Identification of a GII.P21-GII.13 recombinant norovirus strain, mutations shift its binding spectra to host receptor glycans

Identification of a GII.P21-GII.13 recombinant norovirus strain, mutations shift its binding spectra to host receptor glycans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of a GII.P21-GII.13 recombinant norovirus strain, mutations shift its binding spectra to host receptor glycans yongxin Yu, Yunfei Chen, Zexian Zhou, Lei Dong, Miao Jin, Yongjie Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4986214/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract Norovirus is a pervasive pathogen that causes global outbreaks of viral gastroenteritis. Previous studies suggest that histo-blood group antigens (HBGAs) can interact with norovirus, facilitating its entry of host cells and significantly impacting its evolution. In this study, a complete genome of recombinant GII.13[GII.P21] norovirus from fecal samples was analyzed, revealing a weak effect of genomic recombination on the replication efficiency of GII.13[GII.P21]. Molecular dynamics simulations of GII.13 norovirus P proteins from 1978 to 2019 showed changes in binding capacity with HBGAs. Initially, GII.13 proteins bound A or B/H-type HBGAs, but subsequent residue mutations resulted in a loss of this binding capacity, favoring binding to the HBGA type I precursor (Lec) over A or B/H and Lewis antigens. norovirus recombination HBGAs binding affinity molecular dynamics simulation GII.13[P21] Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Norovirus is a highly contagious human pathogen that is accountable for the majority of viral gastroenteritis outbreaks on a global scale[ 1 ]. They are small, non-enveloped viruses of Caliciviridae family, with a single positive-strand RNA genome of approximately 7400 to 7700 nucleotides, typically comprising three open reading frames (ORFs) known as ORF1-3. ORF1 encodes a polyprotein of about 200 kDa, which can be processed to yield six non-structural proteins: P48 (NS1/2), NTPase (NS3), P22 (NS4), VPg (NS5), Pro (NS6), and Pol (NS7). ORF2 and ORF3 encode the major and minor structural capsid proteins, VP1 and VP2, respectively[ 2 ]. Genetic variations in the RNA-dependent RNA polymerase (RdRp) and VP1 regions categorize noroviruses into genogroups and genotypes[ 3 ]. Within each genogroup, norovirus can be further classified into diverse P-types and genotypes based on dual typing nomenclature[ 3 ]. The RdRp is responsible for replicating and synthesizing genomic RNA in norovirus, utilizing genomic RNA templates to synthesize antisense RNA. Genomic RNAs are subsequently generated through the synthesis of antisense RNAs. During genomic RNA replication, subgenomic RNA encoding ORF2 and ORF3 (3' co-terminal to the viral genome) is also synthesized[ 4 ]. In all caliciviruses , a conserved stem loop is positioned 6 nucleotides downstream of the subgenomic RNA start site, thereby ensuring the viability of norovirus. This stem-loop sequence is sufficient for initiating viral RNA synthesis by recombinant norovirus RdRp[ 5 ]. The icosahedral capsid of norovirus consists of ninety VP1 dimers, each VP1 protein contain a shell domain (S domain) and a protruding domain (P domain) that binds to host cell surface receptors[ 6 ]. Specifically, the P domain proteins bind to histo-blood group antigens (HBGAs), which serve as crucial recognition coreceptors or attachment factors for norovirus to enter human host cells. Notably, HBGAs, found in human erythrocytes, saliva, and epithelial cells, not only determine various blood types but also facilitate the binding and transmission of human norovirus[ 7 – 9 ]. X-ray crystal structure analysis of the structural domains of the GII.4 P variant, bound to different HBGAs, revealed a binding pocket in the P2 structural domain responsible for HBGA receptor binding[ 10 ]. Furthermore, the region directly beneath the HBGA binding pocket exhibited minimal variation, while the neighboring residues demonstrated moderate amino acid substitutions[ 11 ]. Different norovirus genotypes exhibit varying binding affinities to HBGAs[ 12 ]. The interaction of norovirus with HBGAs was characterized by saliva binding assay, revealing seven distinct binding modes. For instance, GII.4 variant US95/96 VLPs (VA387 and GrV) bound to saliva samples from individuals with different A/B/O blood groups[ 13 ]. GII.5 (MOH) and GII.3 (MxV and PiV) specifically bound to type A/B saliva but not type O or non-secretory saliva. On the other hand, GII.2 (BUDS), GI.1 (Boxer), and GII.9 (VA207) exhibited strong binding to non-secretory and A/O blood group saliva[ 14 ]. Evolutionary processes have led to modifications in norovirus binding capacity to HBGAs. For instance, the P protein of the historically prevailing GII.4 strain has shown a progressive decrease in its binding pocket dimensions, resulting in enhanced affinity for A/B/H-type HBGAs[ 11 ]. Similarly, the GII.17 P protein, initially lacking binding affinity towards A/B/H saliva samples, developed the ability to bind to them in 2014/15 due to mutations in its binding pocket[ 15 ]. GII.13, closely related to GII.21 and GII.17, it has been shown to bind to A/B/O saliva samples and interact with HBGA antigen type I precursors Lec and Lactose, research indicating a unique binding site recognizing glycans with beta-galactose termini[ 16 ]. Noroviruses have been observed to undergo genetic recombination at the ORF1/ORF2 junction region[ 17 ]. While most recombinant viruses circulate at low levels in the human population, some epidemic viruses have emerged due to recombination events. For instance, different viruses associated with the GII.P16 polymerase type have caused outbreaks worldwide[ 18 ]. In the course of nearly two decades of recombinant sequence analysis and epidemiologic studies of noroviruses, some full-length genome analyses has been reported, but the altered replication and binding capacities resulting from recombination and mutation are worthy of further study. Here, we uncovered the complete genome of a GII.P21-GII.13 norovirus recombinant strain found in a human fecal sample and analyzed its recombination sites. Additionally, through molecular dynamic simulation, we investigated the evolutionary and molecular mechanisms of mutations on the GII.P21-GII.13 norovirus P protein on its binding affinity to HBGAs. The results revealed that mutations in key residues and associated conformational adjustments led to changes in the binding affinity of HBGAs during the evolution of GII.13 noroviruses. 2. Materials and Methods 2.1 Stool samples The norovirus GII.13 positive stool samples (Norovirus Hu/GII/57590/Beijing/04/2009/CHN) were kindly provided by Chinese Center for Disease Control and Prevention, which were collected in 2009 from the Gastroenterology Department of the Xuanwu Hospital of the Capital Medical University as part of a large study “Clinical characteristics and genetic diversity of noroviruses in adults with acute gastroenteritis in Beijing of China of 2008–2009”[ 19 ]. 2.2 Viral RNA extraction A volume of 140 µL of stool suspensions (10% w/v) was used for viral RNA extraction using a QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Extracted RNA was immediately stored at − 80°C until further use. 2.3 Whole genome sequence (WGS) and assembly Double-stranded cDNA sequencing libraries were constructed from RNA samples using a TruSeq stranded mRNA prep kit (Illumina), the quality of the generated libraries was evaluated using the TapeStation (Agilent) and the Qubit (ThermoFisher). Barcoded libraries were pooled together and sequenced on the HiSeq platform (Illumina) generating paired-end 150 bp reads. The quality control of the raw WGS-reads was performed by Fastp software with default parameters[ 20 ]. Low quality reads and adapter sequences were removed prior to further analysis. The processed high-quality reads (50–250 bp) were then de novo assembled into contigs using Megahit v1.2.9 ( https://github.com/voutcn/megahit ). To obtain complete genome sequence, the spliced contigs were reassembled by Align/Assemble tool in Geneious 2021 ( https://www.geneious.com/ ). Finally, the genome sequence was validated through BLASTN search against NCBI non-redundant database ( https://blast.ncbi.nlm.nih.gov/ ). Subsequently, the genotype of norovirus sequences was confirmed using the online Norovirus Typing Tool v2.0 ( https://www.genomedetective.com/app/typingtool/nov/ ). 2.4 Recombination analysis A recombinant sequence dataset was constructed using the strategy outlined in our previous studies[ 21 ]. Specifically, we utilized the keyword "Norovirus" to retrieve norovirus nucleotide sequences from the GenBank nucleotide database (as of September 2022), resulting in 3979 sequences longer than 3000nt being downloaded in FASTA format ( https://www.ncbi.nlm.nih.gov/genbank/ ). These sequences were then genotyped using the Norovirus Typing Tool v2.0, which identified 926 sequences as recombinant. Subsequently, the Mafft v7.505 program was used to align all the recombinant sequences[ 22 ], followed by the employment of RDP4 software to identify their parental sequences[ 23 ]. The putative recombination breakpoints were analyzed using the Simplot v3.5.1 software with a window size of 200 and step size of 20[ 24 ]. 2.5 Phylogenetic analysis Phylogenetic analyses of GII.13 strain (Norovirus Hu/GII/57590/Beijing/04/2009/CHN) and related sequences were performed on the RdRp (1536 bp) and capsid (1629 bp) sequences to determine norovirus P-types and genotypes by MEGA 11 package, respectively. Initially, the norovirus nucleotide sequences were alignment using the Tamura-Nei model and maximum-likelihood method. Bootstrap was calculated with 1000 pseudoreplicate data sets. The distance scale represents the number of nucleotide substitution per position. 2.6 Homology modeling The tertiary structure of P protein of GII.13 was modeled by SWISS-MODEL online server ( https://swissmodel.expasy.org/ ), with homology models generated based on the crystal structure of the GII.13 P domain dimer (PDB accession: 5ZV9) as a template. These models were then meticulously examined and edited using PyMOL 2.5 ( https://www.pymol.org/ ) for refinement and analyze further. 