Ethics statement
The virus isolation procedures in this study were approved by the Institutional Review Board of National Institute for Infectious Diseases (approval ID: 1178) and Tokyo Metropolitan Institute of Public Health (approval ID: 3KenKenKen-466) according to the Declaration of Helsinki 2013.
All experiments with hamsters were performed in accordance with the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments. The protocols were approved by the Institutional Animal Care and Use Committee of National University Corporation Hokkaido University (approval numbers 20-0123 and 20-0060).
Epidemiological and viral sequence data of Omicron
The data of the 7-day average of new COVID-19 cases per day in South Africa and the UK by December 18, 2021 were downloaded from the Our World in Data (https://ourworldindata.org/covid-cases) on December 20, 2021. The numbers of Omicron sequences reported and the countries reported the Omicron sequences by December 24, 2021 (Extended Data Table 2) were obtained from outbreak.info (https://outbreak.info) on December 25, 2021
Estimation of viral transmissibility
We estimated the relative transmissibility of each SARS-CoV-2 lineage in South Africa (Fig. 1b) and the UK (Fig. 1d) according to the lineage dynamics determined by viral genomic surveillance data. The dynamics of five most predominant lineages in each country from January 1, 2021 to December 10, 2021 were analysed. For South Africa, the dynamics of Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2 and AY lineages), Omicron (B.1.1.529, BA.1, and BA.2), and the C.1.2 lineage were used. For the UK, the dynamics of Alpha, Beta, Delta, Omicron, and the B.1.177 lineage (including its sublineages) were used.
We prepared the input data to estimate the relative transmissibility of each viral lineage for each country. The metadata of the sequenced SARS-CoV-2 strains (e.g., the collection date, collection place and PANGO lineage) were downloaded from the GISAID database (https://www.gisaid.org/) on December 20, 2021. The viral strains belonging to the lineages above were used for the following analysis. The number of isolated strains in each viral lineage in each day was counted and subsequently summarized in three-day bins. Finally, the count matrix representing the abundance of respective viral lineages (viral lineage ID k ∈ {1, 2, …, K}; K = 5) in respective time bins (t ∈ {1, 2, …, T}; T = 114) for each country was constructed.
We modelled the transition of the relative frequency of K types of viral lineages with a Bayesian multinomial logistic model as below:
Parameter estimation was performed by the framework of Bayesian statistical inference with Markov chain Monte Carlo methods (MCMC) implemented in Stan [CmdStan v2.28.1] (https://mc-stan.org). Non-informative priors were set for all parameters. Four independent MCMC chains were run with 2,000 and 3,000 steps of warmup and sampling iterations, respectively. We confirmed that all estimated parameters had <1.01 R-hat convergence diagnostic and >1,000 effective sampling size values, indicating that the MCMC runs were successfully convergent. The fitted model well reconstructed the observed data of the viral lineage dynamics in South Africa (R2 = 0.994; Extended Data Fig. 1b) and the UK (R2 = 0.99995; Extended Data Fig. 1d). The analyses above were performed in R v3.6.3.
Cell culture
HEK293 cells (a human embryonic kidney cell line; ATCC CRL-1573) and HEK293-ACE2/TMPRSS2 (HEK293 cells stably expressing human ACE2 and TMPRSS2)19 were maintained in Dulbecco’s modified Eagle's medium (DMEM) (high glucose) (Wako, Cat# 044-29765) containing 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). A549 (a human lung epithelial cell line; ATCC CCL-185) and A549-ACE2 cells (A549 cells stably expressing human ACE2)19 were maintained in Ham’s F-12K medium (Wako, Cat# 080-08565) containing 10% FBS and 1% PS. Vero cells [an African green monkey (Chlorocebus sabaeus) kidney cell line; JCRB0111] were maintained in Eagle’s minimum essential medium (EMEM) (Wako, Cat# 051-07615) containing 10% FBS and 1% PS. VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human TMPRSS2)40 were maintained in DMEM (low glucose) (Wako, Cat# 041-29775) containing 10% FBS, G418 (1 mg/ml; Nacalai Tesque, Cat# G8168-10ML) and 1% PS. Calu-3 cells (a human lung epithelial cell line; ATCC HTB-55) were maintained in EMEM (Sigma-Aldrich, Cat# M4655-500ML) containing 10% FBS and 1% PS. Calu-3/DSP1-7 cells (Calu-3 cells stably expressing DSP1-7 cells)41 was maintained in EMEM (Wako, Cat# 056-08385) supplemented with 20% FBS and 1% PS. HeLa-ACE2/TMPRSS2 cells (HeLa cells stably expressing human ACE2 and TMPRSS2)42 were maintained in DMEM (low glucose) (Wako, Cat# 041-29775) containing 10% FBS, G418 (1 mg/ml; Nacalai Tesque, Cat# G8168-10ML) and 1% PS.
