Emergence of Human–Bovine Reassortant G8P[8] Rotaviruses in South Korea: Whole-Genome and Antigenic Characterization Highlighting Vaccine Mismatch Potential

Emergence of Human–Bovine Reassortant G8P[8] Rotaviruses in South Korea: Whole-Genome and Antigenic Characterization Highlighting Vaccine Mismatch Potential | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 11 August 2025 V1 Latest version Share on Emergence of Human–Bovine Reassortant G8P[8] Rotaviruses in South Korea: Whole-Genome and Antigenic Characterization Highlighting Vaccine Mismatch Potential Authors : Thoi Truong , Heekuk Park , Young Rok Kim , and Wonyong Kim 0000-0001-9649-3919 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175492642.20064567/v1 249 views 95 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Rotaviruses remain the leading cause of acute gastroenteritis in children under five years of age. In South Korea, two licensed vaccines—RotaTeq ® and Rotarix TM (RV1)—have significantly reduced the burden of rotavirus-related hospitalizations. However, vaccine-driven immune pressure has coincided with the emergence of uncommon genotypes, including G8P[8], now increasingly detected in pediatric cases. This study reports the complete genome characterization of G8P[8] strains isolated from one vaccinated and two unvaccinated children in Seoul. The strains possessed a DS-1-like genomic constellation and showed evidence of reassortment in the VP7, NSP2, and NSP4 gene segments with bovine-origin strains from Asia and Africa. Amino acid substitutions were identified in the key antigenic regions of VP7 and VP4 when compared with those of the vaccine strains. Three-dimensional structural remodeling was performed to assess the potential structural consequences of these changes. VP7 models showed moderate divergence from the RV1 strain, whereas VP4 exhibited greater conformational shifts, particularly in the VP8 domain. These findings suggest that structural and sequence-level variations may contribute to antigenic drift, potentially reducing vaccine-induced immunity. This study presents the first structure-based antigenic characterization of G8P[8] strains in South Korea, underscoring the need for continued genomic surveillance and consideration of updated vaccine formulations. 1. Introduction Group A rotaviruses (RVAs) (family Sedoreoviridae , genus Rotavirus , species Rotavirus alphagastroenteritidis ) are a leading cause of acute gastroenteritis and diarrhea-related hospitalization in children under five years of age, contributing to over 450,000 annual deaths, predominantly in developing countries 1 2 , . In response, two live-attenuated oral vaccines—Rotarix TM (RV1, monovalent G1P[8]) and RotaTeq ® (RV5, pentavalent G1–G4, P[8])—were introduced in 2006, demonstrating significant effectiveness in reducing severe cases of disease 2 . However, these vaccines do not confer complete protection against heterotypic or emerging rotavirus genotypes that are not represented in their compositions 3 . RVAs possess a segmented genome comprising 11 double-stranded RNA segments that encode six structural (VP1–VP4, VP6, and VP7) and up to six nonstructural proteins (NSP1–NSP5/6). Among them, VP7 and VP4 are outer capsid proteins that elicit neutralizing antibodies and are used for dual genotyping, designated as GxP[x] (e.g., G1P[8]) 4 . In addition to G/P typing, the Rotavirus Classification Working Group proposed a full-genome genotype constellation system encompassing all gene segments, resulting in genotype profiles such as the Wa-like (I1-R1-C1-M1-A1-N1-T1-E1-H1) and DS-1-like (I2-R2-C2-M2-A2-N2-T2-E2-H2) backbones 5 6 , . The G8 genotype was initially identified in cattle and subsequently reported in human infections in Indonesia in 1984 7 8 , . Over time, G8 rotaviruses have spread globally in combination with various P types, including P[4], P[6], and P[8] 9-11 . While sporadically detected in Africa and Europe throughout the early 2000s 12 13 , , the G8P[8] genotype has gained prominence in Asia since 2013, with reports from Thailand, Vietnam, and Japan 3 14 , . These strains often possess DS-1-like genetic backbones and may result from interspecies reassortment between human and animal rotaviruses G8P[8] strains are partially heterotypic