2.7 Molecular docking AutoDock Vina 1.2.0 ( https://github.com/ccsb-scripps/AutoDock-Vina ) was used for molecular docking. The structures of A, B, H, Le a , and Le b HBGAs molecules were obtained from Pubchem ( https://pubchem.ncbi.nlm.nih.gov/ ) using specific accession numbers (49852448, 49852447, 5288275, 22806751, and 45480569, respectively). The type I precursor Lec was sourced from the crystal structure of the GII.13 P domain co-crystallized with Lec (6JYN) from the PDB database ( https://www.rcsb.org/ ). These ligands underwent hydrogenation to establish torsional bonds, and the proteins were hydrogenated and selected as rigid acceptors. A Grid Box of dimensions 30 × 30 × 30 ų was positioned within the binding pocket region of the norovirus P domain to facilitate docking. The AutoDock Vina program generated nine binding conformations, from which the conformation with the most binding activity was selected for further molecular dynamics simulation. 2.8 Norovirus-HBGA binding affinity simulation In order to reveal the molecular mechanism for the change of the binding affinity of GII.13 with HBGAs, the structure of P domain in complex with HBGA was constructed for different GII.13 P domains and then the Molecular Dynamic (MD) simulation combined with the Molecular Mechanics / Poisson Boltzmann Surface Area (MM-PBSA) method was used to investigate the interactions between the P domains and the HBGAs. In this study, six representative GII.13 norovirus P proteins (MW305577/1978, AY113106/1998, AB809974/2008, OR713748/2009, MK753008/2013, MN394543/2019) were combined with A/B/H-type and Lewis-type HBGAs and the HBGAs type I precursor Lec for MD simulations. Hydrogen atoms were added to the ligand conformation results using Avogadro v1.1.1 software[ 25 ], and topology files were generated using Sobtop v1.0 based on GAFF force field parameters ( http://sobereva.com/soft/Sobtop ). The Gromacs 2022 pdb2gmx command was used to generate protein receptor topology files[ 26 ], and the Amber ff99SB-ILDN and GAFF force field parameters were used for the norovirus P domains and the HBGAs, respectively. The complex was placed in a hexahedral solvent box and solvated in a water box using the TIP3P model. The total simulated system consisted of approximately 68,000 atoms. After energy minimization and heating, MD simulations without positional constraints were performed for 100 ns. The MM-PBSA method was used to calculate the binding free energy between the norovirus P domain and the HBGA molecule based on the simulation trajectories. The last 10 ns trajectory for each MD simulation was used to calculate the binding free energy, and the relative binding affinity of HBGAs bound with different GII.13 norovirus strains was compared to study the evolution of norovirus-HBGA interactions. The contribution of each residue to the binding free energy and the hydrogen bonding interactions around the binding pocket were analyzed in detail to explore the molecular mechanism of the interaction of GII.13 norovirus with HBGAs[ 11 ]. 3. Results 3.1 Full-length genome sequence After quality control, a contig (K141_161950) of 7572 nt in length was assembled and identified to be GII.P21-GII.13 norovirus. Subsequently, this GII.P21- GII.13 was identified as an isolate of Hu/GII/ 57590/ Beijing/2009/CHN with GenBank accession number OR713748. The full-length genome of this stain OR713748/2009 contains three ORFs: ORF1 (1–5100; 5100 nt), ORF2 (5081–6709; 1629 nt), and ORF3 (6709–7440; 732 nt). ORF1 and ORF2 had an overlap of 20 nt, whereas ORF2 and ORF3 had a single-nucleotide overlap. Sequence analysis in BLASTn demonstrated that this sequence had 96.54% (ORF1) and 97.85% (ORF2) nucleotide identity with other norovirus strains from the Japan (AB242256) and United States (JN899242), respectively. 3.2 Phylogenetic analysis In the ORF1 tree (Figure 1A), OR713748/2009 clustered with GII.P21 reference strain (MW305600/2004/Argentina), while in the ORF2 tree (Figure 1B), it clustered with GII.13 reference strain (JN899242/2010/USA). This indicates that OR713748/2009 was identified as an intergenotype recombinant strain of GII.P21-GII.13 based on phylogenetic analysis. Notably, all the GII.13 capsid sequences exhibited significant diversity, and forming at least five distinct clusters representing an evident time-ordered evolutionary process (Figure 1B). These clusters spanned different time periods: 1978-1983, 1998–2008, 2009, 2010, and 2012–2019 (Figure 1B). Two un-rooted trees were reconstructed using RdRp and VP1 nucleotide sequences. Reference strains were downloaded from GenBank and labelled with their accession number followed by genotype, country and year. Phylogenetic analysis was based on RdRp (1536 bp) and capsid (1629 bp) sequence. Sequence alignments were performed using the MEGA 11 package. Initially, the norovirus nucleotide sequences were alignment using the Tamura-Nei model and maximum-likelihood method. Bootstrap was calculated with 1000 pseudoreplicate data sets. 3.3 Recombination site analysis A simplot analysis was conducted to further characterize the potential recombination event of the OR713748/2009 strain. The complete genome sequences of the study strain were aligned with those of closely related types, revealing a breakpoint at nt 5135, at the 5’ end of ORF2 (Figure 2B). Additionally, to determine the novelty of this recombination site (nt 5135), a total of 926 recombinant sequences were analyzed by RDP4, of which only 148 sequences were able to predict parental sequences. As depicted in Figure 2A (0 is the start nucleic acid of ORF2, -100~0 is the RdRp region of ORF1, 0-20 is the overlap region of ORF1 and ORF2, 20~110 is the N-terminal arm region of ORF2), it was observed that most of the breakpoints were located within the RdRp (79%, n=117), and fewer were distributed in the overlap (11.5%, n=17) and capsid (9.5%, n=14) region. Notably, the majority of recombination sites were found in close proximity to the RNA binding site (44%, n=65)[2]. Coincidentally, the RNA binding site region (-9~6) functions as the promoter of the norovirus subgenome[27], and it has been demonstrated that genome recombination frequently occurs in this region[17]. However, we found that there was no mutation in the Motif functional gene by sequence alignment analysis, so it means that recombination has no effect on the synthesis of subgenome. Norovirus recombination sites distribution analysis was conducted on 148 representative recombinant sequences (shown in blue-green) capable of predicting parental sequences. Among them, 16 were identified as GII.13 recombinant strains, depicted in dark blue. ORF2 begins at position 0, -100~0 represents the RdRp region of ORF1, 0-20 represents the overlap region of ORF1 and ORF2, 20~110 represents the N-terminal arm region of ORF2. The parental sequences are identified as MW305678/2013/Japan and LC122738/2003/Japan in figure B. 3.4 Sequence variations of the GII.13 P domain To investigate the molecular mechanism of amino acid mutations on the P domain of GII.13 norovirus of the norovirus-HBGA interaction, the query strain OR713748/2009/GII.P21-GII.13 along with other five representative GII.13 capsid sequences (MW305577/1978/GII.P41-GII.13, AY113106/1998/GII.P13-GII.13, AB809974/2008/GII.P13-GII.13, MK753008/2013/GII.P16-GII.13,and MN394543/2019/GII.P21-GII.13) were firstly selected on the base of temporal distributions (Figure 1B) and subjected to molecular dynamics simulations and residue energy decomposition. Then a structure-based sequence alignment of the P domain (amino acid) was performed to examine any potential differences among these six GII.13 P domain sequences. Overall, P1 subdomain sequences were relatively conserved (Figure 3), however, significant sequence differences were observed in the outer loop regions in the P2 subdomain that includes the B-loop (aa 291 to 299), P-loop (aa 339 to 354), and A-loop (aa 376 to 384) (Figure 3). In addition, slight changes were also found in the S-loop (aa 439 to 446) located in the P1 subdomain, and in the U-loop (aa 409 to 418) located in the P2 subdomains (Figure 3). The structure-based sequence alignment was performed for the P domains of MW305577/1978, AY113106/1998, AB809974/2008, OR713748/2009, MK753008/2013 and MN394543/2019. The regions spanning P1 and P2 subdomains are indicated by arrows. Identical residues are highlighted in blue and similar residues are highlighted in red. Specific amino acid ranges are also highlighted, including B-loop (aa 291 to 299), P-loop (aa 339 to 354), N-loop (aa 358 to 365), A-loop (aa 376 to 384), T-loop (aa 396 to 404), U-loop (aa 409 to 418), S-loop (aa 439 to 446), HBS I (aa 345 to 348), HBS II (aa 379), and HBS III (aa 443 to 444). 3.5 Structure shift of the GII.13 P domain The comparison of the overall structures of the six GII.13 P domains revealed that most amino acid substitutions occurred in the P2 subdomain, while the P1 subdomain remained relatively conserved (Figure 4A). To elucidate the specific changes observed in the loops, superpositions of the P domain structures of the six representative GII.13 P domains were constructed (Figure 4B). Notably, significant differences in the structure of the A-, B-, P-loop, and U-loop are evident on the top surface (Figure 4A). These structural alterations have implications for the integrity of the HBGAs binding interface, primarily formed by the A-loop, B-loop and HBS II (Figure 4A), and are expected to affect the binding ability of GII.13 to HBGAs. The most prominent difference was observed in the B-loop (Figure 4B), where three distinct structures were identified among the six GII.13 P domains. The MW305577/1978/GII.P41-GII.13, AY113106/1998/GII.P13-GII.13, MK753008/2013/GII.P16-GII.13, and MN394543/2019/GII.P21-GII.13 P domains shared the same structure, while the OR713748/2009/GII.P21-GII.13 and AB809974/2008/GII.P13-GII.13 P domains each presented a unique structure (Figure 4B). The A-loop, B-loop, P-loop, S-loop, T-loop, U-loop, N-loop and HBS site are shown in magenta, green, blue, tan, cyan, orange, yellow and red, respectively. The position of the HBGA binding interface is marked in red. The area enclosed by the yellow dotted circle represents the region where the structure of the upper surface of the P protein is apparently altered. The structures of the seven surface binding loops located on P domain were compared by superposition, with the color scheme as follows: MW305577/1978 in orange, AY113106/1998 in blue, AB809974/2008 in green, OR713748/2009 in yellow, MK753008/2013 in cyan, and MN394543.1/2019 in violet red. 3.6 Difference in binding ability of GII.13 P domain to HBGAs To investigate the molecular mechanism responsible for the discrepancies in HBGA-binding affinity for different GII.13 P domains, atomic interactions between the corresponding P domains and the receptor HBGAs were analyzed using MD simulations and MM-PBSA calculations. Initially, 36 combinations of GII.13 P domains (n=6) and HBGAs (n=6) were simulated, and the RMSD plot identified that only 18 out of 36 simulated complex structures remained in a stable state (Figure S1). Subsequently, the calculation of total binding energy using MM-PBSA indicated that only 9 complex structures exhibited binding activity (Figure S1). In the case of positive control (6JYN), only lec polysaccharide showed moderate binding affinity to this P domain, consistent with previous ELISA binding experimental results(!!! INVALID CITATION !!! 