SARS-CoV-2 preparation and titration
To isolate an Omicron variant (BA.1 lineage, strain TY38-873; GISAID ID: EPI_ISL_7418017), saliva was collected from a traveller arrived at Japan, and RT-qPCR testing for SARS-CoV-2 was performed in an airport quarantine station, Japan. The sample was subjected to whole genome sequencing based on a modified ARTIC Network protocol43, and the near full-length SARS-CoV-2 genome sequence was deposited in GISAID (GISAID ID: EPI_ISL_6913953). Virus isolation was performed as previously described40. In brief, the saliva was inoculated into VeroE6/TMPRSS2 cells and cytopathic effect (CPE) was observed 4 d after inoculation. Then, the supernatant was harvested and stored at –80°C as an original virus (GISAID ID: EPI_ISL_7418017). After one more passage in VeroE6/TMPRSS2 cells, the virus was obtained from National Institute of Infectious Diseases, Japan. An early pandemic D614G-bearing isolate (B.1.1 lineage, strain TKYE610670; GISAID ID: EPI_ISL_479681) and a Delta isolate (B.1.617.2 lineage, strain TKYTK1734; GISAID ID: EPI_ISL_2378732) were used in the previous study2.
Virus preparation and titration was performed as previously described2,19. To prepare the working virus stock, 100 μl of the seed virus was inoculated into VeroE6/TMPRSS2 cells (5 × 106 cells in a T-75 flask). One hour after infection, the culture medium was replaced with DMEM (low glucose) (Wako, Cat# 041-29775) containing 2% FBS and 1% PS. At 3 d.p.i., the culture medium was harvested and centrifuged, and the supernatants were collected as the working virus stock. The viral genome sequences of working viruses were verified as described below.
The titre of the prepared working virus was measured as the 50% tissue culture infectious dose (TCID50). Briefly, one day before infection, VeroE6/TMPRSS2 cells (10,000 cells) were seeded into a 96-well plate. Serially diluted virus stocks were inoculated into the cells and incubated at 37°C for 4 d. The cells were observed under microscopy to judge the CPE appearance. The value of TCID50/ml was calculated with the Reed–Muench method44.
SARS-CoV-2 infection
One day before infection, Vero cells (10,000 cells), VeroE6/TMPRSS2 cells (10,000 cells), Calu-3 cells (10,000 cells), HeLa-ACE2/TMPRSS2 cells (10,000 cells), A549-ACE2 cells (10,000 cells) and A549 cells (10,000 cells) were seeded into a 96-well plate. SARS-CoV-2 (1,000 or 100 TCID50) was inoculated and incubated at 37°C for 1 h. The infected cells were washed, and 180 µl of culture medium was added. The culture supernatant (10 µl) was harvested at the indicated time points and used for RT–qPCR to quantify the viral RNA copy number (see below). To monitor the syncytium formation in infected cell culture, bright-field photos were obtained using an All-in-One Fluorescence Microscope BZ-X800 (Keyence).
Immunofluorescence staining
Immunofluorescence staining was performed as previously described2. Briefly, one day before infection, VeroE6/TMPRSS2 cells (10,000 cells) were seeded into 96-well, glass bottom, black plates and infected with SARS-CoV-2 (100 TCID50). At 24 h.p.i., the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (Nacalai Tesque, Cat# 09154-85) for 1 h at 4°C. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 1 h, blocked with 10% FBS in PBS for 1 h at 4°C. The fixed cells were then stained using rabbit anti-SARS-CoV-2 N polyclonal antibody (GeneTex, Cat# GTX135570, 1:1,000) for 1 h. After washing three times with PBS, cells were incubated with an Alexa 488-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific, Cat# A-11008, 1:1,000) for 1 h. Nuclei were stained with DAPI (Thermo Fisher Scientific, Cat# 62248). Fluorescence microscopy was performed on an All-in-One Fluorescence Microscope BZ-X800 (Keyence).
