Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 clinical isolates

doi:10.21203/rs.3.rs-1820048/v1

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Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 clinical isolates

https://doi.org/10.21203/rs.3.rs-1820048/v1

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published 02 Nov, 2022

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The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern (VOC). Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral infectivity, raising concerns that the infectivity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here, we evaluated the replicative ability and pathogenicity of authentic BA.4 and BA.5 isolates in wild-type Syrian hamsters and human ACE2 (hACE2) transgenic hamsters. In contrast to recent data with a recombinant chimeric virus possessing the spike protein of BA.4/BA.5 in the background of a BA.2 strain, we observed no obvious differences among BA.2, BA.4, and BA.5 isolates in growth ability or pathogenicity in hamsters, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2. Our study highlights the importance of using authentic isolates when evaluating virological features.

The Omicron (B.1.1.529 lineage) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for coronavirus disease 2019 (COVID-19) (WHO dashboard https://covid19.who.int), has spread rapidly worldwide and become the dominant variant circulating globally. Omicron variants have been classified into at least five different sublineages: BA.1, BA.2, BA.3, BA.4, and BA.5. As of June 2022, BA.2 has become the most prevalent sublineage, replacing the previously dominant BA.1 sublineage, in most countries around the world; however, BA.4 and BA.5 are predominant in South Africa and Botswana, and Portugal (https://covariants.org/per-variant). In addition, the prevalence of BA.5 is increasing rapidly in many European countries. Preliminary data suggest that BA.4 and BA.5 may be more transmissible than the other Omicron variants⁶.

Recently, we and others have demonstrated that both the BA.1 and BA.2 sublineages are less pathogenic in animal models than previously circulating variants of concern (VOC), consistent with preliminary clinical data in humans^1-4,7-9. Compared with BA.2, both BA.4 and BA.5 possess three additional changes (69-70del, L452R, and F486V) and one reversion mutation (R493Q) in their spike (S) proteins, which play a pivotal role in viral infectivity. The BA.1 subvariants also do not have the two additional mutations L452R, and F486V. Previous studies have shown that the L452R mutation increases the fusogenicity and infectivity of SARS-CoV-2 variants including Omicron^10,11. Notably, a recent study reported that a recombinant chimeric virus possessing the BA.4/BA.5 S gene in the background of a BA.2 strain is more pathogenic than the parental recombinant BA.2 virus⁵. This finding suggests that the infectivity and pathogenicity of BA.4 and BA.5 may be higher than those of BA.1 and BA.2. However, the functional activity of BA.4 and BA.5 clinical isolates from patients is unknown. Here, we assessed the replicative capacity and pathogenicity of authentic BA.4 and BA.5 isolated from COVID-19 patients in established COVID-19 animal models.

BA.4 and BA.5 infection in hamsters

To characterize the BA.4 and BA.5 variants in vivo, we amplified the following BA.4 and BA.5 clinical isolates: hCoV-19/USA/MD/HP30386/2022 (BA.4: HP30386) and SARS-CoV-2/human/USA/COR-22-063113/2022 (BA.5: COR-22-063113), propagated in Vero E6-TMPRSS2-T2A-ACE2 cells; and hCoV-19/Japan/TY41-703/2022 (BA.4: TY41-703), hCoV-19/Japan/TY41-702/2022 (BA.5: TY41-702), and hCoV-19/Japan/TY41-704/2022 (BA.5: TY41-704), propagated in VeroE6/TMPRSS2 cells. We confirmed that all five isolates contained the five additional amino acid changes (i.e., 69-70del, L452R, F486V, and reversion mutation R493Q), compared to a BA.2 isolate (hCoV-19/Japan/UT-NCD1288-2N/2022; NCD1288) (Extended Data Table 1). However, the two BA.4 isolates (HP30386 and TY41-703) had a V3G substitution in the signal peptide region of S, in addition to the five mutations. One BA.5 isolate (COR-22-063113) encoded a T76I substitution in the N-terminal domain (NTD), and another BA.5 isolate (TY41-704) possessed a mixed viral population encoding either R or W at position 682.