16). For the oldest strain MW305577/1978/GII.P41-GII.13, only type A trisaccharide HBGA exhibited low binding affinity to this P domain (Table S1), with a binding free energy of -21.65 kJ/mol (Table 1). On the other hand, AY113106/1998/GII.P13-GII.13 displayed weak binding activity towards type B HBGA and a strong binding activity towards type H HBGA (Table S1), with binding free energies of -26.39 kJ/mol and -64.37 kJ/mol, respectively (Table 1). Notably, AY113106/1998/GII.P13-GII.13, AB809974/2008/GII.P13-GII.13, OR713748/2009/GII.P21-GII.13, and MK753008/2013/GII.P16-GII.13 all exhibited a strong ability to bind HBGAs type I antigen precursor Lec polysaccharide. However, MN394543/2019/GII.P21-GII.13 could not bind with any HBGAs, including Lec polysaccharide (Table S1). These results indicate that the change of the crucial loops on the surface of the GII.13 P domain may have a significant influence on its binding capacity to HBGA polysaccharides. 3.7 Contribution of amino acid residue in binding affinity To investigate the molecular mechanism underlying the enhancement of norovirus-HBGAs binding strength, a residue energy decomposition analysis was conducted to identify the key residues that contribute to the binding affinity between the P proteins and HBGAs. The results presented in Figure 5 showed that the residue His 378 in HBS II exhibited a strong binding affinity when binding to A/B-type HBGAs, with binding free energies of -3.6 kJ/mol and -3.3 kJ/mol, respectively (Table 1). However, upon conducting sequence comparison analysis, it was discovered that residue His 378 underwent mutation in 2008 (from H to D) (Table S2). Residue energy decomposition analysis revealed that the binding of early GII.13 norovirus to type A and type B HBGAs was primarily attributed to the binding affinity of residues 373-379 (Table 1). Among these residues, residue His 378 was identified as the traditional HBGAs binding site with a strong binding affinity (Table 1). Molecular docking demonstrated that residue His 378 also contributed in the binding of MW305577/1978 to type-A HBGAs (Figure 6C). A salt bridge was formed within the protein, and it serves a crucial role in both the structure and function of the protein (Figure 6B). Furthermore, it was noted that this mutation greatly affected the binding affinity of GII.13 to HBGAs. Based on the binding energy contribution of the residues, it can be observed that the binding site of GII.13 P protein to Lec exhibits a relatively stable interaction (Table S1). Sequences MW305577/1978 and MN394543/2019 were unable to bind to Lec (Table S1). Residue 298 was found to have a crucial role in this binding activity. The binding site between MK753008/2013 and Lec differed from other GII.13 strains, and it had a stronger binding affinity to Lec polysaccharide (Table 1). (A), (B), and (C) represent the residue contribution of MW30557 binding to A-type HBGA, the energy contribution of AY113106/1998 binding to B-type HBGA, and the energy contribution of AY113106/1998 binding to H-type HBGA, respectively; (D), (E), and (F) correspond to the binding free energies contributed by the HBS I (residues 347-350), HBS II (residues 374-380), and HBS III (residues 442-445) binding sites, respectively. 4. Discussion Recombination and genetic drift are pivotal mechanisms driving the observed evolution and diversity within norovirus[ 28 ]. The norovirus RdRp can shape viral evolution by altering viral fidelity and replication rate[ 29 ]. Mahar et al. demonstrated that the evolution of capsid genes is linked to intergenic recombination and the interchange of RdRp genotypes, resulting in accelerated mutation rates[ 30 ]. RdRp switching may be a mechanism to increase the mutation rate by acquiring RdRp with lower fidelity or enhanced replication competence. This divergent evolution might enable norovirus to evade herd immunity and expand their target population by recognizing new host factors. For instance, in 2012, the GII.4 Sydney_2012 variant emerged with a completely different RdRp (GII.P31). This variant replaced all others and has been circulating for over a decade in the world. In 2015, this variant was recombined with a GII.P16-GII.2, acquired a new RdRp (GII.P16), GII.P16-GII.4 Sydney_2012, which quickly predominated in the next few years[ 31 ]. Notably, GII.P16 RdRp has five unique amino acid substitutions, potentially leading to improved adaptability and rapid dominance in the Asian epidemic[ 32 , 33 ]. Moreover, the motif region in RdRp is crucial for norovirus replication, while the stem-loop structure on the negative-sense RNA strand is essential for subgenome replication, as demonstrated by previous studies[ 2 , 5 ], we believe that intergenic recombination of noroviruses contributes to their increased mutation rate of capsid proteins and thus easier escape from host immune recognition. Our sequence alignment analysis and prediction of the amino acid secondary structure revealed that both the motif domain and the stem-loop region of the RdRp gene of strain OR713748/2009 have remained unchanged (data not shown). This suggests that genetic recombination may not impact the replication ability of OR713748/2009. However, the study's limitation lies in the exclusive focus on mutations in the RdRp and stem-loop regions of OR713748/2009, warranting further consideration of the overall replacement of the ORF1 region and its effects on the sequence evolution rate. Perhaps, as Tohma K et al. reported, the non-structural proteins NS1 / 2 and NS4 of norovirus also do enhanced the epidemic potential of some viruses, warranting further research[ 34 ]. Notably, intergenic recombination was not the sole force driving norovirus evolution; evidence of antigenic drift and selective pressures also played crucial roles[ 30 ]. Structural analysis of the HBGA binding interface revealed that norovirus recognize HBGAs through a conserved, genome-specific binding interface, indicating strong selection by HBGAs for the evolution of norovirus[ 35 ]. A significant number of amino acid mutations were identified in the VP1 region through sequence alignment analysis. In this study, we investigated the molecular mechanism of the change in binding affinity between GII.13 norovirus P protein and HBGAs across different time periods. Based on the relative binding affinities and residue energy decomposition results, it was indicated that AY113106/1998 had a broad binding range to HBGAs (Table S1 ). However, energetic decomposition analysis revealed that the residues involved in direct interactions with B-, H-, and Lea-type HBGA antigens, as well as the type I precursor substance Lec, were conserved in the GII.13 sequences. The structural analysis highlighted the essential role of the A-loop and B-loop in the GII.13 P domain region for creating the binding pocket, indicating that and any alterations in these loops could potentially affect the binding of HBGAs to GII.13 (Fig. 6 A). It is postulated that non-interacting residues might have undergone mutations to regulate the binding affinity. Studies have shown that Lec contains β-galactose, which is modified by FUT2 and FUT3 enzymes to form A/B/H HBGA antigen[ 36 ]. The structure of Lec was found to be much simpler than that of the HBGA antigen, and the β-galactose-binding epitope remained unaffected, enabling Lec to bind to GII.13[ 16 ]. Prior to 1998, GII.13 exhibited binding to A or B/H HBGAs, while from 2008 to 2016, it bound to Lec. However, in 2019, it did not bind to the six HBGAs in the binding experiment, indicating a gradual reduction in the binding spectra of GII.13 (Fig. 7 ). Surprisingly, the binding experiments revealed that the binding ability of GII.13 to ABO saliva remained consistent from 1983 to 2016[ 16 ], suggesting a shift in the binding substances utilized by GII.13. This loss of the ability to bind to A/B/H HBGAs may not necessarily impact the spread of GII.13. The GII.13 strain lacks binding affinity towards HBGAs but retains the ability to infect human hosts, suggesting its capacity to recognize various molecules beyond HBGAs as potential attachment factors. Previous research has indicated that the high mutation rate of the viral RNA genome could contribute to human norovirus's ability to evade herd immunity and broaden its target population by identifying novel host factors. For example, the GII.17 strain exhibits weak or no binding affinity towards ABH-type HBGA, and the presence of lactose effectively inhibits its binding. The prevalence of the GII.17 variant increased in 2014/2015, showing a notable affinity for HBGA, no longer impeded by lactose[ 16 , 37 ]. Analysis of the data suggests that GII.13 can bind to Lec but not general HBGAs. Given that GII.13 norovirus has a unique HBGA binding site and a close evolutionary relationship to GII.17[ 15 ], GII.13 also has the potential to expand its target population through amino acid mutations, posing a potential risk of widespread outbreaks