Comparative analysis of the RVA VP7 and VP4 antigenic epitopes circulating in Iran and the Rotarix and RotaTeq vaccines

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In this study, circulating lineages of VP4 and VP7 proteins of human RVA isolates from children under 5 years of age were analyzed and their cytotoxic T cell and antigenic epitopes were compared to the RotaTeq and Rotarix vaccine strains. Methods Viral RNA was extracted from 51 positive samples and amplified using specific primers. Sequencing was performed and multiple sequence alignments were done in MEGA and phylogenetic trees were constructed. Similarity of VP7 and VP4 amino acids with the vaccine stains and structural analysis were performed using the UCSF Chimera-Molecular Modeling System. Results The Iranian strains clustered in the G1/II, G2/IV, G3/I, G4/I, G9/III, P[8]/III, P[4]/IV, and P[6]/I lineages. Comparative analysis of VP7 antigenic epitopes showed that G1/II strains are completely conserved, but G2/IV, G3/I, G4/I, G6, G9/III strains contained 2, 3-5, 2, 4 and 9 amino acids substitutions, respectively. P[8]/III genotypes differed by 3 amino acids, while P[6]/I genotypes had the most substitutions. CTL epitopes were completely conserved in G3/I strains, but other genotypes differed by 1-4 amino acids in comparison to the vaccine strains. Conclusions Considering the diversity of circulating RVA genotypes and the observed mutations in the neutralizing and CTL epitopes, immune escape by some of the strains is likely in Iran. This finding underscores the importance of evaluating the effect of rotavirus vaccines on local genotypes and related lineages before implementing the vaccination program. Rotavirus Vaccine VP4 VP7 Lineage Antigenic epitopes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Rotavirus is a leading cause of diarrheal disease, accounting for approximately 29.3% of global deaths related to diarrhea in children under 5 years of age [1]. Over 90% of these mortalities occur in low- to middle-income countries. Despite a significant drop in the global burden of rotavirus over the past three decades, mainly due to the introduction of rotavirus vaccines, the prevalence of rotavirus remains high in Africa, Oceania, South Asia, and the Middle-East [2,3]. The most common rotavirus genotypes found in children worldwide vary by region and time period. This diversity is linked to the virus’s genomic structure and its geographic evolution, influenced by common host factors and transmission routes. The infectious RVA particle has an icosahedral structure with three layers, consisting of 11 segments of double-stranded RNA encoding six structural proteins (VP) and six non-structural proteins (NSP) [4]. To date, 35 G genotypes and 50 P genotypes have been described for group A rotaviruses. The most prevalent genotypes worldwide are G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] [5]. These genotypes are determined based on the two outer capsid proteins, VP7 (G type) and VP4 (P type), which function as specific binding sites for neutralizing antibodies [4]. The introduction of rotavirus vaccines has been associated with changes in the distribution of rotavirus genotypes in certain areas, resulting in a decline in the prevalence of vaccine-targeted genotypes and an increase in the prevalence of genotypes not covered by the vaccines [6]. Recent studies have revealed variations in the distribution of RVA genotypes across different age groups. In Canada and Greece, a higher prevalence of G12P[8] and G9P[8] genotypes was observed among older children, specifically those aged between 24 and 59 months [7,8]. In children aged 0 to 12 months, other genotypes like G1P[8] and G9P[4] genotypes were found to be more common [9,10]. Several factors may contribute to this occurrence, including diversity in the expression of certain receptors in the intestinal tract at different age groups, exposure to common genotypes in various geographic areas, induced immunity, and host genetics [11–13]. Similarly, there is evidence of a correlation between rotavirus genotypes and disease symptoms. Studies have shown that different genotypes of rotavirus are associated with varying clinical characteristics and disease severity. In one study, G1P[8] and G9P[8] were the most common genotypes detected in children with moderate and severe acute gastroenteritis (AGE), respectively [9]. A significantly higher frequency of fever in children infected with the G3P[8] genotype was reported in study of Mathew et al. [14]. Additionally, this correlation was detected in the case of emerging genotypes, such as G8P[8], and G9P[4] [15]. The interaction between RVA antigens of various genotypes and the host immune system offers an explanation for variations in disease outcomes among the immunized and non-immunized children. This interaction primarily depends on the immunogenic domains within the VP4 and VP7 proteins. Notably, the VP7 gene, encompassing 326 amino acids, contains nine variable regions, with four serving as antigenic epitopes, specifically identified as 7-1a, 7-1b, and 7 − 2. Activation of the VP4 protein, spanning 776 amino acids, requires proteolytic cleavage into two distinct segments: VP8 and VP5, each containing four (8 − 1–8 − 4) and five (5 − 1–5–5) antigenic epitopes, respectively [4]. Due to the diversity of immunogenic motifs within these proteins, multiple lineages and sub-lineages have been defined. According to the study of Motamedi-Rad et al., within the predominant G-types, there are eleven, six, four, six, and six lineages for G1, G2, G3, G4, and G9 lineage, respectively. In terms of P serotypes, four, five, and five distinct lineages were reported for P[8], P[4], and P[6], respectively [16]. RV vaccination is the most effective strategy for significantly reducing the incidence of severe infections and the number of deaths in children. Rotarix (GlaxoSmithKline) consists of a monovalent G1P[8] strain derived from a single human G1P[8] strain. Conversely, RotaTeq (Merck), is a human-bovine reassortant vaccine, composed of five strains derived from human (G1, G2, G3, and G4) and bovine (P[8]) strains [17]. Both RVA vaccines have demonstrated the ability to elicit homotypic and heterotypic immune responses, resulting in a notable decline in morbidity and mortality. However, the RVA vaccine has proven to be more effective in high-income countries (80–90%) against severe rotavirus disease compared to low- to middle-income countries (40–70%) [18]. Various factors contribute to this disparity, including the genotypic and lineage diversity of RVA strains, which are driven by recombination, rearrangement, reassortment, and mutations. Additionally, host genetic factors, malnutrition, gut microbiota dysbiosis, co-infections, environmental enteropathy, and the passive transfer of maternal antibodies have been suggested as potential determinants of differences in RVA vaccine efficacy between high- and low-income countries [16]. RVA lineages with distinct antigenic properties could potentially allow RVA strains to evade vaccine-induced immunity [19]. With the increasing diversity of RVA strains and the incidence of uncommon fully and partially heterotypic genotypes, such as G1P[6] or G9P[4], particularly in countries without RVA vaccination programs, concerns are growing regarding the reduced effectiveness of approved vaccines against the emerging variants [18]. In this current cross-sectional study, the VP7 and VP4 lineages of human rotavirus A (RVA) strains circulating in children under the age of five with diarrhea were analyzed. Additionally, the amino acid sequences of these RVAs and their potential antigenic distinctions and cytotoxic T cell epitopes compared to the vaccine strains were assessed. Materials and Methods Sample selection In order to determine the phylogenetic and antigenic epitope associations of VP7 and VP4 among the Iranian and vaccine strains, a total of 51 rotavirus strains were included in this study. This study was approved by the research ethics committees of Research Institute of Children’s Health at Shahid Beheshti University of Medical Sciences (IR.SBMU.RICH.REC.1401.021), and parental consent was obtained for all the children’s samples. RNA extraction, RT-PCR, and Amplification of VP7 and VP4 Viral RNA was extracted from 10% stool suspensions of the selected rotavirus-positive fecal specimens using the High Pure Viral Nucleic Acid Extraction kit (Roche, Mannheim, Germany). The RNA extracts were denatured at 95°C for five minutes, and the AddScript cDNA Synthesis kit (Addbio, Daejeon, South Korea) was used to generate cDNAs through RT-PCR using primers Beg9 and End9 (VP7), and Con3 and Con2 (VP4) following WHO protocols [20]. This cDNA served as a template for amplifying the of full-length VP7 (1062 base pairs) and partial-length VP4 (VP8 region, 876 base pairs) genes of RVA strains, as described previously [21]. The PCR products were verified using electrophoresis in a 1.5% agarose gel and sequencing as described below. Nucleic acid sequencing Sequencing was performed using the same forward primers that were used in the RT-PCR. The obtained sequences were analyzed using Chromas 2.2.6. The sequences were manually checked and compared to other sequences in Genbank. Subsequently, their genotypes were confirmed using the Web-based Rotavirus A genotyping tool ( https://www.rivm.nl/mpf/typingtool/rotavirusa/ ). The nucleotide sequences were then submitted to GenBank, where they were assigned accession numbers OQ789844-OQ789866 for VP7 and OQ789867- OQ789894 for VP4. Phylogenetic Analysis For the phylogenetic analysis, nucleotide sequences of the VP7 and VP4 genes of related strains, as well as Rotarix and RotaTeq vaccine strains, were obtained from GenBank. Multiple sequence alignments were done using CLUSTALW in MEGA software Version X. Phylogenetic trees were constructed using the Neighbor-Joining method and Tamura-3-parameter model in MEGA X and validated by the bootstrap method with 1000 replicates. VP7 and VP4 amino acid sequence similarities with vaccine sequences were calculated using ClustalW in Clustal Omega online tool [22]. Analysis of