Biological characteristics of an Enterovirus 71 subgroup C4 strain isolated in China

Biological characteristics of an Enterovirus 71 subgroup C4 strain isolated in China | 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 Biological characteristics of an Enterovirus 71 subgroup C4 strain isolated in China Tianli Ma, Huan Li, Yunfang Li, Weishi Lin, Zhengying Yu, Lizhong Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7479059/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted 11 You are reading this latest preprint version Abstract Background Hand, foot, and mouth disease (HFMD) is a widespread infectious disease primarily affecting infants and young children. Enterovirus 71 (EV71) comprises seven genogroups, among which subgroup C4 is the dominant viral agent in China and is frequently associated with HFMD and central nervous system infections. We analyzed the genetic characteristics of an EV71 subgroup C4 strain through whole-genome sequencing and investigated its biological characteristics, including infectivity, replication, and cytotoxicity, in human rhabdomyosarcoma (RD) and African green monkey kidney (Vero) cells. Methods We examined the genetic and biological features of a clinical EV71 GD10 strain isolated in China. Its relationships with strains listed in GenBank were evaluated using phylogenetic analysis. Viral infectivity and replication were assessed in RD and Vero cells. Cytotoxicity was evaluated by measuring cell viability, lactate dehydrogenase (LDH) release, and ATP levels. Effects on blood–brain barrier (BBB) integrity were investigated in vitro by assessing transendothelial electrical resistance and viral load across the barrier. Results Sequence analysis confirmed that GD10 belonged to subgroup C4 and closely resembled strains from China. GD10 infection induced a pronounced cytopathic effect and elevated viral RNA levels in RD cells but not in Vero cells. The infection time-dependently increased LDH release and reduced ATP levels. GD10 compromised BBB integrity and crossed the cellular barrier in vitro . Conclusion The GD10 strain demonstrated strong adaptability to RD cells and impaired BBB function. Our results improve the understanding of virus–host interactions and may support efforts towards EV71 vaccine development. subgroup C4 viral kinetics cytotoxicity blood–brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease (HFMD), predominantly infects infants and young children. Patients with HFMD often develop rashes on their palms and soles, oral ulcers, and fever [ 1 ]. Although HFMD is self-limiting, EV71 infection occasionally causes severe neurological complications [ 2 ]. EV71 was first isolated in 1969 from a child diagnosed with encephalitis in the USA and has since been sporadically reported in America and Europe [ 3 – 10 ]. In 1997, a large HFMD outbreak occurred in Malaysia, causing 34 fatalities [ 11 ]. The following year, another extensive outbreak occurred in Taiwan, with over 100,000 reported HFMD cases [ 12 ]. Since its emergence, EV71 has consistently caused epidemics across the Asia-Pacific region [ 13 – 19 ], causing two outbreaks in Shandong and Anhui provinces between 2007 and 2008 and subsequently becoming endemic in mainland China [ 20 , 21 ]. EV71 currently comprises seven groups based on the nucleotide sequences of VP1 [ 22 ]. The BrCr strain, the sole member of Group A, was initially identified in the USA in 1969 and re-emerged in China in 2008 [ 3 , 23 ]. Groups B and C are further divided into five subgroups. Subgroups B1 and B2 primarily circulated in the Netherlands, Hungary, Bulgaria, and the USA between 1960 and 1989; subgroups B3–B5 circulated in the Asia-Pacific region beginning in 1990 [ 24 – 26 ]. Group C rapidly spread globally, causing numerous outbreaks, particularly across Asia-Pacific countries [ 11 , 12 , 27 ]. Subgroup C4 emerged as the predominant EV71 strain in China from 2007 onwards and continues circulating to date [ 28 – 30 ]. Additional genotypes were recently identified; Groups D and G circulated solely in India from 2008, without documented outbreaks [ 31 , 32 ]. Groups E and F were identified in Africa in 2003 and Madagascar in 2004, respectively [ 33 , 34 ]. Since HFMD outbreaks were reported in Shandong and Anhui between 2007 and 2008, subgroup C4 of EV71 has become the predominant