and necessitating further attention. Overall, this study examined the distribution of recombination sites in norovirus and the effects of recombination on the replication efficiency and binding capacity of GII.P21-GII.13 norovirus. The findings suggest that alterations in the non-structural proteins of the OR713748/2009 had a weak effect on the replication efficiency of the virus, but that proteins other than RdRp may have influenced the mutation rate of VP1, which providing a possible explanation for the GII13 norovirus's inability to bind HBGA while still maintaining its ability to infect the human host. Furthermore, gaining a comprehensive understanding of the molecular mechanism underlying the binding of GII.13 P protein to HBGAs, and analyzing the residues involved in the binding process, may be valuable for the development of drugs and vaccines targeting GII.13 noroviruses. Declarations Acknowledgements We acknowledge all of the clinicians who collected data and samples. This work was supported by the National Natural Science Foundation of China (31601570, 32370151) and Plan of Action for Scientific and Technological innovation of Science and Technology Commission of Shanghai Municipality (22N31900700). The authors have no conflict of interest to declare. References Patel MM, Widdowson M-A, Glass RI et al (2008) Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg Infect Dis 14:1224–1231. https://doi.org/10.3201/eid1408.071114 Deval J, Jin Z, Chuang Y-C, Kao CC (2017) Structure(s), function(s), and inhibition of the RNA-dependent RNA polymerase of noroviruses. Virus Res 234:21–33. https://doi.org/10.1016/j.virusres.2016.12.018 Chhabra P, de Graaf M, Parra GI et al (2019) Updated classification of norovirus genogroups and genotypes. J Gen Virol 100:1393–1406. https://doi.org/10.1099/jgv.0.001318 Alhatlani B, Vashist S, Goodfellow I (2015) Functions of the 5’ and 3’ ends of calicivirus genomes. 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Front Microbiol 10:1280. https://doi.org/10.3389/fmicb.2019.01280 Ao Y, Cong X, Jin M et al (2018) Genetic Analysis of Reemerging GII.P16-GII.2 Noroviruses in 2016–2017 in China. J Infect Dis 218:133–143. https://doi.org/10.1093/infdis/jiy182 Jin M, Wu S, Kong X et al (2020) Norovirus Outbreak Surveillance, China, 2016–2018. Emerg Infect Dis 26:437–445. https://doi.org/10.3201/eid2603.191183 Tohma K, Lepore CJ, Martinez M et al Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints. PLOS PATHOGENS Singh BK, Leuthold MM, Hansman GS (2015) Human noroviruses’ fondness for histo-blood group antigens. J Virol 89:2024–2040. https://doi.org/10.1128/JVI.02968-14 Barbé L, Le Moullac-Vaidye B, Echasserieau K et al (2018) Histo-blood group antigen-binding specificities of human rotaviruses are associated with gastroenteritis but not with in vitro infection. Sci Rep 8:12961. https://doi.org/10.1038/s41598-018-31005-4 Zuo Y, Xue L, Gao J et al (2021) Evolutionary Mechanism of Immunological Cross-Reactivity Between Different GII.17 Variants. Front Microbiol 12:653719. https://doi.org/10.3389/fmicb.2021.653719 Table Table 1 is available in the Supplementary Files section. Supplementary Files floatimage1.jpeg Table 1. Energy contribution of amino acid residues in the binding of norovirus-HBGAs SupplementarymaterialsChenetal.docx Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2025 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 08 Sep, 2024 Reviewers invited by journal 08 Sep, 2024 Editor assigned by journal 02 Sep, 2024 First submitted to journal 02 Sep, 2024 Editorial decision: Major Revision 31 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4986214","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":351073657,"identity":"0b724f7d-99aa-4afa-87a7-0597ea601a63","order_by":0,"name":"yongxin Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYLCCDwwHwLQE0ToYZ5CshZmHJC0GN3LMpG3+3JEzOMB88DYPg10eYS1nzphJ57Y9MzY4wJZszcOQXExYy/EeoJaGw4kbDvCYSQNdmNhAUMthoEqLPyAt/N+I1AKyhYENbAsbcVokzxwrtuxtO2wseZjN2HKOQTJhLXw3kjfe+PHnsBzf8eaHN95U2BHWonCAwwDCYga7k5B6IJBvYH9AhLJRMApGwSgY0QAAdTg9UHSBe5cAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1447-2880","institution":"Shanghai Ocean University College of Food Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"yongxin","middleName":"","lastName":"Yu","suffix":""},{"id":351073658,"identity":"efa42b7f-e175-46d9-bb79-9a363ad3ccb5","order_by":1,"name":"Yunfei Chen","email":"","orcid":"","institution":"Shanghai Ocean University College of Food Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunfei","middleName":"","lastName":"Chen","suffix":""},{"id":351073659,"identity":"61f74bc1-d336-4792-9611-463698de7f6e","order_by":2,"name":"Zexian Zhou","email":"","orcid":"","institution":"Shanghai Ocean University College of Food Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zexian","middleName":"","lastName":"Zhou","suffix":""},{"id":351073660,"identity":"bfa2dbe5-76db-45bc-9393-6a394c3c7915","order_by":3,"name":"Lei Dong","email":"","orcid":"","institution":"Shanghai Ocean University College of Food Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Dong","suffix":""},{"id":351073661,"identity":"7567e53b-0866-4919-b429-34cbfd4ecd87","order_by":4,"name":"Miao Jin","email":"","orcid":"","institution":"China CDC: Chinese Center for Disease Control and Prevention","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Jin","suffix":""},{"id":351073662,"identity":"4daab563-11c9-4227-aafc-53cd0f4a685f","order_by":5,"name":"Yongjie Wang","email":"","orcid":"","institution":"Shanghai Ocean University College of Food Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongjie","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-08-27 17:32:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4986214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4986214/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06277-7","type":"published","date":"2025-04-01T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66386343,"identity":"f77d3ee8-40f0-47cf-ad19-1817a32838e1","added_by":"auto","created_at":"2024-10-11 08:13:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":272027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic trees of norovirus (A) RdRp and (B) ORF2. \u003c/strong\u003eTwo un-rooted trees were reconstructed using RdRp and VP1 nucleotide sequences. Reference strains were downloaded from GenBank and labelled with their accession number followed by genotype, countryand year. Phylogenetic analysis was based on RdRp (1536 bp) and capsid (1629 bp) sequence. Sequence alignments were performed using the MEGA 11 package. Initially, the norovirus nucleotide sequences were alignment using the Tamura-Nei model and maximum-likelihood method. Bootstrap was calculated with 1000 pseudoreplicate data sets.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/4d50624ee1de2540814eaa66.png"},{"id":66386784,"identity":"54daad26-1174-46c8-957d-677e8fe28578","added_by":"auto","created_at":"2024-10-11 08:21:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) Distribution of norovirus recombination sites\u003c/strong\u003e. Norovirus recombination sites distribution analysis was conducted on 148 representative recombinant sequences (shown in blue-green) capable of predicting parental sequences. Among them, 16 were identified as GII.13 recombinant strains, depicted in dark blue. ORF2 begins at position 0, -100~0 represents the RdRp region of ORF1, 0-20 represents the overlap region of ORF1 and ORF2, 20~110 represents the N-terminal arm region of ORF2;\u003cstrong\u003e (B) Simplot analysis of strain OR713748\u003c/strong\u003e. The parental sequences are identified as MW305678/2013/Japan and LC122738/2003/Japan.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/9f4ec01f84c2a8a98bc10973.png"},{"id":66386345,"identity":"8c6f5a94-9c0b-48a7-9b73-e9fcab9d3892","added_by":"auto","created_at":"2024-10-11 08:13:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":247587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence alignment of GII.13 P domains (amino acids)\u003c/strong\u003e. The structure-based sequence alignment was performed for the P domains of MW305577/1978, AY113106/1998, AB809974/2008, OR713748/2009, MK753008/2013 and MN394543/2019. The regions spanning P1 and P2 subdomains are indicated by arrows. Identical residues are highlighted in blue and similar residues are highlighted in red. Specific amino acid ranges are also highlighted, including B-loop (aa 291 to 299), P-loop (aa 339 to 354), N-loop (aa 358 to 365), A-loop (aa 376 to 384), T-loop (aa 396 to 404), U-loop (aa 409 to 418), S-loop (aa 439 to 446), HBS I (aa 345 to 348), HBS II (aa 379), and HBS III (aa 443 to 444).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/3f151ddfd31387ffe0535b68.png"},{"id":66387960,"identity":"1029d3b4-d880-4a91-80ad-42a3b317c03d","added_by":"auto","created_at":"2024-10-11 08:29:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":315737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructures of GII.13 norovirus P dimer. (A) Top surface topology alterations in the GII.13 norovirus P dimers\u003c/strong\u003e. The A-loop, B-loop, P-loop, S-loop, T-loop, U-loop, N-loop and HBS site are shown in magenta, green, blue, tan, cyan, orange, yellow and red, respectively. The position of the HBGA binding interface is marked in red. The area enclosed by the yellow dotted circle represents the region where the structure of the upper surface of the P protein is apparently altered. \u003cstrong\u003e(B) Comparison of the seven major loops of GII.13 norovirus\u003c/strong\u003e. The structures of the seven surface binding loops located on P domain were compared by superposition, with the color scheme as follows: MW305577/1978 in orange, AY113106/1998 in blue, AB809974/2008 in green, OR713748/2009 in yellow, MK753008/2013 in cyan, and MN394543.1/2019 in violet red.