the VP7 and VP4 antigenic epitopes To further investigate the discrepancies found in the antigenic epitopes of the circulating rotavirus strains in Iran, the amino acid changes within the antigenic epitope of the RVA outer-capsid proteins of VP7 and VP4 in Iranian strains were compared with the Rotarix and RotaTeq vaccine strains. Structural analysis of VP7 (PBD 3FMG) and VP8 (PDB 1KQR) was performed using the UCSF Chimera-Molecular Modeling System software version 1.17.1. Results Phylogenetic analysis and comparison of VP7 proteins of circulating Iranian RVA strains and vaccine strains A VP7 phylogenetic tree depicting the circulating Iranian strains along with those of RotaTeq, Rotarix and reference strains is presented in Fig. 1 . The analysis revealed that the Iranian RVA sequences corresponded to G-types of G1 (n = 11), G2 (n = 1), G3 (n = 5), (n = 2), G6 (n = 1) and G9 (n = 5) that confirmed through similarity with reference isolates. G6 was characterized in this study for the first time in Iran. The phylogenetic analysis indicated that G1 strains clustered in lineage I (Fig. 1 ). These strains exhibited amino acid sequence identities of 96.39–96.92% compared with Rotarix G1 (lineage II) and 93.77–94.30% in comparison to RotaTeq G1 (lineage III) (Table 1 ). The G2 strain belonged to lineage IV (Fig. 1 ), which was relatively distant from Rotarix G1/II, but similar to RotaTeq G2/II, with 73.95% and 94.86% similarity, respectively (Table 1 ). While the Iranian G3 strains were in different lineages compared to the vaccine strains (lineage II as opposed to lineage I), they showed high similarity, with 80.57–81.60% and 96.39–97.24% of similarity to the G1/II strain of Rotarix and G3 lineage II strain of RotaTeq, respectively (Table 1 ). The G6 strain was relatively closely related to G1/II and G6 of Rotarix and RotaTeq strains, with 82.67% and 93% amino acid identities, respectively. The G4/I Iranian strains clustered in the same lineage as the RotaTeq G4 vaccine strain and demonstrated higher amino acid homology (96.32%) compared to the G1/II of Rotarix (76.99%). Additionally, G9 strains were included in lineage III (Fig. 1 ), and showed 80.19–81.65% and 79.55–86.15% identity with the G1 of Rotarix and G1-G4 and G6 of RotaTeq (Due to the absence of G9 in both vaccines), respectively. Notably, the G3/I strains exhibited the highest amino acid identity with the G3/II of RotaTeq (96.39–97.24%) (Table 1 ). Table 1 Amino Acid Sequence Identity Matrix for Comparing G and P Types of Iranian Strains with Vaccine Strains G and P genotype/lineage G1/II Rotarix GI/III RotaTeq G2/II RotaTeq G3/II RotaTeq G4/I RotaTeq G6 RotaTeq P[8]/I Rotarix P[8]/II RotaTeq G1/II (n = 9) 96.39–96.92 93.77–94.30 74.43–75.69 79.02–80.31 76.10–76.90 79.61–80.31 G2/IV (n = 1) 73.95 74.60 94.86 73.95 70.74 75.24 G3/I (n = 5) 80.57–81.60 81.21–81.90 75.08–76.07 96.39–97.24 74.52–75.41 82.80-83.44 G4/I (n = 2) 76.99 76.07 72.70 74.85 96.32 78.22 G6 (n = 1) 82.67 82.00 76.67 85.67 79.00 93.00 G9/III (n = 5) 80.19–81.65 79.55–80.98 77.92–79.14 85.25–86.15 78.53–79.75 83.12–83.86 P[8]/III (n = 13) 92.34–93.09 94.35–94.95 P[4]/IV (n = 5) 85.61–85.92 87.27–87.41 P[6]/I (n = 10) 70.07–71.73 68.00-69.61 Phylogenetic analysis and comparison of VP4 (VP8) proteins of circulating Iranian RVA strains and vaccine strains A Neighbor-Joining tree was generated using partial genome sequences from the VP4 gene segment. The total number of nucleotide sequences acquired for P-types were 13, 5, and 10 for P[8], P[4], and P[6] respectively. The constructed phylogenetic tree revealed that Iranian P[8] strains fell into lineage III (Fig. 2 ), in contrast to Rotarix and RotaTeq P[8] strains, which are related to lineages I and II, respectively. These strains showed a higher similarity to the P[8] strain of RotaTeq, with similarities ranging from 94.35–94.95%, than the Rotarix strain (92.34–93.09%) (Table 1 ). Iranian P[4] strains, which belonged to lineage IV, were rather distantly related to both Rotarix and RotaTeq P[8] strains compared to P[8] strains, with amino acid similarities of 85.61–85.92% to the former and 87.27–87.41% to the latter (Table 1 ). Additionally, the detected P[6] strains clustered in lineage I and displayed low amino acid resemblance, ranging from 70.07–71.73% when compared to P[8] strains of Rotarix and 68–69.61% when compared to RotaTeq respectively. Comparative analysis of VP7 neutralizing epitopes between Iranian strains and vaccine strains The results of the amino acid comparison between the G-type strains in this study and the corresponding vaccine strains can be found in Table S1 . When analyzing the VP7 protein, the isolates exhibited a relatively high percentage of amino acid variations across all subunits of the VP7 epitopes (Fig. 3 ). Among the 29 amino acid residues in the VP7 neutralizing epitopes as described by Zeller et al. [23], only five amino acids (Positions 98, 104, 201, 264 and 291) were completely conserved among the circulating Iranian strains when compared to the vaccine strains. Upon aligning the Iranian G1 serotypes from this study with the G1 strains of RotaTeq and Rotarix vaccine strains, a substantial similarity in amino acid residues was observed. All the G1 strains displayed absolutely identical VP7 antigenic epitopes to Rotarix G1 strains, whereas two amino acid substitutions in 7-1a and 7 − 2 epitopes were found in all of them in comparison with G1 of RotaTeq (D97E and S147N). Regarding the G2, when compared to G2 of RotaTeq, there were few amino acid variations in antigenic epitopes in the 7-1a and 7-1b epitopes (A87T, D96N, S213D and S242N). Conversely, this genotype showed 18 amino acid changes in all three antigenic regions when compared to G1 of Rotarix. In the case of G3, in comparison to RotaTeq, the G3 strains demonstrated three to five amino acid differences (See Additional file 1). The OQ789845/G3/I strain showed the least substitutions (A212T, K238N and D242N) in the 7-1a and 7-1b regions, while the OQ789859/G3/I strain displayed the most replacements (D123N, A212T, K238N, A221D and D242N) across all regions. The remaining three strains had only four mutations (A212T, K238N, A221D and D242N) in the 7-1b and 7 − 2 epitopes. Interestingly, each of these G3 strains contained the K238N mutation, which provides a potential N-linked glycosylation site that does not exist at the corresponding position in RotaTeq G3 [23]. In addition, G3 showed 11–12 amino acid substitutions across all three epitopes when compared to G1 of Rotarix. The VP7 epitopes of both G4 strains differed by four amino acid substitutions when compared to the epitopes in the G4 strain of RotaTeq (D130E, R143K, A145T, and D211N), all of which were present in all three antigenic epitopes. Conversely, these strains had 16 amino acid discrepancies when compared to the G1 strain of Rotarix. As for the newly identified G6 strain, there were 7 and 15 amino acid distinctions compared to the G6 and G1 strains of RotaTeq and Rotarix in all three regions respectively. Furthermore, Iranian G9 strains showed a total of 13 amino acid differences compared to RotaTeq G1-G4 and G6 strains and a sum of 14 variations in comparison to G1 of Rotarix. These variances were found within the three neutralizing antigen epitopes (7-1a, 7-1b, and 7 − 2). Comparative analysis of VP8 neutralizing epitopes between Iranian strains and vaccine strains VP4 typically undergoes cleavage into two fragments known as VP8 and VP5. The VP8 fragment encompasses four surface-exposed antigens, namely 8–1, 8–2, 8–3, and 8–4, which collectively consist of 25 amino acids [23]. All the Iranian P[8] strains shared 19 out of 25 residues that were identical to those present in the VP4 antigenic epitopes of Rotarix and RotaTeq. Notably, epitopes 8 − 2 and 8 − 4 were conserved among all of these serotypes (See Additional file 2). It is important to note that all P[8] strains featured E150D and D195G/N195G substitutions, and four of them had a N194D mutation. This means that the polar asparagine in Rotarix and aspartic acid in RotaTeq have been replaced by a non-polar glycine. As for P[4] strains, there were a greater number of amino acid distinctions across three antigenic epitopes, namely 8 − 1, 8 − 3 and 8 − 4, compared to P[8] strains, with 10 and 11 amino acid discrepancies compared to RotaTeq and Rotarix P[8] strains, respectively. Furthermore, similar to P[8] strains, 8 − 2 epitope was conserved among all P[4] strains. Upon mapping these variations within the VP4 epitopes of the vaccine strains and Iranian strains, it was revealed that they appeared relatively inconsistent along the molecule's front and back (Fig. 4 ). P[6] strains showed the most divergence from both P[8] vaccine strains, with 15 amino acid differences from Rotarix, and 16 from RotaTeq. These mutations were concentrated in the 8 − 3 and 8 − 4 epitopes. Comparative analysis of Cytotoxic T Lymphocyte epitopes of Iranian and vaccine strains Among the 26 amino acids found in CTL epitopes, only five were completely conserved in Iranian G-types (21Y, 23L, 24K, 51Q, and 52N) (See Additional file 3). A comparison of G1 vaccine and Iranian strains revealed three amino acid differences in CTL epitopes at positions L19F, R28Q, and Y/T41S. The G2 serotypes differed by 4 and 14 amino acid substitutions from G1 of Rotarix and G2 of RotaTeq, respectively. Iranian G3 and G4 strains displayed the fewest number of substitutions within their CTL epitopes when compared to the G3 and G4 of RotaTeq, with only one mutation in the former and two discrepancies in the latter. Discussion Genetic drift, reassortment, animal to human transmission, and rearrangement are among mechanisms that lead to the emergence of novel rotavirus variants worldwide [24]. Members of known lineages in each genotype contain accumulated mutations that have been introduced in each replication cycle [23]. Some G and P genotypes exhibit significant amino acid differences in their antigenic epitopes compared to the vaccine strains, which might be responsible for escape from induced neutralizing antibodies [25]. In the present study, the amino acid sequences and the antigenic epitopes of