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. Author manuscript; available in PMC: 2026 Feb 24.
Published in final edited form as: Nature. 2022 Nov 2;612(7940):540–545. doi: 10.1038/s41586-022-05482-7

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

Ryuta Uraki 1,2,*, Peter J Halfmann 3,*, Shun Iida 4,*, Seiya Yamayoshi 1,2,*, Yuri Furusawa 1,2, Maki Kiso 1, Mutsumi Ito 1, Kiyoko Iwatsuki-Horimoto 1, Sohtaro Mine 4, Makoto Kuroda 3, Tadashi Maemura 3, Yuko Sakai-Tagawa 1, Hiroshi Ueki 1,2, Rong Li 5, Yanan Liu 5, Deanna Larson 5, Shuetsu Fukushi 6, Shinji Watanabe 7, Ken Maeda 8, Andrew Pekosz 9, Ahmed Kandeil 10, Richard J Webby 10, Zhongde Wang 5, Masaki Imai 1,2,, Tadaki Suzuki 4,, Yoshihiro Kawaoka 1,2,3,
PMCID: PMC12927073  NIHMSID: NIHMS2142991  PMID: 36323336

Summary paragraph

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)1–4. Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral entry, raising concerns that the replication capacity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here, we evaluated the replicative ability and pathogenicity of BA.4 and BA.5 isolates in wild-type Syrian hamsters, human ACE2 (hACE2) transgenic hamsters, and hACE2 transgenic mice. we observed no obvious differences among BA.2, BA.4, and BA.5 isolates in growth ability or pathogenicity in rodent models, 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 clinical isolates have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2.

Introduction

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 were 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. A preliminary data suggests that BA.4 and BA.5 may be more transmissible than the other Omicron variants5.

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 clinical data in humans1–4,6–8. 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 entry. 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 Omicron9–11. However, the biological properties of BA.4 and BA.5 isolates are unknown. Here, we assessed the replicative capacity and pathogenicity of BA.4 and BA.5 isolated from COVID-19 patients in established COVID-19 animal models.

Results

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 model12–14 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 105 PFU of BA.2 (NCD1288) or B.1.617.2 (hCoV-19/USA/WI-UW-5250/2021: UW5250). Intranasal infection with B.1.617.2 resulted in remarkable 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.2, BA.4, or BA.5 isolates gained weight over the 10-day experiment, similar to the mock-infected animals. Compared with the B.1.617.2-infected group, significant differences in weight changes were observed in the BA.4 (TY41-703)- and BA.5 (COR-22-063113, TY41-702, or TY41-704)-infected groups at some timepoints, but not the BA.4 (HP30386)-infected group. No significant differences in weight changes were found among the BA.2-, BA.4-, and BA.5-infected groups. 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 (Fig. 1b). 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 compared with the mock-infected group at any timepoint after infection. In contrast, infection with B.1.617.2 caused remarkable changes in Penh and Rpef at 5 dpi.

Figure 1. The replication and pathogenicity of BA.2, BA.4, and BA.5 isolates in wild-type hamsters.

Figure 1.

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a,b, Wild-type Syrian hamsters 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), B.1.617.2 (UW5250), or PBS (mock). a, Body weights of virus-infected (n =5) and mock-infected hamsters (n =7) were monitored daily for 10 days. b, Pulmonary function analyses in virus-infected (n =5) and mock-infected hamsters (n =7). Penh and Rpef were measured by using whole-body plethysmography. Data are presented as the mean ± s.e.m. Data were analyzed by using a two-way ANOVA followed by Tukey’s multiple comparisons. c, Virus replication in infected Syrian hamsters. Hamsters (n =10) were intranasally inoculated with 105 PFU in 30 μL of BA.2 (NCD1288), BA.4 (HP30386), BA.5(COR-22-063113), or B.1.617.2 (UW5250) and euthanized at 3 and 6 dpi for virus titration (n =5/day). Virus titers in the nasal turbinates and lungs were determined by performing plaque assays with Vero E6-TMPRSS2-T2A-ACE2 cells. Vertical bars show the mean ± s.e.m. Points indicate data from individual hamsters. The lower limit of detection is indicated by the horizontal dashed line. Data were analyzed by using a one-way ANOVA with Tukey’s multiple comparisons test (titers in the lungs at 3 dpi and nasal turbinates at 3 and 6 dpi) or the Kruskal-Wallis test followed by Dunn’s test (titers in the lungs at 6 dpi). P values of < 0.05 were considered statistically significant. Data are from one experiment.

