Beyond Rotavirus: Seasonal Dynamics and Genetic Diversity of Non-Polio Enteroviruses in Hospitalized Children with Acute Gastroenteritis in Pune, India

Beyond Rotavirus: Seasonal Dynamics and Genetic Diversity of Non-Polio Enteroviruses in Hospitalized Children with Acute Gastroenteritis in Pune, India | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Beyond Rotavirus: Seasonal Dynamics and Genetic Diversity of Non-Polio Enteroviruses in Hospitalized Children with Acute Gastroenteritis in Pune, India Sachin Pal, Rishabh Waghchaure, Sunithakumari V S, Pooja A Umare, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8115337/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Acute gastroenteritis (AGE) in children is primarily associated with pathogens such as Rotavirus, Norovirus, Adenovirus, Astrovirus, and increasingly, human enteroviruses (EVs). EVs are non-enveloped RNA viruses belonging to the Picornaviridae family, comprising over 100 subtypes classified into four species. Despite their clinical relevance, the prevalence of EVs in AGE remains underexplored in India, resulting in limited data on their epidemiology and molecular diversity. Methods: A total of 450 stool samples were collected from hospitalized children under five years of age with AGE in Pune, India, between 2017 and 2023. Molecular characterization was subsequently performed to identify and confirm the types of enteroviruses present. Results: EVs were detected in 11.3% (51/450) of cases; 58.8% (30/51) among them were mono-infections. Rotavirus emerged as the most frequently identified pathogen, while co-infections involving Norovirus, Adenovirus, Astrovirus, and Parechovirus were documented. Eighteen distinct EV genotypes were characterized, with EV-B being the most dominant (64%). EVs were found circulating throughout the year, with a notable peak in the monsoon (June-August), and were predominantly detected in children below two years of age. Conclusion: Association of EVs in more than 10% of AGE cases highlights the importance of incorporating EV screening in AGE cases and understanding the burden caused by this important group of viruses. Acute severe gastroenteritis Children Pathogens Rotavirus Norovirus Adenovirus Astrovirus Enterovirus Genotypes Prevalence Figures Figure 1 Figure 2 Figure 3 Figure 4 Key Points This study investigated 450 paediatric gastroenteritis cases in Pune, India (2017–2023), detecting enteroviruses in 11.3% with 18 diverse genotypes, predominantly EV-B. Infections peaked during monsoon, mainly affecting children under two. Findings highlight enteroviruses’ underrecognized role in acute gastroenteritis burden in India Introduction Acute gastroenteritis (AGE) is a major cause of illness and mortality in infants and young children worldwide, especially in developing nations 1 . Viruses such as rotavirus, norovirus, astrovirus, and adenovirus are recognized as the leading causes of diarrhoea in children. Viruses account for more than 70% of AGE cases, with norovirus, rotavirus, astrovirus, adenovirus, and sapovirus being the leading viral culprits 2 . Additionally, several viral agents found in the stools of AGE patients have not yet been fully identified, suggesting a possible link to diarrheal disease. In 2021, diarrheal diseases were responsible for over 37,874 deaths among children under five years old from India (Our World in Data: Diarrheal Diseases https://ourworldindata.org/diarrheal-diseases assessed on 5.8.2025). Several recent studies have shown a rising prevalence of enteroviruses (EVs) in AGE patients globally, with rates ranging from 5.3% to 26.3% 3–7 . EVs, are members of the Picornaviridae family and consist of 13 species, including 10 EVs (EV-A to -J) and 3 rhinoviruses (RV-A to -C) ( http://www.picornaviridae.com ). Seven of these species, including four EVs (EV-A to -D) and three RVs, affect humans. EV is a small, non-enveloped virus with a single-stranded RNA genome and four structural proteins (VP1–VP4), with VP1 containing a neutralization site used for typing. There are 159 identified human EV types ( http://www.picornaviridae.com ). EVs cause various diseases in the gastrointestinal, respiratory, and central nervous systems, and although many infections are asymptomatic, they can lead to severe illness, particularly in children. Enteroviruses were initially associated with severe diseases like hand, foot, and mouth disease (HFMD), myocarditis, pericarditis, and neonatal sepsis. Recently, Picornaviridae family members, including EVs, Parechoviruses (HPeVs), and Aichivirus (AiV), have been linked to AGE 8 . Previously, EV intestinal presence was thought to be coincidental rather than causal, but now enteroviruses are recognized as significant agents of AGE. Though most EV infections are asymptomatic, still 1.0% of infections could lead to severe infections with high mortality and morbidity among infants 9 . The variable rates (2.5–16.6%) of EV infection have been reported in AGE cases from different parts of the world. CV A1, CV B6, E1 and E6 were reported in association with gastroenteritis 10 , 11 . Recent studies have reported on EV infection concerning diarrheal disease and the molecular characteristics of EV 3,4,12 . An Outbreak of gastroenteritis was reported in Japan caused due to E6. Novel Serotypes of EVs (EV-76-91) were detected in isolates of acute gastroenteritis from Bangladesh 13 – 14 . This study aimed to determine the incidence of EVs in AGE patients, focusing on whether they are more prevalent in young children (under 5 years) in India. Molecular characterization will provide insights into strain circulation patterns and genetic variations, essential for developing diagnostics, antivirals, and vaccines. Identifying common enterovirus types will also help predict potential future epidemics in the region. Materials and methods 2.1 Specimen collection: Stool samples were collected from pediatric patients under the age of five who were hospitalized with AGE at various hospitals across Pune city, India, between January 2017 and December 2023. All collected samples were preserved at − 70°C until further analysis. The study received ethical clearance from the ICMR-NIV Institutional Ethics Committee (Approval No. MP-24A-7N dated 11.03.2024). Written informed consent was obtained from the parents or legal guardians of each participant. Clinical and demographic information was documented using case reporting forms (CRFs). Each child was assessed for the presence of fever, and the frequency and duration of diarrhoea and vomiting episodes. The severity of illness was classified using the Vesikari scoring system as mild (0–5), moderate (6–10), severe (11–15), or very severe (16–20). Frequency of symptoms is given in Table-1. Table 1 Signs and symptoms of patients in the AGE cases from 2017–2023 (n = 450) Symptoms % of children with the symptom (N) [Total participants, n = 450] Diarrhea 439/450 (97.5%) 97.5 (439) Vomiting 356/450 (79.1%) Dehydration 407/450 (90.4%) Lethargy 415/450 (92.2%) Abdominal pain 363/450 (80.4%) 2.2. Virus nucleic acid extraction and detection of viruses by real-time RT-PCR Stool samples were processed as 10% suspensions in PBS, and viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen). Enteroviruses were detected by real-time PCR (BIORAD CFX-96) targeting the conserved 5′ NCR region, using published primers and a FAM-labeled TaqMan probe with the ABI Fast Virus Kit under standard cycling conditions 15 . 2.3 Molecular Characterization by VP1/VP2 Genotyping Faecal samples that tested positive for EVs were subjected to genotyping by amplifying the partial VP1 region. Nested-PCR amplification was carried out using outer primers 224 and 222, followed by inner primers AN89 and AN88 (Table 2 ) as previously described 16 , 17 . The PCR reagents and thermal cycling conditions were consistent with the EV detection protocol, except for the annealing temperatures, which were set to 42°C for the first round and 55°C for the second round. Additionally, the number of amplification cycles in the second round was extended to 40 cycles. The amplicon sizes for the first and second rounds of PCR were 993 and 350 base pairs, respectively. In addition to EV testing, all stool samples were screened for other common viral pathogens associated with diarrhoea, including Adenovirus (AdV), Norovirus (NoV), Rotavirus A (RVA), Human Astrovirus (HAstV), and Human Parechovirus (HPeV), using real-time RT-PCR protocols standardized and validated in our laboratory. Table 2 Primer pairs used for the amplification of VP1/ VP2 region Region Primer Sequence 5’ to 3’ Position Product size Reference VP3- VP1 VP1 224F GCIATGYTIGGIACICAYRT 1977–1996 958 bp 16,17 VP1 222R CICCIGGIGGIAYRWACAT 2935 − 2917 VP1 AN 89F CCAGCACTGACAGCAGYNGARAYNGG 2602–2677 384 bp AN 88R TACTGGACCACCTGGNGGNAYRWACAT 2977 − 2951 2.4 Nucleotide sequencing and phylogenetic analysis PCR-amplified VP1/VP2 regions from EV-positive samples were purified (QIAquick Kit, Qiagen) and sequenced using the BigDye Terminator v3.1 Kit on an ABI 3100 sequencer. Forward and reverse reads were cleaned (Dye-Ex 2.0 Kit, Qiagen) and analyzed via BLAST against GenBank reference sequences. Genotypes were assigned using the Enterovirus Genotyping Tool v1.0. For further characterization, partial VP1 sequences were aligned with reference strains (top BLAST hits) using MAFFT. Phylogenetic trees were generated in MEGA v11.0 (Neighbor-Joining, Kimura 2-parameter model) with 1,000 bootstrap replicates to assess robustness. 