VP7 and VP4 of circulating Iranian strains were compared with Rotarix and RotaTeq vaccine strains to determine potential disparities that could affect vaccine efficacy and their implications for future vaccination programs. Based on VP7 amino acid sequence analysis, Iranian strains clustered in the G1/II, G2/IV, G3/I, G4/I, G6 and G9/III lineages. All G1/II strains shared the greatest degree of amino acid similarity and were completely conserved in all three antigenic epitopes when compared to Rotarix G1 strain. Additionally, G1 strains showed a high amino acid identity with G1 of RotaTeq (G1/II), but all of them displayed two amino acid changes in neutralizing epitopes, D97E and S147N [26], which had been reported before in Serbian and Chinese G1 strains [18,27]. The G2 genotype belonged to lineage IV and was more closely related to G2/II of RotaTeq than G1/II of Rotarix. The strain displayed just four amino acid changes at positions 87, 96, 213 and 242 compared to G2 epitopes of RotaTeq. G2 strains are typically associated with P[4] genotypes, and protection against this genotype by RotaTeq primarily relies on the G2 (VP7) element of the vaccine. Some mutations at neutralizing epitopes of this genotype are related to escape from vaccine immunity. Accordingly, amino acid alteration at position 96 in antigenic region 7-1a in G2P[4] led to an outbreak in Australia [28]. The G3 strain of RotaTeq and the Iranian G3 strain were closely related, but clustered in different lineages. Among the studied strains, three to five amino acid differences were found in the 7-1b region in comparison to the G3 of RotaTeq. Notably, the study revealed the presence of a K238N amino acid substitution in all the strains, which may indicate a N-linked glycosylation site, consistent with results reported in China, Qatar and Italy [17,18,29]. The K238N mutation can enhance viral replication, diminish the efficacy of monoclonal antibodies, and prevent neutralizing antibody activity [30,31]. Furthermore, the shift in charge may impact the chemical properties of the protein, and the change in polarity implies that the epitope may become more inaccessible due to its increased hydrophobicity [32]. Epidemiological data from Australia obtained during the post-vaccine era suggest a possible increase in G3 strains where RotaTeq has been administered [33]. In addition, after the introduction of RotaTeq in the United States, the G3 genotype became predominant in some seasons [34]. The two Iranian G4 strains were closely related to RotaTeq G4 and belonged to the same lineage (G4/I). Four discrepancies were found between the RotaTeq and Iranian G4 antigenic epitopes. The existence of these mutations among Iranian RVA-G4 strains may indicate the emergence of a variant with a capacity of immune escape [16]. Additionally, an alignment of their VP7 amino acids revealed insertion of an asparagine residue at position 76, which can affect glycosylation and, in turn, modify antigenic characteristics. This is because the asparagine insertion at this position, situated in a hydrophilic region, has the potential to enhance the region's hydrophilicity [35]. Regarding the G6 and G9 Iranian strains, both were distantly related to Rotarix and RotaTeq VP7 strains, with G9 strains showing a relatively closer similarity to G3 of RotaTeq. This was consistent with a previous study in Iran [16]. Comparison of the VP7 epitopes of the G6 strain to the G6 of RotaTeq showed seven amino acid differences distributed across all three antigenic epitopes, which did not represent any radical changes in the antigenicity of VP7 epitopes, as previously reported in a US study [31]. As for the five G9 strains in this study, there was a high frequency of mutations across all three regions when compared to VP7 regions of both vaccines. One G9 strain (OQ789847/G9/III) exhibited a D100N mutation, which as reported in Chinese G9 strains, represents as an escape mutant [36]. The VP4 spike protein plays a crucial role in viral neutralization due to its several structural and functional roles, including virus particle binding, penetration, and maturation [27]. All the analyzed sequences of VP8* region of the Iranian P[8] strains belonged to lineage III, which were relatively related to P[8]/lineages I and II of Rotarix and RotaTeq, respectively. However, VP8* epitopes of the P[8] strains differed significantly from the Rotarix and RotaTeq counterparts. Similar patterns of amino acid substitutions in VP8* epitopes of the P[8] strains between circulating and vaccine strains in our study were reported from Serbia, China and Qatar [17,18,27]. The amino acid changes identified at positions S131R and N135D in this study could result in polarity changes, which play a role in RV’s escape from the host immunity [27]. Taken together, although both vaccines have shown to be very effective in Europe and the United States, there is increasing concern about the evolution of resistant strains [37]. Regarding the high diversity of RVA genotypes in Iran and detected mutations in most of their neutralizing epitopes, escape from induced immunity of RotaTeq and Rotarix seems to be highly probable after vaccination program. However, as was shown in a study in Belgium, the introduction of vaccine can effectively decrease dominant genotypes from the same lineage, G1/II-P[8]/I strains, compared to unrelated lineages [23], which could highly reduce the infection rate among children. Presentation of a specific amino acid sequence of structural antigens to B- or T-cells can mediate immunity to RVA. Although, the precise mechanism by which vaccination confers protection against rotavirus is not well known, neutralizing antibodies specific to VP4 and VP7 proteins seems to be the key factors of protection, which are considered for development of the RotaTeq vaccines [37]. In this study, similarity of the two known CTL epitopes of the VP7 protein was analyzed in the Iranian G-types compared with Rotarix and RotaTeq strains. G1 strains in our isolates showed 3 amino acid substitutions within VP7 CTL epitopes compared to the vaccine strains, which is consistent with a previous study in Africa [38]. In the case of other genotypes, in comparison to RotaTeq strain, G2 strains showed the highest number of discrepancies, while G3 and G4 strains displayed the least number of differences across both epitopes. This discrepancy was also reported in a study in Russia [39]. According to our knowledge, there are no available data on the effects of these mutations on the immunity induced by CTLs. Impact of the characterized mutations in VP7 CTL epitopes of the circulating strains in Iran on the processing and presentation of RVA antigens to immune cells and RVA clearance should be further studied. This study had some limitations. While the data were sufficient for differentiating RVA genotypes and their lineages, sub-genotypic lineages could not be identified. This was primarily due to the partial sequencing of VP7 and VP4 gene segments and budgetary constraints. The isolates analyzed in this study belonged to pediatric patients admitted to a referral hospital in Tehran. To obtain a better overview of lineage diversity for effective vaccine implementation, a comprehensive multicenter study is necessary. Conclusion This study provides crucial insights into the genetic and antigenic characteristics of Iranian strains compared to the vaccine strains. Regarding the high diversity of RVA genotypes in Iran and detected mutations in neutralizing and CTL epitopes, escape from induced immunity of RotaTeq and Rotarix seems to be probable after vaccination program. To fully show the importance of these differences and their implications on vaccine efficacy, further studies on the intragenotype antigenic variability of RVA are necessary prior to the introduction of Rotarix and RotaTeq into the national immunization program. Declarations Acknowledgements Not applicable. Author contributions M.A., R.M.G, and A.K. conceptualized the study, formed the methodology, conducted the investigation and supervised the study. T.F. performed the analysis and wrote the original draft. M.A. reviewed and edited the draft. S.M.Z. and S.M. supported the study officially and coordinated the study. M.A. and R.M.G are both corresponding authors. Funding details This work was supported by the World Health Organization under grand 202700981 Data availability The raw data required to reproduce these findings are available from the corresponding author upon request. Ethics approval and consent to participate This study was approved by the research ethics committees of Research Institute of Children’s Health at Shahid Beheshti University of Medical Sciences (IR.SBMU.RICH.REC.1401.021), and parental consent was obtained for all admitted children. Competing Interests The authors declare no competing interests. References Kraay ANM, Chaney DM, Deshpande A, Pitzer VE, Lopman BA. 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Eurosurveillance. 2022;27:1–12. doi: 10.2807/1560-7917.ES.2022.27.47.2101133 Gibory M, Bruun T, Flem E, Dembinski JL, Haltbakk I, Størdal K, et al. Genetic diversity of rotavirus strains circulating in Norway before and after the introduction of rotavirus vaccination in children. J Med Virol. 2022;94:2624–31. doi: 10.1002/jmv.27484 Giri S, Kumar CPG, Khakha SA, Chawla-Sarkar M, Gopalkrishna V, Chitambar SD, et al. Diversity of rotavirus genotypes circulating in children < 5 years of age hospitalized for acute gastroenteritis in India from 2005 to 2016: Analysis of temporal and regional genotype variation. BMC Infect Dis. 2020;20. doi: 10.1186/s12879-020-05448-y Ramani S, Hu L, Venkataram Prasad B V, Estes MK. Diversity in Rotavirus-Host Glycan Interactions: A “Sweet” Spectrum. Cell Mol Gastroenterol Hepatol. 2016;2:263–73. doi: 10.1016/j.jcmgh.2016.03.002 Andersson M, Lindh M. Rotavirus genotype shifts among Swedish children and adults-Application of a real-time PCR genotyping. 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J Med Virol. 