We next assessed levels of infection in the respiratory tract of wild-type hamsters infected with 105 PFU of BA.4 (HP30386), BA.5 (COR-22-063113), BA.2 (NCD1288), or B.1.617.2 (UW5250) (Fig. 1c). BA.4, BA.5, and BA.2 replicated in the lungs and nasal turbinates of the infected animals with no significant differences in viral titers at both timepoints examined. However, at 3 dpi, the virus titers were significantly lower in the respiratory tract of animals infected with BA.4 or BA.5, compared with animals infected with B.1.617.2 [mean differences in viral titer = 4.7 and 2.9 log10 (PFU/g) and 0.95 and 0.94 log10 (PFU/g) for the viral titers in the lungs and nasal turbinates, respectively]. At 6 dpi, the virus titers in the lungs and nasal turbinates of the BA.4- or BA.5-infected group were also 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], although the differences in the lungs were not statistically significant between the BA.5- and B.1.617.2-infected groups. 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.

Histopathology in infected hamsters

Syrian hamsters inoculated with BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113), or B.1.617.2 (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. 2a). 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. 2a). 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. 2a). Histopathological scores of inflammation in the alveoli were comparable among the BA.2-, BA.4-, and BA.5-inoculated groups at 6 dpi, whereas worse scores were obtained for the B.1.617.2-inoculated group at the same timepoint (Fig. 2b). 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 clear decrease in antigen-positive cells over time (Fig. 2a and 2c). 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. 2a and 2d); the distribution of viral RNA, determined by in situ hybridization, was similar for all three inoculated groups (Fig. 2a). 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. 2a, 2c and 2d).

Figure 2. Histopathological findings of in hamsters inoculated with SARS-CoV-2 Omicron sublineage viruses.

Figure 2.

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Wild-type Syrian hamsters (n =5, each per group) were inoculated with 105 PFU of BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113), or B.1.617.2 (UW5250) viruses and sacrificed at 3 and 6 dpi for histopathological examinations. Representative images of the bronchi/bronchioles and alveoli are shown. Upper rows, hematoxylin and eosin (H&E) staining. Middle rows, immunohistochemistry that detects the SARS-CoV-2 nucleocapsid protein. Lower rows, in situ hybridization targeting the nucleocapsid gene of SARS-CoV-2. Scale bars, 100 μm. b, Histopathological scores of inflammation in the alveoli. The scores were determined based on the percentage of alveolar inflammation in a given area of a pulmonary section collected from each animal in each group by using the following scoring system: 0, no inflammation; 1, affected area (≤1%); 2, affected area (>1%, ≤10%); 3, affected area (>10%, ≤50%); 4, affected area (>50%). An additional point was added when pulmonary edema and/or alveolar hemorrhage was observed. Therefore, the scores for individual animals ranged from 0 to 5. c, d, Immunohistochemistry scores of bronchial/bronchiolar regions (c) and alveolar regions (d). The scores were determined based on the percentage of viral antigen-positive cells, detected by immunohistochemistry, in a high-power field by using the following scoring system: 0, no positive cells; 1, positive cells (≤10%); 2, positive cells (>10%, ≤25%); 3, positive cells (>25%, ≤50%); 4, positive cells (>50%). Immunohistochemistry scores for each animal were calculated for bronchial/bronchiolar and alveolar regions, respectively, as a total score for the three high-power fields with the highest positivity rate on the section; the scores for individual animals ranged from 0 to 12. Vertical bars show the mean ± s.e.m. Points indicate data from individual hamsters. Data were analyzed by using a one-way ANOVA with Tukey’s multiple comparisons test. P values of < 0.05 were considered statistically significant. Data are from one experiment.

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.