Plaque Assay
Plaque assay was performed as previously described2,19. Briefly, one day before infection, VeroE6/TMPRSS2 cells (100,000 cells) were seeded into a 24-well plate and infected with SARS-CoV-2 (10,000 TCID50) at 37°C. At 2 h.p.i, mounting solution containing 3% FBS and 1.5% carboxymethyl cellulose (Wako, Cat# 039-01335) was overlaid, followed by incubation at 37°C. At 3 d.p.i., the culture medium was removed, and the cells were washed with PBS three times and fixed with 4% paraformaldehyde phosphate (Nacalai Tesque, Cat# 09154-85). The fixed cells were washed with tap water, dried, and stained with staining solution [0.1% methylene blue (Nacalai Tesque, Cat# 22412-14) in water] for 30 m. The stained cells were washed with tap water and dried, and the size of plaques was measured using ImageJ.
RT–qPCR
RT–qPCR was performed as previously described2,19. Briefly, 5 μl of culture supernatant was mixed with 5 μl of 2 × RNA lysis buffer [2% Triton X-100, 50 mM KCl, 100 mM Tris-HCl (pH 7.4), 40% glycerol, 0.8 U/μl recombinant RNase inhibitor (Takara, Cat# 2313B)] and incubated at room temperature for 10 min. RNase-free water (90 μl) was added, and the diluted sample (2.5 μl) was used as the template for real-time RT-PCR performed according to the manufacturer’s protocol using the One Step TB Green PrimeScript PLUS RT-PCR kit (Takara, Cat# RR096A) and the following primers: Forward N, 5'-AGC CTC TTC TCG TTC CTC ATC AC-3'; and Reverse N, 5'-CCG CCA TTG CCA GCC ATT C-3'. The viral RNA copy number was standardized with a SARS-CoV-2 direct detection RT-qPCR kit (Takara, Cat# RC300A). Fluorescent signals were acquired using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific), a CFX Connect Real-Time PCR Detection system (Bio-Rad), an Eco Real-Time PCR System (Illumina), a qTOWER3 G Real-Time System (Analytik Jena) or a 7500 Real Time PCR System (Applied Biosystems).
Plasmid construction
Plasmids expressing the SARS-CoV-2 S proteins of the D614G-bearing early pandemic SARS-CoV-2 (pC-SARS2-S D614G) and Delta (pC-SARS2-S Delta) were prepared in our previous study2,19. A plasmid expressing the SARS-CoV-2 Omicron S protein (pC-SARS2-S Omicron) was generated by overlap extension PCR using pC-SARS2-S D614G2,19 and pC-SARS2-S Alpha2 as the templates and the primers listed in Supplementary Table 1. The resulting PCR fragment was digested with KpnI and NotI and inserted into the KpnI-NotI site of the pCAGGS vector45.
SARS-CoV-2 S-based fusion assay
The SARS-CoV-2 S-based fusion assay was performed as previously described2,19. This assay utilizes a dual split protein (DSP) encoding Renilla luciferase (RL) and GFP genes; the respective split proteins, DSP8-11 and DSP1-7, are expressed in effector and target cells by transfection. Briefly, on day 1, effector cells (i.e., S-expressing cells) and target cells (see below) were prepared at a density of 0.6–0.8 × 106 cells in a 6-well plate. To prepare effector cells, HEK293 cells were cotransfected with the S expression plasmids (400 ng) and pDSP8-11 (400 ng) using TransIT-LT1 (Takara, Cat# MIR2300). To prepare target cells, HEK293 cells were cotransfected with pC-ACE2 (200 ng) and pDSP8-11 (400 ng). Target HEK293 cells in selected wells were cotransfected with pC-TMPRSS2 (40 ng) in addition to the plasmids above. VeroE6/TMPRSS2 cells were transfected with pDSP1-7 (400ng). On day 3 (24 h.p.i), 16,000 effector cells were detached and reseeded into 96-well black plates (PerkinElmer, Cat# 6005225), and target cells (HEK293, VeroE6/TMPRSS2 or Calu-3/DSP1-7 cells) were reseeded at a density of 1,000,000 cells/2 ml/well in 6-well plates. On day 4 (48 h.p.i.), target cells were incubated with EnduRen live cell substrate (Promega, Cat# E6481) for 3 h and then detached, and 32,000 target cells were added to a 96-well plate with effector cells. RL activity was measured at the indicated time points using a Centro XS3 LB960 (Berthhold Technologies). To measure the surface expression level of S protein, effector cells were stained with rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-112048, 1:100) or mouse anti-SARS-CoV-2 S monoclonal antibody (clone 1A9, GeneTex, Cat# GTX632604, 1:100). Normal rabbit IgG (SouthernBiotech, Cat# 0111-01, 1:100) or purified mouse IgG1 isotype control antibody (clone MG1-45, BioLegend, Cat# 401401, 1:100) or was used as negative controls, and APC-conjugated goat anti-mouse or rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 115-136-146, 1:50 or Cat# 111-136-144, 1:50) was used as a secondary antibody. Surface expression level of S proteins was analysed using FACS Canto II (BD Biosciences) and FlowJo software v10.7.1 (BD Biosciences). Gating strategy for flow cytometry is shown in Supplementary Fig. 1. RL activity was normalized to the MFI of surface S proteins, and the normalized values are shown as fusion activity.