2020;92:1110–23. doi: 10.1002/jmv.25642 Mathew S, Al Khatib HA, Al Ibrahim M, Al Ansari K, Smatti MK, Nasrallah GK, et al. Vaccine evaluation and genotype characterization in children infected with rotavirus in Qatar. Pediatr Res. 2023;1–9. doi: 10.1038/s41390-023-02468-7 Mao T, Wang M, Wang J, Ma Y, Liu X, Wang M, et al. Phylogenetic analysis of the viral proteins VP4/VP7 of circulating human rotavirus strains in China from 2016 to 2019 and comparison of their antigenic epitopes with those of vaccine strains. Front Cell Infect Microbiol. 2022;12:927490. doi: 10.3389/fcimb.2022.927490 Latifi T, Eybpoosh S, Afchangi A, Jalilvand S, Shoja Z. Genetic characterization of P[8] rotavirus strains circulated in Iran between 2009 and 2017. J Med Virol. 2022;94:3561–9. doi: 10.1002/jmv.27766 WHO. Manual of rotavirus detection and characterization methods Manual of rotavirus detection and characterization methods Immunization , Vaccines and Biologicals. Published online 2009. Jin Q, Ward RL, Knowlton DR, Gabbay YB, Linhares AC, Rappaport R, et al. Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses. Arch Virol. 1996;141:2057–76. doi: 10.1007/BF01718215 Madeira F, Pearce M, Tivey ARN, Basutkar P, Lee J, Edbali O, et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 2022;50:W276—W279. doi: 10.1093/nar/gkac240 Zeller M, Patton JT, Heylen E, De Coster S, Ciarlet M, Van Ranst M, et al. Genetic analyses reveal differences in the VP7 and VP4 antigenic epitopes between human rotaviruses circulating in Belgium and rotaviruses in Rotarix and RotaTeq. J Clin Microbiol. 2012;50:966–76. doi: 10.1128/JCM.05590-11 Kirkwood CD. Genetic and Antigenic Diversity of Human Rotaviruses: Potential Impact on Vaccination Programs. J Infect Dis. 2010;202:S43–8. doi: 10.1086/653548 Gupta S, Gauhar M, Bubber P, Ray P. Phylogenetic analysis of VP7 and VP4 genes of the most predominant human group A rotavirus G12 identified in children with acute gastroenteritis in Himachal Pradesh, India during 2013-2016. J Med Virol. 2021;93:6200–9. doi: 10.1002/jmv.27142 Aoki ST, Settembre EC, Trask SD, Greenberg HB, Harrison SC, Dormitzer PR. Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab. Science. 2009;324:1444–7. doi: 10.1126/science.1170481 Patić A, Vuković V, Kovačević G, Petrović V, Ristić M, Djilas M, et al. Detection and Molecular Characterization of Rotavirus Infections in Children and Adults with Gastroenteritis from Vojvodina, Serbia. Microorganisms. 2022;10. doi: 10.3390/microorganisms10102050 Donato CM, Pingault N, Demosthenous E, Roczo-Farkas S, Bines JE. Characterisation of a G2P[4] Rotavirus Outbreak in Western Australia, Predominantly Impacting Aboriginal Children. Pathog (Basel, Switzerland). 2021;10. doi: 10.3390/pathogens10030350 Bonura F, Bányai K, Mangiaracina L, Bonura C, Martella V, Giammanco GM, et al. Emergence in 2017–2019 of novel reassortant equine-like G3 rotavirus strains in Palermo, Sicily. Transbound Emerg Dis. 2022;69:813–35. doi: 10.1111/tbed.14054 Zao CL, Yu WN, Kao CL, Taniguchi K, Lee CY, Lee CN. Sequence analysis of VP1 and VP7 genes suggests occurrence of a reassortant of G2 rotavirus responsible for an epidemic of gastroenteritis. J Gen Virol. 1999;80 ( Pt 6):1407–15. doi: 10.1099/0022-1317-80-6-1407 Esona MD, Gautam R, Katz E, Jaime J, Ward ML, Wikswo ME, et al. Comparative genomic analysis of genogroup 1 and genogroup 2 rotaviruses circulating in seven US cities, 2014-2016. Virus Evol. 2021;7:veab023. doi: 10.1093/ve/veab023 Betts MJ, Russell RB. Amino Acid Properties and Consequences of Substitutions. Bioinforma Genet. John Wiley & Sons, Ltd; 2003. p. 289–316. doi: 10.1002/0470867302 Kirkwood CD, Boniface K, Barnes GL, Bishop RF. Distribution of rotavirus genotypes after introduction of rotavirus vaccines, Rotarix® and RotaTeq®, into the National Immunization Program of Australia. Pediatr Infect Dis J. 2011;30:S48-53. doi: 10.1097/INF.0b013e3181fefd90 Hull JJ, Teel EN, Kerin TK, Freeman MM, Esona MD, Gentsch JR, et al. United States rotavirus strain surveillance from 2005 to 2008: genotype prevalence before and after vaccine introduction. Pediatr Infect Dis J. 2011;30:S42-7. doi: 10.1097/INF.0b013e3181fefd78 Berois M, Libersou S, Russi J, Arbiza J, Cohen J. Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo-Uruguay from 1996-1999. J Med Virol. 2003;71:456–62. doi: 10.1002/jmv.10511 Peng R, Li D, Wang J, Xiong G, Wang M, Liu D, et al. Reassortment and genomic analysis of a G9P[8]-E2 rotavirus isolated in China. Virol J. 2023;20:135. doi: 10.1186/s12985-023-02064-5 Ward RL, Clark HF, Offit PA. Influence of potential protective mechanisms on the development of live rotavirus vaccines. J Infect Dis. 2010;202 Suppl:S72-9. doi: 10.1086/653549 Mwangi PN, Mogotsi MT, Seheri ML, Mphahlele MJ, Peenze I, Esona MD, et al. Whole Genome In-Silico Analysis of South African G1P[8] Rotavirus Strains before and after Vaccine Introduction over a Period of 14 Years. Vaccines. 2020;8. doi: 10.3390/vaccines8040609 Morozova O V, Sashina TA, Fomina SG, Novikova NA. Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines. Arch Virol. 2015;160:1693–703. doi: 10.1007/s00705-015-2439-6 Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additionalfile2.docx Additionalfile3.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jun, 2024 Read the published version in Heliyon → Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3842299","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266030324,"identity":"43789239-b784-4434-971b-6b4eaf7009b6","order_by":0,"name":"Tina Fallah","email":"","orcid":"","institution":"Alzahra University","correspondingAuthor":false,"prefix":"","firstName":"Tina","middleName":"","lastName":"Fallah","suffix":""},{"id":266030325,"identity":"15023ac3-3910-4fa9-8f0e-0606465128a8","order_by":1,"name":"Roxana Mansour Ghanaie","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Roxana","middleName":"Mansour","lastName":"Ghanaie","suffix":""},{"id":266030326,"identity":"4fc13d1c-f6b7-4c87-90f7-505b80fd2898","order_by":2,"name":"Abdollah Karimi","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Abdollah","middleName":"","lastName":"Karimi","suffix":""},{"id":266030327,"identity":"9961ac2b-d73e-4e08-997b-fef20d138630","order_by":3,"name":"Seyed Mohsen Zahraei","email":"","orcid":"","institution":"Ministry of Health and Medical Education","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Mohsen","lastName":"Zahraei","suffix":""},{"id":266030328,"identity":"eb6929c1-977b-4836-95fe-2545822492bf","order_by":4,"name":"Sussan Mahmoudi","email":"","orcid":"","institution":"Ministry of Health and Medical Education","correspondingAuthor":false,"prefix":"","firstName":"Sussan","middleName":"","lastName":"Mahmoudi","suffix":""},{"id":266030329,"identity":"d895b797-a10d-4b41-9f37-e001ffa02fe3","order_by":5,"name":"Masoud Alebouyeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYNACNgYGPgYeMDOBH0wWEKGFDaZFsgFEGpCixeAAiMKjRbf9+MPHPGUM8mzsvQcf87bZ5BmfX5344YEBgzy/2AGsWszO5Bgb85xjMGzjOZdszNuWVmx24+1mCaDDDGfOTsCu5UAOmzRvGwNjm0SOGZBxOHHbjbMbQFoSDG7j0HL++TOQFvs2+TcgLf8TN884u/kHXi03EkAqGRLbJHhAjAOJG/h7t+G35cYbY8M55ySS23jykoGM5MQZN3i3WSQYSOD2y/n0hw/elNnY9rOfPQhk2CX295/dfPNHhY08vzR2LVAgAaEY2UDsBCQRwuAPEPMfIFb1KBgFo2AUjBAAAL1WXcKgMiVNAAAAAElFTkSuQmCC","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Masoud","middleName":"","lastName":"Alebouyeh","suffix":""}],"badges":[],"createdAt":"2024-01-07 10:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3842299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3842299/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.heliyon.2024.e33887","type":"published","date":"2024-07-01T03:13:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49435909,"identity":"899ca196-9c1d-432d-81ea-5fda90cdcc01","added_by":"auto","created_at":"2024-01-10 20:05:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215991,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of the VP7 proteins of circulating Iranian strains and vaccine strains (Rotarix G1 and RotaTeq G1-G4, and G6). Neighbor-Joining trees were constructed using VP7 coding nucleotide sequences. AF426162 porcine strain was used as an outgroup. Bootstrap values (1000 replicates) of over 70% are indicated.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/3f5838e32e754d5cef37a5a4.png"},{"id":49436839,"identity":"60430f68-e809-442e-be7e-907356d1b3c9","added_by":"auto","created_at":"2024-01-10 20:13:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155230,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of the VP4 proteins of circulating Iranian strains and vaccine strains (P[8] strains of Rotarix and RotaTeq). Neighbor-Joining trees were constructed using VP4 coding \u0026nbsp;nucleotide sequences. AF426162 porcine strain was used as an outgroup. Bootstrap values (1000 replicates) of \u0026nbsp;over 70% are indicated.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/3d5e1f7df40bd2a813ac2492.png"},{"id":49436838,"identity":"8869a38d-c43b-491d-bfe7-22e4624288b8","added_by":"auto","created_at":"2024-01-10 20:13:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262627,"visible":true,"origin":"","legend":"\u003cp\u003eA three-dimensional illustration of the amino acid substitutions identified in the VP7 protein of RVA strains. VP7 3D structure (first image). Antigenic epitopes (7-1a, 7-1b, and 7-2) are represented by yellow, green, and blue, respectively. Surface-exposed residues that differ between circulating strains in Iran and the strains contained in Rotarix or RotaTeq are shown in red.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/156672a3b31bdef0a3d26585.png"},{"id":49437108,"identity":"86be53a7-6dbb-4761-9749-5ebc552e37df","added_by":"auto","created_at":"2024-01-10 20:21:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":319443,"visible":true,"origin":"","legend":"\u003cp\u003eA three-dimensional illustration of the amino acid substitutions identified in the VP4 protein of RVA strains. VP4 3D structure (first image). Antigenic epitopes (8-1, 8-1, 8-3 and 8-4) are represented by yellow, green, blue, and purple respectively. Surface-exposed residues that differ between circulating strains in Iran and the strains contained in Rotarix or RotaTeq are shown in red.