BA.4 and BA.5 infection in hACE2 rodents

We investigated the replication and pathogenicity of BA.4 and BA.5 by using a more susceptible rodent models, that is, hACE2-expressing transgenic hamsters 15. B.1.617.2 infection in hACE2-expressing hamsters resulted in marked body weight loss by 5 dpi (Fig. 3a) and 100% mortality at 5 dpi (Fig. 3b). By contrast, the hamsters infected with BA.2 (NCD1288), BA.4 (HP30386), or BA.5 (COR-22-063113) exhibited less weight loss than those infected with B.1.617.2 (Fig. 3a) and all of them survived (Fig. 3b). At 5 dpi, BA.4, BA.5, and BA.2 replicated in the respiratory organs of the infected hamsters with no significant differences in viral titers. The viral titers in the nasal turbinates of hamsters infected with BA.4 or BA.5 were significantly lower than those of hamsters infected with B.1.617.2 [mean differences in viral titer =2.5 and 3.3 log10 (PFU/g), respectively]. The lung titers in the BA.4- or BA.5-infected groups were also substantially lower than those in the B.1.617.2-infected group [mean differences in viral titer =4.4 and 3.2 log10 (PFU/g), respectively], although these differences did not reach statistical significance (Fig. 3c). These results indicate that the replicative abilities of BA.4 and BA.5 are similar to those of BA.2, and are attenuated compared with those of B.1.617.2 in hACE2-expressing hamsters, consistent with our findings in wild-type hamsters.

Figure 3. The replication and pathogenicity of BA.4 and BA.5 in hACE2-expressing hamsters.

Figure 3.

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a-c, hACE2-expressing Syrian hamsters (n =4) were intranasally inoculated with 105 PFU in 30 μL of BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113) or B.1.617.2 (UW5250). Body weights (a) and survival (b) were monitored daily for 5 days. The data are presented as the mean percentages of the starting weight ± s.e.m. Body weight data were analyzed by using a two-way ANOVA followed by Tukey’s multiple comparisons. c, Infected hamsters were euthanized at 5 dpi for virus titration (BA.2, n =4; BA.4, n =4; BA.5, n =4; B.1.617.2, n =3). Virus titers in the nasal turbinates and lungs were determined by performing plaque assays with Vero E6-TMPRSS2-T2A-ACE2 cells. Vertical bars show the mean ± s.e.m. Points indicate data from individual hamsters. The lower limit of detection is indicated by the horizontal dashed line. Data were analyzed by using a one-way ANOVA with Tukey’s multiple comparisons test (titers in the nasal turbinates) or the Kruskal-Wallis test followed by Dunn’s test (titers in the lungs). P values of < 0.05 were considered statistically significant. Data are from one experiment.

To further investigate the replication and pathogenicity of BA.4 and BA.5 isolates in vivo, we inoculated hACE2-expressing mice with BA.4 (HP30386) or BA.5 (COR-22-063113) 16,17. Mice infected with B.1.617.2 experienced marked body weight loss (Extended Data Fig. 1a) and 100% mortality at 10 dpi (Extended Data Fig. 1b). By contrast, all mice infected with BA.2 (NCD1288), BA.4 (HP30386), or BA.5 (COR-22-063113) survived, consistent with our observations in hACE2-expressing hamsters. Although the animals infected with BA.2 showed substantial body weight loss, the animals infected with BA.4 or BA.5 gained body weight (Extended Data Fig. 1a and b). The lung titers of mice infected with BA.2, BA.4, or BA.5 were significantly lower than those of mice infected with B.1.617.2 at both 2 dpi and 5 dpi (Extended Data Fig. 1c). No marked differences in viral titers in the lungs were observed among BA.2-, BA.4-, and BA.5-infected animals at either timepoint examined. We also histopathologically analyzed the lungs of hACE2-expressing mice infected with BA.4 or BA.5. This evaluation revealed no obvious peribronchial/peribronchiolar inflammation in the lungs of virus-inoculated hACE2-expressing mice (Extended Data Fig. 1d). At 2 dpi, no obvious pneumonia was detected in any groups, although small foci of inflammatory cell infiltration in alveolar regions were observed in some animals (Extended Data Fig. 1d). Notably, at 5 dpi, mild and scattered inflammation in the alveoli was observed not only in the B.1.617.2-inoculated group, but also in all the Omicron sublineage virus-inoculated groups. The extent of inflammatory cell infiltration varied among the individual animals; however, there were no clear differences among the groups (Extended Data Fig. 1d).