We first evaluated the pathogenicity of the two BA.4 and three BA.5 isolates in wild-type Syrian hamsters, a well-established small animal model^12-14 for the study of COVID-19. Syrian hamsters were intranasally inoculated with 10⁵ plaque-forming units (PFU) of BA.4 (HP30386 or TY41-703) or BA.5 (COR-22-063113, TY41-702, or TY41-704). For comparison, additional hamsters were infected with clinical isolates of BA.2 (10⁵ PFU of NCD1288) and B.1.617.2 (10⁵ PFU of hCoV-19/USA/WI-UW-5250/2021 (Delta: UW5250)). Intranasal infection with B.1.617.2 resulted in significant body weight loss by 7 days post-infection (dpi) (-7.6%) (Fig. 1a), consistent with our previous observations¹. By contrast, all or most of the animals infected with the BA.4 or BA.5 isolates gained weight over the 10-day experiment, similar to BA.2 (NCD1288)-infected animals. Compared to the BA.2 (NCD1288)-infected group, no differences in weight changes were observed in the BA.4- and BA.5-infected groups, with the exception of a difference in weight change between the BA.5 (COR-22-063113)- and the BA.2 (NCD1288)-infected groups at 5 dpi. We also assessed pulmonary function in the infected hamsters by measuring Penh and Rpef, which are surrogate markers for bronchoconstriction and airway obstruction, respectively, by using a whole-body plethysmography system. Infection with the BA.2 (NCD1288), BA.4 (HP30386 or TY41-703), or BA.5 (COR-22-063113, TY41-702, or TY41-704) isolates did not cause substantial changes in either Penh or Rpef at any timepoint post-infection compared to the mock-infected group. In contrast, infection with B.1.617.2 caused a remarkable increase in Penh and a significant decrease in Rpef in comparison with BA.2 (Fig. 1b).

We next assessed levels of infection in the respiratory tract of wild-type Syrian hamsters (Fig. 1c). Hamsters were intranasally infected with 10⁵ PFU of BA.4 (HP30386), BA.5 (COR-22-063113), BA.2 (NCD1288), or B.1.617.2 (Delta: UW5250); at 3 and 6 dpi, the animals were sacrificed, and nasal turbinates and lungs were collected for virus titration. The virus titers were determined by use of plaque assays on Vero E6-TMPRSS2-T2A-ACE2 cells. At 3 dpi, BA.4 (HP30386), BA.5 (COR-22-063113), and BA.2 (NCD1288) replicated in the lungs [mean titers = 5.0, 6.8, and 5.5 log₁₀(PFU/g), respectively] and nasal turbinates [mean titers = 8.4, 8.4, and 8.3 log₁₀(PFU/g), respectively] of the infected animals with no significant differences in viral titers. However, the virus titers in the lungs and nasal turbinates were significantly lower in the respiratory tract of animals infected with BA.4, BA.5, or BA.2, compared to animals infected with B.1.617.2 [mean differences in viral titer = 4.7, 2.9, and 4.2 log₁₀(PFU/g) and 0.95, 0.94, and 1.1 log₁₀(PFU/g) for the viral titers in the lungs and nasal turbinates, respectively]. At 6 dpi, the lung titers in the BA.4 (HP30386)-infected group [mean titers = 2.2 log₁₀(PFU/g)] were significantly lower than those in the B.1.617.2-infected groups [mean titers = 5.8 log₁₀(PFU/g)]; no other marked differences in viral titers in these respiratory organs were found among the BA.4 (HP30386)-, BA.5 (COR-22-063113)-, and BA.2 (NCD1288)-infected groups. The virus titers in the lungs and nasal turbinates of the BA.4-, BA.5-, or BA.2-infected group were lower than those in the B.1.617.2-infected group [mean differences in viral titer = 1.3-3.6 log₁₀(PFU/g) and 1.3-2.4 log₁₀(PFU/g) in the lungs and nasal turbinates, respectively]. These results demonstrate that the replicative abilities of BA.4 and BA.5 are similar to those of BA.2, and attenuated compared with those of B.1.617.2.