agent circulating in China [ 20 , 21 ]. Determining the genetic and biological characteristics of the prevalent EV71 strain can improve the understanding of its molecular epidemiological characteristics and facilitate the development of effective vaccines. However, few studies have examined the full-genome characteristics of subgroup C4 strains along with their cytopathogenic effects and ability to cross the blood–brain barrier (BBB) in vitro . In this study, we characterized the genetic properties of an EV71 subgroup C4 strain and evaluated its biological characteristics, including infectivity, viral replication, and cytotoxicity in human rhabdomyosarcoma (RD) and African green monkey kidney (Vero) cells. Furthermore, we assessed whether the isolate could infect and cross the BBB using an established in vitro BBB model. Materials and methods Cell lines and viruses RD and Vero cells were maintained in Dulbecco's modified Eagle’s medium (DMEMGibco, no C11995500BT) containing 10% fetal bovine serum (FBS). The human brain microvessel endothelial cell line (hCMEC/D3) was purchased from Cellverse (iCell-h070; Shanghai, China) and cultured in DMEM containing 10% FBS and 40 µg/mL endothelial cell growth supplement (E2759; Sigma-Aldrich, St. Louis, MO, USA). The EV71 prototype strain BrCr was preserved in our laboratory. The EV71 clinical strain GD10 (GenBank accession number: KJ004559.1) was kindly provided by the Chinese Center for Disease Control and Prevention. The viral titer of these stocks was measured as the 50% tissue culture infective dose (TCID 50 ) per milliliter (mL) according to the Reed and Muench method, as previously described [ 35 ]. Viral genome sequencing and phylogenetic analysis Total viral RNAs were extracted from GD10 strain-infected cell culture supernatants using an RNA extraction kit, following the manufacturer’s instructions. All mRNAs were subjected to RNA sequencing using an Illumina MiSeq v2 instrument (San Diego, CA, USA) with 300-bp paired-end reads. After removing host-derived reads, virus-specific reads were included in genome alignment and assembly. All sequence reads were mapped to the selected reference EV71 virus using CLC Bio’s clc_ref_assemble_long program. We obtained the genome of the isolated EV71 (GD10 strain). Comparative sequence analyses, including sequence alignments and estimation of genetic distances, were performed using MEGA software (version 11.0; ClustalW, Molecular Evolutionary Genetic Analysis software). Phylogenetic trees were constructed using the neighbor-joining method with Kimura in MEGA, and branch support was calculated based on 1,000 bootstrap replicates. Viral infection Vero and RD cells were seeded into 24-well plates and incubated overnight. Cells at 80% confluence were infected with the BrCr or GD10 strain at a multiplicity of infection (MOI) of 0.01 for 1 h. Following inoculation, the viral suspension was removed, and the cells were washed with phosphate-buffered saline (PBS) before adding fresh culture medium. Cytopathic effect (CPE) was observed, and cell morphology was recorded using a Carl Zeiss Axio Vert.A1 microscope (Oberkochen, Germany). Infected cells were harvested at various time points for RNA extraction for reverse transcription-quantitative PCR (RT-qPCR) and protein extraction for western blotting. RT-qPCR analysis Infected cells were trypsinized, and total RNA was extracted using an RNA Easy Fast Tissue/Cell Kit (DP451; Tiangen, Shanghai, China). RNA was reverse-transcribed into cDNA using a FastKing RT Kit (KR116; Tiangen, China). RT-qPCR was performed with EV71 VP1-specific primers (Table S1 ) using a SuperReal PreMix Plus (SYBR Green) Kit (FP205; Tiangen, China) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). EV71 expression was normalized to that of GAPDH and presented as the fold-change (EV71/GAPDH). Cell cytotoxicity and viability assay Vero and RD cells were seeded into 96-well plates and infected with 0.01 MOI of the BrCr or GD10 strain. Cell proliferation was assessed using a CCK-8 assay kit (40203ES60; Yeasen, Shanghai, China). At the indicated time points post-infection, CCK-8 reagent was added and incubated in the dark. Absorbance was measured at 450 nm using a microplate reader (SYNERGY HTX; BioTek, Winooski, VT, USA). Cell viability was assessed with the CellTiter-Glo Luminescent