for 10 min at 37°C. The digest was then sheared by using 25gauge needle and strained over a 40 μm cell strainer. We seeded 150,000 cells onto Transwell insert (Corning, https://www.corning.com) pre-coated with rat tail collagen 1 (Corning). The cells were cultured in a mixture of airway organoid growth medium (5) and Pneumacult-ALI (Stemcell, https://www.stemcell.com) complete base medium at a ratio of 1:1 at 37°C for 3-4 days. Once the cells reached confluency, they were cultured at air-liquid interface (ALI) in Pneumacult-ALI Maintenance Medium (Stemcell). The medium was changed every 3 days. The 2-dimensional transwell cultures were used for infection after 3 weeks of differentiation. Cells were infected with the coronaviruses at a multiplicity of infection of 2 at the apical side for 1 h at 37°C. Cells were washed with PBS and culture at ALI in the same growth medium.

Viral Titration by TCID50 assay
Vero-E6 cells were seeded on 96-well tissue culture plates 1 day before the virus titration (TCID50) assay. Cells were washed once with PBS and replenished with 2% DMEM medium supplemented with 100 units/mL of penicillin and 100 μg/mL of streptomycin. Serial dilutions of virus supernatant, from 0.5-7 log, were performed before adding the virus dilutions onto the plates in quadruplicate. The plates were observed daily for cytopathic effects. The endpoint of viral dilution leading to CPE in 50% of inoculated wells was estimated by using the Karber method (7). Area-under-curve (AUC) was calculated by integrating infectious virus titers at 24-96 hpi in ex vivo bronchus and lung tissues.

Immunohistochemical Staining
Immunohistochemical staining of the respiratory tract tissue was carried out for the SARS-CoV-2 nucleoprotein, as previously described (2). The fixed paraffin-embedded ex vivo cultures of human tissues were stained with SARS-CoV nucleoprotein (4D11) (4,5,8). The tissue sections were first microwaved in 10 mmol citrate buffer, blocked with 10% normal horse serum at room temperature. The sections were then incubated with 4D11 antibody followed by alkaline phosphatase (AP) conjugated antimouse antibody (Vector Laboratories, Inc., https://vectorlabs.com ) and developed with VectorRed (VR) (Vector Laboratories, Inc.).

Real-time PCR assay
The RNA of infected cells were extracted at 48 h post infection by using a MiniBEST Universal RNA Extraction Kit (TaKaRa Bio, Inc., http://www.takara-bio.com). RNA was reverse-transcribed by using Oligo-dT primers with Advantage RT-for-PCR Kit (TaKaRa Bio, Inc.). mRNA expression of target genes was performed by using ViiA7 Real-Time PCR System (Applied Biosystems, https://www.thermofisher.com). The gene expression profiles were quantified and normalized with β-actin, as previously described (9-11).

Cytometric Bead Array
Protein concentration of interferon gamma-induced protein-10 in the supernatants collected from apical and basolateral chambers was determined by bead-based immunoassays, BD Cytometric Bead Array (BD Bioscience, https://www.bdbiosciences.com) according to the manufacturer's protocol. In brief, 50 μL of cell culture supernatant and a 10-point standard curve (ranging from 0-2,500 pg/mL) was used for the measurement of each cytokine and chemokine.
The samples were analyzed by using a BD LSR Fortessa Analyzer (BD Bioscience). Standard curves for the cytokines and chemokines were built and the fluorescence intensity concentrations were calculated by using FlowJo version 7.6.1 (https://www.flowjo.com).

Biosafety and Ethics
All infection work was carried out in a Biosafety Level-3 facility. Informed consent was obtained from all subjects and approval was granted by the Institutional Review Board of the