We then investigated the infectivity and pathogenicity of BA.4 and BA.5 by using a more susceptible hamster model, that is, transgenic hamsters expressing hACE2 (line M41) under the control of an epithelial cytokeratin-18 promoter¹⁵. Intranasal inoculation of hamsters with 10⁵ PFU of B.1.617.2 (UW5250) virus caused significant weight loss within 5 dpi (Fig. 1d) and resulted in 100% mortality at 5 dpi (Fig. 1e). In contrast, all animals infected with BA.4 (HP30386) or BA.5 (COR-22-063113) survived. At 5 dpi, the virus titers in the nasal turbinates of hamsters infected with BA.4 or BA.5 [mean titers = 3.1 and 4.0 log₁₀(PFU/g), respectively] were significantly lower than those infected with B.1.617.2 [mean titer = 6.4 log₁₀(PFU/g), respectively]. The lung titers in the BA.4- and BA.5-infected groups were also lower than those in the B.1.617.2-infected group [mean differences in viral titer = 4.4, 3.2 log₁₀(PFU/g), respectively] (Fig. 1f). These results indicate that BA.4 and BA.5 have lower replicative abilities than B.1.617.2 in hACE2 transgenic hamsters, consistent with wild-type hamsters.

Overall, our data show that lung infection and disease after BA.4 and BA.5 infection is attenuated compared to that after infection with B.1.617.2 in hamsters.

Histopathological findings in the airways and lungs of SARS-CoV-2 Omicron sublineage-inoculated Syrian hamsters

Syrian hamsters inoculated with BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113), or B.1.617.2 (Delta: UW5250) were euthanized at 3 and 6 dpi, respectively, for histopathological analysis of the airways and lungs. In animals inoculated with any of the Omicron sublineage viruses, no obvious inflammation was observed in the peripheral airways or alveolar region at 3 dpi (Fig. 2). At 6 dpi, infiltration of inflammatory cells (i.e., mononuclear cells and neutrophils) was observed with these omicron viruses in the peribronchial and peribronchiolar regions (Fig. 2). Although small foci of inflammatory cell infiltration into the alveolar spaces were observed in some animals at 6 dpi, there was no evidence of pneumonia at either timepoint examined. By contrast, in B.1.617.2-inoculated animals, peribronchial/peribronchiolar inflammation was observed at 3 dpi, and massive infiltration of inflammatory cells with hemorrhage was prominent in the alveolar regions at 6 dpi (Fig. 2). Immunohistochemistry revealed that SARS-CoV-2 antigen was present on the bronchial/bronchiolar epithelium in the BA.2-, BA.4-, or BA.5-inoculated animals at 3 dpi, with a significant decrease in antigen-positive cells over time (Fig. 2). In the BA.2-, BA.4-, or BA.5-inoculated animals, viral antigen was rarely detected in the alveolar regions at either timepoint examined (Fig. 2); the distribution of viral RNA, determined by in situ hybridization, was similar for all three inoculated groups (Fig. 2). In contrast, viral antigen and RNA were detected not only in the bronchial/bronchiolar regions but also in the alveolar regions at 3 dpi with the B.1.617.2 virus, both of which decreased over time (Fig. 2).

In summary, there were no significant differences in the distribution of viral antigen/RNA-positive cells and extension of inflammation, which was limited to the peribronchial/peribronchiolar regions, among the BA.2-, BA.4-, and BA.5-inoculated groups. These Omicron sublineage viruses also showed attenuated pathogenicity compared with the B.1.617.2 virus in the hamster model. These findings are consistent with previous reports on the BA.2 variant⁴.