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/c0a2d071c0f8fd69cc52bfcf.png"},{"id":66386782,"identity":"ae75bf2f-65c5-4d2c-954f-945ba6b3c87c","added_by":"auto","created_at":"2024-10-11 08:21:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe per-residue energy decomposition analysis\u003c/strong\u003e. (A), (B), and (C) represent the residue contribution of MW30557 binding to A-type HBGA, the energy contribution of AY113106/1998 binding to B-type HBGA, and the energy contribution of AY113106/1998 binding to H-type HBGA, respectively; (D), (E), and (F) correspond to the binding free energies contributed by the HBS I (residues 347-350), HBS II (residues 374-380), and HBS III (residues 442-445) binding sites, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/b0af32c28f15cc0a98bc7cac.png"},{"id":66386351,"identity":"a81bfbac-4fd5-4aa5-b455-1b2fb1b663f0","added_by":"auto","created_at":"2024-10-11 08:13:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":260835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding interface of MW305577/1978 with HBGA and its interaction with A-type HBGA\u003c/strong\u003e. (A) A close-up of the binding site and pocket of the complex of MW305577/1978 sequence P-dimer and A-type HBGA. (B) Binding sites and bonds of A-type HBGA in the binding pocket. Hydrogen bonds (shown in blue), salt bridges (shown in yellow).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/2b5fa28933bf7ba65a97507e.png"},{"id":66386783,"identity":"85c063ba-fcc6-4baf-89ee-461e1bab062f","added_by":"auto","created_at":"2024-10-11 08:21:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":166637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary changes of the conventional HBGA binding site (Red) among members of the GII.13 genotype\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/7a6cad1f89329c70355c7dfb.png"},{"id":80082020,"identity":"41bf6569-59d7-4d82-bb98-bffc4053ac4c","added_by":"auto","created_at":"2025-04-07 16:05:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2224253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/1b4b1fae-c754-42b7-8018-516197681847.pdf"},{"id":66386349,"identity":"4ed64486-353b-449d-b207-5dd005621ae1","added_by":"auto","created_at":"2024-10-11 08:13:48","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":400093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cstrong\u003eEnergy contribution of amino acid residues in the binding of norovirus-HBGAs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/b0b2179e9fe98d8e5c20f8f1.jpeg"},{"id":66386352,"identity":"fdaa5ab4-1262-4c61-8857-0da825fff993","added_by":"auto","created_at":"2024-10-11 08:13:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":455095,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialsChenetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4986214/v1/61d5d28e735e6c5a1ed01c9b.docx"}],"financialInterests":"","formattedTitle":"Identification of a GII.P21-GII.13 recombinant norovirus strain, mutations shift its binding spectra to host receptor glycans","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNorovirus is a highly contagious human pathogen that is accountable for the majority of viral gastroenteritis outbreaks on a global scale[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are small, non-enveloped viruses of \u003cem\u003eCaliciviridae\u003c/em\u003e family, with a single positive-strand RNA genome of approximately 7400 to 7700 nucleotides, typically comprising three open reading frames (ORFs) known as ORF1-3. ORF1 encodes a polyprotein of about 200 kDa, which can be processed to yield six non-structural proteins: P48 (NS1/2), NTPase (NS3), P22 (NS4), VPg (NS5), Pro (NS6), and Pol (NS7). ORF2 and ORF3 encode the major and minor structural capsid proteins, VP1 and VP2, respectively[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Genetic variations in the RNA-dependent RNA polymerase (RdRp) and VP1 regions categorize noroviruses into genogroups and genotypes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Within each genogroup, norovirus can be further classified into diverse P-types and genotypes based on dual typing nomenclature[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe RdRp is responsible for replicating and synthesizing genomic RNA in norovirus, utilizing genomic RNA templates to synthesize antisense RNA. Genomic RNAs are subsequently generated through the synthesis of antisense RNAs. During genomic RNA replication, subgenomic RNA encoding ORF2 and ORF3 (3' co-terminal to the viral genome) is also synthesized[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In all \u003cem\u003ecaliciviruses\u003c/em\u003e, a conserved stem loop is positioned 6 nucleotides downstream of the subgenomic RNA start site, thereby ensuring the viability of norovirus. This stem-loop sequence is sufficient for initiating viral RNA synthesis by recombinant norovirus RdRp[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe icosahedral capsid of norovirus consists of ninety VP1 dimers, each VP1 protein contain a shell domain (S domain) and a protruding domain (P domain) that binds to host cell surface receptors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Specifically, the P domain proteins bind to histo-blood group antigens (HBGAs), which serve as crucial recognition coreceptors or attachment factors for norovirus to enter human host cells. Notably, HBGAs, found in human erythrocytes, saliva, and epithelial cells, not only determine various blood types but also facilitate the binding and transmission of human norovirus[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. X-ray crystal structure analysis of the structural domains of the GII.4 P variant, bound to different HBGAs, revealed a binding pocket in the P2 structural domain responsible for HBGA receptor binding[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, the region directly beneath the HBGA binding pocket exhibited minimal variation, while the neighboring residues demonstrated moderate amino acid substitutions[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent norovirus genotypes exhibit varying binding affinities to HBGAs[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The interaction of norovirus with HBGAs was characterized by saliva binding assay, revealing seven distinct binding modes. For instance, GII.4 variant US95/96 VLPs (VA387 and GrV) bound to saliva samples from individuals with different A/B/O blood groups[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. GII.5 (MOH) and GII.3 (MxV and PiV) specifically bound to type A/B saliva but not type O or non-secretory saliva. On the other hand, GII.2 (BUDS), GI.1 (Boxer), and GII.9 (VA207) exhibited strong binding to non-secretory and A/O blood group saliva[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Evolutionary processes have led to modifications in norovirus binding capacity to HBGAs. For instance, the P protein of the historically prevailing GII.4 strain has shown a progressive decrease in its binding pocket dimensions, resulting in enhanced affinity for A/B/H-type HBGAs[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, the GII.17 P protein, initially lacking binding affinity towards A/B/H saliva samples, developed the ability to bind to them in 2014/15 due to mutations in its binding pocket[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. GII.13, closely related to GII.21 and GII.17, it has been shown to bind to A/B/O saliva samples and interact with HBGA antigen type I precursors Lec and Lactose, research indicating a unique binding site recognizing glycans with beta-galactose termini[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNoroviruses have been observed to undergo genetic recombination at the ORF1/ORF2 junction region[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While most recombinant viruses circulate at low levels in the human population, some epidemic viruses have emerged due to recombination events. For instance, different viruses associated with the GII.P16 polymerase type have caused outbreaks worldwide[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the course of nearly two decades of recombinant sequence analysis and epidemiologic studies of noroviruses, some full-length genome analyses has been reported, but the altered replication and binding capacities resulting from recombination and mutation are worthy of further study.\u003c/p\u003e \u003cp\u003eHere, we uncovered the complete genome of a GII.P21-GII.13 norovirus recombinant strain found in a human fecal sample and analyzed its recombination sites. Additionally, through molecular dynamic simulation, we investigated the evolutionary and molecular mechanisms of mutations on the GII.P21-GII.13 norovirus P protein on its binding affinity to HBGAs. The results revealed that mutations in key residues and associated conformational adjustments led to changes in the binding affinity of HBGAs during the evolution of GII.13 noroviruses.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Stool samples\u003c/h2\u003e \u003cp\u003eThe norovirus GII.13 positive stool samples (Norovirus Hu/GII/57590/Beijing/04/2009/CHN) were kindly provided by Chinese Center for Disease Control and Prevention, which were collected in 2009 from the Gastroenterology Department of the Xuanwu Hospital of the Capital Medical University as part of a large study \u0026ldquo;Clinical characteristics and genetic diversity of noroviruses in adults with acute gastroenteritis in Beijing of China of 2008\u0026ndash;2009\u0026rdquo;[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Viral RNA extraction\u003c/h2\u003e \u003cp\u003eA volume of 140 \u0026micro;L of stool suspensions (10% w/v) was used for viral RNA extraction using a QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Extracted RNA was immediately stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Whole genome sequence (WGS) and assembly\u003c/h2\u003e \u003cp\u003eDouble-stranded cDNA sequencing libraries were constructed from RNA samples using a TruSeq stranded mRNA prep kit (Illumina), the quality of the generated libraries was evaluated using the TapeStation (Agilent) and the Qubit (ThermoFisher). Barcoded libraries were pooled together and sequenced on the HiSeq platform (Illumina) generating paired-end 150 bp reads.