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/20c0103a756fccaa80ca7366.png"},{"id":59756582,"identity":"c264c5ac-1f73-4512-9337-c853754725e2","added_by":"auto","created_at":"2024-07-06 03:13:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1498362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/51610331-ade3-4382-a3b6-ba5e3a5dff06.pdf"},{"id":49435915,"identity":"b5974ab9-f02a-483e-8cc4-349a7412b3e4","added_by":"auto","created_at":"2024-01-10 20:05:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21584,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/4af22a875109a627de2fbbc0.docx"},{"id":49435910,"identity":"bf6f21d1-28c0-4eb9-a48e-0f98fdd5ff56","added_by":"auto","created_at":"2024-01-10 20:05:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17400,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/cb013076cfe82445701b5034.docx"},{"id":49435912,"identity":"6cb9e3e8-4e77-4144-88d5-f31d1794a699","added_by":"auto","created_at":"2024-01-10 20:05:16","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18155,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3842299/v1/977d2d5da40f020c4eebef1a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative analysis of the RVA VP7 and VP4 antigenic epitopes circulating in Iran and the Rotarix and RotaTeq vaccines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRotavirus is a leading cause of diarrheal disease, accounting for approximately 29.3% of global deaths related to diarrhea in children under 5 years of age [1]. Over 90% of these mortalities occur in low- to middle-income countries. Despite a significant drop in the global burden of rotavirus over the past three decades, mainly due to the introduction of rotavirus vaccines, the prevalence of rotavirus remains high in Africa, Oceania, South Asia, and the Middle-East [2,3].\u003c/p\u003e \u003cp\u003eThe most common rotavirus genotypes found in children worldwide vary by region and time period. This diversity is linked to the virus\u0026rsquo;s genomic structure and its geographic evolution, influenced by common host factors and transmission routes. The infectious RVA particle has an icosahedral structure with three layers, consisting of 11 segments of double-stranded RNA encoding six structural proteins (VP) and six non-structural proteins (NSP) [4]. To date, 35 G genotypes and 50 P genotypes have been described for group A rotaviruses. The most prevalent genotypes worldwide are G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] [5]. These genotypes are determined based on the two outer capsid proteins, VP7 (G type) and VP4 (P type), which function as specific binding sites for neutralizing antibodies [4]. The introduction of rotavirus vaccines has been associated with changes in the distribution of rotavirus genotypes in certain areas, resulting in a decline in the prevalence of vaccine-targeted genotypes and an increase in the prevalence of genotypes not covered by the vaccines [6].\u003c/p\u003e \u003cp\u003eRecent studies have revealed variations in the distribution of RVA genotypes across different age groups. In Canada and Greece, a higher prevalence of G12P[8] and G9P[8] genotypes was observed among older children, specifically those aged between 24 and 59 months [7,8]. In children aged 0 to 12 months, other genotypes like G1P[8] and G9P[4] genotypes were found to be more common [9,10]. Several factors may contribute to this occurrence, including diversity in the expression of certain receptors in the intestinal tract at different age groups, exposure to common genotypes in various geographic areas, induced immunity, and host genetics [11\u0026ndash;13]. Similarly, there is evidence of a correlation between rotavirus genotypes and disease symptoms. Studies have shown that different genotypes of rotavirus are associated with varying clinical characteristics and disease severity. In one study, G1P[8] and G9P[8] were the most common genotypes detected in children with moderate and severe acute gastroenteritis (AGE), respectively [9]. A significantly higher frequency of fever in children infected with the G3P[8] genotype was reported in study of Mathew et al. [14]. Additionally, this correlation was detected in the case of emerging genotypes, such as G8P[8], and G9P[4] [15].\u003c/p\u003e \u003cp\u003eThe interaction between RVA antigens of various genotypes and the host immune system offers an explanation for variations in disease outcomes among the immunized and non-immunized children. This interaction primarily depends on the immunogenic domains within the VP4 and VP7 proteins. Notably, the VP7 gene, encompassing 326 amino acids, contains nine variable regions, with four serving as antigenic epitopes, specifically identified as 7-1a, 7-1b, and 7\u0026thinsp;\u0026minus;\u0026thinsp;2. Activation of the VP4 protein, spanning 776 amino acids, requires proteolytic cleavage into two distinct segments: VP8 and VP5, each containing four (8\u0026thinsp;\u0026minus;\u0026thinsp;1\u0026ndash;8\u0026thinsp;\u0026minus;\u0026thinsp;4) and five (5\u0026thinsp;\u0026minus;\u0026thinsp;1\u0026ndash;5\u0026ndash;5) antigenic epitopes, respectively [4]. Due to the diversity of immunogenic motifs within these proteins, multiple lineages and sub-lineages have been defined. According to the study of Motamedi-Rad et al., within the predominant G-types, there are eleven, six, four, six, and six lineages for G1, G2, G3, G4, and G9 lineage, respectively. In terms of P serotypes, four, five, and five distinct lineages were reported for P[8], P[4], and P[6], respectively [16].\u003c/p\u003e \u003cp\u003eRV vaccination is the most effective strategy for significantly reducing the incidence of severe infections and the number of deaths in children. Rotarix (GlaxoSmithKline) consists of a monovalent G1P[8] strain derived from a single human G1P[8] strain. Conversely, RotaTeq (Merck), is a human-bovine reassortant vaccine, composed of five strains derived from human (G1, G2, G3, and G4) and bovine (P[8]) strains [17]. Both RVA vaccines have demonstrated the ability to elicit homotypic and heterotypic immune responses, resulting in a notable decline in morbidity and mortality. However, the RVA vaccine has proven to be more effective in high-income countries (80\u0026ndash;90%) against severe rotavirus disease compared to low- to middle-income countries (40\u0026ndash;70%) [18]. Various factors contribute to this disparity, including the genotypic and lineage diversity of RVA strains, which are driven by recombination, rearrangement, reassortment, and mutations. Additionally, host genetic factors, malnutrition, gut microbiota dysbiosis, co-infections, environmental enteropathy, and the passive transfer of maternal antibodies have been suggested as potential determinants of differences in RVA vaccine efficacy between high- and low-income countries [16]. RVA lineages with distinct antigenic properties could potentially allow RVA strains to evade vaccine-induced immunity [19].\u003c/p\u003e \u003cp\u003eWith the increasing diversity of RVA strains and the incidence of uncommon fully and partially heterotypic genotypes, such as G1P[6] or G9P[4], particularly in countries without RVA vaccination programs, concerns are growing regarding the reduced effectiveness of approved vaccines against the emerging variants [18]. In this current cross-sectional study, the VP7 and VP4 lineages of human rotavirus A (RVA) strains circulating in children under the age of five with diarrhea were analyzed. Additionally, the amino acid sequences of these RVAs and their potential antigenic distinctions and cytotoxic T cell epitopes compared to the vaccine strains were assessed.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample selection\u003c/h2\u003e \u003cp\u003eIn order to determine the phylogenetic and antigenic epitope associations of VP7 and VP4\u003c/p\u003e \u003cp\u003eamong the Iranian and vaccine strains, a total of 51 rotavirus strains were included in this study. This study was approved by the research ethics committees of Research Institute of Children\u0026rsquo;s Health at Shahid Beheshti University of Medical Sciences (IR.SBMU.RICH.REC.1401.021), and parental consent was obtained for all the children\u0026rsquo;s samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, RT-PCR, and Amplification of VP7 and VP4\u003c/h2\u003e \u003cp\u003eViral RNA was extracted from 10% stool suspensions of the selected rotavirus-positive fecal specimens using the High Pure Viral Nucleic Acid Extraction kit (Roche, Mannheim, Germany). The RNA extracts were denatured at 95\u0026deg;C for five minutes, and the AddScript cDNA Synthesis kit (Addbio, Daejeon, South Korea) was used to generate cDNAs through RT-PCR using primers Beg9 and End9 (VP7), and Con3 and Con2 (VP4) following WHO protocols [20]. This cDNA served as a template for amplifying the of full-length VP7 (1062 base pairs) and partial-length VP4 (VP8 region, 876 base pairs) genes of RVA strains, as described previously [21]. The PCR products were verified using electrophoresis in a 1.5% agarose gel and sequencing as described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNucleic acid sequencing\u003c/h2\u003e \u003cp\u003eSequencing was performed using the same forward primers that were used in the RT-PCR. The obtained sequences were analyzed using Chromas 2.2.6. The sequences were manually checked and compared to other sequences in Genbank. Subsequently, their genotypes were confirmed using the Web-based Rotavirus A genotyping tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rivm.nl/mpf/typingtool/rotavirusa/\u003c/span\u003e\u003cspan address=\"https://www.rivm.nl/mpf/typingtool/rotavirusa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The nucleotide sequences were then submitted to GenBank, where they were assigned accession numbers OQ789844-OQ789866 for VP7 and OQ789867- OQ789894 for VP4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic Analysis\u003c/h2\u003e \u003cp\u003eFor the phylogenetic analysis, nucleotide sequences of the VP7 and VP4 genes of related strains, as well as Rotarix and RotaTeq vaccine strains, were obtained from GenBank. Multiple sequence alignments were done using CLUSTALW in MEGA software Version X. Phylogenetic trees were constructed using the Neighbor-Joining method and Tamura-3-parameter model in MEGA X and validated by the bootstrap method with 1000 replicates. VP7 and VP4 amino acid sequence similarities with vaccine sequences were calculated using ClustalW in Clustal Omega online tool [22].