Cell Viability Assay (G7570; Promega, Madison, WI, USA) based on intracellular ATP quantification. Cell death was evaluated using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (G1780; Promega) by measuring lactate dehydrogenase (LDH) release, following the manufacturer’s instructions. Western blotting Cells were lysed in cold PIRA buffer (P0013B; Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (P1045; Beyotime, China) on ice, followed by centrifugation at 4°C. The protein concentration was quantified using a Pierce BCA Protein Assay Kit (23225; Thermo Fisher Scientific, Waltham, MA, USA). Proteins were resolved by 10% -polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (0.22 µm, Millipore, Billerica, MA, USA). The membranes were blocked in blocking buffer (PBS containing 5% milk and 0.05% Tween-20) at room temperature, washed with PBST (PBS containing 0.05% Tween-20), and incubated with primary antibodies at 4°C overnight. After PBST, the membranes were incubated with secondary antibodies. The blots were visualized using a SuperSignal West Femto Trial Kit (34094; Thermo Fisher Scientific) and imaged with a ChemiDoc MP Imaging System (Bio-Rad). A polyclonal antibody against EV71 was generated in-house from rabbits immunized with VP1 protein. Rabbit anti-GAPDH (5174S) was purchased from Cell Signaling Technology (Danvers, MA, USA). In vitro BBB model and transendothelial electrical resistance (TEER) measurement hCMEC/D3 cells were seeded on the apical side of transwell inserts (3462; Corning, Inc., Corning, NY, USA) with hCMEC/D3 complete medium in the upper and lower chambers. Cells were incubated at 37°C with 5% CO 2 until confluence and fresh media was replaced daily. The apical chamber was exposed to EV71 strains at MOI 6.6 for 1 h, then replaced with fresh medium. TEER was measured using the Millicell®-ER system (#MERS00002; Merck Millipore, Kenilworth, NJ, USA), as described previously [ 36 ]. Briefly, the electrode was disinfected with 70% ethanol and equilibrated in pre-warmed medium. The longer arm of the electrode was placed in the lower chamber, and the shorter in the upper chamber. Triplicate readings were recorded for each insert. TEER values were calculated by multiplying the average resistance (Ω) by the membrane area (1.12 cm 2 ). The background TEER value was used as a mock control. Statistical analysis All experiments were performed with at least three replicates, and data are presented as the means ± standard deviation (SD). GraphPad Prism software (version 8.0.3, GraphPad, Software Inc., San Diego, CA, USA) was used for statistical analysis. A p- value < 0.05 was considered statistically significant. The statistical methods are noted in the figure legends. Results Viral genome and phylogenetic tree The GD10 strain sequence consisted of 7415 bp and contained a single open reading frame (ORF) encoding a 2193-amino acid polypeptide, deposited in GenBank under accession number KJ004559.1. Phylogenetic analyses were performed to assess genetic relationships between this strain and 22 reference strains isolated worldwide and deposited in GenBank. A phylogenetic tree was constructed based on full-genome nucleotide sequences. Phylogenetic analysis assigned the GD10 strain to EV71 subgroup C4. Various EV71 groups have emerged in China, including genogroups A, B, and C (Fig. 1 ). Groups A and B exhibited limited circulation, whereas Group C showed extensive dissemination. The GD10 strain shared 88% nucleotide identity with other EV71 strains from Beijing, indicating close genetic relatedness and high homology. These findings confirm that EV71 subgroup C4 remains the predominant subgroup circulating in mainland China. Infection and replication of the GD10 strain We initially evaluated CPE in Vero and RD cells to assess the infectivity of the GD10 clinical isolate. Both cell types were infected with the GD10 strain at an MOI of 0.01; the BrCr strain served as a positive control. The BrCr strain induced evident CPE in both cell lines at 36–48 h post-infection (hpi) (Fig. 2 ). However, GD10 infection induced CPE in RD cells beginning at 36 hpi, whereas no evident cell death occurred in Vero cells. To evaluate replication kinetics, viral RNA expression was measured over time. The BrCr strain replicated robustly in both cell lines (Figs. 3 A–B). In RD cells, GD10 