to vaccine strains and may contribute to reduced vaccine effectiveness, particularly in regions where they have become predominant. In South Korea, the first detection of human G8P[8] occurred in 2008 17 . After a period of apparent absence, a nationwide surveillance in 2017 reported the re-emergence and subsequent predominance of G8P[8] strains. In this study, the complete genome sequences of three G8P[8] strains isolated from one vaccinated and two unvaccinated children in Korea during 2017 were analyzed. Their genetic constellation, phylogenetic relationships, and antigenic differences in VP7 and VP4 proteins were examined to characterize these strains. Additionally, structure prediction using AlphaFold2 and structural comparisons were conducted to assess the potential impact on vaccine recognition, providing new insights into the evolution and immunogenic divergence of emerging G8P[8] strains. 2. Materials and Methods 2.1. Patient Information, Sample Collection and Analysis, and Ethical Approval Stool samples were collected from pediatric patients hospitalized with acute gastroenteritis at Chung-Ang University Hospital (Seoul, South Korea) in early 2018. Clinical information and vaccination histories were obtained through medical chart review and guardian interviews. Patient CAU17L-79 was a 27-month-old girl who presented with vomiting (twice/day), diarrhea (twice/day), fever (38.5 °C), and abdominal pain. The vaccination history included two doses of the Rotarix TM vaccine administered at 2 and 4 months of age. The patient was hospitalized for four days, during which laboratory results revealed a white blood cell (WBC) count of 8,230 cells/μL and a hemoglobin level of 12.2 g/dL. Patient CAU17L-103 was a 17-month-old unvaccinated boy who experienced severe symptoms, including vomiting (10 times/day), watery diarrhea (once/day), and fever of 39 °C. Laboratory results revealed a hemoglobin level of 12.2 g/dL and a WBC count of 8,260 cells/μL. Patient CAU17L-110 was a 41-month-old unvaccinated girl who experienced watery diarrhea (10 times/day), vomiting (once/day), and fever of 39 °C. Laboratory results for patient CAU17L-110 revealed a hemoglobin level of 11.5 g/dL and a WBC count of 4,560 cells/μL. The stool samples were collected following protocol (#1710-009-303) approved by the Human Subjects Institutional Review Board of Chung-Ang University College of Medicine (Seoul, South Korea). Participation was voluntary, and written informed consent was obtained from the parents or legal guardians. This consent included authorization to use the data for future research purposes. Rotavirus infection was confirmed in all three stool samples (CAU17L-79, CAU17L-103, and CAU17L-110) using the RIDASCREEN Rotavirus ELISA kit (R-Biopharm AG, Darmstadt, Germany), according to the manufacturer’s instructions. All samples tested negative for other viral pathogens, including astrovirus, enteric adenovirus, and norovirus, using the Seeplex Diarrhea-V ACE Detection Assay (Seegene, Seoul, South Korea). 2.2. Rotavirus Isolation and Propagation Stool samples confirmed positive for rotavirus infection were further processed for virus isolation using MA104 cells, as previously described 18 . For this purpose, each sample was prepared as a 10% (w/v) suspension in phosphate-buffered saline (pH 7.4), followed by centrifugation at 10,000 × g for 10 min at 4 °C to remove particulate debris. Clarified suspensions were used to infect confluent monolayers of MA104 cells cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Prior to infection, the cells were washed and incubated with a 200-μL virus suspension containing 10 μg/mL trypsin (type IX; Sigma-Aldrich, St. Louis, MO, USA) to enhance viral infectivity. Cultures were monitored daily at 37 °C for cytopathic effects. Infected cells were passaged every five days until clear cytopathic effects were observed, typically after 2–4 passages. To confirm successful viral replication following cell culture, reverse transcription-polymerase chain reaction was performed on the culture supernatants using VP6-specific primers and stored at −80 °C for downstream genomic and antigenic analyses. 