Within-Host Fitness and Antigenicity Shift Are Key Factors Influencing the Prevalence of Within-Host Variations in the SARS-CoV-2 S GeneXi, Hua, Jiang
et alViruses (2025) 17 (3)
Abstract: Within-host evolution plays a critical role in shaping the diversity of SARS-CoV-2. However, understanding the primary factors contributing to the prevalence of intra-host single nucleotide variants (iSNVs) in the viral population remains elusive. Here, we conducted a comprehensive analysis of over 556,000 SARS-CoV-2 sequencing data and prevalence data of different SARS-CoV-2 S protein amino acid mutations to elucidate key factors influencing the prevalence of iSNVs in the SARS-CoV-2 S gene. Within-host diversity analysis revealed the presence of mutational hotspots within the S gene, mainly located in NTD, RBD, TM, and CT domains. Additionally, we generated a single amino acid resolution selection status map of the S protein. We observed a significant variance in within-host fitness among iSNVs in the S protein. The majority of iSNVs exhibited low to no within-host fitness and displayed low alternate allele frequency (AAF), suggesting that they will be eliminated due to the narrow transmission bottleneck of SARS-CoV-2. Notably, iSNVs with moderate AAFs (0.06-0.12) were found to be more prevalent than those with high AAFs. Furthermore, iSNVs with the potential to alter antigenicity were more prevalent. These findings underscore the significance of within-host fitness and antigenicity shift as two key factors influencing the prevalence of iSNVs in the SARS-CoV-2 S gene.
Serological Assays Reveal No Evidence of Natural SARS-CoV-2 Infection in US CattleRamasamy, Quraishi, Mukherjee
et alMicroorganisms (2025) 13 (3)
Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to pose a significant threat to public health. Notably, SARS-CoV-2 demonstrates the capacity to infect various non-human animal species, including both captive and free-living animals. Earlier experimental studies revealed low susceptibility of domestic cattle (Bos taurus) to ancestral B.1 lineage; however, recent experimental findings indicate greater permissiveness of cattle to SARS-CoV-2 Delta variant. While some studies detected evidence of SARS-CoV-2 infection in cattle in Italy, Germany, India, and Nigeria, currently, there is no evidence of SARS-CoV-2 infections in US cattle. We have investigated over 600 samples, including pre-pandemic and pandemic cattle sera collected from Pennsylvania for the presence of SARS-CoV-2 antibodies. Since serological tests have inherent problems of false positives and negatives, we conducted a comprehensive assessment of multiple serological assays. As there are no known SARS-CoV-2 positive cattle serum samples, we used hyperimmune serum raised in cattle with SARS-CoV-2-spike receptor binding domain (RBD) as positive control for the test validation. We found that pseudovirus neutralization assays with a luciferase reporter system can produce false positive results, and care must be taken to interpret serological diagnosis using these assays. We found no serological evidence of natural SARS-CoV-2 infection or transmission among cattle in the US. This study underscores the importance of robust evaluation when employing serological assays for SARS-CoV-2 detection in cattle populations.
The Use of Heterologous Antigens for Biopanning Enables the Selection of Broadly Neutralizing Nanobodies Against SARS-CoV-2Aripov, Zaykovskaya, Mechetina
et alAntibodies (Basel) (2025) 14 (1)
Abstract: Background: Since the emergence of SARS-CoV-2 in the human population, the virus genome has undergone numerous mutations, enabling it to enhance transmissibility and evade acquired immunity. As a result of these mutations, most monoclonal neutralizing antibodies have lost their efficacy, as they are unable to neutralize new variants. Antibodies that neutralize a broad range of SARS-CoV-2 variants are of significant value in combating both current and potential future variants, making the identification and development of such antibodies an ongoing critical goal. This study discusses the strategy of using heterologous antigens in biopanning rounds. Methods: After four rounds of biopanning, nanobody variants were selected from a phage display library. Immunochemical methods were used to evaluate their specificity to the S protein of various SARS-CoV-2 variants, as well as to determine their competitive ability against ACE2. Viral neutralization activity was analyzed. A three-dimensional model of nanobody interaction with RBD was constructed. Results: Four nanobodies were obtained that specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and exhibit neutralizing activity against various SARS-CoV-2 strains. Conclusions: The study demonstrates that performing several rounds of biopanning with heterologous antigens allows the selection of nanobodies with a broad reactivity spectrum. However, the fourth round of biopanning does not lead to the identification of nanobodies with improved characteristics.
Neutralization and spike stability of JN.1-derived LB.1, KP.2.3, KP.3, and KP.3.1.1 subvariantsLi, Faraone, Hsu
et almBio (2025)
Abstract: During the summer of 2024, coronavirus disease 2019 (COVID-19) cases surged globally, driven by variants derived from JN.1 subvariants of severe acute respiratory syndrome coronavirus 2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and spike stability of these subvariants-LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2 and increased resistance to elevated temperatures. Molecular modeling suggests that DelS31 enhances the NTD-receptor-binding domain (RBD) interaction, favoring the RBD down conformation and reducing accessibility to ACE2 and specific nAbs. Moreover, DelS31 introduces an N-linked glycan at N30, shielding the NTD from antibody recognition. These findings underscore the role of NTD mutations in immune evasion, spike stability, and viral infectivity, highlighting the need to consider DelS31-containing antigens in updated COVID-19 vaccines.IMPORTANCEThe emergence of novel severe acute respiratory syndrome coronavirus 2 variants continues to pose challenges for global public health, particularly in the context of immune evasion and viral stability. This study identifies a key N-terminal domain (NTD) mutation, DelS31, in JN.1-derived subvariants that enhances neutralizing antibody escape while reducing infectivity and cell-cell fusion. The DelS31 mutation stabilizes the spike protein conformation, limits S1 shedding, and increases thermal resistance, which possibly contribute to prolonged viral persistence. Structural analyses reveal that DelS31 enhances NTD-receptor-binding domain interactions by introducing glycan shielding, thus decreasing antibody and ACE2 accessibility. These findings emphasize the critical role of NTD mutations in shaping viral evolution and immune evasion, underscoring the urgent need for updated coronavirus disease 2019 vaccines that account for these adaptive changes.
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