2.6. Isolation of representative EV-positive samples: Human Rhabdomyosarcoma (RD) cells were cultured in MEM supplemented with 10% FBS, L-glutamine, and antibiotics at 37°C with 5% CO₂. Clinical specimens were inoculated onto RD monolayers in 24-well plates and monitored for 4–5 days for cytopathic effect (CPE). Wells showing complete CPE were harvested, freeze–thawed thrice, centrifuged, and the clarified supernatant stored at − 70°C. The supernatant was used for up to three passages to enhance virus recovery. 2.7. Sequence Assembly and Annotation of EVs: Twenty-two Indian enterovirus genomes were assembled using the CLC Genomics Workbench [ https://digitalinsights.qiagen.com/technical-support/system-requirements/ ]. The finalized and annotated sequences were submitted to GenBank via the BANKIT submission tool. Results 3.1. Prevalence of EV infection in AGE patients In this study, a total of 450 samples were tested, with 51 samples identified as positive for EV through real-time RT-PCR amplification of the 5’ UTR region, resulting in an overall positivity rate of 11.3%. The prevalence of EV infection fluctuated annually, ranging from 7.8% to 31.3%, while no positivity was observed during the COVID-19 pandemic due to a lack of available samples, as shown in Fig. 1B. The highest detection rate was recorded in 2017 (31.3%), and the lowest in 2020 (0%), likely due to the reduced number of samples collected during the pandemic. Overall, the positivity rate for females across all years was 45% (23/51), compared to 54.9% for males (28/51). 3.2 Prevalence of EV infection in different age groups In terms of age, the highest prevalence of EV detection was observed in the 0–12 months age group (41.2%, 21/51), followed by the 13–24 months group (27.4%, 14/51), and the lowest in the 49–60 months group (5.8%, 3/51) (Fig. 1A). 3.3 Seasonal distribution of EV infection The seasonal distribution from 2017 to 2023, depicted in Fig. 2 , showed that enterovirus cases occurred year-round, with a peak in the monsoon months. Incidence increased from 7.7% in March to 15.3% in August, reaching a maximum of 23% in July. The majority of EV-positive cases (27 out of 51; 52.94.%) were recorded from April to October, with the peak occurring in July (Fig. 2 ). This seasonal trend aligns with other studies that reported the highest prevalence of EV infections in monsoon with increase in the humidity accompanied by heavy/moderate rainfall 3 , 4 . 3.4. Prevalence of EV infection and co-infection in AGE patients Among the positive samples, specifically, mono-infection with EV accounted for 58.8% (30/51) of the cases, while co-infections with other enteric viruses, including rotavirus, norovirus, adenovirus, human astrovirus, and human parechovirus, were observed in 41.2% (21/51) of the cases. Among the co-infections, double infections occurred in 39.2% (20/51) of cases, and triple infections were seen in one case (1.96%). In terms of clinical characterization, most patients experienced diarrhoea along with other symptoms such as abdominal pain, nausea, vomiting, or fever. No severe cases were reported. 3.5 Distribution of EV species and genotyping Molecular characterization of EV strains from 30 positive samples was performed by sequencing the partial VP1 or VP2 gene. Of the 51 positive samples identified in this study, the VP1 gene was successfully amplified in 24 samples, while 6 samples that did not amplify for VP1 were characterized using the VP2 gene. Of the 24 samples characterized by VP1, 33.3% (8/24) were identified as EV-A, 50% (12/24) as EV-B, and 16.7% (4/24) as EV-C. The 6 samples characterized by VP2 amplification all belonged to the EV-B species. This resulted in the following detection pattern: EV-B (60%, 18/30) > EV-A (26.6%, 8/30) > EV-C (13.3%, 4/30). In total, 16 different genotypes of EV were identified, as summarized in Table 3 . A subset of EV-positive samples (41.2%, 21/51) could not be characterized due to failure to produce amplicons or issues with genotyping, and in two cases, genotyping was not performed due to the limited sample quantity. The distribution of EV species and genotypes identified in this study is shown in Table 3 . Table 3 Different types of EV associated with AGE in children during 2017–2023 Species Serotypes No of Characterized Samples (% of total) Enterovirus-A CVA-5 2 (6.7) CVA-6 1 (3.3) CVA-10 2 (6.7) CVA-16 1 (3.3) CVB-4 1 (3.3) EV-71 1 (3.3) Subtotal 8 (26.6) Enterovirus-B CVA-9 3 (10) CVB-3 2 (6.7) CVB-4 2 (6.7) E-5 1 (3.4) E-1 2 (6.7) E-7 2 (6.7) E-11 4 (13.3) E-14 1 (3.4) E-25 1 (3.4) Subtotal 18 (60) Enterovirus-C CVA-13 2 (6.7) Total 28 (93.3) 3.6 Phylogenetic Analysis and characterization of EV strains To explore the genetic relationships between the 28 EV strains identified in this study and previously reported EV reference strains, phylogenetic trees for each EV species (A, B and C) were constructed, as illustrated in Table 4 ; Fig. 3 . The results showed that all EV strains from this study clustered closely with their respective reference strains based on genotype. Each of the 22 EV genotypes formed distinct branches alongside their corresponding reference strains, validating the genotyping results obtained through BLAST and the Enterovirus Genotyping Tool. Nearly all EV strains exhibited over 90% nucleotide sequence identity with their corresponding reference genotypes, except for CV-A10, CV-A5, CV-A16, and Echovirus 25, which showed lower sequence identities (75–85%) compared to their reference sequences. Sequences from 22 enterovirus genotypes submitted in NCBI rest 6 sequences yielded partial sequences with several ambiguous regions, which did not fulfill the criteria for NCBI submission. However, the reliable portions were analyzed and utilized for phylogenetic and identification purposes. Table 4 Sequence data submitted to NCBI Sr. No. Sample Genotype Collection Date Accession No. 1 1727766 Coxsackievirus A-10 14-06-2017 PV611641.1 2 1728229 Echovirus − 25 28-06-2017 PV637434.1 3 1728841 Coxsackievirus A − 6 11-07-2017 PV611636.1 4 1730321 Echovirus − 11 31-07-2017 PV637430.1 5 1734322 Coxsackievirus A − 5 27-09-2017 PV611648.1 6 1850025 Coxsackievirus A − 5 02-05-2018 PV611649.1 7 1850027 Enterovirus − 71 16-05-2018 PV584128.1 8 1850082 Echovirus − 11 13-07-2018 PV637431.1 9 1850137 Coxsackievirus A − 16 20-08-2018 PV611645.1 10 1850156 Coxsackievirus B − 3 04-09-2018 PV611646.1 11 198147 Coxsackievirus B − 3 14-08-2019 PV611647.1 12 198141 Coxsackievirus A − 13 08-08-2019 PV611643.1 13 211435 Coxsackievirus A − 6 07-06-2021 PV611637.1 14 220647 Coxsackie Virus A − 13 26-02-2022 PV611644.1 15 220777 Echovirus − 11 04-03-2022 PV637432.1 16 220875 Coxsackievirus B − 4 24-03-2022 PV611634.1 17 220881 Coxsackievirus A − 9 24-03-2022 PV611638.1 18 240130 Coxsackievirus A − 9 17-05-2023 PV611639.1 19 2315608 Echovirus − 11 12-08-2023 PV637433.1 20 2318987 Coxsackievirus B − 4 17-10-2023 PV611635.1 21 2319307 Coxsackievirus A − 10 22-10-2023 PV611642.1 22 2411207 Coxsackievirus A − 9 12-02-2024 PV611640.1 Phylogenetic tree based on partial VP1 sequences (~ 300 bp) from Indian isolates associated with AGE cases and reference sequences retrieved from GenBank; (a) EV-A, (b) EV-B, and (c) EV-C species. Trees were generated using the neighbor-joining method with the Kimura 2-parameter model implemented in MEGA v.11 Branch support was assessed through 1000 bootstrap replicates, with bootstrap values above 75% shown at the corresponding nodes. Filled circles indicate the enteroviruses identified in the present study. 3.7 Isolation of representative EV-positive samples: Among the characterized enteroviruses, six distinct virus isolates were selected for attempted isolation. The selection criteria were based on cycle threshold (Ct) values, indicative of high viral load, and the specific EV species (CVA-10, CVB-4, E-7. E-11, E-14 and CVA-16) identified during molecular characterization. Each selected sample corresponded to a unique EV species. The six EV strains were propagated in monolayer cultures of the RD cell line, with the first three passages conducted in 24-well plates and the fourth passage performed in T-25 flasks. All strains induced a cytopathic effect (CPE) within 2–4 days post-inoculation. The CPE was consistently observed across all passages, confirming successful viral replication in the RD cell line (Fig. 4 ). 