2.3. RNA Extraction and Whole-Genome Sequencing Double-stranded RNA was extracted from 140 μL of 10% (w/v) fecal suspensions using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. The purified RNA was eluted in 50 μL of RNase-free water and stored at −80 °C until further analysis. For whole-genome sequencing, reverse transcription and second-strand synthesis were performed to generate double-stranded cDNA. Sequencing libraries were prepared using the Ligation Sequencing Kit and Native Barcoding Expansion Kit (SQK-LSK109 and EXP-NBD104, respectively; Oxford Nanopore Technologies, Oxford, UK), according to the manufacturer’s instructions. Barcoded libraries were pooled and loaded onto a FLO-MIN106D (R9.4.1) flow cell for sequencing using a MinION Mk1B device. Basecalling was performed using Guppy (v.4.5.4) in high-accuracy mode, and barcode demultiplexing was carried out using qcat. Reads were assembled via a reference-guided approach using the Medaka pipeline (https://github.com/nanoporetech/medaka; Oxford Nanopore Technologies), followed by manual curation. Genotypes were assigned using the RotaC (v.2.0) online classification tool (http://rotac.regatools.be). Complete genome sequences of the Korean G8P[8] strains—CAU17L-79, CAU17L-103, and CAU17L-110—were deposited in the GenBank database under accession numbers MN058730–MN058762. 2.4 Phylogenetic Analysis Nucleotide sequences for each of the 11 rotavirus gene segments (VP1–4, VP6, VP7, and NSP1–NSP5/6) were aligned using the MUSCLE algorithm implemented in MEGA v.6.0 (MEGA6) 1920, . Phylogenetic trees were independently constructed for each segment using three methods: neighbor-joining, maximum-likelihood, and maximum-parsimony 21 . The best-fitting substitution models for maximum-likelihood analyses were selected based on the lowest Bayesian Information Criterion score calculated in MEGA6. The robustness of each tree topology was evaluated via bootstrap analysis with 1,000 replicates. Consensus trees were visualized using MEGA6 with default parameters, and reference sequences were retrieved from the National Center for Biotechnology Information GenBank database based on genotype, lineage, and geographic relevance. Lineage assignments were performed in accordance with established criteria for genogroup classification. A complete list of the reference strains and their accession numbers is provided in Supplementary Table 1. 2.5. Epitope Comparison and Structural Modeling Epitope-level sequence analyses were performed using RV1 and RV5 vaccine strains to identify amino acid substitutions in antigenic regions of the Korean G8P[8] strains. For three-dimensional (3D) structural comparisons, the RV1 strain—representing the Wa-like G1P[8] genotype—was selected as the vaccine reference, considering that patient CAU17L-79 had been vaccinated with RV1. Structural models of both the RV1 and Korean G8P[8] strains were generated using AlphaFold2 via the ColabFold platform 22 23 , . Structural alignments and root-mean-square deviation ( RMSD) analyses were performed using the bio3d package in R 24 . Residue-level visualization and mutation mapping were carried out using the r3dmol package, enabling precise annotation of antigenic domains and structural localization of substitutions. All structural models and comparisons were generated in silico and were not validated through experimental methods. 3. Results 3.1. Genotyping and Genomic Backbone Classification Whole-genome sequencing of the three rotavirus isolates—CAU17L-79, CAU17L-103, and CAU17L-110—confirmed that all strains belonged to the G8P[8] genotype. Genotyping based on all 11 rotavirus gene segments revealed a consistent constellation of G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2, corresponding to the DS-1-like genomic backbone. Genotype assignments were performed using the RotaC classification tool and supported by phylogenetic comparisons with reference strains retrieved from GenBank (Table 1, Supplementary Table 1). VP7 and VP4, the principal surface antigens, were examined for genetic and antigenic divergence from current vaccine strains to assess potential implications for vaccine effectiveness. 