replicated significantly but with lower viral RNA levels than those in BrCr (Fig. 3 B). However, GD10 RNA was undetectable in Vero cells (Fig. 3 A). These results suggest that RD cells more effectively support GD10 replication. We also detected VP1 expression at the protein level using western blotting. Consistent with the RT-qPCR data, VP1 protein expression was increased in both cell types after BrCr infection but was absent following GD10 infection (Figs. 3 C–F). These results suggest that antigenic variation within the VP1 protein prevented binding by the polyclonal antibody. Collectively, these data show that GD10 infection induced CPE and support efficient replication in RD cells but not in Vero cells. Cell viability and cytotoxicity following GD10 infection Given the observed CPE and viral replication (Figs. 2 and 3 ), we next assessed the cell death response to GD10 in Vero and RD cells. CCK-8 assay revealed a marked decrease in Vero cell viability following BrCr infection, whereas GD10 did not induce such a decrease (Fig. 4 A). In RD cells, both BrCr and GD10 caused significant reductions in viability, consistent with the observed CPE (Fig. 2 A). Cytotoxicity was further assessed by measuring LDH release into the supernatant and ATP levels. In Vero cells, BrCr infection led to increased LDH release starting at 12 hpi, with progressive elevation through 48 hpi (Fig. 4 C). ATP levels also declined significantly (Fig. 4 E). In contrast, GD10 infection did not affect LDH or ATP levels in Vero cells. In RD cells, both BrCr and GD10 increased LDH release and decreased ATP levels. These results indicate that GD10 did not induce detectable cytotoxicity in Vero cells. Ability of the GD10 strain to cross the BBB To assess the effects of GD10 on the integrity of the BBB, we infected hCMEC/D3 cells cultured in transwell inserts to establish an in vitro BBB model. Both BrCr and GD10 infection significantly reduced TEER relative to that in the mock control from the initial infection stage (Fig. 5 A). TEER values progressively declined, reaching a minimum at 12 hpi. Although BrCr-induced TEER reduction was more pronounced than that induced by GD10, the difference was not significant. Concordantly, inactivated EV71 did not affect TEER, indicating that only active infection compromised BBB function. Viral RNA in the bottom compartment was measured to evaluate BBB penetration (Fig. 5 B). At 12 hpi, the BrCr viral titer were significantly higher than that of GD10; however, both strains showed comparable viral titers by 24 hpi (Fig. 5 B). These results suggest that GD10 compromised BBB integrity and crossed the barrier early during infection in vitro . Discussion In this study, we described the genetic diversity and biological characteristics of the clinical EV71 subgroup C4 strain GD10. Compared with the BrCr strain, GD10 infection induced severe CPE in RD cells but not in Vero cells. Additionally, RD cells supported GD10 replication, whereas Vero cells did not. EV71 reportedly infects various cell lines, including human neuroblastoma (SK-N-SH), colorectal (HT29), neural (SF268), and microvascular endothelial (HMEC-1) cells [ 37 – 41 ]. A previous study showed that five C4 strains from different Chinese regions induce CPE and replicate in Vero cells [ 42 ]. However, our findings indicate that Vero cells do not efficiently support GD10 infection. Although BrCr and GD10 exhibited similar replication patterns in RD cells, BrCr viral RNA levels were significantly higher, suggesting a more rapid replication rate. We did not detect VP1 protein expression in RD cells following GD10 infection, possibly because the anti-EV71 antibody could not bind to the GD10 strain owing to the different antigenic epitopes residing in the BrCr strain. We further assessed cell viability and cytotoxicity, demonstrating that GD10 induced cell damage comparable with that caused by BrCr, with increased LDH release and decreased ATP production. These data indicate that GD10 infection induces lytic cell death. Collectively, GD10 exhibits distinct host-cell susceptibility relative to previously reported subgroup strains. The prototype BrCr strain (genogroup A) was isolated from a patient with central nervous system (CNS) disease in the USA [ 3 ]. Subgroup C4 was identified as a major public health concern in China