3.8 Molecular characterization of isolates: RNA extraction was performed for all viral isolates across successive passages, followed by confirmation of viral presence using real-time reverse transcription polymerase chain reaction (RT-PCR). The assay targeted the 5′ non-coding region (NCR) of Enteroviruses (EV) using specific primers and probes. Viral RNA in the culture supernatant was detected, and the viral load at each passage (Table-5) was quantified by extrapolating copy numbers from a standard curve generated using known positive controls. Ct values and corresponding viral genome copy numbers were determined by quantitative RT-PCR at each passage (P1–P4). A progressive decrease in Ct values with increasing passage reflects enhanced viral replication and higher viral load. Data are shown for Coxsackievirus A10 (CVA-10), Coxsackievirus B4 (CVB-4), Echovirus 7 (E-7), Echovirus 11 (E-11), Echovirus 14 (E-14), and Coxsackievirus A16 (CVA-16) (Table-5). All viral isolates showing cytopathic effects were confirmed positive for the NCR via real-time RT-PCR and were subsequently subjected to molecular typing and characterization using VP1 gene-specific primers and further confirmed the presence of the target viral sequences. Table 5 Replication Kinetics of Enteroviruses Across Serial Passages in RD cell line Virus Name Passage − 01 Passage − 02 Passage − 03 Passage − 04 Ct Copy Number Ct Copy Number Ct Copy Number Ct Copy Number CVA-10 29.31 3.40x10 3 18.15 1.75 x10 6 14.93 1.49 x10 7 14.02 2.89 x10 7 CVB-4 34.13 2.52 x10 2 31.10 1.48 x10 3 15.66 1.33 x10 7 14.10 3.06 x10 7 E-7 30.14 2.63 x10 3 20.93 6.26 x10 5 15.79 1.18 x10 7 14.53 2.36 x10 7 E-11 24.37 9.11 x10 4 16.67 6.68 x10 6 16.69 6.90 x10 6 13.13 5.51 x10 7 E-14 29.14 4.66 x10 3 20.47 7.46 x10 5 15.08 1.03 x10 7 13.38 4.54 x10 7 CVA 16 32.21 7.65 x10 2 16.51 8.40 x10 6 18.13 2.77 x10 6 13.23 5.01 x10 7 Discussion AGE is an intestinal disease that remains a major cause of high morbidity and considerable mortality rates worldwide, particularly in developing countries where the risk of infection is elevated and hygiene, sanitary conditions, and nutritional status of children are poor. Gastrointestinal infections pose a significant challenge to health services globally due to their high morbidity and mortality rates. Testing patients only for the most common pathogens results in diagnoses for only half of these cases. In our study, we have tested the samples for EV, AstV, AdV, NoV, HPeV and RVA and the prevalence of EV is relatively high as compared to RVA, this could be attributed to the higher use of vaccines against RVA in regular immunization schedule of infants in Pune. EVs are frequently implicated in AGE, especially in pediatric populations. Although these viruses are predominantly linked to respiratory infections, hand-foot-and-mouth disease, and neurological disorders, they also manifest with gastrointestinal symptoms. Observations have indicated a higher incidence of enteroviral infections in cases of acute diarrhoea compared to non-diarrheal illnesses from India 18 . This study describes the epidemiological, seasonal, and molecular characteristics of EVs detected in children suffering from AGE from western India, Pune from 2017 to 2023. The prevalence of EV in our study was 11.3% ranging between 0% in year 2020 to 31.3% in year 2017, there is no such pattern of apparent increase and decrease in year-wise positivity, but the null positivity in year 2020 could be explained by limited sample collection due to COVID-19 pandemic. These kinds of prevalence percentages have been reported from different countries in similar other studies 5 , 18 – 20 . The incidence of EVs observed in this study aligns with findings from recent research conducted across diverse geographical regions, reinforcing the concept that enteroviruses can be significant etiological agents in gastrointestinal disorders. This is further substantiated by the notably high positivity rate (58.8%, 30/51) of EVs detected in stool samples from patients with gastroenteritis who tested negative or coinfected with other enteric viruses. Historical data from outbreaks of acute gastroenteritis in Western India have similarly reported a higher prevalence of enteroviral infections relative to other enteric pathogens 4 . Initially, the typing of EV strains mainly relies on virus isolation followed by serotyping based on neutralization with pooled antisera, but now various molecular typing and sequencing-based methods are available for enterovirus typing. Several different RT-PCR methods targeting different regions of the EV genome, particularly those encoding the structural period of the virus have been used 21 – 22 . In this present study, the EV-positive samples have been typed by VP1/VP2 gene amplification. Eighteen distinct genotypes of EV have been identified in this study, with EV-B showing the highest relative prevalence at 60%, followed by EV-A at 27%, and EV-C at 13.3%. These results are consistent with previous studies, which also demonstrated a higher frequency of EV-B species compared to EV-A and EV-C in paediatric cases of AGE 7 , 20 , 23 . Few studies have suggested that Echoviruses and coxsackie viruses have a role in causing diarrhoea and AGE in children 3 , 24 . In our study, E-11 and CVA-9 were the most predominant detected genotypes, Outbreaks of diarrhoea caused by E-11 have been reported from Malaysia 25 and southern India 26 . In our study, approximately 41.2% of the samples were co-infected with additional diarrhoea-associated viruses, suggesting that the observed symptoms or disease progression in these cases might be attributable to these co-infecting viruses rather than enterovirus alone. Furthermore, children whose samples tested negative for enteroviruses or other known viruses causing AGE continued to exhibit symptoms. Additionally, since bacterial and parasitic infections were not assessed in this study, the aetiology of these samples should also be taken into account. In conclusion, this study provides evidence for the presence and circulation of various EV serotypes among patients with AGE. The ongoing circulation of predominant enterovirus types within the population highlights the potential risk of future outbreaks of gastroenteritis. The isolation and characterization of these predominant strains are crucial for the development of serological diagnostic tools and for conducting seroepidemiological studies. The current low success rate in the etiological diagnosis of AGE underscores the necessity of identifying new pathogens. While viruses from the Caliciviridae family, such as Sapovirus and type II Norovirus, are now regarded as secondary potential agents in AGE, our findings suggest that enteroviruses should be given serious consideration due to their high incidence and effective replication, particularly in children under five years of age. Future research should focus on defining the role of enteroviruses in AGE by evaluating viral load markers and assessing the impact of various enterovirus genotypes and polio vaccine strains. This research is vital for improving clinical management, patient care, and public health strategies, given that viral gastroenteritis remains a significant global health issue, particularly in regions with challenges related to hygiene and sanitation. Declarations Funding This study was supported by the ICMR (Diarr/Adhoc/3/2022 ECD-II) and ICMR-National Institute of Virology, Pune, Maharashtra, India (No. EVE 2201). Ethical Statement: This study was reviewed and approved by the Ethics Committee of the ICMR-National Institute of Virology, Pune (Approval No. MP-24A-7N dated 11.03.2024), in accordance with established ethical guidelines for biomedical research involving human samples. Competing Interests The authors declare that they have no conflict of interest. Data availability Full genome sequence of this case viral variants has been deposited in the GenBank NCBI (Accession no: Table 4) using the BANKIT submission tool. Author’s contribution ML: Conceptualization, methodology, project administration, resources, supervision, writing, review and editing. SP and RW: Contributed to the experimentation, data curation, formal analysis, writing original draft, investigation and methodology. SP and PU: Contributed to the experimentations. SC, SK and JK: Supported in the analysis of data and phylogenetic analysis. 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Silva PA, Stark K, Mockenhaupt FP, Reither K, Weitzel T, Ignatius R, et al. Molecular characterization of enteric viral agents from children in northern region of Ghana. J Med Virol 2008; 80 : 1790–8. Bina Rai S, Wan Mansor H, Vasantha T, Norizah I, Chua KB. An outbreak of echovirus 11 amongst neonates in a confinement home in Penang, Malaysia. Med J Malaysia 2007; 62 : 223–6. Patel JR, Daniel J, Mathan VI. An epidemic of acute diarrhoea in rural southern India associated with echovirus type 11 infection. Journal of Hygiene 1985; 95 : 483–92. