SARS-CoV-2, which causes COVID-19, continues to circulate around the world, making it necessary to study the impact of rapidly emerging mutations on escape from neutralizing antibodies and pathogenesis. While RBD mutations are well characterized, mutations in the N-terminal domain (NTD) of the spike protein remain comparatively understudied despite their relevance to antibody recognition. This study investigates two phenotypically distinct SARS-CoV-2 mutants, which exhibited differences in plaque morphology on Vero cells. Whole-genome sequencing via Illumina identified a novel 12-nucleotide insertion in the spike NTD. This insertion induced a frameshift, introducing five new amino acids potentially altering viral behavior, receptor interactions, and antibody detection in ELISAs. The study further explores the pathogenicity of these variants in a hamster model. These findings underscore the importance of monitoring NTD mutations, which may contribute to immune evasion and influence therapeutic antibody efficacy, highlighting gaps in current research on SARS-CoV-2 evolution.

Introduction. Alongshan virus is a representative of the unclassified group of Jingmenviruses (Flaviviridae), which is detected in Ixodes persulcatus, Ixodes ricinus ticks and various mosquito species in Russia, China, Finland and France. Unlike traditional orthoflaviviruses, the Alongshan virus genome is represented by 4 positive-sense RNA segments. The first and third segments of the genome encode proteins homologous to proteins of the replicative machinery of orthoflaviviruses, the remaining segments encode putative structural proteins that have no known homologues: segment 2 — VP1a (envelope protein), VP1b and NuORF; segment 4 — VP2 (capsid) and VP3 (membrane). Human cases of Alongshan virus-associated disease have been described. The aim of this study is to develop a system for expression and purification of recombinant VP2 and hydrophilic site VP3 proteins to test their antigenic properties. Materials and methods. Miass527 strain of Alongshan virus was used to produce hyperimmune mouse sera and recombinant proteins in a bacterial expression system. Bioinformatic analysis of sequences encoding target proteins and genetically engineered cloning were carried out in this study. Western blotting and enzyme immunoassay (ELISA) were performed to control the results. Results. Recombinant proteins of Alongshan virus have been used in a laboratory diagnostic test system to determine the presence of antibodies to the virus. The obtained recombinant VP2 protein is able to detect antibodies in all tested sera of infected mice, as well as antibodies in human sera both in Western blotting and in enzyme immunoassay. At the same time, antibodies to the recombinant region of VP3 protein were detected irregularly in antiviral immune sera. Conclusion. The detection of antibodies to Alongshan virus in patients confirms the necessity for further investigation of this group of viruses.

Variants of the SARS-CoV-2 virus continue to evolve. Rapidly emerging mutations, particularly in the Spike glycoprotein, may increase transmissibility and infectivity, and also lead to evasion of the immune response. The Spike protein contains two immunodominant epitopes: NTD and RBD. Numerous neutralizing antibodies against NTD are found in patient sera, and most of these target “supersite” epitopes. Consequently, mutations outside this region receive less attention. Our work continues the investigation of the effect of insertions outside the “supersite” on the phenotypic properties of SARS-CoV-2. Previously we characterized two closely related SARS-CoV-2 Delta variants differing by a 12-nucleotide insertion in the N-terminal domain of the Spike glycoprotein. In the present study, we modeled these two Spike glycoproteins and used them to perform spatial alignment analyses of S-protein structures in complex with antibodies against the NTD to assess the potential impact of the insertion on virus binding by neutralizing antibodies. We identified four potential scenarios, how the insertion can impact the binding of neutralizing antibodies targeting the NTD. Our study confirms the importance of investigating rapidly emerging mutations in the NTD, not only within the “supersite” but also beyond it. These mutations may contribute to immune evasion; consequently, their investigation is crucial for assessing the efficacy of commercially available monoclonal antibodies and vaccine preparations.