Population immune pressure from vaccination and prior infection continues to drive the evolution of SARS-CoV-2. Systematic characterization of RBD mutations under complex immune backgrounds is essential for understanding viral adaptation and evolutionary trajectories. Here, we applied a deep mutational scanning (DMS) to comprehensively map the neutralization escape landscape of the Omicron variant JN.1 and its descendant lineage XEC, under immune pressure from individuals who experienced Omicron breakthrough infections following three doses of inactivated vaccines. A neutralization escape map for the single amino acid substitutions in the RBD of JN.1 or XEC was generated, and the escape efficiency of each mutation was determined. The results show that RBD escape mutations are hierarchically organized: low-intensity signals are widespread, whereas high-intensity escape is confined to a few key sites. These escape mutations are not confined solely to the receptor-binding motif (RBM) but are broadly distributed across the entire RBD. Many escape sites could accommodate multiple amino acid substitutions. Integration of DMS data with genomic surveillance of circulating variants from 2024 to 2025 revealed significant overlap between experimentally identified escape sites and mutations observed in natural isolates. This overlap increased substantially in 2025, with site concordance rising from 27.17% and 26.81% to 45.09% and 47.10% for JN.1 and XEC, respectively. The natural prevalence of these escape mutations is further shaped by factors such as receptor-binding affinity, protein stability, and epistatic interactions. Overall, our findings suggest that SARS-CoV-2 antigenic evolution follows the pattern of multiple pathways within a constrained space, providing new insights into the adaptive mechanisms of Omicron-derived variants under hybrid immune pressure.

Objectives: Norovirus genotype GII.3[P12] strains have been an important pathogen for sporadic gastroenteritis infection. In previous studies of GII.3[P12], the number of specimens and time span are relatively small, which is difficult to truly reflect the infection and evolution of this type of norovirus. Here we report a molecular epidemiological study of the NoVs prevalent in Jiangsu between 2010 and 2019 to investigate the evolution of the GII.3[P12] strains in China. Methods: In this study 60 GII.3[P12] norovirus strains were sequenced and analyzed for evolution, recombination, and selection pressure using bioanalysis software. Results: The GII.3[P12] strains were continuously detected during the study period, which showed a high constituent ratio in males, among children aged 0–11 months and winter. A time-scaled evolutionary tree showed that both GII.P12 RdRp and GII.3 VP1 sequences were grouped into three major clusters (Cluster I-III). Most GII.3[P12] strains were mainly located in sub-cluster (SC) II of Cluster III. A SimPlot analysis identified GII.3[P12] strain to be as an ORF1-intragenic recombinant of GII.4[P12] and GII.3[P21]. The RdRp genes of the GII.3[P12] showed a higher mean substitution rate than those of all GII.P12, while the VP1 genes of the GII.3[P12] showed a lower mean substitution rate than those of all GII.3. Alignment of the GII.3 capsid sequences revealed that three HBGA binding sites of all known GII.3 strains remained conserved, while several amino acid mutations in the predicted antibody binding sites were detected. The mutation at 385 was within predicted antibody binding regions, close to host attachment factor binding sites. Positive and negative selection sites were estimated. Two common positively selected sites (sites 385 and 406) were located on the surface of the protruding domain. Moreover, an amino acid substitution (aa204) was estimated to be near the active site of the RdRp protein. These results suggested that evolution of the GII.3[P12] noroviruses was possibly driven by intergenic recombination and mutations in some key amino acid sites. Conclusions: The GII.3[P12] recombinant strain was dominant for the GII.P12 and GII.3 noroviruses in China, which is therefore a major concern for causing gastrointestinal infection.

Group A rotavirus (RVA) is the leading cause of acute gastroenteritis in infants and young children worldwide. To elucidate the molecular epidemiology of G2P[4] rotavirus in China and the protective effects of vaccines, whole-genome analysis of 13 G2P[4] RVA strains collected from China in 2022 was performed. Twelve strains possessed the typical DS-1-like genome constellation G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Only GS2265 possessed the genome constellation G2-P[4]-12-R2-C2-M2-A2-N2-T2-E1-H2. With the exception of the NSP4 segment of GS2265, all other sequences of the 13 G2P[4] RVA strains clustered within the same lineage on phylogenetic analysis. However, QD2210 and SX2205 were grouped into different branches compared to the other strains. In the VP7 antigenic epitopes, four residues differed from the RotaTeq G2 strain; specifically, A87T and D96N in the 7-1a region and S213D and S242N in the 7-1b region. Comparison of the current G2P[4] RVA strains circulating in China with those circulating globally revealed a high degree of sequence identity. High genetic variability among the newly characterized G2P[4] RVA strains suggest the strains evolve fast. Finally, our data suggest that the multivalent RotaTeq vaccine could have provided better protection than the monovalent Rotarix and LLR.