in 2007 [ 43 ]. EV71-associated HFMD often causes severe neurological complications in children aged < 5 years, indicating that EV71 can cross the BBB and invade the CNS [ 2 ]. Three subgroup C4 EV71 strains induced pathological lesions in neonatal mice and rhesus monkeys [ 44 ]. Previously, we showed that GD10 infection caused severe clinical symptoms, including substantial weight loss and hindlimb paralysis, in both BALB/c and C57 mice, confirming its neurovirulence [ 45 ]. Using an in vitro model, we demonstrated that GD10 could cross the BBB, as indicated by reduced TEER values. Furthermore, BrCr infection produced greater barrier disruption than GD10, suggesting that BrCr possesses higher neurovirulence. This study had some limitations. First, we did not compare GD10 with other Chinese subgroup C4 isolates. Second, few cell types were employed to characterize viral biology. Nevertheless, we thoroughly evaluated GD10 infectivity and replication at the cellular level, along with BBB disruption in vitro , facilitating future studies on virus–host interactions and CNS pathogenesis. In conclusion, we analyzed the genetic and biological properties of a clinically isolated EV71 subgroup C4 strain. GD10 exhibited adaptation for infection, replication, and induction of cell death in RD cells, and demonstrated BBB penetration in vitro . Our findings improve the understanding of EV71 pathogenesis and may inform the development of multivalent EV71 vaccines. Abbreviations HFMD: Hand, foot, and mouth disease; EV71: Enterovirus 71; LDH: lactate dehydrogenase; BBB: blood–brain barrier; CPE: cytopathic effect; MOI: multiplicity of infection; TCID 50 : tissue culture infective dose 50; TEER: transendothelial electrical resistance. Declarations Ethics approval and consent to participate Not applicable Clinical Trial Not applicable Consent for publication Not applicable. Availability of data and materials The data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by the National Key Research & Development Program of China [grant number 2022YFC2603800]. Author’s contributions JX, HS, and LJ conceived and designed the study. TM, HL, and YL performed the experiments. TM, WL, ZY, LL and WZ analyzed the data. JX wrote the manuscript. All authors have read and approved the final manuscript. Acknowledgments Not applicable References Tee HK, Zainol MI, Sam I-C, Chan YF. Recent advances in the understanding of enterovirus A71 infection: a focus on neuropathogenesis. Expert Rev Anti Infect Ther. 2021;19:733–47. https://doi.org/10.1080/14787210.2021.1851194 . Ooi MH, Wong SC, Lewthwaite P, Cardosa MJ, Solomon T. Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol. 2010;9:1097–105. https://doi.org/10.1016/S1474-4422(10)70209-X . Schmidt NJ, Lennette EH, Ho HH. An Apparently New Enterovirus Isolated from Patients with Disease of the Central Nervous System. 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Supplementary Files TableS1.docx Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2025 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 30 Sep, 2025 Reviews received at journal 29 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 16 Sep, 2025 Editor invited by journal 03 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Submission checks completed at journal 01 Sep, 2025 First submitted to journal 28 Aug, 2025 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-7479059","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":518245536,"identity":"a4f4f86d-6d74-4715-8b29-0600bd08766d","order_by":0,"name":"Tianli Ma","email":"","orcid":"","institution":"Center for Disease Control and Prevention of PLA","correspondingAuthor":false,"prefix":"","firstName":"Tianli","middleName":"","lastName":"Ma","suffix":""},{"id":518245537,"identity":"202f6339-edf5-418e-9b0e-71cda02a8778","order_by":1,"name":"Huan Li","email":"","orcid":"","institution":"Center for Disease Control and Prevention of 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01:41:39","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121175,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/5cca9509e7a65583862de811.html"},{"id":92127165,"identity":"66591d50-1483-4074-b6dd-d425c956241c","added_by":"auto","created_at":"2025-09-25 01:41:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of enterovirus 71 whole genome sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA phylogenetic tree was constructed using the neighbor-joining method with Kimura in MEGA with 1000 bootstrap replicates. The percentage of trees is shown next to the number of branches. The red dots indicate the GD10 strain used in this study.