Overall, our data show that the pathogenicity of BA.4 and BA.5 is similar to that of BA.2, and is attenuated compared to that of B.1.617.2 in hamsters and mice.

The replicative fitness of BA.4 and BA.5

To further compare the replicative fitness of BA.4 and BA.2, 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 assessed by Next Generation Sequencing (NGS) to determine the proportion of each virus. 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. 4a).

Figure 4. Replicative fitness of BA.4 and BA.5 compared with that of BA.2 in hamsters.

Figure 4.

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a,b, BA.4 (HP30386) and BA.2 (NCD1288) (a) or BA.5 (COR-22-063113) and BA.2 (NCD1288) (b) were mixed at an equal ratio on the basis of their infectious titers, and the virus mixture (total 2 × 105 PFU in 60 μL) was intranasally inoculated into wild-type hamsters (total n =10, n =5/day). Nasal turbinates and lungs were collected from the infected animals at 2 and 4 dpi and analyzed using next generation sequencing (NGS). The proportion of BA.2 and BA.4 or BA.2 and BA.5 was calculated from the five amino acid differences in the S gene between BA.2 and BA.4 or BA.2 and BA.5. c, d, BA.2 (NCD1288) and BA.5 (COR-22-063113) were mixed at a 1:1 (c) or 3:1 (d) ratio on the basis of their infectious titers, and the virus mixture (total 2 × 105 PFU in 60 μL) was intranasally inoculated into hACE2 transgenic hamsters (n =5/group). Nasal turbinates and lungs were collected from the infected animals at 3 dpi and analyzed by using NGS. The proportion of BA.2 and BA.5 was calculated from the five amino acid differences in the S gene between the two viruses. Shown are the relative proportions of BA.2 and BA.4 (a) or BA.2 and BA.5 (b-d) in the infected animals. Data are from one experiment.

We next compared the viral fitness of BA.5 (COR-22-063113) and BA.2 (NCD1288) in wild-type hamsters (Fig. 4b). 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. A similar trend was observed for hACE2 hamsters inoculated with 2 x 105 PFU of a mixture of BA.2 (NCD1288) and BA.5 (TY41-702) at ratios of 1:1 or 3:1 (Fig. 4c and 4d). At 3 dpi, the proportion of BA.5 had increased in the nasal turbinates and lungs of all infected animals compared to that in each inoculum for either ratio, except for the lung sample from hamster 1. Importantly, BA.5 became dominant in the lungs of eight (#s 2, 3, 4, 5, 6, 7, 8, and 9) and in the nasal turbinates of seven (#s 1, 2, 3, 4, 5, 7, and 10) of the ten animals (Fig. 4c and 4d).

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.

Discussion

Our data indicate that the sublineages BA.4 and BA.5 of SARS-CoV-2 Omicron variants are less pathogenic than B.1.617.2 in wild-type and hACE2-expressing hamsters, and hACE2-expressing mice. We observed that the clinical isolates of BA.4 and BA.5 were limited in their replication in the lungs of hamsters and mice, compared to a B.1.617.2 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.21,4,18. These findings indicate that Omicron variants replicate less efficiently in the alveolar epithelial cells of rodents compared with B.1.617.2, which might contribute to the lower disease severity of Omicron variants seen in animal models.

We observed that BA.4 and BA.5 replicate efficiently in the nasal turbinates of hamsters, but not in the lungs (Fig.1c), similar to BA.1 and BA.2 1–4. In human nasal epithelial cultures, Omicron and Delta variants have been reported to replicate with similar efficiency; however, in lower airway organoids and Calu-3 lung cells, Omicron variants replicate to lower titers than Delta variants 19. Viral entry into host cells is mediated by the S protein. The S protein must be proteolytically activated by the human protein transmembrane protease serine 2 (TMPRSS2) at the cell surface or by cathepsin proteases within the endosomal cell compartment 20–23. Interestingly, recent studies have suggested that the entry of Omicron variants is less dependent on TMPRSS2 activities but more sensitive to a cathepsin inhibitor compared with Delta variants 19,24. This finding suggests that the TMPRSS2/cathepsin usage of the S protein may affect the viral replication efficiency in the lungs of humans and hamsters.