The replicative fitness of BA.4 and BA.5 compared with that of BA.2 in hamsters

To further compare the replicative fitness of BA.4 and BA.2, ten wild-type hamsters were intranasally inoculated with 2 x 10⁵ PFU of a mixture (1:1) of BA.4 (HP30386) and BA.2 (NCD1288). At 2 and 4 dpi, the nasal turbinates and lungs of the infected hamsters were harvested and assessed by Next Generation Sequencing (NGS) to determine the proportion of each virus. The proportion was calculated on the basis of the differences between these two viruses across five regions in the S protein. At 2 dpi, the proportion of BA.4 in the lungs and nasal turbinates of all five infected animals had slightly increased compared to that in the inoculum, except for the lung sample from hamster #2. At 4 dpi, the proportion of BA.4 had increased in the nasal turbinates of all five animals; however, the proportion in the lungs increased in only two of the five animals (Fig. 3a).

We next compared the viral fitness of BA.5 (COR-22-063113) and BA.2 (NCD1288) in hamsters. The proportion was calculated on the basis of the differences between these two viruses across five regions in the S protein (Fig. 3b). BA.5 was dominant in the nasal turbinates of all five infected animals at both 2 and 4 dpi. The lung samples also showed a greater proportion of BA.5 at both 2 and 4 dpi, except for the sample from animal #4 at 2 dpi. Taken together, these results suggest that BA.5 may have greater replicative fitness than BA.2. In addition, our data suggest that the viral fitness of BA.4 is comparable or slightly greater than that of BA.2.

Our data indicate that the sublineages BA.4 and BA.5 of SARS-CoV-2 Omicron variants are less pathogenic in both wild-type and hACE2 transgenic hamsters. We observed that the clinical isolates of BA.4 and BA.5 were limited in their infectivity of the lungs of hamsters compared to a prior SARS-CoV-2 variant isolate (Fig. 1a-c). Moreover, histopathological examination revealed that viral antigens were rarely present in the alveoli of hamsters after infection with BA.4 or BA.5 (Fig. 2). Similar findings were obtained with clinical isolates of BA.1 and BA.2^1,4,16. Thus, the limited infectivity of Omicron variants including BA.1, BA.2, BA.4, and BA.5 in the lung might contribute to the lower disease severity seen in animal models.

The BA.4 and BA.5 isolates, but not the BA.1 and BA.2 isolates, contain the L452R mutation in their S protein. A previous study demonstrated that the L452R mutation enhances the infectivity of a pseudovirus carrying the BA.1 S protein in K18-hACE2 transgenic mice¹¹. Notably, a recent paper (Kimura et al.⁵) reported that a recombinant chimeric virus possessing the BA.4 or BA.5 S gene in the background of a BA.2 strain is more pathogenic than the parental recombinant BA.2 virus in hamsters, suggesting that the S sequence differences between BA.2 and BA.4 or BA.5 are responsible for the difference in pathogenicity between these chimeric viruses; based on these data, Kimura et al. concluded that BA.4 and BA.5 are more pathogenic than BA.2. However, in authentic viruses isolated from patients, the genomes of BA.2, BA.4, and BA.5 differ at locations other than the S gene. Of the isolates we tested, the S protein sequence of the BA.5 isolate (TY41-703) was identical to that of the recombinant chimeric virus these authors tested (Extended Data Table 1). Infection with this BA.5 isolate did not cause substantial changes in either body weight or lung function in hamsters compared with the BA.2-infected group (Fig. 1a and b). Therefore, differences at locations other than the S gene could offset differences in the pathogenic potential of the S gene.