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the VP7 and VP4 antigenic epitopes\u003c/h2\u003e \u003cp\u003eTo further investigate the discrepancies found in the antigenic epitopes of the circulating rotavirus strains in Iran, the amino acid changes within the antigenic epitope of the RVA outer-capsid proteins of VP7 and VP4 in Iranian strains were compared with the Rotarix and RotaTeq vaccine strains. Structural analysis of VP7 (PBD 3FMG) and VP8 (PDB 1KQR) was performed using the UCSF Chimera-Molecular Modeling System software version 1.17.1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis and comparison of VP7 proteins of circulating Iranian RVA strains and vaccine strains\u003c/h2\u003e \u003cp\u003eA VP7 phylogenetic tree depicting the circulating Iranian strains along with those of RotaTeq, Rotarix and reference strains is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The analysis revealed that the Iranian RVA sequences corresponded to G-types of G1 (n\u0026thinsp;=\u0026thinsp;11), G2 (n\u0026thinsp;=\u0026thinsp;1), G3 (n\u0026thinsp;=\u0026thinsp;5), (n\u0026thinsp;=\u0026thinsp;2), G6 (n\u0026thinsp;=\u0026thinsp;1) and G9 (n\u0026thinsp;=\u0026thinsp;5) that confirmed through similarity with reference isolates. G6 was characterized in this study for the first time in Iran.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phylogenetic analysis indicated that G1 strains clustered in lineage I (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These strains exhibited amino acid sequence identities of 96.39\u0026ndash;96.92% compared with Rotarix G1 (lineage II) and 93.77\u0026ndash;94.30% in comparison to RotaTeq G1 (lineage III) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The G2 strain belonged to lineage IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which was relatively distant from Rotarix G1/II, but similar to RotaTeq G2/II, with 73.95% and 94.86% similarity, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While the Iranian G3 strains were in different lineages compared to the vaccine strains (lineage II as opposed to lineage I), they showed high similarity, with 80.57\u0026ndash;81.60% and 96.39\u0026ndash;97.24% of similarity to the G1/II strain of Rotarix and G3 lineage II strain of RotaTeq, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The G6 strain was relatively closely related to G1/II and G6 of Rotarix and RotaTeq strains, with 82.67% and 93% amino acid identities, respectively. The G4/I Iranian strains clustered in the same lineage as the RotaTeq G4 vaccine strain and demonstrated higher amino acid homology (96.32%) compared to the G1/II of Rotarix (76.99%). Additionally, G9 strains were included in lineage III (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and showed 80.19\u0026ndash;81.65% and 79.55\u0026ndash;86.15% identity with the G1 of Rotarix and G1-G4 and G6 of RotaTeq (Due to the absence of G9 in both vaccines), respectively. Notably, the G3/I strains exhibited the highest amino acid identity with the G3/II of RotaTeq (96.39\u0026ndash;97.24%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmino Acid Sequence Identity Matrix for Comparing G and P Types of Iranian Strains with Vaccine Strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG and P genotype/lineage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG1/II Rotarix\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGI/III RotaTeq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG2/II RotaTeq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG3/II RotaTeq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eG4/I RotaTeq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eG6 RotaTeq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP[8]/I Rotarix\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP[8]/II RotaTeq\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG1/II (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96.39\u0026ndash;96.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e93.77\u0026ndash;94.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e74.43\u0026ndash;75.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79.02\u0026ndash;80.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e76.10\u0026ndash;76.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e79.61\u0026ndash;80.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG2/IV (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e73.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e75.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG3/I (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80.57\u0026ndash;81.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e81.21\u0026ndash;81.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.08\u0026ndash;76.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e96.39\u0026ndash;97.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e74.52\u0026ndash;75.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e82.80-83.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG4/I (n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e76.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e74.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e78.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG6 (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e82.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e79.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG9/III (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80.19\u0026ndash;81.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e79.55\u0026ndash;80.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e77.92\u0026ndash;79.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85.25\u0026ndash;86.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e78.53\u0026ndash;79.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e83.12\u0026ndash;83.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP[8]/III (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e92.34\u0026ndash;93.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e94.35\u0026ndash;94.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP[4]/IV (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e85.61\u0026ndash;85.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e87.27\u0026ndash;87.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP[6]/I (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e70.07\u0026ndash;71.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e68.00-69.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis and comparison of VP4 (VP8) proteins of circulating Iranian RVA strains and vaccine strains\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA Neighbor-Joining tree was generated using partial genome sequences from the VP4 gene segment. The total number of nucleotide sequences acquired for P-types were 13, 5, and 10 for P[8], P[4], and P[6] respectively. The constructed phylogenetic tree revealed that Iranian P[8] strains fell into lineage III (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), in contrast to Rotarix and RotaTeq P[8] strains, which are related to lineages I and II, respectively. These strains showed a higher similarity to the P[8] strain of RotaTeq, with similarities ranging from 94.35\u0026ndash;94.95%, than the Rotarix strain (92.34\u0026ndash;93.09%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Iranian P[4] strains, which belonged to lineage IV, were rather distantly related to both Rotarix and RotaTeq P[8] strains compared to P[8] strains, with amino acid similarities of 85.61\u0026ndash;85.92% to the former and 87.27\u0026ndash;87.41% to the latter (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, the detected P[6] strains clustered in lineage I and displayed low amino acid resemblance, ranging from 70.07\u0026ndash;71.73% when compared to P[8] strains of Rotarix and 68\u0026ndash;69.61% when compared to RotaTeq respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparative analysis of VP7 neutralizing epitopes between Iranian strains and vaccine strains\u003c/h3\u003e\n\u003cp\u003eThe results of the amino acid comparison between the G-type strains in this study and the corresponding vaccine strains can be found in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. When analyzing the VP7 protein, the isolates exhibited a relatively high percentage of amino acid variations across all subunits of the VP7 epitopes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the 29 amino acid residues in the VP7 neutralizing epitopes as described by Zeller et al. [23], only five amino acids (Positions 98, 104, 201, 264 and 291) were completely conserved among the circulating Iranian strains when compared to the vaccine strains. Upon aligning the Iranian G1 serotypes from this study with the G1 strains of RotaTeq and Rotarix vaccine strains, a substantial similarity in amino acid residues was observed. All the G1 strains displayed absolutely identical VP7 antigenic epitopes to Rotarix G1 strains, whereas two amino acid substitutions in 7-1a and 7\u0026thinsp;\u0026minus;\u0026thinsp;2 epitopes were found in all of them in comparison with G1 of RotaTeq (D97E and S147N). Regarding the G2, when compared to G2 of RotaTeq, there were few amino acid variations in antigenic epitopes in the 7-1a and 7-1b epitopes (A87T, D96N, S213D and S242N). Conversely, this genotype showed 18 amino acid changes in all three antigenic regions when compared to G1 of Rotarix. In the case of G3, in comparison to RotaTeq, the G3 strains demonstrated three to five amino acid differences (See Additional file 1). The OQ789845/G3/I strain showed the least substitutions (A212T, K238N and D242N) in the 7-1a and 7-1b regions, while the OQ789859/G3/I strain displayed the most replacements (D123N, A212T, K238N, A221D and D242N) across all regions. The remaining three strains had only four mutations (A212T, K238N, A221D and D242N) in the 7-1b and 7\u0026thinsp;\u0026minus;\u0026thinsp;2 epitopes. Interestingly, each of these G3 strains contained the K238N mutation, which provides a potential N-linked glycosylation site that does not exist at the corresponding position in RotaTeq G3 [23]. In addition, G3 showed 11\u0026ndash;12 amino acid substitutions across all three epitopes when compared to G1 of Rotarix.