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 entry of a pseudovirus carrying the BA.1 S protein in K18-hACE2 transgenic mice 10. Notably, a recent preprint article (Kimura et al.25) reported that hamsters infected with 104 TCID50 of a recombinant chimeric virus possessing the BA.4 or BA.5 S gene in the background of a BA.2 strain exhibited about 10% body weight loss and severe lung inflammation whereas hamsters infected with parental recombinant BA.2 virus gained weight and experienced less inflammation in the lungs, 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 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 (TY41-702) 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 animals, 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 similar replication and pathogenicity to the strains tested; (2) our study was conducted in healthy young animals; however, individuals aged 65 years and above and people with underlying medical conditions are at increased risk of severe COVID-19. Therefore, it remains unclear whether our data reflect the clinical outcomes in high-risk groups for COVID-19; and (3) our study was also performed in immunologically naïve animals; however, many people have already acquired SARS-CoV-2-specific immunity through natural infection and/or vaccination. Therefore, it remains uncertain whether our data reflect the clinical outcome in patients with immunity to SARS-CoV-2. Recent preprint papers have reported that the severity of BA.5 in humans was similar to that of BA.1 or BA.2, consisting with our data 26–28; however, more clinical data from patients infected with BA.4 or BA.5 are needed to corroborate the findings in the rodent models.

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 clinical isolates.

Materials and Methods

Cells.

VeroE6/TMPRSS2 (JCRB 1819) cells29 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 °C 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)4,30, 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)1,31 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.

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-332, 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.2(NCD1288) (gender: 2 males and 2 females), 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.

For co-infection studies in wild-type hamsters, 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. For co-infection studies in K18-hACE2 transgenic female hamsters, BA.2 (NCD1288) was mixed with BA.5 (COR-22-063113) at a 1:1 or 3:1 ratio on the basis of their titers, and each virus mixture (total 2 x 105 PFU in 60 μL) was inoculated into five K18-hACE2 transgenic hamsters. At 3 dpi, all five animals were euthanized and nasal turbinates and lungs were collected.

Experimental infection of K18-hACE2 mice.

Ten-week-old female K18-hACE2 mice (Jackson Laboratory) were used in this study. Baseline body weights were measured before infection. Under isoflurane anesthesia, five mice per group were intranasally inoculated with 105 PFU (in 25 μL) of BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113), or B.1.617.2 (UW5250). Body weight was monitored daily for 10 days. For virological examination, five mice per group were intranasally infected with 105 PFU (in 25 μL) of BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113), BA.5(TY41-702), or B.1.617.2 (UW5250); 2 and 5 dpi, five animals were euthanized and lungs were collected. The virus titers in the 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 described1,4. 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.

Histopathological scores of inflammation in the alveolar regions were determined based on the percentage of alveolar inflammation in a given area of a pulmonary section collected from each animal in each group by using the following scoring system: 0, no inflammation; 1, affected area (≤1%); 2, affected area (>1%, ≤10%); 3, affected area (>10%, ≤50%); 4, affected area (>50%). An additional point was added when pulmonary edema and/or alveolar hemorrhage was observed. Therefore, histopathological scores of inflammation in the alveoli for individual animals ranged from 0 to 5. Immunohistochemistry scores were determined based on the percentage of viral antigen-positive cells, detected by immunohistochemistry, in a high-power field by using the following scoring system: 0, no positive cells; 1, positive cells (≤10%); 2, positive cells (>10%, ≤25%); 3, positive cells (>25%, ≤50%); 4, positive cells (>50%). Immunohistochemistry scores for each animal were calculated for bronchial/bronchiolar and alveolar regions, respectively, as a total score for the three high-power fields with the highest positivity rate on the section; the scores for individual animals ranged from 0 to 12.

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 added33,34. 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 primers35 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.