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8115337","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550461696,"identity":"80e0ef2c-1bb8-4200-bd34-f0cec8618b68","order_by":0,"name":"Sachin Pal","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Sachin","middleName":"","lastName":"Pal","suffix":""},{"id":550461697,"identity":"be2055cb-bb18-4cf5-bfa2-a8bee976d681","order_by":1,"name":"Rishabh Waghchaure","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Rishabh","middleName":"","lastName":"Waghchaure","suffix":""},{"id":550461698,"identity":"5c16af35-2f87-4502-9ce2-a5c8e7d00c4c","order_by":2,"name":"Sunithakumari V S","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Sunithakumari","middleName":"V","lastName":"S","suffix":""},{"id":550461699,"identity":"fd802c27-123f-49e1-9fc3-e637bfd1b392","order_by":3,"name":"Pooja A Umare","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Pooja","middleName":"A","lastName":"Umare","suffix":""},{"id":550461700,"identity":"c65a6aab-5aee-4236-9cb2-6268883ac51a","order_by":4,"name":"Jithin Kunjumon","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Jithin","middleName":"","lastName":"Kunjumon","suffix":""},{"id":550461701,"identity":"4fac7da9-b5de-4e48-8525-5d925d4f4600","order_by":5,"name":"P Sruthy","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"P","middleName":"","lastName":"Sruthy","suffix":""},{"id":550461702,"identity":"6aeb5883-567b-4be9-9eb3-5f35b181f1f0","order_by":6,"name":"Sarah Cherian","email":"","orcid":"","institution":"ICMR-National Institute of Virology","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Cherian","suffix":""},{"id":550461703,"identity":"4a7e7541-0c7f-4ab3-ac09-45f1f890307f","order_by":7,"name":"Mallika 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14:12:50","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99731,"visible":true,"origin":"","legend":"","description":"","filename":"4db9bedd726e49869f793fde6e5b92321structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/cd51c6c57f3d45d17a9b821f.xml"},{"id":96847650,"identity":"e21e9358-983b-4076-8353-2245ec6c5fa6","added_by":"auto","created_at":"2025-11-26 16:51:24","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109178,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/d4e32eba976f44629e066856.html"},{"id":96919157,"identity":"10e5807f-cf56-48b8-b6e7-be9db7403311","added_by":"auto","created_at":"2025-11-27 14:13:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296208,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/bcac7103bbd4a0de53129edf.png"},{"id":96847635,"identity":"83844b4d-1c73-4b18-b64a-24e195245b8a","added_by":"auto","created_at":"2025-11-26 16:51:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186528,"visible":true,"origin":"","legend":"\u003cp\u003eMonthly distribution of EV infection in pediatric patients in Pune from 2017 to 2023\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/cd59ca8b94e1c340e4092d27.png"},{"id":96847641,"identity":"86387633-4bb7-41b7-b313-0012c64cad8f","added_by":"auto","created_at":"2025-11-26 16:51:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":604790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis based on partial VP1 sequences from Indian isolates associated with AGE cases and other sequences available at the GenBank database.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/32fe3e722ba4ae9ac443ce4a.png"},{"id":96847640,"identity":"02af32ad-420f-4db5-8653-edaed05ecc91","added_by":"auto","created_at":"2025-11-26 16:51:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1176914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytopathic effect of different enteroviruses in RD cell lines. \u003c/strong\u003eRepresentative images show CPE induced by (A) CVA-10, (B) CVB-4, (C) E-7, (D) E-11, (E) E-14, and (F) CVA-16, compared with (G) uninfected control RD Cell line\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/c8e78174914662cd833af52f.png"},{"id":99788339,"identity":"21666b92-ad2d-4272-a330-ea67c31cc519","added_by":"auto","created_at":"2026-01-08 12:46:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3258836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8115337/v1/bd06fd2b-ed54-4b2d-9c53-bd2ce1bf1b8c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBeyond Rotavirus: Seasonal Dynamics and Genetic Diversity of Non-Polio Enteroviruses in Hospitalized Children with Acute Gastroenteritis in Pune, India\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Key Points","content":"\u003cp\u003eThis study investigated 450 paediatric gastroenteritis cases in Pune, India (2017\u0026ndash;2023), detecting enteroviruses in 11.3% with 18 diverse genotypes, predominantly EV-B. Infections peaked during monsoon, mainly affecting children under two.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFindings highlight enteroviruses\u0026rsquo; underrecognized role in acute gastroenteritis burden in India\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAcute gastroenteritis (AGE) is a major cause of illness and mortality in infants and young children worldwide, especially in developing nations\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Viruses such as rotavirus, norovirus, astrovirus, and adenovirus are recognized as the leading causes of diarrhoea in children. Viruses account for more than 70% of AGE cases, with norovirus, rotavirus, astrovirus, adenovirus, and sapovirus being the leading viral culprits\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Additionally, several viral agents found in the stools of AGE patients have not yet been fully identified, suggesting a possible link to diarrheal disease. In 2021, diarrheal diseases were responsible for over 37,874 deaths among children under five years old from India (Our World in Data: Diarrheal Diseases \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ourworldindata.org/diarrheal-diseases\u003c/span\u003e\u003cspan address=\"https://ourworldindata.org/diarrheal-diseases\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e assessed on 5.8.2025). Several recent studies have shown a rising prevalence of enteroviruses (EVs) in AGE patients globally, with rates ranging from 5.3% to 26.3%\u003csup\u003e3\u0026ndash;7\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEVs, are members of the \u003cem\u003ePicornaviridae\u003c/em\u003e family and consist of 13 species, including 10 EVs (EV-A to -J) and 3 rhinoviruses (RV-A to -C) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.picornaviridae.com\u003c/span\u003e\u003cspan address=\"http://www.picornaviridae.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Seven of these species, including four EVs (EV-A to -D) and three RVs, affect humans. EV is a small, non-enveloped virus with a single-stranded RNA genome and four structural proteins (VP1\u0026ndash;VP4), with VP1 containing a neutralization site used for typing. There are 159 identified human EV types (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.picornaviridae.com\u003c/span\u003e\u003cspan address=\"http://www.picornaviridae.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). EVs cause various diseases in the gastrointestinal, respiratory, and central nervous systems, and although many infections are asymptomatic, they can lead to severe illness, particularly in children. Enteroviruses were initially associated with severe diseases like hand, foot, and mouth disease (HFMD), myocarditis, pericarditis, and neonatal sepsis. Recently, \u003cem\u003ePicornaviridae\u003c/em\u003e family members, including EVs, Parechoviruses (HPeVs), and Aichivirus (AiV), have been linked to AGE\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Previously, EV intestinal presence was thought to be coincidental rather than causal, but now enteroviruses are recognized as significant agents of AGE. Though most EV infections are asymptomatic, still 1.0% of infections could lead to severe infections with high mortality and morbidity among infants\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The variable rates (2.5\u0026ndash;16.6%) of EV infection have been reported in AGE cases from different parts of the world. CV A1, CV B6, E1 and E6 were reported in association with gastroenteritis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recent studies have reported on EV infection concerning diarrheal disease and the molecular characteristics of EV\u003csup\u003e3,4,12\u003c/sup\u003e. An Outbreak of gastroenteritis was reported in Japan caused due to E6. Novel Serotypes of EVs (EV-76-91) were detected in isolates of acute gastroenteritis from Bangladesh\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study aimed to determine the incidence of EVs in AGE patients, focusing on whether they are more prevalent in young children (under 5 years) in India. Molecular characterization will provide insights into strain circulation patterns and genetic variations, essential for developing diagnostics, antivirals, and vaccines. Identifying common enterovirus types will also help predict potential future epidemics in the region.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Specimen collection:\u003c/h2\u003e\u003cp\u003eStool samples were collected from pediatric patients under the age of five who were hospitalized with AGE at various hospitals across Pune city, India, between January 2017 and December 2023. All collected samples were preserved at \u0026minus;\u0026thinsp;70\u0026deg;C until further analysis. The study received ethical clearance from the ICMR-NIV Institutional Ethics Committee (Approval No. MP-24A-7N dated 11.03.2024). Written informed consent was obtained from the parents or legal guardians of each participant. Clinical and demographic information was documented using case reporting forms (CRFs). Each child was assessed for the presence of fever, and the frequency and duration of diarrhoea and vomiting episodes. The severity of illness was classified using the Vesikari scoring system as mild (0\u0026ndash;5), moderate (6\u0026ndash;10), severe (11\u0026ndash;15), or very severe (16\u0026ndash;20). Frequency of symptoms is given in Table-1.