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/3ff5ef87ec6c930c2b29542e.png"},{"id":92126543,"identity":"d8feb0aa-437f-48a3-a598-3d08c1e8715a","added_by":"auto","created_at":"2025-09-25 01:33:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15558027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGD10 strain induced CPE in RD cells but not in Vero cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVero (A) and RD (B) cells were infected with the BrCr or GD10 strains at an MOI of 0.01. CPE was examined, and images were captured at different time points after infection. Images are shown at 200× magnification.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/6901b996400ae4f346bed0d6.png"},{"id":92126528,"identity":"7c8e950f-5c27-49a9-815e-287439cd54bc","added_by":"auto","created_at":"2025-09-25 01:33:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2870828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral replication and VP1 protein expression of GD10 in Vero and RD cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVero and RD cells were infected with BrCr (blue) or GD10 strains (red) at an MOI of 0.01 and harvested at different time points. Viral RNA in the lysates of Vero (A) and RD (B) cells was examined using RT-qPCR. The expression levels of VP1 protein in cell lysate were examined by western blotting in Vero (C, D) and RD (E, F) cells. All data are mean values ±SD. The experiments were performed in triplicate and repeated at least twice. Statistical significance was analyzed using a two-way ANOVA \u0026nbsp;(*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/d1490b6441094d44a6ab0cb4.png"},{"id":92126532,"identity":"ed22c898-4ebc-4798-95e5-be5b4bacb7cb","added_by":"auto","created_at":"2025-09-25 01:33:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":677765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGD10 strain causes cell damage in RD cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVero and RD cells were infected with BrCr (blue) or GD10 strains (red) at an MOI of 0.01. Cell viability (A, B), LDH-release-based cell death (C, D), and ATP cell viability (E, F) were assessed at different time points after infection. All data are mean values ± SD. The experiments were performed in triplicate and repeated at least twice. Statistical significance was analyzed using a two-way ANOVA (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/afe1e332b81a759e1077809f.png"},{"id":92126526,"identity":"0c9bfbdf-13db-4cf9-b47a-0690619ffc7f","added_by":"auto","created_at":"2025-09-25 01:33:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":274354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGD10 strain could cross the BBB model\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHuman brain microvascular endothelial cells (hCMEC/D3) were cultured in transwell inserts with growth medium and infected with BrCr (blue), GD10 strain (red), and EV71-heated strain (Gray) at an MOI of 6.6 for 1 h. Bare PTFE membrane transwell inserts under the same conditions were used as mock controls. The permeability of the BBB was examined using TEER values at different time points after infection (A). Statistical analyses were performed using the unpaired \u003cem\u003et\u003c/em\u003e‐tests. The culture medium in the bottom compartment was collected at 12 and 24 hpi, respectively, for quantification of viral load using the TCID\u003csub\u003e50\u003c/sub\u003e assay. Statistical significance was determined using one-way ANOVA (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Each assay was performed in triplicate.