Coculture experiment
One day before transfection, effector cells (i.e., S-expressing cells) were seeded on the cover glass put in 12-well plate, and target HEK293-ACE2/TMPRSS2 cells were prepared at a density of 1.0 x 105 cells in a 12 well plate. To prepare effector cell, HEK293 cells were cotransfected with the expression plasmids for the parental D614G S, Delta S, Omicron S (500 ng) and pEGFP-C1 (500 ng) using PEI Max (Polysciences, Cat# 24765-1). To prepare target cells, HEK293 cells and HEK293-ACE2/TMPRSS2 cells were transfected with pmCherry-C1 (1000 ng). At 24 h post-transfection, target cells were detached and cocultured with effector cells. At 24 h post-coculture (at 48 h post-transfection), cells were fixed with 4% paraformaldehyde in PBS (Nacalai Tesque, cat# 09154-85) for 15 m at room temperature. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, Cat# H3570). The coverslips were mounted on glass slides using Fluoromount-G (Southern Biotechnology, Cat# 0100-01) with Hoechst 33342 and observed using an A1Rsi confocal microscope (Nikon). The size of syncytium (yellow area) was measured using Fiji software v2.0.0-rc-69/1.52p (ImageJ)46.
Western blot
Western blotting was performed as previously described2,19. To quantify the level of the cleaved S2 protein in the cells, the harvested cells were washed and lysed in lysis buffer [25 mM HEPES (pH 7.2), 20% glycerol, 125 mM NaCl, 1% Nonidet P40 substitute (Nacalai Tesque, Cat# 18558-54), protease inhibitor cocktail (Nacalai Tesque, Cat# 03969-21)]. After quantification of total protein by protein assay dye (Bio-Rad, Cat# 5000006), lysates were diluted with 2 × sample buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 12% β-mercaptoethanol, 20% glycerol, 0.05% bromophenol blue] and boiled for 10 m. Then, 10 μl samples (50 μg of total protein) were subjected to Western blotting. To quantify the level of the cleaved S2 protein in the virions, 900 μl of the culture medium containing the pseudoviruses was layered onto 500 μl of 20% sucrose in PBS and centrifuged at 20,000 × g for 2 h at 4°C. Pelleted virions were resuspended in 1× NuPAGE LDS sample buffer (Thermo Fisher Scientific, Cat# NP0007) containing 2% β-mercaptoethanol, and the lysed virions were subjected to Western blotting. For protein detection, the following antibodies were used: mouse anti-SARS-CoV-2 S monoclonal antibody (clone 1A9, GeneTex, Cat# GTX632604, 1:10,000), rabbit anti-SARS-CoV-2 N monoclonal antibody (clone HL344, GeneTex, Cat# GTX635679, 1:5,000), rabbit anti-beta actin (ACTB) monoclonal antibody (clone 13E5, Cell Signalling, Cat# 4970, 1:5,000), mouse anti-alpha tubulin (TUBA) monoclonal antibody (clone DM1A, Sigma-Aldrich, Cat# T9026, 1:10,000), horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 711-035-152, 1:10,000) and HRP-conjugated donkey anti-mouse IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 715-035-150, 1:10,000). Chemiluminescence was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat# 34095) or Western BLoT Ultra Sensitive HRP Substrate (Takara, Cat# T7104A) according to the manufacturers’ instructions. Bands were visualized using an Amersham Imager 600 (GE Healthcare), and the band intensity was quantified using Image Studio Lite v5.2 (LI-COR Biosciences) or ImageJ v2.2.0. Uncropped blots are shown in Supplementary Fig. 2.
Animal experiments
Syrian hamsters (male, 4 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan). Baseline body weights were measured before infection. For the virus infection experiments, hamsters were euthanized by intramuscular injection of a mixture of 0.15 mg/kg medetomidine hydrochloride (Domitor®, Nippon Zenyaku Kogyo), 2.0 mg/kg midazolam (Dormicum®, FUJIFILM Wako Chemicals) and 2.5 mg/kg butorphanol (Vetorphale®, Meiji Seika Pharma). The B.1.1 virus, Delta or Omicron (104 TCID50 in 100 µl) were intranasally infected under anaesthesia. Oral swabs were daily collected under anaesthesia with isoflurane (Sumitomo Dainippon Pharma). Body weight, enhanced pause (Penh, see below) and subcutaneous oxygen saturation (SpO2, see below) were monitored at 1, 3, 5 and 7 d.p.i. Lung tissues were collected at 1, 3, 5 and 7 d.p.i, and viral RNA levels in lung tissues were determined by qRT-PCR. These tissues were also used for histopathological analysis (see below).