Extended Data

Extended Data Figure 1. The replication and pathogenicity of BA.4 and BA.5 in K18-hACE2 mice.

Extended Data Figure 1

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a-c, K18-hACE2 mice (n =5) were intranasally inoculated with 105 PFU in 25 μL of BA.2 (NCD1288), BA.4 (HP30386), BA.5 (COR-22-063113), B.1.617.2 (UW5250), or PBS. Body weights (a) and survival (b) were monitored daily for 10 days. The data are presented as the mean percentages of the starting weight ± s.e.m. c, Infected mice were euthanized at 2 and 5 dpi for virus titration (n =5/group). Virus titers in the lungs were determined by performing plaque assays with Vero E6-TMPRSS2-T2A-ACE2 cells. Vertical bars show the mean ± s.e.m. Points indicate data from individual mice. The lower limit of detection is indicated by the horizontal dashed line. Data were analyzed by using a one-way ANOVA with Tukey’s multiple comparisons test (titers at 2 dpi and 5 dpi). P values of < 0.05 were considered statistically significant. d, Infected mice were euthanized at 2 and 5 dpi for histopathological examinations. Representative images of the lungs are shown. Upper rows, the lungs at low magnification. Middle rows, bronchial/bronchiolar regions. Lower rows, alveolar regions. Scale bars: 500 μm in the upper rows, 100 μm in the middle and lower rows. Data are from one experiment.

Extended Data Table 1.

Amino acid mutations in the clinical isolates of BA.2, BA.4, and BA.5 used in this study