\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\u003eSigns and symptoms of patients in the AGE cases from 2017\u0026ndash;2023 (n\u0026thinsp;=\u0026thinsp;450)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSymptoms\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e% of children with the symptom (N)\u003c/p\u003e\u003cp\u003e[Total participants, n\u0026thinsp;=\u0026thinsp;450]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiarrhea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e439/450 (97.5%) 97.5 (439)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVomiting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e356/450 (79.1%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDehydration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e407/450 (90.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLethargy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e415/450 (92.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbdominal pain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e363/450 (80.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Virus nucleic acid extraction and detection of viruses by real-time RT-PCR\u003c/h2\u003e\u003cp\u003eStool samples were processed as 10% suspensions in PBS, and viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen). Enteroviruses were detected by real-time PCR (BIORAD CFX-96) targeting the conserved 5\u0026prime; NCR region, using published primers and a FAM-labeled TaqMan probe with the ABI Fast Virus Kit under standard cycling conditions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Molecular Characterization by VP1/VP2 Genotyping\u003c/h2\u003e\u003cp\u003eFaecal samples that tested positive for EVs were subjected to genotyping by amplifying the partial VP1 region. Nested-PCR amplification was carried out using outer primers 224 and 222, followed by inner primers AN89 and AN88 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The PCR reagents and thermal cycling conditions were consistent with the EV detection protocol, except for the annealing temperatures, which were set to 42\u0026deg;C for the first round and 55\u0026deg;C for the second round. Additionally, the number of amplification cycles in the second round was extended to 40 cycles. The amplicon sizes for the first and second rounds of PCR were 993 and 350 base pairs, respectively.\u003c/p\u003e\u003cp\u003eIn addition to EV testing, all stool samples were screened for other common viral pathogens associated with diarrhoea, including Adenovirus (AdV), Norovirus (NoV), Rotavirus A (RVA), Human Astrovirus (HAstV), and Human Parechovirus (HPeV), using real-time RT-PCR protocols standardized and validated in our laboratory.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer pairs used for the amplification of VP1/ VP2 region\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequence 5\u0026rsquo; to 3\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePosition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eProduct size\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVP3- VP1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVP1 224F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCIATGYTIGGIACICAYRT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1977\u0026ndash;1996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e958 bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003csup\u003e16,17\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVP1 222R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCICCIGGIGGIAYRWACAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2935\u0026thinsp;\u0026minus;\u0026thinsp;2917\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVP1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAN 89F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCAGCACTGACAGCAGYNGARAYNGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2602\u0026ndash;2677\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e384 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAN 88R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACTGGACCACCTGGNGGNAYRWACAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2977\u0026thinsp;\u0026minus;\u0026thinsp;2951\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Nucleotide sequencing and phylogenetic analysis\u003c/h2\u003e\u003cp\u003ePCR-amplified VP1/VP2 regions from EV-positive samples were purified (QIAquick Kit, Qiagen) and sequenced using the BigDye Terminator v3.1 Kit on an ABI 3100 sequencer. Forward and reverse reads were cleaned (Dye-Ex 2.0 Kit, Qiagen) and analyzed via BLAST against GenBank reference sequences. Genotypes were assigned using the Enterovirus Genotyping Tool v1.0. For further characterization, partial VP1 sequences were aligned with reference strains (top BLAST hits) using MAFFT. Phylogenetic trees were generated in MEGA v11.0 (Neighbor-Joining, Kimura 2-parameter model) with 1,000 bootstrap replicates to assess robustness.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Isolation of representative EV-positive samples:\u003c/h2\u003e\u003cp\u003eHuman Rhabdomyosarcoma (RD) cells were cultured in MEM supplemented with 10% FBS, L-glutamine, and antibiotics at 37\u0026deg;C with 5% CO₂. Clinical specimens were inoculated onto RD monolayers in 24-well plates and monitored for 4\u0026ndash;5 days for cytopathic effect (CPE). Wells showing complete CPE were harvested, freeze\u0026ndash;thawed thrice, centrifuged, and the clarified supernatant stored at \u0026minus;\u0026thinsp;70\u0026deg;C. The supernatant was used for up to three passages to enhance virus recovery.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Sequence Assembly and Annotation of EVs:\u003c/h2\u003e\u003cp\u003eTwenty-two Indian enterovirus genomes were assembled using the CLC Genomics Workbench [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://digitalinsights.qiagen.com/technical-support/system-requirements/\u003c/span\u003e\u003cspan address=\"https://digitalinsights.qiagen.com/technical-support/system-requirements/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. The finalized and annotated sequences were submitted to GenBank via the BANKIT submission tool.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Prevalence of EV infection in AGE patients\u003c/h2\u003e\u003cp\u003eIn this study, a total of 450 samples were tested, with 51 samples identified as positive for EV through real-time RT-PCR amplification of the 5\u0026rsquo; UTR region, resulting in an overall positivity rate of 11.3%. The prevalence of EV infection fluctuated annually, ranging from 7.8% to 31.3%, while no positivity was observed during the COVID-19 pandemic due to a lack of available samples, as shown in Fig.\u0026nbsp;1B. The highest detection rate was recorded in 2017 (31.3%), and the lowest in 2020 (0%), likely due to the reduced number of samples collected during the pandemic. Overall, the positivity rate for females across all years was 45% (23/51), compared to 54.9% for males (28/51).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Prevalence of EV infection in different age groups\u003c/h2\u003e\u003cp\u003eIn terms of age, the highest prevalence of EV detection was observed in the 0\u0026ndash;12 months age group (41.2%, 21/51), followed by the 13\u0026ndash;24 months group (27.4%, 14/51), and the lowest in the 49\u0026ndash;60 months group (5.8%, 3/51) (Fig.\u0026nbsp;1A).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Seasonal distribution of EV infection\u003c/h2\u003e\u003cp\u003eThe seasonal distribution from 2017 to 2023, depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, showed that enterovirus cases occurred year-round, with a peak in the monsoon months. Incidence increased from 7.7% in March to 15.3% in August, reaching a maximum of 23% in July. The majority of EV-positive cases (27 out of 51; 52.94.%) were recorded from April to October, with the peak occurring in July (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This seasonal trend aligns with other studies that reported the highest prevalence of EV infections in monsoon with increase in the humidity accompanied by heavy/moderate rainfall \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Prevalence of EV infection and co-infection in AGE patients\u003c/h2\u003e\u003cp\u003eAmong the positive samples, specifically, mono-infection with EV accounted for 58.8% (30/51) of the cases, while co-infections with other enteric viruses, including rotavirus, norovirus, adenovirus, human astrovirus, and human parechovirus, were observed in 41.2% (21/51) of the cases. Among the co-infections, double infections occurred in 39.2% (20/51) of cases, and triple infections were seen in one case (1.96%). In terms of clinical characterization, most patients experienced diarrhoea along with other symptoms such as abdominal pain, nausea, vomiting, or fever. No severe cases were reported.