3.2. Phylogenetic Analysis of VP7, VP4, VP6, and Internal Genes 3.2.1. Capsid Gene Segments (VP7, VP4, and VP6) The VP7 genes of all three Korean strains clustered within lineage II of the G8 genotype and were thus closely related to human/bovine-like strains from Asia, including RVN1149 (Vietnam), SSKT-269 (Thailand), and S01162 (Japan) (Fig. 1A). Nucleotide and amino acid identities with these strains ranged from 98.95–99.81% and 99.06–99.69%, respectively. In contrast, lineage I strains, such as DRC88, showed a lower similarity (83.90–86.65% nt; 95.63–96.93% aa). Similarly, the VP4 genes clustered within P[8]-II, exhibiting 99.76–100% nucleotide identity among the Korean strains and clustering with Asian reassortant strains (Fig. 1B). In contrast, sequence identity with classical Wa-like P[8]-I strains, including RV1 and RV5, was notably lower (89.9–90.2% nt; 93.9–94.0% aa). The VP6 genes, which define the I-genotype, classified all three Korean strains as I2, showing a high similarity (>99% nt) to other G8P[8] DS-1-like reassortants (Fig. 1C), while exhibiting only modest similarity to bovine strains from India (93.3–93.5% nt). 3.2.2. Internal and Nonstructural Gene Segments (VP1–3, NSP1, NSP3, and NSP5) The VP1–VP3, NSP1, NSP3, and NSP5 genes consistently clustered within the DS-1-like genogroup, with each segment classified as follows: R2 (VP1), C2 (VP2), M2 (VP3), A2 (NSP1), T2 (NSP3), and H2 (NSP5). Sequence identities among the Korean strains and with reference strains, such as RVN1149, ERN8263, and HC12016, exceeded 99% at both nucleotide and amino acid levels (Fig. 2A–F; Supplementary Table 1). Comparisons with the DS-1 prototype strain showed a high sequence identity (>91% nt; >96% aa), whereas alignment with RV5-associated segments showed reduced similarity (85.5–86.2% nt; 3.2.3. Sequence Divergence of NSP2 and NSP4 Genes The NSP2 gene exhibited a divergence pattern: strains CAU17L-79 and CAU17L-110 clustered within N2-II, closely related to RVN1149 (Vietnam) and MRC-DPRU456 (South Africa), whereas strain CAU17L-103 clustered within N2-IV, aligning with G1P[8] and G3P[4] strains from Japan and Hungary, respectively (Fig. 3A). This intra-genotypic variation may indicate a distinct reassortment event. All three NSP4 genes were classified within E2-I, exhibiting high nucleotide similarity (99.4–99.8%) to previously reported G8P[8] strains from Asia. Additional similarity (93.3–95.0% nt) was observed with Indian bovine strains (E2-II), whereas a lower identity (<92%) was noted with the COD/DRC88 prototype and the original DS-1 strains (Fig. 3B; Supplementary Table 1). 3.3. Antigenic and Structural Comparison of VP7 and VP4 with Vaccine Strains To evaluate the antigenic divergence of Korean G8P[8] strains from vaccine strains, both sequence-based epitope analyses and in silico structural modeling were performed for the VP7 and VP4 proteins. 3.3.1. VP7 Antigenic and Structural Comparison Amino acid sequence alignment revealed 25–26 differences in VP7 proteins of the Korean strains when compared with those of RV1 and RV5. Among the 29 residues constituting the three major neutralizing epitopes (7-1a, 7-1b, and 7-2), 14 substitutions were identified relative to RV1, and four substitutions relative to RV5. Comparison with RV1 showed six mutations in the 7-1a epitope (including N94A, G96S, and others), three in 7-1b (N211D, D213T), and five in 7-2 (D145N, Q146A, and others), whereas the comparison with RV5 included fewer substitutions. Despite these epitope changes, the AlphaFold2-predicted 3D structures of VP7 showed high structural conservation, with an RMSD of 7.80 Å between the Korean strain (CAU17L-79) and RV1. Mutations were localized to exposed loop regions without disrupting the overall protein fold, suggesting that although antigenic drift is present, the structural stability is largely maintained (Fig. 4A). 