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/99fde4dd7b7ab53c802daf06.png"},{"id":97724038,"identity":"729f418a-d076-492b-918d-52173a674eaf","added_by":"auto","created_at":"2025-12-08 16:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19645469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/281dbdd8-e719-4698-aad8-ec02435d7ac4.pdf"},{"id":92126522,"identity":"9ef11934-c347-43bf-99f3-c4a0ccbfc979","added_by":"auto","created_at":"2025-09-25 01:33:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17424,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7479059/v1/a9be177d8b5a6b0ea3d41df3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological characteristics of an Enterovirus 71 subgroup C4 strain isolated in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease (HFMD), predominantly infects infants and young children. Patients with HFMD often develop rashes on their palms and soles, oral ulcers, and fever [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although HFMD is self-limiting, EV71 infection occasionally causes severe neurological complications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. EV71 was first isolated in 1969 from a child diagnosed with encephalitis in the USA and has since been sporadically reported in America and Europe [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In 1997, a large HFMD outbreak occurred in Malaysia, causing 34 fatalities [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The following year, another extensive outbreak occurred in Taiwan, with over 100,000 reported HFMD cases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Since its emergence, EV71 has consistently caused epidemics across the Asia-Pacific region [\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], causing two outbreaks in Shandong and Anhui provinces between 2007 and 2008 and subsequently becoming endemic in mainland China [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEV71 currently comprises seven groups based on the nucleotide sequences of \u003cem\u003eVP1\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The BrCr strain, the sole member of Group A, was initially identified in the USA in 1969 and re-emerged in China in 2008 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Groups B and C are further divided into five subgroups. Subgroups B1 and B2 primarily circulated in the Netherlands, Hungary, Bulgaria, and the USA between 1960 and 1989; subgroups B3\u0026ndash;B5 circulated in the Asia-Pacific region beginning in 1990 [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Group C rapidly spread globally, causing numerous outbreaks, particularly across Asia-Pacific countries [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Subgroup C4 emerged as the predominant EV71 strain in China from 2007 onwards and continues circulating to date [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Additional genotypes were recently identified; Groups D and G circulated solely in India from 2008, without documented outbreaks [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Groups E and F were identified in Africa in 2003 and Madagascar in 2004, respectively [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Since HFMD outbreaks were reported in Shandong and Anhui between 2007 and 2008, subgroup C4 of EV71 has become the predominant agent circulating in China [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Determining the genetic and biological characteristics of the prevalent EV71 strain can improve the understanding of its molecular epidemiological characteristics and facilitate the development of effective vaccines. However, few studies have examined the full-genome characteristics of subgroup C4 strains along with their cytopathogenic effects and ability to cross the blood\u0026ndash;brain barrier (BBB) \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we characterized the genetic properties of an EV71 subgroup C4 strain and evaluated its biological characteristics, including infectivity, viral replication, and cytotoxicity in human rhabdomyosarcoma (RD) and African green monkey kidney (Vero) cells. Furthermore, we assessed whether the isolate could infect and cross the BBB using an established \u003cem\u003ein vitro\u003c/em\u003e BBB model.