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Distribution of EV species and genotyping\u003c/h2\u003e\u003cp\u003eMolecular characterization of EV strains from 30 positive samples was performed by sequencing the partial VP1 or VP2 gene. Of the 51 positive samples identified in this study, the VP1 gene was successfully amplified in 24 samples, while 6 samples that did not amplify for VP1 were characterized using the VP2 gene. Of the 24 samples characterized by VP1, 33.3% (8/24) were identified as EV-A, 50% (12/24) as EV-B, and 16.7% (4/24) as EV-C. The 6 samples characterized by VP2 amplification all belonged to the EV-B species. This resulted in the following detection pattern: EV-B (60%, 18/30)\u0026thinsp;\u0026gt;\u0026thinsp;EV-A (26.6%, 8/30)\u0026thinsp;\u0026gt;\u0026thinsp;EV-C (13.3%, 4/30). In total, 16 different genotypes of EV were identified, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A subset of EV-positive samples (41.2%, 21/51) could not be characterized due to failure to produce amplicons or issues with genotyping, and in two cases, genotyping was not performed due to the limited sample quantity. The distribution of EV species and genotypes identified in this study is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferent types of EV associated with AGE in children during 2017\u0026ndash;2023\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSerotypes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo of Characterized Samples (% of total)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eEnterovirus-A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVA-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVA-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVA-10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVA-16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVB-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEV-71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSubtotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e8 (26.6)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003eEnterovirus-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVA-9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (10)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVB-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVB-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (13.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (3.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSubtotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e18 (60)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnterovirus-C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCVA-13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (6.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e28 (93.3)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Phylogenetic Analysis and characterization of EV strains\u003c/h2\u003e\u003cp\u003eTo explore the genetic relationships between the 28 EV strains identified in this study and previously reported EV reference strains, phylogenetic trees for each EV species (A, B and C) were constructed, as illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The results showed that all EV strains from this study clustered closely with their respective reference strains based on genotype. Each of the 22 EV genotypes formed distinct branches alongside their corresponding reference strains, validating the genotyping results obtained through BLAST and the Enterovirus Genotyping Tool. Nearly all EV strains exhibited over 90% nucleotide sequence identity with their corresponding reference genotypes, except for CV-A10, CV-A5, CV-A16, and Echovirus 25, which showed lower sequence identities (75\u0026ndash;85%) compared to their reference sequences. Sequences from 22 enterovirus genotypes submitted in NCBI rest 6 sequences yielded partial sequences with several ambiguous regions, which did not fulfill the criteria for NCBI submission. However, the reliable portions were analyzed and utilized for phylogenetic and identification purposes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequence data submitted to NCBI\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCollection Date\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAccession No.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1727766\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A-10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14-06-2017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611641.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1728229\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEchovirus \u0026minus;\u0026thinsp;25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28-06-2017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV637434.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1728841\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11-07-2017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611636.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1730321\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEchovirus \u0026minus;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31-07-2017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV637430.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1734322\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27-09-2017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611648.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1850025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e02-05-2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611649.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1850027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEnterovirus \u0026minus;\u0026thinsp;71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16-05-2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV584128.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1850082\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEchovirus \u0026minus;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13-07-2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV637431.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1850137\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20-08-2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611645.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1850156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus B \u0026minus;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e04-09-2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611646.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e198147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus B \u0026minus;\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14-08-2019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611647.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e198141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e08-08-2019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611643.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e211435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e07-06-2021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611637.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e220647\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackie Virus A \u0026minus;\u0026thinsp;13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26-02-2022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611644.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e220777\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEchovirus \u0026minus;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e04-03-2022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV637432.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e220875\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus B \u0026minus;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24-03-2022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611634.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e220881\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24-03-2022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611638.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e240130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17-05-2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611639.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2315608\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEchovirus \u0026minus;\u0026thinsp;11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12-08-2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV637433.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2318987\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus B \u0026minus;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17-10-2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611635.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2319307\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22-10-2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611642.