\u003c/p\u003e \u003cp\u003eThe quality control of the raw WGS-reads was performed by Fastp software with default parameters[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Low quality reads and adapter sequences were removed prior to further analysis. The processed high-quality reads (50\u0026ndash;250 bp) were then \u003cem\u003ede novo\u003c/em\u003e assembled into contigs using Megahit v1.2.9 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/voutcn/megahit\u003c/span\u003e\u003cspan address=\"https://github.com/voutcn/megahit\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To obtain complete genome sequence, the spliced contigs were reassembled by Align/Assemble tool in Geneious 2021 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneious.com/\u003c/span\u003e\u003cspan address=\"https://www.geneious.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Finally, the genome sequence was validated through BLASTN search against NCBI non-redundant database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, the genotype of norovirus sequences was confirmed using the online Norovirus Typing Tool v2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genomedetective.com/app/typingtool/nov/\u003c/span\u003e\u003cspan address=\"https://www.genomedetective.com/app/typingtool/nov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Recombination analysis\u003c/h2\u003e \u003cp\u003eA recombinant sequence dataset was constructed using the strategy outlined in our previous studies[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Specifically, we utilized the keyword \"Norovirus\" to retrieve norovirus nucleotide sequences from the GenBank nucleotide database (as of September 2022), resulting in 3979 sequences longer than 3000nt being downloaded in FASTA format (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These sequences were then genotyped using the Norovirus Typing Tool v2.0, which identified 926 sequences as recombinant. Subsequently, the Mafft v7.505 program was used to align all the recombinant sequences[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], followed by the employment of RDP4 software to identify their parental sequences[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The putative recombination breakpoints were analyzed using the Simplot v3.5.1 software with a window size of 200 and step size of 20[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Phylogenetic analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic analyses of GII.13 strain (Norovirus Hu/GII/57590/Beijing/04/2009/CHN) and related sequences were performed on the RdRp (1536 bp) and capsid (1629 bp) sequences to determine norovirus P-types and genotypes by MEGA 11 package, respectively. Initially, the norovirus nucleotide sequences were alignment using the Tamura-Nei model and maximum-likelihood method. Bootstrap was calculated with 1000 pseudoreplicate data sets. The distance scale represents the number of nucleotide substitution per position.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Homology modeling\u003c/h2\u003e \u003cp\u003eThe tertiary structure of P protein of GII.13 was modeled by SWISS-MODEL online server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with homology models generated based on the crystal structure of the GII.13 P domain dimer (PDB accession: 5ZV9) as a template. These models were then meticulously examined and edited using PyMOL 2.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pymol.org/\u003c/span\u003e\u003cspan address=\"https://www.pymol.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for refinement and analyze further.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Molecular docking\u003c/h2\u003e \u003cp\u003eAutoDock Vina 1.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ccsb-scripps/AutoDock-Vina\u003c/span\u003e\u003cspan address=\"https://github.com/ccsb-scripps/AutoDock-Vina\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for molecular docking. The structures of A, B, H, Le\u003csup\u003ea\u003c/sup\u003e, and Le\u003csup\u003eb\u003c/sup\u003e HBGAs molecules were obtained from Pubchem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using specific accession numbers (49852448, 49852447, 5288275, 22806751, and 45480569, respectively). The type I precursor Lec was sourced from the crystal structure of the GII.13 P domain co-crystallized with Lec (6JYN) from the PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These ligands underwent hydrogenation to establish torsional bonds, and the proteins were hydrogenated and selected as rigid acceptors. A Grid Box of dimensions 30 \u0026times; 30 \u0026times; 30 \u0026Aring;\u0026sup3; was positioned within the binding pocket region of the norovirus P domain to facilitate docking. The AutoDock Vina program generated nine binding conformations, from which the conformation with the most binding activity was selected for further molecular dynamics simulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Norovirus-HBGA binding affinity simulation\u003c/h2\u003e \u003cp\u003eIn order to reveal the molecular mechanism for the change of the binding affinity of GII.13 with HBGAs, the structure of P domain in complex with HBGA was constructed for different GII.13 P domains and then the Molecular Dynamic (MD) simulation combined with the Molecular Mechanics / Poisson Boltzmann Surface Area (MM-PBSA) method was used to investigate the interactions between the P domains and the HBGAs. In this study, six representative GII.13 norovirus P proteins (MW305577/1978, AY113106/1998, AB809974/2008, OR713748/2009, MK753008/2013, MN394543/2019) were combined with A/B/H-type and Lewis-type HBGAs and the HBGAs type I precursor Lec for MD simulations. Hydrogen atoms were added to the ligand conformation results using Avogadro v1.1.1 software[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and topology files were generated using Sobtop v1.0 based on GAFF force field parameters (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sobereva.com/soft/Sobtop\u003c/span\u003e\u003cspan address=\"http://sobereva.com/soft/Sobtop\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The Gromacs 2022 pdb2gmx command was used to generate protein receptor topology files[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and the Amber ff99SB-ILDN and GAFF force field parameters were used for the norovirus P domains and the HBGAs, respectively.\u003c/p\u003e \u003cp\u003eThe complex was placed in a hexahedral solvent box and solvated in a water box using the TIP3P model. The total simulated system consisted of approximately 68,000 atoms. After energy minimization and heating, MD simulations without positional constraints were performed for 100 ns. The MM-PBSA method was used to calculate the binding free energy between the norovirus P domain and the HBGA molecule based on the simulation trajectories. The last 10 ns trajectory for each MD simulation was used to calculate the binding free energy, and the relative binding affinity of HBGAs bound with different GII.13 norovirus strains was compared to study the evolution of norovirus-HBGA interactions. The contribution of each residue to the binding free energy and the hydrogen bonding interactions around the binding pocket were analyzed in detail to explore the molecular mechanism of the interaction of GII.13 norovirus with HBGAs[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Full-length genome sequence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter quality control, a contig (K141_161950) of 7572 nt in length was assembled and identified to be GII.P21-GII.13 norovirus. Subsequently, this GII.P21- GII.13 was identified as an isolate of Hu/GII/ 57590/ Beijing/2009/CHN with GenBank accession number OR713748. The full-length genome of this stain OR713748/2009 contains three ORFs: ORF1 (1–5100; 5100 nt), ORF2 (5081–6709; 1629 nt), and ORF3 (6709–7440; 732 nt). ORF1 and ORF2 had an overlap of 20 nt, whereas ORF2 and ORF3 had a single-nucleotide overlap. Sequence analysis in BLASTn demonstrated that this sequence had 96.54% (ORF1) and 97.85% (ORF2) nucleotide identity with other norovirus strains from the Japan (AB242256) and United States (JN899242), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Phylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the ORF1 tree (Figure 1A),\u0026nbsp;OR713748/2009 clustered with GII.P21 reference strain (MW305600/2004/Argentina), while in the ORF2 tree (Figure 1B), it clustered with GII.13 reference\u0026nbsp;strain (JN899242/2010/USA). This indicates that OR713748/2009 was identified as an intergenotype recombinant strain of GII.P21-GII.13 based on phylogenetic analysis.\u003c/p\u003e\n\u003cp\u003eNotably, all the GII.13 capsid sequences exhibited significant diversity, and forming at least five distinct clusters representing an evident time-ordered evolutionary process (Figure 1B). These clusters spanned different time periods: 1978-1983, 1998–2008, 2009, 2010, and 2012–2019 (Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo un-rooted trees were reconstructed using RdRp and VP1 nucleotide sequences. Reference strains were downloaded from GenBank and labelled with their accession number followed by genotype, country and year. Phylogenetic analysis was based on RdRp (1536 bp) and capsid (1629 bp) sequence. Sequence alignments were performed using the\u0026nbsp;MEGA 11 package. Initially, the norovirus nucleotide sequences were alignment using the Tamura-Nei model and maximum-likelihood method. Bootstrap was calculated with 1000 pseudoreplicate data sets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Recombination site analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA simplot analysis was conducted to further characterize the potential recombination event of the OR713748/2009 strain. The complete genome sequences of the study strain were aligned with those of closely related types, revealing a breakpoint at nt 5135, at the 5’ end of ORF2 (Figure 2B). Additionally, to determine the novelty of this recombination site (nt 5135), a total of 926 recombinant sequences were analyzed by RDP4, of which only 148 sequences were able to predict parental sequences. As depicted in Figure 2A\u0026nbsp;(0 is the start nucleic acid of ORF2, -100~0 is the RdRp region of ORF1, 0-20 is the overlap region of ORF1 and ORF2, 20~110 is the N-terminal arm region of ORF2),\u0026nbsp;it was observed that most of the breakpoints were located within the RdRp (79%, n=117), and fewer were distributed in the overlap (11.5%, n=17) and capsid (9.5%, n=14) region. Notably, the majority of recombination sites were found in close proximity to the RNA binding site (44%, n=65)[2]. Coincidentally, the RNA binding site region (-9~6) functions as the promoter of the norovirus subgenome[27], and it has been demonstrated that genome recombination frequently occurs in this region[17]. However, we found that there was no mutation in the Motif functional gene by sequence alignment analysis, so it means that recombination has no effect on the synthesis of subgenome.