3.3.2. VP4 Antigenic and Structural Comparison VP4 analysis identified 17 amino acid differences between the Korean G8P[8] and vaccine strains. Mutations were primarily located within established antigenic domains: the VP8* region (including E150D, N113D, S125N, and N135D) and in VP5* (L388I). Compared to RV1, six amino acid substitutions were found across antigenic domains 8-1 and 8-3, and the comparison with RV5 revealed three. Structural modeling of VP4 proteins revealed a more pronounced conformational divergence. The predicted VP4 model of CAU17L-79 exhibited an RMSD of 27.6 Å when compared to the RV1 model, indicating substantial structural differences. Mutation sites were mapped onto VP8* (residues 1–247) and VP5* (residues 248–776), with the majority of substitutions localized within VP8*, which may affect receptor binding and antibody recognition (Fig. 4B). Notably, no antigenic differences were observed among the three Korean G8P[8] strains for both VP7 and VP4 proteins, regardless of the vaccination status of the patient. The observed mutations were consistently localized within or adjacent to defined neutralizing epitopes, underscoring their potential impact on vaccine cross-protection. 4. Discussion Rotaviruses continue to be a major cause of severe gastroenteritis in children under five years of age, particularly in developing countries, despite the global implementation of vaccines 17 . Since the introduction of RV1 and RV5 in 2006, a marked decline in rotavirus-related morbidity and mortality was observed 1 . However, vaccine effectiveness varies by genotype, with RV1 reported to be 61–88% effective and RV5 88–95% effective in preventing severe gastroenteritis 1 2 , . Breakthrough infections and genotype shifts following vaccination have raised concerns regarding the emergence of vaccine-escape variants 3 . In South Korea, the genotype landscape has shifted over time. Prior to vaccine introduction (2004–2007), the predominant genotypes were G1P[8], G3P[8], and G4P[6] 25 . After 2008, genotypes such as G9P[8], G3P[8], and the re-emerged G2P[4] were detected between 2007 and 2015 26–27 . Since 2017, G8P[8] has become the predominant genotype in Seoul, mirroring trends observed in Vietnam, Thailand, and Japan 3 14 , . G8P[8] is an unusual genotype historically associated with bovine hosts, but has increasingly been identified in human infections 8-10 . Whole-genome analyses performed in the present study revealed that the Korean G8P[8] strains possess a DS-1-like genomic constellation: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis showed close genetic relatedness between Korean G8P[8] strains and human–bovine reassortant strains from Asia (RVN1149, SSKT-269, and S01162) and with select African bovine strains (such as MRC-DPR456/2009) 3 14 , . Gene segments VP7, NSP2, and NSP4 exhibited varying degrees of similarity to those of bovine-origin strains from India and South Africa, supporting the hypothesis that these Korean strains may have emerged through multiple reassortment events involving interspecies transmission. Reassortment between human and animal rotaviruses is a well-established mechanism contributing to their evolution and antigenic diversity 26 . In the post-vaccine era, immune pressure may select for antigenically divergent variants, facilitating the emergence of non-vaccine genotypes such as G8P[8] 27 . VP7 and VP4 constitute the primary targets of neutralizing antibodies, and mutations within their antigenic regions may influence vaccine effectiveness. Sequence-level epitope analysis revealed multiple amino acid substitutions in both proteins when compared to the RV1 and RV5 strains. VP7 showed 14 substitutions relative to RV1 and four relative to RV5, which were distributed across all three major epitopes (7-1a, 7-1b, 7-2) 28 29 , . Similarly, VP4 mutations were identified within the VP8* (regions 8-1, 8-3) and VP5* (region 5-1) domains 30 31 , . To better understand the structural implications of epitope variation, VP7 and VP4 proteins of the Korean G8P[8] and RV1 vaccine strains were modeled using AlphaFold2. Structural alignment revealed a moderate difference in VP7 (RMSD: 7.8 Å), whereas the VP4 comparison yielded a substantially higher RMSD value of 27.6 Å, primarily attributable to conformational divergence in the VP8* domain. These results suggest that even modest amino acid substitutions in surface proteins may contribute to antigenic drift, potentially affecting vaccine-mediated protection. Interestingly, no epitope-level differences in VP7 or VP4 were observed among the three Korean isolates, regardless of the vaccination status of the children. This raises the possibility that current vaccines, particularly RV1, may offer limited protection against these emerging G8P[8] strains. These findings suggest that the observed amino acid