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2411207\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCoxsackievirus A \u0026minus;\u0026thinsp;9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12-02-2024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePV611640.1\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\u003ePhylogenetic tree based on partial VP1 sequences (~\u0026thinsp;300 bp) from Indian isolates associated with AGE cases and reference sequences retrieved from GenBank; (a) EV-A, (b) EV-B, and (c) EV-C species. Trees were generated using the neighbor-joining method with the Kimura 2-parameter model implemented in MEGA v.11 Branch support was assessed through 1000 bootstrap replicates, with bootstrap values above 75% shown at the corresponding nodes. Filled circles indicate the enteroviruses identified in the present study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Isolation of representative EV-positive samples:\u003c/h2\u003e\u003cp\u003eAmong the characterized enteroviruses, six distinct virus isolates were selected for attempted isolation. The selection criteria were based on cycle threshold (Ct) values, indicative of high viral load, and the specific EV species (CVA-10, CVB-4, E-7. E-11, E-14 and CVA-16) identified during molecular characterization. Each selected sample corresponded to a unique EV species.\u003c/p\u003e\u003cp\u003eThe six EV strains were propagated in monolayer cultures of the RD cell line, with the first three passages conducted in 24-well plates and the fourth passage performed in T-25 flasks. All strains induced a cytopathic effect (CPE) within 2\u0026ndash;4 days post-inoculation. The CPE was consistently observed across all passages, confirming successful viral replication in the RD cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Molecular characterization of isolates:\u003c/h2\u003e\u003cp\u003eRNA extraction was performed for all viral isolates across successive passages, followed by confirmation of viral presence using real-time reverse transcription polymerase chain reaction (RT-PCR). The assay targeted the 5\u0026prime; non-coding region (NCR) of Enteroviruses (EV) using specific primers and probes. Viral RNA in the culture supernatant was detected, and the viral load at each passage (Table-5) was quantified by extrapolating copy numbers from a standard curve generated using known positive controls. Ct values and corresponding viral genome copy numbers were determined by quantitative RT-PCR at each passage (P1\u0026ndash;P4). A progressive decrease in Ct values with increasing passage reflects enhanced viral replication and higher viral load. Data are shown for Coxsackievirus A10 (CVA-10), Coxsackievirus B4 (CVB-4), Echovirus 7 (E-7), Echovirus 11 (E-11), Echovirus 14 (E-14), and Coxsackievirus A16 (CVA-16) (Table-5). All viral isolates showing cytopathic effects were confirmed positive for the NCR via real-time RT-PCR and were subsequently subjected to molecular typing and characterization using VP1 gene-specific primers and further confirmed the presence of the target viral sequences.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eReplication Kinetics of Enteroviruses Across Serial Passages in RD cell line\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVirus Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003ePassage \u0026minus;\u0026thinsp;01\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003ePassage \u0026minus;\u0026thinsp;02\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003ePassage \u0026minus;\u0026thinsp;03\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003ePassage \u0026minus;\u0026thinsp;04\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCt\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCopy\u003c/p\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCt\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCopy\u003c/p\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCt\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCopy\u003c/p\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCt\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eCopy\u003c/p\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCVA-10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.40x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.75 x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.49 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.89 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCVB-4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.52 x10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.48 x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.33 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.06 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE-7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.63 x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.26 x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.18 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.36 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE-11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.11 x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.68 x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.90 x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.51 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE-14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.66 x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.46 x10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.03 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.54 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCVA 16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.65 x10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.40 x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.77 x10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.01 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAGE is an intestinal disease that remains a major cause of high morbidity and considerable mortality rates worldwide, particularly in developing countries where the risk of infection is elevated and hygiene, sanitary conditions, and nutritional status of children are poor. Gastrointestinal infections pose a significant challenge to health services globally due to their high morbidity and mortality rates. Testing patients only for the most common pathogens results in diagnoses for only half of these cases. In our study, we have tested the samples for EV, AstV, AdV, NoV, HPeV and RVA and the prevalence of EV is relatively high as compared to RVA, this could be attributed to the higher use of vaccines against RVA in regular immunization schedule of infants in Pune.\u003c/p\u003e\u003cp\u003eEVs are frequently implicated in AGE, especially in pediatric populations. Although these viruses are predominantly linked to respiratory infections, hand-foot-and-mouth disease, and neurological disorders, they also manifest with gastrointestinal symptoms. Observations have indicated a higher incidence of enteroviral infections in cases of acute diarrhoea compared to non-diarrheal illnesses from India \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study describes the epidemiological, seasonal, and molecular characteristics of EVs detected in children suffering from AGE from western India, Pune from 2017 to 2023. The prevalence of EV in our study was 11.3% ranging between 0% in year 2020 to 31.3% in year 2017, there is no such pattern of apparent increase and decrease in year-wise positivity, but the null positivity in year 2020 could be explained by limited sample collection due to COVID-19 pandemic. These kinds of prevalence percentages have been reported from different countries in similar other studies \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe incidence of EVs observed in this study aligns with findings from recent research conducted across diverse geographical regions, reinforcing the concept that enteroviruses can be significant etiological agents in gastrointestinal disorders. This is further substantiated by the notably high positivity rate (58.8%, 30/51) of EVs detected in stool samples from patients with gastroenteritis who tested negative or coinfected with other enteric viruses. Historical data from outbreaks of acute gastroenteritis in Western India have similarly reported a higher prevalence of enteroviral infections relative to other enteric pathogens \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInitially, the typing of EV strains mainly relies on virus isolation followed by serotyping based on neutralization with pooled antisera, but now various molecular typing and sequencing-based methods are available for enterovirus typing. Several different RT-PCR methods targeting different regions of the EV genome, particularly those encoding the structural period of the virus have been used \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this present study, the EV-positive samples have been typed by VP1/VP2 gene amplification. Eighteen distinct genotypes of EV have been identified in this study, with EV-B showing the highest relative prevalence at 60%, followed by EV-A at 27%, and EV-C at 13.3%. These results are consistent with previous studies, which also demonstrated a higher frequency of EV-B species compared to EV-A and EV-C in paediatric cases of AGE\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Few studies have suggested that Echoviruses and coxsackie viruses have a role in causing diarrhoea and AGE in children \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In our study, E-11 and CVA-9 were the most predominant detected genotypes, Outbreaks of diarrhoea caused by E-11 have been reported from Malaysia \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and southern India \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our study, approximately 41.2% of the samples were co-infected with additional diarrhoea-associated viruses, suggesting that the observed symptoms or disease progression in these cases might be attributable to these co-infecting viruses rather than enterovirus alone. Furthermore, children whose samples tested negative for enteroviruses or other known viruses causing AGE continued to exhibit symptoms. Additionally, since bacterial and parasitic infections were not assessed in this study, the aetiology of these samples should also be taken into account.