We note several limitations in this study: (1) In hACE2 transgenic hamsters, we characterized one strain of BA.4 (i.e., HP30386) and one strain of BA.5 (i.e., COR-22-063113). Therefore, additional studies may be required to determine whether other BA.4 and/or BA.5 strains exhibit infectivity and pathogenicity similar to the strains tested; (2) the in vivo competition assay was performed with the combination of BA.2 and BA.4 or BA.2 and BA.5. Since the prevalence of BA.4 and BA.5 is increasing all over the world, data on the comparative viral fitness of these two subvariants would be valuable; and (3) Since we do not have available clinical data, it is unclear whether attenuation of BA.4 or BA.5 in hamsters correlates with data in humans. Clinical data of patients infected with BA.4 or BA.5 are needed to corroborate the findings in the hamster model.

In summary, our data suggest that the sublineages BA.4 and BA.5 of SARS-CoV-2 Omicron variants have similar pathogenicity to that of the BA.2 sublineage in rodent models. Our results highlight the importance of evaluating viral replication and pathogenesis using authentic clinical isolates.

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Cells.

VeroE6/TMPRSS2 (JCRB 1819) cells¹⁷ were propagated in the presence of 1 mg/ml geneticin (G418; Invivogen) and 5 μg/ml plasmocin prophylactic (Invivogen) in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Fetal Calf Serum (FCS). Vero E6-TMPRSS2-T2A-ACE2 cells (provided by Dr. Barney Graham, NIAID Vaccine Research Center) were cultured in DMEM supplemented with 10% FCS, 10 mM HEPES pH 7.3, 100 U/mL of penicillin–streptomycin, and 10 μg/mL puromycin. VeroE6/TMPRSS2 and Vero E6-TMPRSS2-T2A-ACE2 cells were maintained at 37 ℃ with 5% CO₂. The cells were regularly tested for mycoplasma contamination by using PCR, and confirmed to be mycoplasma-free.

Viruses.

hCoV-19/Japan/UT-NCD1288-2N/2022 (BA.2; NCD1288)^4,18, hCoV-19/Japan/TY41-703/2022 (BA.4; TY41-703), hCoV-19/Japan/TY41-702/2022 (BA.5; TY41-702), hCoV-19/Japan/TY41-704/2022 (BA.5; TY41-704), and hCoV-19/USA/WI-UW-5250/2021 (B.1.617.2; UW5250)^1,19 were propagated in VeroE6/TMPRSS2 cells in VP-SFM (Thermo Fisher Scientific). hCoV-19/USA/MD/HP30386/2022 (BA.4; HP30386) and SARS-CoV-2/human/USA/COR-22-063113/2022 (BA.5; COR-22-063113) were propagated in Vero E6-TMPRSS2-T2A-ACE2 cells in VP-SFM (Thermo Fisher Scientific). TY41-703 (BA.4), COR-22-063113 (BA.5), and TY41-704 (BA.5) were subjected to next generation sequencing (NGS) (see Whole genome sequencing); amino acid differences between these viruses and the reference sequence (Wuhan/Hu-1/2019) are shown in Extended Data Table 1. All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo and the National Institute of Infectious Diseases, Japan, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan, or in BSL3 agriculture containment laboratories at the University of Wisconsin-Madison, which are approved for such use by the Centers for Disease Control and Prevention and by the US Department of Agriculture.

Animal experiments and approvals.

Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number PA19-75) and the Institutional Animal Care and Use Committee at the University of Wisconsin, Madison (assurance number V006426). Virus inoculations were performed under isoflurane, and all efforts were made to minimize animal suffering. In vivo studies were not blinded, and animals were randomly assigned to infection groups. No sample-size calculations were performed to power each study. Instead, sample sizes were determined based on prior in vivo virus challenge experiments.

Experimental infection of Syrian hamsters.

Six-week-old male wild-type Syrian hamsters (Japan SLC Inc., Shizuoka, Japan) were used in this study. Baseline body weights were measured before infection. Under isoflurane anesthesia, five hamsters per group were intranasally inoculated with 10⁵ PFU (in 30 μL) of BA.2 (NCD1288), BA.4 (HP30386 or TY41-703), BA.5 (COR-22-063113, TY41-702 or TY41-704), or B.1.617.2 (UW5250). Body weight was monitored daily for 10 days. For virological and pathological examinations, ten hamsters per group were intranasally infected with 10⁵ PFU (in 30 μL) of BA.2 (NCD1288), BA.4 (HP30386), BA.5(COR-22-063113), or B.1.617.2 (UW5250); 3 and 6 dpi, five animals were euthanized and nasal turbinates and lungs were collected. The virus titers in the nasal turbinates and lungs were determined by use of plaque assays on Vero E6-TMPRSS2-T2A-ACE2 cells.