substitutions, particularly within structurally and antigenically important regions of VP4 and VP7, may influence the antigenic landscape of G8P[8] strains. Although the structural divergence revealed through RMSD analysis highlights the potential for immune evasion, recognizing the limitations of computational modeling in capturing full immunological complexity is important. Nevertheless, this study offers the first structure-informed antigenic characterization of G8P[8] strains in South Korea and emphasizes the need for continued molecular surveillance, integration of structural tools in genotype monitoring, and periodic reassessment of vaccine strain composition to ensure broad and sustained protection. Taken together, the study findings suggest that structural and sequence-level alterations in VP7 and VP4 may affect antigenic recognition by vaccine-induced antibodies. Although computational modeling provided insights into potential immune evasion, experimental validation, such as serological assays and antibody-binding studies, is essential to confirm these predictions. This study underscores the increasing importance of integrating genomic surveillance with structural virology to guide vaccine design, especially as genotype shifts continue to reshape the post-vaccine rotavirus landscape. 5. Conclusions This study provides the first complete genome characterization of G8P[8] rotavirus strains in South Korea. The isolates from both vaccinated and unvaccinated children exhibited a DS-1-like genotype constellation and shared identical antigenic profiles. Comparative analysis with the RV1 and RV5 vaccine strains revealed multiple amino acid substitutions within key epitope regions of VP7 and VP4. These findings suggest that current vaccines exhibit limited cross-protection against emerging strains and highlight the need for continued genomic surveillance and antigenic monitoring of emerging rotavirus strains. Author Contributions Conceptualization and study design: Wonyong Kim. Methodology: Thoi Cong Truong. Data analysis: Heekuk Park, Young Rok Kim, and Wonyong Kim. Contributed reagents, materials, and analytical tools: Wonyong Kim. Writing–original draft, manuscript review, and editing: and Thoi Cong Truong, Heekuk Park, Wonyong Kim. All authors reviewed and approved the final article. References 1. Matthijnssens J, Ciarlet M, McDonald SM, et al. Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Arch Virol. 2011;156(8):1397-1413.2. Tate JE, Burton AH, Boschi-Pinto C, Parashar UD, World Health Organization-Coordinated Global Rotavirus Surveillance N. 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Genetic Analyses Reveal Differences in the VP7 and VP4 Antigenic Epitopes between Human Rotaviruses Circulating in Belgium and Rotaviruses in Rotarix and RotaTeq. Journal of Clinical Microbiology. 2012;50(3):966-976.28. Lazdins I, Coulson BS, Kirkwood C, et al. Rotavirus Antigenicity Is Affected by the Genetic Context and Glycosylation of Vp7. Virology. 1995;209(1):80-89.29. Coulson BS, Kirkwood C. Relation of Vp7 Amino-Acid-Sequence to Monoclonal-Antibody Neutralization of Rotavirus and Rotavirus Monotype. Journal of Virology. 1991;65(11):5968-5974.30. McDonald SM, Matthijnssens J, McAllen JK, et al. Evolutionary Dynamics of Human Rotaviruses: Balancing Reassortment with Preferred Genome Constellations. Plos Pathog. 2009;5(10).31. Patton JT. Rotavirus Diversity and Evolution in the Post-Vaccine World. Discov Med. 2012;13(68):85-97. Figure legends Figure 1. Neighbor-joining phylogenetic trees of the VP7 (A), VP4 (B), and VP6 (C) segments from the Korean G8P[8] and reference strains available on GenBank. Different genotypes within each tree are indicated. Phylogenetic analysis was conducted using the MEGA v.6.0 program with the neighbor-joining method and 1,000 bootstrap replicates. The strains analyzed in this study are highlighted in red. Figure 2. Phylogenetic analysis of the VP1 (A), VP2 (B), and VP3 (C) genomic segments using the same methodology as that described in Figure 1. Korean G8P[8] strains are marked in red. Figure 3. Phylogenetic trees of the NSP1–NSP5 (A–E) genomic segments constructed using the neighbor-joining method with 1,000 bootstrap replicates in MEGA v.6.0. Strains used in this study are shown in