Protein Position Virus (Pango lineage)
Early SARS-CoV-2 (A) hCoV-19/Japan/UT-NCD1288-2N-2022 (BA.2) hCoV-19/USA/
MD/HP30386/ 2022 (BA.4)		 hCoV-19/Japan/TY41-703/2022 (BA.4) SARS-CoV-2/human/USA/COR-22-063113/2022 (BA.5) hCoV-19/Japan/TY41-702/2022 (BA.5) hCoV-19/Japan/TY41-704/2022 (BA.5)
ORF1a 135 Ser Arg Arg Arg Arg Arg Arg
141 Lys Lys dela del Lys Lys Lys
142 Gly Gly del del Gly Gly Gly
143 Phe Phe del del Phe Phe Phe
556 Gln Gln Gln Gln Gln Lys Gln
842 Thr Ile Ile Ile Ile Ile Ile
1307 Gly Ser Ser Ser Ser Ser Ser
2139 Ala Ala Ala Thr Ala Ala Ala
2153 Thr Thr Ile Thr Thr Thr Thr
2648 Thr Thr Thr Thr Thr Thr Ile
3027 Leu Phe Phe Phe Phe Phe Phe
3090 Thr Ile Ile Ile Ile Ile Ile
3201 Leu Phe Leu Leu Leu Leu Leu
3255 Thr Ile Ile Ile Ile Ile Ile
3395 Pro His His His His His His
3515 Pro Pro Pro Pro Leu (62%) Pro Pro
3557 Phe Phe Phe Phe Phe Phe Leu
3650 Phe Phe Phe Phe Phe Leu (85%) Phe
3675 Ser del del del del del del
3676 Gly del del del del del del
3677 Phe del del del del del del
3753 Phe Val(20%) Phe Phe Phe Phe Phe
3829 Leu Leu Leu Phe (71%) Phe (33%) Leu Leu
4159 Thr Thr Ile Thr Thr Thr Thr
4391 Leu Phe(19%) Leu Leu Leu Leu Leu
ORF1b 314 Pro Leu Leu Leu Leu Leu Leu
662 Gly Val Gly Gly Gly Gly Gly
959 Ser Pro Ser Ser Ser Ser Ser
1156 Met Met Met Met Met Ile Met
1161 Pro Ser(27%) Pro Pro Pro Pro Pro
1315 Arg Cys Cys Cys Cys Cys Cys
1566 Ile Val Val Val Val Val Val
2163 Thr Ile Ile Ile Ile Ile Ile
2497 Leu Leu Phe (73%) Leu Leu Leu Leu
2513 Ala Val(18%) Ala Ala Ala Ala Ala
S S1 (1-684) SP (1-12) 3 Val Val Gly Gly Val Val Val
NTD (13-304) 19 Thr Ile Ile Ile Ile Ile Ile
24 Leu del del del del del del
25 Pro del del del del del del
26 Pro del del del del del del
27 Ala Ser Ser Ser Ser Ser Ser
69 His His del del del del del
70 Val Val del del del del del
76 Thr Thr Thr Thr Ile Thr Thr
142 Gly Asp Asp Asp Asp Asp Asp
213 Val Gly Gly Gly Gly Gly Gly
RBD (331-527) 339 Gly Asp Asp Asp Asp Asp Asp
371 Ser Phe Phe Phe Phe Phe Phe
373 Ser Pro Pro Pro Pro Pro Pro
375 Ser Phe Phe Phe Phe Phe Phe
376 Thr Ala Ala Ala Ala Ala Ala
405 Asp Asn Asn Asn Asn Asn Asn
408 Arg Ser Ser Ser Ser Ser Ser
417 Lys Asn Asn Asn Asn Asn Asn
440 Asn Lys Lys Lys Lys Lys Lys
452 Leu Leu Arg Arg Arg Arg Arg
477 Ser Asn Asn Asn Asn Asn Asn
478 Thr Lys Lys Lys Lys Lys Lys
484 Glu Ala Ala Ala Ala Ala Ala
486 Phe Phe Val Val Val Val Val
493 Gln Arg Gln Gln Gln Gln Gln
498 Gln Arg Arg Arg Arg Arg Arg
501 Asn Tyr Tyr Tyr Tyr Tyr Tyr
505 Tyr His His His His His His
SD1/2 (528-684) 614 Asp Gly Gly Gly Gly Gly Gly
655 His Tyr Tyr Tyr Tyr Tyr Tyr
679 Asn Lys Lys Lys Lys Lys Lys
681 Pro His His His His His His
682 Arg Arg Arg Arg Arg Arg Trp (10%)
S2 (685-1210) 764 Asn Lys Lys Lys Lys Lys Lys
796 Asp Tyr Tyr Tyr Tyr Tyr Tyr
954 Gln His His His His His His
969 Asn Lys Lys Lys Lys Lys Lys
ORF3a 223 Thr Ile Ile Ile Ile Ile Ile
E 9 Thr Ile Ile Ile Ile Ile Ile
14 Val Val del (57%) Val del (48%) Val Val
M 3 Asp Asp Asp Asp Asn Asn Asp
19 Gln Glu Glu Glu Glu Glu Glu
63 Ala Thr Thr Thr Thr Thr Thr
131 Arg Arg Arg Arg Arg Arg Thr
ORF6 57 Pro Pro Pro Pro Pro Pro Leu
61 Asp Leu Leu Leu Asp Asp Asp
ORF7b 10 Pro Ser Pro Pro Pro Pro Pro
11 Leu Leu Phe Phe Leu Leu Leu
27 Glu del Glu Glu Glu Glu Glu
28 Asn del Asn Asn Asn Asn Asn
29 Ala del Ala Ala Ala Ala Ala
N 13 Pro Leu Leu Leu Leu Leu Leu
31 Glu del del del del del del
32 Arg del del del del del del
33 Ser del del del del del del
136 Glu Glu Glu Glu Glu Asp Glu
151 Pro Pro Ser Ser Pro Pro Pro
203 Arg Lys Lys Lys Lys Lys Lys
204 Gly Arg Arg Arg Arg Arg Arg
413 Ser Arg Arg Arg Arg Arg Arg
ORF10 29 Gln Gln Gln Stop (22%) Gln Gln Gln
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Compared with the reference strain Wuhan/Hu-1/2019, mutaions are shown. The spike protein is composed of two subunits, S1 and S2. Compared with BA.2 (hCoV-19/Japan/UT-NCD1288-2N/2022), spike protein mutations are shown in bold red for BA.4 and in blod blue for BA.5. ORF, open reading frame; SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; SD1/2, subdomain 1 and 2; E, Envelope; M, Membrane; and N, Nucleocapsid.

dela: deletion

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).

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.

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

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.

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

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