Histopathological analysis
Histopathological analysis was performed as described in the previous study2. Briefly, excised animal tissues were fixed with 4% paraformaldehyde in PBS, and processed for paraffin embedding. The paraffin blocks were sectioned with 3-µm-thickness and then mounted on silane-coated glass slides (MAS-GP, Matsunami). H&E staining was performed according to a standard protocol. For IHC, an Autostainer Link 48 (Dako) was utilized. The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 m at 97°C to activate, and a mouse anti-SARS-CoV-2 N monoclonal antibody (R & D systems, Clone 1035111, Cat# MAB10474-SP, 1:400) was used as a primary antibody. The sections were sensitized using EnVision FLEX (Agilent) for 15 m and visualised by peroxidase-based enzymatic reaction with 3,3’-diaminobenzidine tetrahydrochloride as substrate for 5 m.
Histopathological scoring of lung lesion
Histopathological scoring was performed as described in the previous study2. Briefly, pathological features including bronchitis or bronchiolitis, hemorrhage or congestion, alveolar damage with epithelial apoptosis and macrophage infiltration, presence of type II pneumocytes, and the area of the presence of large type II pneumocytes were evaluated by certified pathologists and the degree of these pathological findings were arbitrarily scored using four-tiered system as 0 (negative), 1 (weak), 2 (moderate), and 3 (severe). Total histopathological score is the sum of these five indices.
Lung function test
Respiratory parameters were measured by using a whole-body plethysmography system (DSI) according to the manufacturer's instructions. In brief, a hamster was placed in an unrestrained plethysmography chamber and allowed to acclimatize for 30 s, then, data were acquired over a 3-m period by using FinePointe Station and Review softwares v2.9.2.12849 (STARR). The state of oxygenation was examined by measuring percutaneous oxygen saturation (SpO2) using pulse oximeter, MouseOx PLUS (STARR). SpO2 was measured by attaching a measuring chip to the neck of hamsters sedated by sedated by 0.25 mg/kg medetomidine hydrochloride.
Viral genome sequencing analysis
The sequences of the working viruses were verified by viral RNA-sequencing analysis. Viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen, Cat# 52906). The sequencing library for total RNA-sequencing was prepared using NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Cat# E7530). Paired-end, 150-bp sequencing was performed using MiSeq (Illumina) with MiSeq reagent kit v3 (Illumina, Cat# MS-102-3001). Sequencing reads were trimmed using fastp v0.21.047 and subsequently mapped to the viral genome sequences of a lineage A isolate (strain WK-521; GISIAD ID: EPI_ISL_408667)40 using BWA-MEM v0.7.1748. Variant calling, filtering, and annotation were performed using SAMtools v1.949 and snpEff v5.0e50.
For the clinical isolates [an Omicron isolate (strain TY38-873; GISAID ID: EPI_ISL_7418017), a Delta isolate (strain TKYTK1734; GISAID ID: EPI_ISL_2378732)2 and a D614G-bearing B.1.1 isolate (strain TKYE610670; GISAID ID: EPI_ISL_479681)2], the detected variants that are present in the original sequences were excluded. Information on the detected mutations is summarized in Supplementary Table 2.
Statistics and reproducibility
Statistical significance was tested using a two-sided Student's t-test or a two-sided Mann–Whitney U-test unless otherwise noted. The tests above were performed using Excel software v16.16.8 (Microsoft) or Prism 9 software v9.1.1 (GraphPad Software).
In the time-course experiments using hamsters (Fig. 3h), to evaluate the difference between experimental conditions thorough all over timepoints, a multiple regression analysis including experimental conditions as explanatory variables and timepoints as qualitative control variables was performed. The P value was calculated by a two-sided Wald test. Subsequently, family-wise error rates were calculated by Holm method. The analyses above were performed in R v3.6.3.
In Fig. 3e–3g, photographs shown are the representative areas of two independent experiments by using 3 hamsters at each timepoint. In Fig. 2b–2d and Extended Data Fig. 3, 4b, assays were performed in triplicate. Photographs shown are the representatives of 20 fields of view taken for each sample.