red; genotypic lineages are labeled. Figure 4. Structural comparison of VP7 and VP4 proteins between the Korean G8P[8] rotavirus and Wa-like vaccine strains. (A) VP7 protein: superimposed structural models of VP7 from the CAU17L-79 (sky blue) and Wa-like reference (gray) strains. Mutations in major antigenic regions are color-coded and labeled according to their locations: red, 7-1a region (residues 94, 96, 97, 125, 129, and 130); yellow, 7-1b region (residues 211, 212, 213, 217, and 221); and green, 7-2 region (residues 145, 146, 147, and 148). These antigenic sites correspond to neutralizing epitopes critical for antibody binding. The structural overlay shows conserved core regions, but surface epitope variability may affect immune recognition. (B) VP4 protein: structural alignment of VP4 proteins from the CAU17L-79 (VP8*: sky blue; VP5*: green) and Wa-like reference (gray) strains. Amino acid mutations within the neutralizing epitope regions are highlighted as follows: red, VP8*-1 region (residues 150 and 195); blue, VP8*-3 region (residues 113, 125, and 135); and yellow, VP5*-1 region (residue 388). The VP4 protein was divided into two functional subunits (VP8* and VP5*) following proteolytic cleavage. Mutations in VP8* are more surface-exposed and implicated in host cell attachment, suggesting potential vaccine escape via altered antigenicity. Table 1. Genotypic characterization of the 11 genome segments of Korean G8P[8] and reference rotavirus strains obtained from GenBank Strains Country of origin Genotype VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5 RVA/Human-wt/Kore/CAU17-79/2017 Korea G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/Kore/CAU17L-103/2017 Korea G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/Kore/CAU17L-110/2017 Korea G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/THA/SSKT-269/2014 Thailand G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/VNM/RVN1149/2014 Vietnam G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/JPN/SO1162/2017 Japan G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/COD/DRC88/2003 Congo G8 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/CR2006/2006 Croatia G8 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-tc/IND/69M/1980 India G8 P[10] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/GHA/GH018-08/2008 Ghana G8 P[6] I2 R2 C2 M2 A2 N2 T2 E2 H3 RVA/Human-tc/USA/DS-1/1976 USA G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/Itali/PR457/2009 Italy G10 P14 I2 R2 C2 M2 A11 N2 T6 E2 H3 RVA/Human-wt/HUN/ERN5523/2012 Hungary G3 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/HUN/ERN8263/2015 Hungary G3 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Human-wt/JPN/HC12016/2012 Japan G1 P[8] I2 R2 C2 M2 A2 N2 T2 E2 H2 RVA/Cow-wt/ZAF/MRC-DPRU456/2009 South Africa G6 P[11] I2 R2 C2 M2 A13 N2 T6 E2 H3 RVA/Cow-wt/USA/WC3/1981 USA G6 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/Bovine-wt/NCDV USA G6 P[6] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/Bovine-wt/India/WB/2011 India G6 P[11] I2 - - - - N2 T6 E2 H3 RVA/Bovine-wt/India/HR/2011/B91 India G1 P[11] I2 - - - - N2 T6 E2 H2 RVA/Bovine-wt/Ind/UP/BE4 India G8 P[10] - - - - - - - E2 - RVA/Cow-wt/IND/86/2007 India G8 P[14 I2 - - - - - - E2 - RVA/Human-tc/USA/Wa/1974 USA G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/USA/Rotarix/2009 USA G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/USA/RotaTeq-WI79-9/1992 USA G1 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/USA/RotaTeq-SC2-9/1992 USA G2 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/USA/RotaTeq-WI78-8/1992 USA G3 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/USA/ RotaTeq-BrB-9/1996 USA G4 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/USA/RotaTeq-WI79-4/1992/G6P8 USA G6 P[8] I2 R2 C2 M2 A3 N2 T6 E2 H3 RVA/Horse-wt/GBR/L338/1991/2012/G1P8 UK G13 P18 I6 R9 C9 M6 A6 N9 T12 E14 H11 Orange-colored cells indicate genome segments derived from the G8 rotavirus strains; blue indicates segments derived from the Wa-like (G1P[8]) rotavirus backbones; and green indicates segments derived from the DS-1-like (G2P[4]) backbones. Bold text indicate information on the G8P[8] rotavirus strains isolated in Korea; “-” indicates data that could not be obtained. Table 