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe VP7 epitopes of both G4 strains differed by four amino acid substitutions when compared to the epitopes in the G4 strain of RotaTeq (D130E, R143K, A145T, and D211N), all of which were present in all three antigenic epitopes. Conversely, these strains had 16 amino acid discrepancies when compared to the G1 strain of Rotarix. As for the newly identified G6 strain, there were 7 and 15 amino acid distinctions compared to the G6 and G1 strains of RotaTeq and Rotarix in all three regions respectively. Furthermore, Iranian G9 strains showed a total of 13 amino acid differences compared to RotaTeq G1-G4 and G6 strains and a sum of 14 variations in comparison to G1 of Rotarix. These variances were found within the three neutralizing antigen epitopes (7-1a, 7-1b, and 7\u0026thinsp;\u0026minus;\u0026thinsp;2).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparative analysis of VP8 neutralizing epitopes between Iranian strains and vaccine strains\u003c/h2\u003e \u003cp\u003eVP4 typically undergoes cleavage into two fragments known as VP8 and VP5. The VP8 fragment encompasses four surface-exposed antigens, namely 8\u0026ndash;1, 8\u0026ndash;2, 8\u0026ndash;3, and 8\u0026ndash;4, which collectively consist of 25 amino acids [23]. All the Iranian P[8] strains shared 19 out of 25 residues that were identical to those present in the VP4 antigenic epitopes of Rotarix and RotaTeq.\u0026nbsp;Notably, epitopes 8\u0026thinsp;\u0026minus;\u0026thinsp;2 and 8\u0026thinsp;\u0026minus;\u0026thinsp;4 were conserved among all of these serotypes (See Additional file 2). It is important to note that all P[8] strains featured E150D and D195G/N195G substitutions, and four of them had a N194D mutation. This means that the polar asparagine in Rotarix and aspartic acid in RotaTeq have been replaced by a non-polar glycine. As for P[4] strains, there were a greater number of amino acid distinctions across three antigenic epitopes, namely 8\u0026thinsp;\u0026minus;\u0026thinsp;1, 8\u0026thinsp;\u0026minus;\u0026thinsp;3 and 8\u0026thinsp;\u0026minus;\u0026thinsp;4, compared to P[8] strains, with 10 and 11 amino acid discrepancies compared to RotaTeq and Rotarix P[8] strains, respectively. Furthermore, similar to P[8] strains, 8\u0026thinsp;\u0026minus;\u0026thinsp;2 epitope was conserved among all P[4] strains. Upon mapping these variations within the VP4 epitopes of the vaccine strains and Iranian strains, it was revealed that they appeared relatively inconsistent along the molecule's front and back (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). P[6] strains showed the most divergence from both P[8] vaccine strains, with 15 amino acid differences from Rotarix, and 16 from RotaTeq.\u0026nbsp;These mutations were concentrated in the 8\u0026thinsp;\u0026minus;\u0026thinsp;3 and 8\u0026thinsp;\u0026minus;\u0026thinsp;4 epitopes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparative analysis of Cytotoxic T Lymphocyte epitopes of Iranian and vaccine strains\u003c/h2\u003e \u003cp\u003eAmong the 26 amino acids found in CTL epitopes, only five were completely conserved in Iranian G-types (21Y, 23L, 24K, 51Q, and 52N) (See Additional file 3). A comparison of G1 vaccine and Iranian strains revealed three amino acid differences in CTL epitopes at positions L19F, R28Q, and Y/T41S. The G2 serotypes differed by 4 and 14 amino acid substitutions from G1 of Rotarix and G2 of RotaTeq, respectively. Iranian G3 and G4 strains displayed the fewest number of substitutions within their CTL epitopes when compared to the G3 and G4 of RotaTeq, with only one mutation in the former and two discrepancies in the latter.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGenetic drift, reassortment, animal to human transmission, and rearrangement are among mechanisms that lead to the emergence of novel rotavirus variants worldwide [24]. Members of known lineages in each genotype contain accumulated mutations that have been introduced in each replication cycle [23]. Some G and P genotypes exhibit significant amino acid differences in their antigenic epitopes compared to the vaccine strains, which might be responsible for escape from induced neutralizing antibodies [25]. In the present study, the amino acid sequences and the antigenic epitopes of VP7 and VP4 of circulating Iranian strains were compared with Rotarix and RotaTeq vaccine strains to determine potential disparities that could affect vaccine efficacy and their implications for future vaccination programs.\u003c/p\u003e \u003cp\u003eBased on VP7 amino acid sequence analysis, Iranian strains clustered in the G1/II, G2/IV, G3/I, G4/I, G6 and G9/III lineages. All G1/II strains shared the greatest degree of amino acid similarity and were completely conserved in all three antigenic epitopes when compared to Rotarix G1 strain. Additionally, G1 strains showed a high amino acid identity with G1 of RotaTeq (G1/II), but all of them displayed two amino acid changes in neutralizing epitopes, D97E and S147N [26], which had been reported before in Serbian and Chinese G1 strains [18,27]. The G2 genotype belonged to lineage IV and was more closely related to G2/II of RotaTeq than G1/II of Rotarix. The strain displayed just four amino acid changes at positions 87, 96, 213 and 242 compared to G2 epitopes of RotaTeq.\u0026nbsp;G2 strains are typically associated with P[4] genotypes, and protection against this genotype by RotaTeq primarily relies on the G2 (VP7) element of the vaccine. Some mutations at neutralizing epitopes of this genotype are related to escape from vaccine immunity. Accordingly, amino acid alteration at position 96 in antigenic region 7-1a in G2P[4] led to an outbreak in Australia [28].\u003c/p\u003e \u003cp\u003eThe G3 strain of RotaTeq and the Iranian G3 strain were closely related, but clustered in different lineages. Among the studied strains, three to five amino acid differences were found in the 7-1b region in comparison to the G3 of RotaTeq.\u0026nbsp;Notably, the study revealed the presence of a K238N amino acid substitution in all the strains, which may indicate a N-linked glycosylation site, consistent with results reported in China, Qatar and Italy [17,18,29]. The K238N mutation can enhance viral replication, diminish the efficacy of monoclonal antibodies, and prevent neutralizing antibody activity [30,31]. Furthermore, the shift in charge may impact the chemical properties of the protein, and the change in polarity implies that the epitope may become more inaccessible due to its increased hydrophobicity [32]. Epidemiological data from Australia obtained during the post-vaccine era suggest a possible increase in G3 strains where RotaTeq has been administered [33]. In addition, after the introduction of RotaTeq in the United States, the G3 genotype became predominant in some seasons [34].\u003c/p\u003e \u003cp\u003eThe two Iranian G4 strains were closely related to RotaTeq G4 and belonged to the same lineage (G4/I). Four discrepancies were found between the RotaTeq and Iranian G4 antigenic epitopes. The existence of these mutations among Iranian RVA-G4 strains may indicate the emergence of a variant with a capacity of immune escape [16]. Additionally, an alignment of their VP7 amino acids revealed insertion of an asparagine residue at position 76, which can affect glycosylation and, in turn, modify antigenic characteristics. This is because the asparagine insertion at this position, situated in a hydrophilic region, has the potential to enhance the region's hydrophilicity [35].\u003c/p\u003e \u003cp\u003eRegarding the G6 and G9 Iranian strains, both were distantly related to Rotarix and RotaTeq VP7 strains, with G9 strains showing a relatively closer similarity to G3 of RotaTeq.\u0026nbsp;This was consistent with a previous study in Iran [16]. Comparison of the VP7 epitopes of the G6 strain to the G6 of RotaTeq showed seven amino acid differences distributed across all three antigenic epitopes, which did not represent any radical changes in the antigenicity of VP7 epitopes, as previously reported in a US study [31]. As for the five G9 strains in this study, there was a high frequency of mutations across all three regions when compared to VP7 regions of both vaccines. One G9 strain (OQ789847/G9/III) exhibited a D100N mutation, which as reported in Chinese G9 strains, represents as an escape mutant [36].\u003c/p\u003e \u003cp\u003eThe VP4 spike protein plays a crucial role in viral neutralization due to its several structural and functional roles, including virus particle binding, penetration, and maturation [27]. All the analyzed sequences of VP8* region of the Iranian P[8] strains belonged to lineage III, which were relatively related to P[8]/lineages I and II of Rotarix and RotaTeq, respectively. However, VP8* epitopes of the P[8] strains differed significantly from the Rotarix and RotaTeq counterparts. Similar patterns of amino acid substitutions in VP8* epitopes of the P[8] strains between circulating and vaccine strains in our study were reported from Serbia, China and Qatar [17,18,27]. The amino acid changes identified at positions S131R and N135D in this study could result in polarity changes, which play a role in RV\u0026rsquo;s escape from the host immunity [27]. Taken together, although both vaccines have shown to be very effective in Europe and the United States, there is increasing concern about the evolution of resistant strains [37]. Regarding the high diversity of RVA genotypes in Iran and detected mutations in most of their neutralizing epitopes, escape from induced immunity of RotaTeq and Rotarix seems to be highly probable after vaccination program. However, as was shown in a study in Belgium, the introduction of vaccine can effectively decrease dominant genotypes from the same lineage, G1/II-P[8]/I strains, compared to unrelated lineages [23], which could highly reduce the infection rate among children.