\u003c/p\u003e\n\u003cp\u003eNorovirus recombination sites distribution analysis was conducted on 148 representative recombinant sequences (shown in blue-green) capable of predicting parental sequences. Among them, 16 were identified as GII.13 recombinant strains, depicted in dark blue.\u0026nbsp;ORF2 begins at position 0, -100~0 represents the RdRp region of ORF1, 0-20 represents the overlap region of ORF1 and ORF2, 20~110 represents the N-terminal arm region of ORF2. The parental sequences are identified as\u0026nbsp;MW305678/2013/Japan and LC122738/2003/Japan in figure B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Sequence variations of the GII.13 P domain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanism of amino acid mutations on the P domain of GII.13 norovirus of the norovirus-HBGA interaction, the query strain OR713748/2009/GII.P21-GII.13 along with other five representative GII.13 capsid sequences (MW305577/1978/GII.P41-GII.13, AY113106/1998/GII.P13-GII.13, AB809974/2008/GII.P13-GII.13, MK753008/2013/GII.P16-GII.13,and MN394543/2019/GII.P21-GII.13) were firstly selected on the base of temporal distributions (Figure 1B) and subjected to molecular dynamics simulations and residue energy decomposition.\u003c/p\u003e\n\u003cp\u003eThen a structure-based sequence alignment of the P domain (amino acid) was performed to examine any potential differences among these six GII.13 P domain sequences. Overall, P1 subdomain sequences were relatively conserved (Figure 3), however, significant sequence differences were observed in the outer loop regions in the P2 subdomain that includes the B-loop (aa 291 to 299), P-loop (aa 339 to 354), and\u0026nbsp;A-loop (aa 376 to 384) (Figure 3). In addition, slight changes were also found in the S-loop (aa 439 to 446) located in the P1 subdomain, and in the U-loop (aa 409 to 418)\u0026nbsp;located in the P2 subdomains (Figure 3).\u003c/p\u003e\n\u003cp\u003eThe structure-based sequence alignment was performed for the P domains of MW305577/1978, AY113106/1998, AB809974/2008, OR713748/2009, MK753008/2013 and MN394543/2019. The regions spanning P1 and P2 subdomains are indicated by arrows. Identical residues are highlighted in blue and similar residues are highlighted in red. Specific amino acid ranges are also highlighted, including\u0026nbsp;B-loop (aa 291 to 299), P-loop (aa 339 to 354), N-loop (aa 358 to 365), A-loop (aa 376 to 384), T-loop (aa 396 to 404), U-loop (aa 409 to 418), S-loop (aa 439 to 446), HBS I (aa 345 to 348), HBS II (aa 379), and HBS III (aa 443 to 444).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Structure shift of the GII.13 P domain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe comparison of the overall structures of the six GII.13 P domains revealed that most amino acid substitutions occurred in the P2 subdomain, while the P1 subdomain remained relatively conserved (Figure 4A). To elucidate the specific changes observed in the loops, superpositions of the P domain structures of the six representative GII.13 P domains were constructed (Figure 4B). Notably, significant differences in the structure of the A-, B-, P-loop, and U-loop are evident on the top surface (Figure 4A). These structural alterations have implications for the integrity of the HBGAs binding interface, primarily formed by the A-loop, B-loop and HBS II (Figure 4A), and are expected to affect the binding ability of GII.13 to HBGAs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe most prominent difference was observed in the B-loop (Figure 4B), where three distinct structures were identified among the six GII.13 P domains. The MW305577/1978/GII.P41-GII.13, AY113106/1998/GII.P13-GII.13, MK753008/2013/GII.P16-GII.13, and MN394543/2019/GII.P21-GII.13 P domains shared the same structure, while the OR713748/2009/GII.P21-GII.13 and AB809974/2008/GII.P13-GII.13 P domains each presented a unique structure (Figure 4B).\u003c/p\u003e\n\u003cp\u003eThe A-loop, B-loop, P-loop, S-loop, T-loop, U-loop, N-loop and HBS site are shown in magenta, green, blue, tan, cyan, orange, yellow and red, respectively. The position of the HBGA binding interface is marked in red. The area enclosed by the yellow dotted circle represents the region where the structure of the upper surface of the P protein is apparently altered.\u003c/p\u003e\n\u003cp\u003eThe structures of the seven surface binding loops located on P domain were compared by superposition, with the color scheme as follows: MW305577/1978 in orange, AY113106/1998 in blue, AB809974/2008 in green, OR713748/2009 in yellow, MK753008/2013 in cyan, and MN394543.1/2019 in violet red.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Difference in binding ability of GII.13 P domain to HBGAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanism responsible for the\u0026nbsp;discrepancies\u0026nbsp;in HBGA-binding affinity for different GII.13 P domains, atomic interactions between the corresponding P domains and the receptor HBGAs were analyzed using MD simulations and MM-PBSA calculations.\u003c/p\u003e\n\u003cp\u003eInitially, 36 combinations of GII.13 P domains (n=6) and HBGAs (n=6) were simulated, and the RMSD plot identified that only 18 out of 36 simulated complex structures remained in a stable state (Figure S1). Subsequently, the calculation of total binding energy using MM-PBSA indicated that only 9 complex structures exhibited binding activity (Figure S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the case of positive control (6JYN), only lec polysaccharide showed moderate binding affinity to this P domain, consistent with previous ELISA binding experimental results(!!! INVALID CITATION !!! 16). For the oldest strain MW305577/1978/GII.P41-GII.13, only type A trisaccharide HBGA exhibited low binding affinity to this P domain (Table S1), with a binding free energy of -21.65 kJ/mol (Table 1). On the other hand, AY113106/1998/GII.P13-GII.13 displayed weak binding activity towards type B HBGA and a strong binding activity towards type H HBGA (Table S1),\u0026nbsp;with binding free energies of -26.39 kJ/mol and -64.37 kJ/mol, respectively (Table 1). Notably, AY113106/1998/GII.P13-GII.13, AB809974/2008/GII.P13-GII.13, OR713748/2009/GII.P21-GII.13, and MK753008/2013/GII.P16-GII.13 all exhibited a strong ability to bind HBGAs type I antigen precursor Lec polysaccharide. However, MN394543/2019/GII.P21-GII.13 could not bind with any HBGAs, including Lec polysaccharide (Table S1). These results indicate that the change of the crucial loops on the surface of the GII.13 P domain may have a significant influence on its binding capacity to HBGA polysaccharides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Contribution of amino acid residue in binding affinity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the molecular mechanism underlying the enhancement of norovirus-HBGAs binding strength, a residue energy decomposition analysis was conducted to identify the key residues that contribute to the binding affinity between the P proteins and HBGAs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results presented in Figure 5 showed that the residue His 378 in HBS II exhibited a strong binding affinity when binding to A/B-type HBGAs, with binding free energies of -3.6 kJ/mol and -3.3 kJ/mol, respectively (Table 1). However, upon conducting sequence comparison analysis, it was discovered that residue His 378 underwent mutation in 2008 (from H to D) (Table S2).\u0026nbsp;Residue energy decomposition analysis revealed that the binding of early GII.13 norovirus to type A and type B HBGAs was primarily attributed to the binding affinity of residues 373-379\u0026nbsp;(Table 1). Among these residues, residue His 378 was identified as the traditional HBGAs binding site with a strong binding affinity\u0026nbsp;(Table 1).\u0026nbsp;Molecular docking demonstrated that residue His 378 also contributed in the binding of MW305577/1978 to type-A HBGAs\u0026nbsp;(Figure 6C).\u0026nbsp;A salt bridge was formed within the protein, and it serves a crucial role in both the structure and function of the protein (Figure 6B). Furthermore, it was noted that this mutation greatly affected the binding affinity of GII.13 to HBGAs.\u003c/p\u003e\n\u003cp\u003eBased on the binding energy contribution of the residues, it can be observed that the binding site of GII.13 P protein to Lec exhibits a relatively stable interaction (Table S1). Sequences MW305577/1978 and MN394543/2019 were unable to bind to Lec (Table S1). Residue 298 was found to have a crucial role in this binding activity. The binding site between MK753008/2013 and Lec differed from other GII.13 strains, and it had a stronger binding affinity to Lec polysaccharide (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A), (B), and (C) represent the residue contribution of MW30557 binding to A-type HBGA, the energy contribution of AY113106/1998 binding to B-type HBGA, and the energy contribution of AY113106/1998 binding to H-type HBGA, respectively; (D), (E), and (F) correspond to the binding free energies contributed by the HBS I (residues 347-350), HBS II (residues 374-380), and HBS III (residues 442-445) binding sites, respectively.