2. Differences in antigenic sites among the RV1 and RV5 vaccine strains and Korean G8P[8] rotaviruses (A) Additional amino acid variations in VP7 9 15 16 34 40 45 50 66 68 70 71 72 73 90 118 119 120 139 189 213 233 235 275 278 G1 RV1 I S I I I F A V A S T Q E S F K E L S D I H P N G1/RV5 I S I I I F A V A S T Q E S F K E L S D I H P N G2 RV5 I S I I V S T V T S T S G K F K D V T S V H P V G3 RV5 V S V I I S A S T S T R E A F K D L S N V H P A G4 RV5 V S F I V S A A A S T Q D P F N E V S A V H P S G6 RV5 I S I I M A A A A S T Q S S F K E L S D V H P T CAU17L-79 T L L L F T S N Q V S T S E L R S I D T V Y L A CAU17L-103 T L L L F T S N Q V S T S E L R S I D T V Y P A CAU17L-110 T L L L F T S N Q V S T S E L R S I D T L Y P A (B) Variations in defined VP7 neutralizing epitopes 87 91 94 96 97 98 99 100 104 123 125 129 130 191 201 211 212 213 238 242 143 145 146 147 148 190 217 221 264 G1 RV1 T T N G E W K D Q S V V D K Q N V D N T K D Q N L S M N G G1 RV5 T T N G D W K D Q S V V D K Q N V D N T K D Q S L S M N G G2 RV5 A N S D E W E N Q D T M N K Q D V S N S R D N T S D I S G G3 RV5 T T N N S W K D Q D A V D K Q D A N K D K D A T L S E A G G4 RV5 S T S T E W K D Q N L I D K Q D T A D T R A S G E S T S G G6 RV5 V N A T E W K D Q D A V E K Q N P D N A K D S T Q S T T G CAU17L-79 T T A S S W K D Q D A I N K Q D T T N T K N A N S S E A G CAU17L-103 T T A S S W K D Q D A I N K Q D T T N T K N A N S S E A G CAU17L-110 T T A S S W K D Q D A I N K Q D T T N T K N A N S S E A G CAU17L-79, CAU17L-103, and CAU17L-110 represent the G8P[8] rotavirus strains isolated in Korea. Table 3. Differences in VP4 amino acid sequences between the RV1 and RV5 vaccine and Korean G8P[8] strains 7 19 78 106 108 120 149 150 245 281 385 560 580 587 597 604 617 P[8] RV1 R H N V I M N F K V Y I I I V L K P[8] RV5 R H I V I T N F K V H I I I V L K P[5] RV5 R S V I I T D G P I Y S S T Q E G CAU17L-79 K Y T I V N S L T I D V V F I V N CAU17L-103 K Y T I V N S L T I D V V F I V N CAU17L-110 K Y T I V N S L T I D V V F I V N Antigenic sites across VP8* and VP5* are included. CAU17L-79, CAU17L-103, and CAU17L-110 represent the G8P[8] rotavirus strains isolated in Korea. Table 4. Differences in antigenic sites among the RV1 and RV5 vaccine and Korean G8P[8] strains (A) VP8* antigenic domain 8-1 8-2 8-3 8-4 100 146 148 150 188 190 192 193 194 195 196 180 183 113 114 115 116 125 131 132 133 135 87 88 89 P[8] RV1 D S Q E S T N L N N I T A N P V D S S N D N N T N P[8] RV5 D S Q E S T N L N D I T A N P V D N R N D D N T N P[5] RV5 G T I G R I T N Y A S E N T S E T S S N A D T G P CAU17L-79 D S Q D S T N L N D I T A D P V D N R N D D N T N CAU17L-103 D S Q D S T N L N D I T A D P V D N R N D D N T N CAU17L-110 D S Q D S T N L N D I T A D P V D N R N D D N T N (B) VP5* antigenic domain 5-1 5-2 5-3 5-4 5-5 384 386 388 393 394 398 440 441 434 459 429 306 P[8] RV1 Y F I W P G R T P E L R P[8] RV5 Y F L W P G R T P E L R P[5] RV5 D S A Q W K T R E R R M CAU17L-79 Y F I W P G R T P E L R CAU17L-103 Y F I W P G R T P E L R CAU17L-110 Y F I W P G R T P E L R CAU17L-79, CAU17L-103, and CAU17L-110 represent the G8P[8] rotavirus strains isolated in Korea. Information & Authors Information Version history V1 Version 1 11 August 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords genetic variation genetics human rotavirus mutation recombination virus classification Authors Affiliations Thoi Truong Chung-Ang University College of Medicine View all articles by this author Heekuk Park Columbia University Division of Infectious Diseases View all articles by this author Young Rok Kim Chung-Ang University College of Medicine View all articles by this author Wonyong Kim 0000-0001-9649-3919 [email protected] Chung-Ang University College of Medicine View all articles by this author Metrics & Citations Metrics Article Usage 249 views 95 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Thoi Truong, Heekuk Park, Young Rok Kim, et al. Emergence of Human–Bovine Reassortant G8P[8] Rotaviruses in South Korea: Whole-Genome and Antigenic Characterization Highlighting Vaccine Mismatch Potential. Authorea . 11 August 2025. DOI: https://doi.org/10.22541/au.175492642.20064567/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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