\u003c/p\u003e \u003cp\u003ePresentation of a specific amino acid sequence of structural antigens to B- or T-cells can mediate immunity to RVA. Although, the precise mechanism by which vaccination confers protection against rotavirus is not well known, neutralizing antibodies specific to VP4 and VP7 proteins seems to be the key factors of protection, which are considered for development of the RotaTeq vaccines [37]. In this study, similarity of the two known CTL epitopes of the VP7 protein was analyzed in the Iranian G-types compared with Rotarix and RotaTeq strains. G1 strains in our isolates showed 3 amino acid substitutions within VP7 CTL epitopes compared to the vaccine strains, which is consistent with a previous study in Africa [38]. In the case of other genotypes, in comparison to RotaTeq strain, G2 strains showed the highest number of discrepancies, while G3 and G4 strains displayed the least number of differences across both epitopes. This discrepancy was also reported in a study in Russia [39]. According to our knowledge, there are no available data on the effects of these mutations on the immunity induced by CTLs. Impact of the characterized mutations in VP7 CTL epitopes of the circulating strains in Iran on the processing and presentation of RVA antigens to immune cells and RVA clearance should be further studied.\u003c/p\u003e \u003cp\u003eThis study had some limitations. While the data were sufficient for differentiating RVA genotypes and their lineages, sub-genotypic lineages could not be identified. This was primarily due to the partial sequencing of VP7 and VP4 gene segments and budgetary constraints. The isolates analyzed in this study belonged to pediatric patients admitted to a referral hospital in Tehran. To obtain a better overview of lineage diversity for effective vaccine implementation, a comprehensive multicenter study is necessary.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides crucial insights into the genetic and antigenic characteristics of Iranian strains compared to the vaccine strains. Regarding the high diversity of RVA genotypes in Iran and detected mutations in neutralizing and CTL epitopes, escape from induced immunity of RotaTeq and Rotarix seems to be probable after vaccination program. To fully show the importance of these differences and their implications on vaccine efficacy, further studies on the intragenotype antigenic variability of RVA are necessary prior to the introduction of Rotarix and RotaTeq into the national immunization program.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.A., R.M.G, and A.K. conceptualized the study, formed the methodology, conducted the investigation and supervised the study. T.F. performed the analysis and wrote the original draft. M.A. reviewed and edited the draft. S.M.Z. and S.M. supported the study officially and coordinated the study. M.A. and R.M.G are both corresponding authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the World Health Organization under grand 202700981\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data required to reproduce these findings are available from the corresponding author upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the research ethics committees of Research Institute of Children\u0026rsquo;s Health at Shahid Beheshti University of Medical Sciences (IR.SBMU.RICH.REC.1401.021), and parental consent was obtained for all admitted children.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKraay ANM, Chaney DM, Deshpande A, Pitzer VE, Lopman BA. 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Detection and Molecular Characterization of Rotavirus Infections in Children and Adults with Gastroenteritis from Vojvodina, Serbia. Microorganisms. 2022;10. doi: 10.3390/microorganisms10102050\u003c/li\u003e\n \u003cli\u003eDonato CM, Pingault N, Demosthenous E, Roczo-Farkas S, Bines JE. Characterisation of a G2P[4] Rotavirus Outbreak in Western Australia, Predominantly Impacting Aboriginal Children. Pathog (Basel, Switzerland). 2021;10. doi: 10.3390/pathogens10030350\u003c/li\u003e\n \u003cli\u003eBonura F, B\u0026aacute;nyai K, Mangiaracina L, Bonura C, Martella V, Giammanco GM, et al. Emergence in 2017\u0026ndash;2019 of novel reassortant equine-like G3 rotavirus strains in Palermo, Sicily. Transbound Emerg Dis. 2022;69:813\u0026ndash;35. doi: 10.1111/tbed.14054\u003c/li\u003e\n \u003cli\u003eZao CL, Yu WN, Kao CL, Taniguchi K, Lee CY, Lee CN. Sequence analysis of VP1 and VP7 genes suggests occurrence of a reassortant of G2 rotavirus responsible for an epidemic of gastroenteritis. J Gen Virol. 1999;80 ( Pt 6):1407\u0026ndash;15. doi: 10.1099/0022-1317-80-6-1407\u003c/li\u003e\n \u003cli\u003eEsona MD, Gautam R, Katz E, Jaime J, Ward ML, Wikswo ME, et al. Comparative genomic analysis of genogroup 1 and genogroup 2 rotaviruses circulating in seven US cities, 2014-2016. Virus Evol. 2021;7:veab023. doi: 10.1093/ve/veab023\u003c/li\u003e\n \u003cli\u003eBetts MJ, Russell RB. Amino Acid Properties and Consequences of Substitutions. Bioinforma Genet. John Wiley \u0026amp; Sons, Ltd; 2003. p. 289\u0026ndash;316. doi: 10.1002/0470867302\u003c/li\u003e\n \u003cli\u003eKirkwood CD, Boniface K, Barnes GL, Bishop RF. Distribution of rotavirus genotypes after introduction of rotavirus vaccines, Rotarix\u0026reg; and RotaTeq\u0026reg;, into the National Immunization Program of Australia. Pediatr Infect Dis J. 2011;30:S48-53. doi: 10.1097/INF.0b013e3181fefd90\u003c/li\u003e\n \u003cli\u003eHull JJ, Teel EN, Kerin TK, Freeman MM, Esona MD, Gentsch JR, et al. United States rotavirus strain surveillance from 2005 to 2008: genotype prevalence before and after vaccine introduction. Pediatr Infect Dis J. 2011;30:S42-7. doi: 10.1097/INF.0b013e3181fefd78\u003c/li\u003e\n \u003cli\u003eBerois M, Libersou S, Russi J, Arbiza J, Cohen J. Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo-Uruguay from 1996-1999. J Med Virol. 2003;71:456\u0026ndash;62. doi: 10.1002/jmv.10511\u003c/li\u003e\n \u003cli\u003ePeng R, Li D, Wang J, Xiong G, Wang M, Liu D, et al. Reassortment and genomic analysis of a G9P[8]-E2 rotavirus isolated in China. Virol J. 2023;20:135. doi: 10.1186/s12985-023-02064-5\u003c/li\u003e\n \u003cli\u003eWard RL, Clark HF, Offit PA. Influence of potential protective mechanisms on the development of live rotavirus vaccines. J Infect Dis. 2010;202 Suppl:S72-9. doi: 10.1086/653549\u003c/li\u003e\n \u003cli\u003eMwangi PN, Mogotsi MT, Seheri ML, Mphahlele MJ, Peenze I, Esona MD, et al. Whole Genome In-Silico Analysis of South African G1P[8] Rotavirus Strains before and after Vaccine Introduction over a Period of 14 Years. Vaccines. 2020;8. doi: 10.3390/vaccines8040609\u003c/li\u003e\n \u003cli\u003eMorozova O V, Sashina TA, Fomina SG, Novikova NA. Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines. Arch Virol. 2015;160:1693\u0026ndash;703. doi: 10.1007/s00705-015-2439-6\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rotavirus, Vaccine, VP4, VP7, Lineage, Antigenic epitopes","lastPublishedDoi":"10.21203/rs.3.rs-3842299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3842299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Group A rotavirus is a leading cause of diarrheal disease, with its prevalence remaining high in low- and middle-income countries. In this study, circulating lineages of VP4 and VP7 proteins of human RVA isolates from children under 5 years of age were analyzed and their cytotoxic T cell and antigenic epitopes were compared to the RotaTeq and Rotarix vaccine strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Viral RNA was extracted from 51 positive samples and amplified using specific primers. Sequencing was performed and multiple sequence alignments were done in MEGA and phylogenetic trees were constructed. Similarity of VP7 and VP4 amino acids with the vaccine stains and structural analysis were performed using the UCSF Chimera-Molecular Modeling System.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e The Iranian strains clustered in the G1/II, G2/IV, G3/I, G4/I, G9/III, P[8]/III, P[4]/IV, and P[6]/I lineages. Comparative analysis of VP7 antigenic epitopes showed that G1/II strains are completely conserved, but G2/IV, G3/I, G4/I, G6, G9/III strains contained 2, 3-5, 2, 4 and 9 amino acids substitutions, respectively. P[8]/III genotypes differed by 3 amino acids, while P[6]/I genotypes had the most substitutions. CTL epitopes were completely conserved in G3/I strains, but other genotypes differed by 1-4 amino acids in comparison to the vaccine strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eConsidering the diversity of circulating RVA genotypes and the observed mutations in the neutralizing and CTL epitopes, immune escape by some of the strains is likely in Iran. This finding underscores the importance of evaluating the effect of rotavirus vaccines on local genotypes and related lineages before implementing the vaccination program.\u003c/p\u003e","manuscriptTitle":"Comparative analysis of the RVA VP7 and VP4 antigenic epitopes circulating in Iran and the Rotarix and RotaTeq vaccines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-10 20:05:11","doi":"10.21203/rs.3.rs-3842299/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a06494ca-fbde-487f-b63d-aa719fa50c02","owner":[],"postedDate":"January 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-06T03:13:14+00:00","versionOfRecord":{"articleIdentity":"rs-3842299","link":"https://doi.org/10.1016/j.heliyon.2024.e33887","journal":{"identity":"heliyon","isVorOnly":true,"title":"Heliyon"},"publishedOn":"2024-07-01 03:13:14","publishedOnDateReadable":"July 1st, 2024"},"versionCreatedAt":"2024-01-10 20:05:11","video":"","vorDoi":"10.1016/j.heliyon.2024.e33887","vorDoiUrl":"https://doi.org/10.1016/j.heliyon.2024.e33887","workflowStages":[]},"version":"v1","identity":"rs-3842299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3842299","identity":"rs-3842299","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}