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRecombination and genetic drift are pivotal mechanisms driving the observed evolution and diversity within norovirus[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The norovirus RdRp can shape viral evolution by altering viral fidelity and replication rate[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Mahar et al. demonstrated that the evolution of capsid genes is linked to intergenic recombination and the interchange of RdRp genotypes, resulting in accelerated mutation rates[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. RdRp switching may be a mechanism to increase the mutation rate by acquiring RdRp with lower fidelity or enhanced replication competence. This divergent evolution might enable norovirus to evade herd immunity and expand their target population by recognizing new host factors. For instance, in 2012, the GII.4 Sydney_2012 variant emerged with a completely different RdRp (GII.P31). This variant replaced all others and has been circulating for over a decade in the world. In 2015, this variant was recombined with a GII.P16-GII.2, acquired a new RdRp (GII.P16), GII.P16-GII.4 Sydney_2012, which quickly predominated in the next few years[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Notably, GII.P16 RdRp has five unique amino acid substitutions, potentially leading to improved adaptability and rapid dominance in the Asian epidemic[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, the motif region in RdRp is crucial for norovirus replication, while the stem-loop structure on the negative-sense RNA strand is essential for subgenome replication, as demonstrated by previous studies[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], we believe that intergenic recombination of noroviruses contributes to their increased mutation rate of capsid proteins and thus easier escape from host immune recognition.\u003c/p\u003e \u003cp\u003eOur sequence alignment analysis and prediction of the amino acid secondary structure revealed that both the motif domain and the stem-loop region of the RdRp gene of strain OR713748/2009 have remained unchanged (data not shown). This suggests that genetic recombination may not impact the replication ability of OR713748/2009. However, the study's limitation lies in the exclusive focus on mutations in the RdRp and stem-loop regions of OR713748/2009, warranting further consideration of the overall replacement of the ORF1 region and its effects on the sequence evolution rate. Perhaps, as Tohma K et al. reported, the non-structural proteins NS1 / 2 and NS4 of norovirus also do enhanced the epidemic potential of some viruses, warranting further research[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, intergenic recombination was not the sole force driving norovirus evolution; evidence of antigenic drift and selective pressures also played crucial roles[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Structural analysis of the HBGA binding interface revealed that norovirus recognize HBGAs through a conserved, genome-specific binding interface, indicating strong selection by HBGAs for the evolution of norovirus[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A significant number of amino acid mutations were identified in the VP1 region through sequence alignment analysis. In this study, we investigated the molecular mechanism of the change in binding affinity between GII.13 norovirus P protein and HBGAs across different time periods. Based on the relative binding affinities and residue energy decomposition results, it was indicated that AY113106/1998 had a broad binding range to HBGAs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, energetic decomposition analysis revealed that the residues involved in direct interactions with B-, H-, and Lea-type HBGA antigens, as well as the type I precursor substance Lec, were conserved in the GII.13 sequences.\u003c/p\u003e \u003cp\u003eThe structural analysis highlighted the essential role of the A-loop and B-loop in the GII.13 P domain region for creating the binding pocket, indicating that and any alterations in these loops could potentially affect the binding of HBGAs to GII.13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). It is postulated that non-interacting residues might have undergone mutations to regulate the binding affinity. Studies have shown that Lec contains β-galactose, which is modified by FUT2 and FUT3 enzymes to form A/B/H HBGA antigen[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The structure of Lec was found to be much simpler than that of the HBGA antigen, and the β-galactose-binding epitope remained unaffected, enabling Lec to bind to GII.13[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrior to 1998, GII.13 exhibited binding to A or B/H HBGAs, while from 2008 to 2016, it bound to Lec. However, in 2019, it did not bind to the six HBGAs in the binding experiment, indicating a gradual reduction in the binding spectra of GII.13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Surprisingly, the binding experiments revealed that the binding ability of GII.13 to ABO saliva remained consistent from 1983 to 2016[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting a shift in the binding substances utilized by GII.13. This loss of the ability to bind to A/B/H HBGAs may not necessarily impact the spread of GII.13.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe GII.13 strain lacks binding affinity towards HBGAs but retains the ability to infect human hosts, suggesting its capacity to recognize various molecules beyond HBGAs as potential attachment factors. Previous research has indicated that the high mutation rate of the viral RNA genome could contribute to human norovirus's ability to evade herd immunity and broaden its target population by identifying novel host factors. For example, the GII.17 strain exhibits weak or no binding affinity towards ABH-type HBGA, and the presence of lactose effectively inhibits its binding. The prevalence of the GII.17 variant increased in 2014/2015, showing a notable affinity for HBGA, no longer impeded by lactose[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Analysis of the data suggests that GII.13 can bind to Lec but not general HBGAs. Given that GII.13 norovirus has a unique HBGA binding site and a close evolutionary relationship to GII.17[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], GII.13 also has the potential to expand its target population through amino acid mutations, posing a potential risk of widespread outbreaks and necessitating further attention.\u003c/p\u003e \u003cp\u003eOverall, this study examined the distribution of recombination sites in norovirus and the effects of recombination on the replication efficiency and binding capacity of GII.P21-GII.13 norovirus. The findings suggest that alterations in the non-structural proteins of the OR713748/2009 had a weak effect on the replication efficiency of the virus, but that proteins other than RdRp may have influenced the mutation rate of VP1, which providing a possible explanation for the GII13 norovirus's inability to bind HBGA while still maintaining its ability to infect the human host. Furthermore, gaining a comprehensive understanding of the molecular mechanism underlying the binding of GII.13 P protein to HBGAs, and analyzing the residues involved in the binding process, may be valuable for the development of drugs and vaccines targeting GII.13 noroviruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe acknowledge all of the clinicians who collected data and samples. This work was supported by the National Natural Science Foundation of China (31601570, 32370151) and Plan of Action for Scientific and Technological innovation of Science and Technology Commission of Shanghai Municipality (22N31900700). The authors have no conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePatel MM, Widdowson M-A, Glass RI et al (2008) Systematic literature review of role of noroviruses in sporadic gastroenteritis. 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Front Microbiol 12:653719. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2021.653719\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2021.653719\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"norovirus, recombination, HBGAs, binding affinity, molecular dynamics simulation, GII.13[P21]","lastPublishedDoi":"10.21203/rs.3.rs-4986214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4986214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNorovirus is a pervasive pathogen that causes global outbreaks of viral gastroenteritis. Previous studies suggest that histo-blood group antigens (HBGAs) can interact with norovirus, facilitating its entry of host cells and significantly impacting its evolution. In this study, a complete genome of recombinant GII.13[GII.P21] norovirus from fecal samples was analyzed, revealing a weak effect of genomic recombination on the replication efficiency of GII.13[GII.P21]. Molecular dynamics simulations of GII.13 norovirus P proteins from 1978 to 2019 showed changes in binding capacity with HBGAs. Initially, GII.13 proteins bound A or B/H-type HBGAs, but subsequent residue mutations resulted in a loss of this binding capacity, favoring binding to the HBGA type I precursor (Lec) over A or B/H and Lewis antigens.\u003c/p\u003e","manuscriptTitle":"Identification of a GII.P21-GII.13 recombinant norovirus strain, mutations shift its binding spectra to host receptor glycans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-11 08:13:43","doi":"10.21203/rs.3.rs-4986214/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-09-08T22:45:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-08T13:46:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-02T14:32:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2024-09-02T04:50:38+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-08-31T11:14:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e151dc03-14ec-41a3-af6b-f231faca1fcd","owner":[],"postedDate":"October 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T16:00:46+00:00","versionOfRecord":{"articleIdentity":"rs-4986214","link":"https://doi.org/10.1007/s00705-025-06277-7","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-04-01 15:57:28","publishedOnDateReadable":"April 1st, 2025"},"versionCreatedAt":"2024-10-11 08:13:43","video":"","vorDoi":"10.1007/s00705-025-06277-7","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06277-7","workflowStages":[]},"version":"v1","identity":"rs-4986214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4986214","identity":"rs-4986214","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}