Virological characteristics of SARS-CoV-2 Omicron BA.5.2.48

Abstract

With the prevalence of sequentially-emerged sublineages including BA.1, BA.2 and BA.5, SARS-CoV-2 Omicron infection has transformed into a regional epidemic disease. As a sublineage of BA.5, the BA.5.2.48 outbroke and evolved into multi-subvariants in China without clearly established virological characteristics. Here, we evaluated the virological characteristics of two isolates of the prevalent BA.5.2.48 subvariant, DY.2 and DY.1.1 (a subvariant of DY.1). Compared to the normal BA.5 spike, the double-mutated DY.1.1 spike demonstrates efficient cleavage, reduced fusogenicity and higher hACE2 binding affinity. BA.5.2.48 demonstrated enhanced airborne transmission capacity than BA.2 in hamsters. The pathogenicity of BA.5.2.48 is greater than BA.2, as revealed in Omicron-lethal H11-K18-hACE2 rodents. In both naïve and convalescent hamsters, DY.1.1 shows stronger fitness than DY.2 in hamster turbinates. Thus regional outbreaking of BA.5.2.48 promotes the multidirectional evolution of its subvariants, gaining either enhanced pathogenicity or a fitness in upper airways which is associated with higher transmission.

Keywords: BA.5; Omicron; SARS-CoV-2; animal models; pathogenicity; spike.

Figure 1

Evolution, prevalence and replicative kinetics of DY.1 and DY.2. (A) Evolutionary origin of BA.5.2.48 sublineages, including DY.1 and its sublineages DY.1.1, DY.2, DY.3 and DY.4. Synonymous mutations in nucleotides and amino acid mutations are shown in regular and italic font, respectively. (B) Prevalence of DY.1 (blue) and DY.2 (orange) in China for one year from September 2022 (2022.09) to August 2023 (2023.08). (C, D) Replicative kinetics of DY.1.1 and DY.2. The viral loads (C) and viral titers (D) of the Omicron BA.1, BA.2 and BA.5.2.48 isolates were determined in Vero E6 cells, HeLa^hACE2+ cells and Calu-3 cells. The significance of the differences in replication between BA.2 (dark red) and BA.1 (green), DY.1.1 (blue) or DY.2-CC1 (orange) are indicated above the lines by the asterisks in colors corresponding to the individual virus. The significance of the differences in replication between DY.1.1 and DY.2 is indicated by the black asterisks below the lines.

Figure 2

Characteristics of the spikes of BA.5.2.48 subvariants. (A) Spike-mediated infection by pseudovirus assay. The infectivity of the strains relative to that of the negative control (NC) is shown. (B) Spike-mediated cell-cell fusion determined by the DSP method. Significant differences between the spikes of previous epidemic variants (including D614G in black, BA.2 in red and BA.5 in orange) and the spikes of BA.5-A570S (A570S, yellow) or BA.5-A570S-K147E (SE, green) are revealed above the lines with asterisks in colors corresponding to the individual variants. (C) Spike procession in authentic SARS-CoV-2 virions, including BJ05P14, Delta, BA.2 and the two BA.5 subvariants DY.1.1 and DY.2. (D, E) Comparative binding affinities, including data (D) and response curves (E), of the BA.5, BA.5-A570S and BA.5-A570S-K147E spike to hACE2.

Figure 3

In vivo virological characteristics of BA.5.2.48 in hamsters. (A) Airborne transmission of BA.5.2.48 and BA.2 in hamsters. Viral titers of the lungs and turbinates (Tb) of the inoculated donor and the acceptor hamsters were indicated in red and black, respectively. WT hamsters were intranasally inoculated with BA.2, DY.1.1 or DY.2; K18-hACE2 hamsters were intranasally inoculated with BA.2, DY.1.1, DY.2 or WT. Five hamsters per group were used to measure the respective parameters (C, D, F–H). Four hamsters per group were euthanized at 3 DPI and used for data collection (E, I). The data (in C, D, F–H) of the mock, BA.2, DY.1.1, DY.2 and WT groups are shown in gray, red, blue, orange and green, respectively (as demonstrated in C and F). (B) After challenge, the WT hamsters survived, while the K18-hACE2 hamsters died. (C, G) Body weights of infected WT (C) or K18-hACE2 (G) hamsters. Significant differences between the mock group and each infected group are revealed above the lines using asterisks in colors corresponding to the respective infected group. The significant differences between DY.1.1 and DY.2 are shown with black asterisks above the lines. (D, H). Relative viral RNA loads in the nasal lavages of infected WT (D) or K18-hACE2 (H) hamsters and baseline viral loads are indicated by dotted gray lines. (E, I) Viral titers in the lungs (dark red) or turbinates (cray) of infected WT (E) or K18-hACE2 (I) hamsters. (F) Percentage survival of infected K18-hACE2 hamsters. (J) H&E staining of the lungs of infected K18-hACE2 hamsters. The selected areas (revealed by a black rectangle in each major panel) are enlarged and shown in inset panels; scale bars: 500 μm (major panels) and 100 μm (inset panels). (K) Distribution of virus in the lungs of infected K18-hACE2 hamsters; scale bars: 200 μm.

Figure 4

In vivo virological characteristics of BA.5.2.48 in K18-hACE2 mice. Mice were intranasally inoculated with BA.2, DY.1.1 or DY.2, and the corresponding results (A–C) are shown in red, blue or orange, respectively (as shown in A). (A–C) Four mice per group were used to measure the percentage of survival (A), body weight (B) and relative viral RNA loads in oral swabs (C). Significant differences in (B) were revealed as in Figure 3B . (D) Four mice per group were euthanized at 3 DPI, after which the viral titers in the lungs (dark red) and turbinates (cray) were measured.

Figure 5

In vivo fitness of the BA.5.2.48 subvariants. (A) Competitive fitness of DY.1.1 (blue) and DY.2 (orange) in the lungs and turbinates of coinfected hamsters at 3 DPI (numbered #1 to #6) and 5 DPI (numbered #7 to #12); the proportions of DY.1.1 and DY.2 viral RNA in the inoculation mixture are indicated in the top left corner. (B) Fitness in nasal lavage fluid from coinfected hamsters (numbered #13 to #17) collected at 2, 4, 6 and 8 DPI. (C) One day after serum collection at 21 DPI, the (mock-) convalescent hamsters were rechallenged with the DY.1.1 and DY.2 mixture, after which tissue samples were collected three days after rechallenge. (D) Relative fitness in the lungs (dark red) or turbinates (cyan) of DY.1.1 compared to DY.2 in mock-, WT-, BA.1- and BA.2- convalesced hamsters. The ratio of DY.1.1 to DY.2 in inoculation is indicated by a dotted gray line (referred to as the inoculation ratio). The ratios of sample ratios to the inoculation ratio were used to determine the relative fitness. Median fitness was demonstrated by lines in boxes above (for fitness in the lungs) or below (for fitness in the turbinates). Significant differences between the convalescent group and the mock group were analyzed.