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides evidence for the presence and circulation of various EV serotypes among patients with AGE. The ongoing circulation of predominant enterovirus types within the population highlights the potential risk of future outbreaks of gastroenteritis. The isolation and characterization of these predominant strains are crucial for the development of serological diagnostic tools and for conducting seroepidemiological studies. The current low success rate in the etiological diagnosis of AGE underscores the necessity of identifying new pathogens. While viruses from the Caliciviridae family, such as Sapovirus and type II Norovirus, are now regarded as secondary potential agents in AGE, our findings suggest that enteroviruses should be given serious consideration due to their high incidence and effective replication, particularly in children under five years of age. Future research should focus on defining the role of enteroviruses in AGE by evaluating viral load markers and assessing the impact of various enterovirus genotypes and polio vaccine strains. This research is vital for improving clinical management, patient care, and public health strategies, given that viral gastroenteritis remains a significant global health issue, particularly in regions with challenges related to hygiene and sanitation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the ICMR (Diarr/Adhoc/3/2022 ECD-II) and ICMR-National Institute of Virology, Pune, Maharashtra, India (No. EVE 2201).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Ethics Committee of the ICMR-National Institute of Virology, Pune (Approval No. MP-24A-7N dated 11.03.2024), in accordance with established ethical guidelines for biomedical research involving human samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull genome sequence of this case viral variants has been deposited in the GenBank NCBI (Accession no: Table 4)\u0026nbsp;using the BANKIT submission tool.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eML: Conceptualization, methodology, project administration, resources, supervision,\u0026nbsp;writing, review and editing.\u0026nbsp;SP and RW: Contributed to the experimentation, data curation, formal analysis, writing original draft, investigation and methodology.\u0026nbsp;SP and PU:\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003eContributed to the experimentations. SC, SK and JK: Supported in the analysis of data and phylogenetic analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuarino A, Aguilar J, Berkley J, Broekaert I, Vazquez-Frias R, Holtz L, Lo Vecchio A, Meskini T, Moore S, Rivera Medina JF, Sandhu B, Smarrazzo A, Szajewska H, Treepongkaruna S. Acute Gastroenteritis in Children of the World: What Needs to Be Done? J Pediatr Gastroenterol Nutr. 2020 May;70(5):694-701.\u003c/li\u003e\n\u003cli\u003eB\u0026aacute;nyai K, Estes MK, Martella V, Parashar UD. Viral gastroenteritis. \u003cem\u003eThe Lancet\u003c/em\u003e 2018; \u003cem\u003e392\u003c/em\u003e : 175\u0026ndash;86. \u003c/li\u003e\n\u003cli\u003eRao DC, Ananda Babu M, Raghavendra A, Dhananjaya D, Kumar S, Maiya PP. 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Rev Med Virol. 2021 Jan;31(1):1-12.\u003c/li\u003e\n\u003cli\u003ePham N, Thongprachum A, Baba T, Okitsu S, Trinh Q, Komine-Aizawa S, \u003cem\u003eet al.\u003c/em\u003e A 3-Month-Old Child with Acute Gastroenteritis with Enterovirus D68 Detected from Stool Specimen. \u003cem\u003eClin Lab\u003c/em\u003e 2017; \u003cem\u003e63\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003ePham NTK, Thongprachum A, Trinh QD, Okitsu S, Komine-Aizawa S, Shimizu H, \u003cem\u003eet al.\u003c/em\u003e Detection and genetic characterization of enterovirus strains circulating among children with acute gastroenteritis in Japan during 2014\u0026ndash;2016. \u003cem\u003eInfection, Genetics and Evolution\u003c/em\u003e 2018; \u003cem\u003e61\u003c/em\u003e : 16\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003ePerera D, Shimizu H, Yoshida H, Van Tu P, Ishiko H, McMinn PC, \u003cem\u003eet al.\u003c/em\u003e A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses. \u003cem\u003eJ Med Virol\u003c/em\u003e 2010; \u003cem\u003e82\u003c/em\u003e : 649\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eOberste MS, Gerber SI. Enteroviruses and Parechoviruses: Echoviruses, Coxsackieviruses, and Others. \u003cem\u003eViral Infections of Humans\u003c/em\u003e. Boston, MA: Springer US; 2014 p. 225\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eChansaenroj J, Tuanthap S, Thanusuwannasak T, Duang-in A, Klinfueng S, Thaneskongtong N, \u003cem\u003eet al.\u003c/em\u003e Human enteroviruses associated with and without diarrhea in Thailand between 2010 and 2016. \u003cem\u003ePLoS One\u003c/em\u003e 2017; \u003cem\u003e12\u003c/em\u003e : e0182078.\u003c/li\u003e\n\u003cli\u003eSilva PA, Stark K, Mockenhaupt FP, Reither K, Weitzel T, Ignatius R, \u003cem\u003eet al.\u003c/em\u003e Molecular characterization of enteric viral agents from children in northern region of Ghana. \u003cem\u003eJ Med Virol\u003c/em\u003e 2008; \u003cem\u003e80\u003c/em\u003e : 1790\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eBina Rai S, Wan Mansor H, Vasantha T, Norizah I, Chua KB. An outbreak of echovirus 11 amongst neonates in a confinement home in Penang, Malaysia. \u003cem\u003eMed J Malaysia\u003c/em\u003e 2007; \u003cem\u003e62\u003c/em\u003e : 223\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003ePatel JR, Daniel J, Mathan VI. An epidemic of acute diarrhoea in rural southern India associated with echovirus type 11 infection. \u003cem\u003eJournal of Hygiene\u003c/em\u003e 1985; \u003cem\u003e95\u003c/em\u003e : 483\u0026ndash;92.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Acute severe gastroenteritis, Children, Pathogens, Rotavirus, Norovirus, Adenovirus, Astrovirus, Enterovirus, Genotypes, Prevalence","lastPublishedDoi":"10.21203/rs.3.rs-8115337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8115337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Acute gastroenteritis (AGE) in children is primarily associated with pathogens such as Rotavirus, Norovirus, Adenovirus, Astrovirus, and increasingly, human enteroviruses (EVs). EVs are non-enveloped RNA viruses belonging to the \u003cem\u003ePicornaviridae\u003c/em\u003efamily, comprising over 100 subtypes classified into four species. Despite their clinical relevance, the prevalence of EVs in AGE remains underexplored in India, resulting in limited data on their epidemiology and molecular diversity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A total of 450 stool samples were collected from hospitalized children under five years of age with AGE in Pune, India, between 2017 and 2023. Molecular characterization was subsequently performed to identify and confirm the types of enteroviruses present.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e EVs were detected in 11.3% (51/450) of cases; 58.8% (30/51) among them were mono-infections. Rotavirus emerged as the most frequently identified pathogen, while co-infections involving Norovirus, Adenovirus, Astrovirus, and Parechovirus were documented. Eighteen distinct EV genotypes were characterized, with EV-B being the most dominant (64%). EVs were found circulating throughout the year, with a notable peak in the monsoon (June-August), and were predominantly detected in children below two years of age.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Association of EVs in more than 10% of AGE cases highlights the importance of incorporating EV screening in AGE cases and understanding the burden caused by this important group of viruses.\u003c/p\u003e","manuscriptTitle":"Beyond Rotavirus: Seasonal Dynamics and Genetic Diversity of Non-Polio Enteroviruses in Hospitalized Children with Acute Gastroenteritis in Pune, India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 16:51:19","doi":"10.21203/rs.3.rs-8115337/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":"458368d4-9f14-44df-a47b-8fe2bad48010","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-31T05:54:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 16:51:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8115337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8115337","identity":"rs-8115337","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}