For co-infection studies, BA.2 (NCD1288) was mixed with BA.4 (HP30386) or BA.5 (COR-22-063113) at an equal ratio on the basis of their titers, and the virus mixture (total 2 x 10⁵ PFU in 60 µL) was inoculated into ten wild-type hamsters. At 2 and 4 dpi, five animals were euthanized and nasal turbinates and lungs were collected.

The K18-hACE2 transgenic hamster lines (line M41) were developed by using a piggyBac-mediated transgenic approach. The K18-hACE2 cassette from the pK18-hACE2 plasmid was transferred into a piggyBac vector, pmhyGENIE-3²⁰, for pronuclear injection¹⁵. Then, 5- to 10-week-old K18-hACE2 homozygous transgenic hamsters, whose hACE2 expression was confirmed, were intranasally inoculated with 10⁵ PFU (in 30 μL) of BA.4 (HP30386) (gender: 1 male and 3 females), BA.5 (COR-22-063113) (gender: 1 male and 3 females), or B.1.617.2 (UW5250) (gender: 2 males and 2 females). Body weight was monitored daily for 5 days. At 5 dpi, the animals were euthanized and nasal turbinates and lungs were collected. The virus titers in the nasal turbinates and lungs were determined by use of plaque assays on Vero E6-TMPRSS2-T2A-ACE2 cells.

Lung function.

Respiratory parameters were measured by using a whole-body plethysmography system (PrimeBioscience) according to the manufacturer’s instructions. In brief, infected hamsters were placed in the unrestrained plethysmography chambers and allowed to acclimate for 1 min before data were acquired over a 3-min period by using FinePointe software.

Histopathology

Histopathological examination was performed as previously described^1,4. Briefly, excised animal lungs were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and processed for paraffin embedding. The paraffin blocks were sliced into 3-µm-thick sections and mounted on silane-coated glass slides, followed by hematoxylin and eosin (H&E) stain for histopathological examination. Tissue sections were also processed for immunohistochemistry with a rabbit polyclonal antibody for SARS-CoV nucleocapsid protein (ProSpec; ANT-180, 1:500 dilution, Rehovot, Israel), which cross-reacts with SARS-CoV-2 nucleocapsid protein. Specific antigen-antibody reactions were visualized by staining with 3,3’-diaminobenzidine tetrahydrochloride and using the Dako Envision system (Dako Cytomation; K4001, 1:1 dilution, Glostrup, Denmark). To detect SARS-CoV-2 RNA, in situ hybridization was performed by using an RNA scope 2.5 HD Red Detection kit (Advanced Cell Diagnostics, Newark, California) with an antisense probe targeting the nucleocapsid gene of SARS-CoV-2 (Advanced Cell Diagnostics) and following the manufacturer's instructions.

Whole genome sequencing

Viral RNA was extracted by using a QIAamp Viral RNA Mini Kit (QIAGEN). The whole genome of SARS-CoV-2 was amplified by using a modified ARTIC network protocol in which some primers were replaced or added^21,22. Briefly, viral cDNA was synthesized from the extracted RNA by using a LunarScript RT SuperMix Kit (New England BioLabs). The DNA was then amplified by performing a multiplexed PCR in two pools using the ARTIC-N5 primers²³ and the Q5 Hot Start DNA polymerase (New England BioLabs). The DNA libraries for Illumina NGS were prepared from pooled amplicons by using a QIAseq FX DNA Library Kit (QIAGEN) and were then analyzed by using the iSeq 100 System (Illumina). To determine the sequence of TY41-703 (BA.4), COR-22-063113 (BA.5) and TY41-704 (BA.5), the reads were assembled by the CLC Genomics Workbench (version 22, Qiagen) with the Wuhan/Hu-1/2019 sequence (GenBank accession no. MN908947) as a reference. The sequences of TY41-703 (BA.4), COR-22-063113 (BA.5), and TY41-704 (BA.5) were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database with Accession IDs: EPI_ISL_13512668, EPI_ISL_13512579, and EPI_ISL_13512582, respectively. For the analysis of the ratio of BA.2 to BA.4 or BA.5 after co-infection, the ratio of BA.2 to BA.4 or BA.5 was calculated from the 5 amino acid differences in the S gene between the two viruses. Samples with more than 300 read-depths were analyzed.

Statistical analysis.

GraphPad Prism software was used to analyze all of the data. Statistical analysis included the Kruskal-Wallis test followed by Dunn’s test, and ANOVA with post-hoc tests. Differences among groups were considered significant for P values < 0.05.

Data availability.

All data supporting the findings of this study are available in the paper. There are no restrictions in obtaining access to the primary data. The source data are provided with this paper. The sequences of the viruses [BA.4 (TY41-703), BA.5 (COR-22-063113), and BA.5 (TY41-704)] were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database with the Accession IDs; EPI_ISL_13512668, EPI_ISL_13512579, and EPI_ISL_13512582, respectively.

Code availability.

No code was used in the course of the data acquisition or analysis.

Acknowledgements

We thank Susan Watson for scientific editing. We also thank Kyoko Yokota, Naoko Mizutani, Kengo Kajiyama, Yuko Sato, and Seiya Ozono for technical assistance. Vero E6-TMPRSS2-T2A-ACE2 cells were provided by Dr. Barney Graham, NIAID Vaccine Research Center. This work was supported by a Research Program on Emerging and Re-emerging Infectious Diseases (JP20fk0108412, and JP22fk0108637), a Project Promoting Support for Drug Discovery (JP20nk0101632), the Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125002) from the Japan Agency for Medical Research and Development (AMED), the National Institutes of Allergy and Infectious Diseases Center for Research on Influenza Pathogenesis (HHSN272201400008C), and the Center for Research on Influenza Pathogenesis and Transmission (CRIPT) (75N93021C00014).

Author contributions

R.U., P.J.H, S.I., S.Y., Y.F., M.Kiso, M.Ito, K.I.-H., S.M., M.Kuroda, T.M. , H.U., M.Imai, and T.S performed the hamster infection experiments, titrated virus in tissues, and analyzed pathology. S.Y. performed next generation sequencing. Z.W., R.L., Y.L., and D.L. generated human ACE2 hamsters. S.Y., Y.S.-T., S.F., S.W., K.M., A.P., A.K., and R.J.W propagated and/or sequenced viruses. R.U., P.J.H, S.I., S.Y., M.Imai, T.S. and Y.K. obtained funding, conceived the study, and/or supervised the research. R.U., Y.F., M.Imai and Y.K. wrote the initial draft, with all other authors providing editorial comments.

Competing interests

Y.K. has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi & Co. LTD, Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio. The remaining authors declare that they have no competing interests.

Corresponding author

Correspondence to Yoshihiro Kawaoka, D.V.M., Ph.D., [email protected]; Tadaki Suzuki, M.D., Ph.D., [email protected]; and Masaki Imai, D.V.M., Ph.D., [email protected]

Yes there is potential Competing Interest. Y.K. has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi & Co. LTD, Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio. The remaining authors declare that they have no competing interests.

Urakietal.ExtendedDataTable1.xlsx
Extended Data Table 1
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Uraki et al.nr-editorial-policy-checklist
Urakietal.nrreportingsummaryBA.4X5.pdf
Uraki et al. nr-reporting-summary

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Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 clinical isolates

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