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and viruses\u003c/h2\u003e\u003cp\u003eRD and Vero cells were maintained in Dulbecco's modified Eagle\u0026rsquo;s medium (DMEMGibco, no C11995500BT) containing 10% fetal bovine serum (FBS). The human brain microvessel endothelial cell line (hCMEC/D3) was purchased from Cellverse (iCell-h070; Shanghai, China) and cultured in DMEM containing 10% FBS and 40 \u0026micro;g/mL endothelial cell growth supplement (E2759; Sigma-Aldrich, St. Louis, MO, USA). The EV71 prototype strain BrCr was preserved in our laboratory. The EV71 clinical strain GD10 (GenBank accession number: KJ004559.1) was kindly provided by the Chinese Center for Disease Control and Prevention. The viral titer of these stocks was measured as the 50% tissue culture infective dose (TCID\u003csub\u003e50\u003c/sub\u003e) per milliliter (mL) according to the Reed and Muench method, as previously described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eViral genome sequencing and phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eTotal viral RNAs were extracted from GD10 strain-infected cell culture supernatants using an RNA extraction kit, following the manufacturer\u0026rsquo;s instructions. All mRNAs were subjected to RNA sequencing using an Illumina MiSeq v2 instrument (San Diego, CA, USA) with 300-bp paired-end reads. After removing host-derived reads, virus-specific reads were included in genome alignment and assembly. All sequence reads were mapped to the selected reference EV71 virus using CLC Bio\u0026rsquo;s clc_ref_assemble_long program. We obtained the genome of the isolated EV71 (GD10 strain). Comparative sequence analyses, including sequence alignments and estimation of genetic distances, were performed using MEGA software (version 11.0; ClustalW, Molecular Evolutionary Genetic Analysis software). Phylogenetic trees were constructed using the neighbor-joining method with Kimura in MEGA, and branch support was calculated based on 1,000 bootstrap replicates.\u003c/p\u003e\n\u003ch3\u003eViral infection\u003c/h3\u003e\n\u003cp\u003eVero and RD cells were seeded into 24-well plates and incubated overnight. Cells at 80% confluence were infected with the BrCr or GD10 strain at a multiplicity of infection (MOI) of 0.01 for 1 h. Following inoculation, the viral suspension was removed, and the cells were washed with phosphate-buffered saline (PBS) before adding fresh culture medium. Cytopathic effect (CPE) was observed, and cell morphology was recorded using a Carl Zeiss Axio Vert.A1 microscope (Oberkochen, Germany). Infected cells were harvested at various time points for RNA extraction for reverse transcription-quantitative PCR (RT-qPCR) and protein extraction for western blotting.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR analysis\u003c/h3\u003e\n\u003cp\u003eInfected cells were trypsinized, and total RNA was extracted using an RNA Easy Fast Tissue/Cell Kit (DP451; Tiangen, Shanghai, China). RNA was reverse-transcribed into cDNA using a FastKing RT Kit (KR116; Tiangen, China). RT-qPCR was performed with EV71 VP1-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) using a SuperReal PreMix Plus (SYBR Green) Kit (FP205; Tiangen, China) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). EV71 expression was normalized to that of GAPDH and presented as the fold-change (EV71/GAPDH).\u003c/p\u003e\n\u003ch3\u003eCell cytotoxicity and viability assay\u003c/h3\u003e\n\u003cp\u003eVero and RD cells were seeded into 96-well plates and infected with 0.01 MOI of the BrCr or GD10 strain. Cell proliferation was assessed using a CCK-8 assay kit (40203ES60; Yeasen, Shanghai, China). At the indicated time points post-infection, CCK-8 reagent was added and incubated in the dark. Absorbance was measured at 450 nm using a microplate reader (SYNERGY HTX; BioTek, Winooski, VT, USA). Cell viability was assessed with the CellTiter-Glo Luminescent Cell Viability Assay (G7570; Promega, Madison, WI, USA) based on intracellular ATP quantification. Cell death was evaluated using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (G1780; Promega) by measuring lactate dehydrogenase (LDH) release, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eCells were lysed in cold PIRA buffer (P0013B; Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (P1045; Beyotime, China) on ice, followed by centrifugation at 4\u0026deg;C. The protein concentration was quantified using a Pierce BCA Protein Assay Kit (23225; Thermo Fisher Scientific, Waltham, MA, USA). Proteins were resolved by 10% -polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (0.22 \u0026micro;m, Millipore, Billerica, MA, USA). The membranes were blocked in blocking buffer (PBS containing 5% milk and 0.05% Tween-20) at room temperature, washed with PBST (PBS containing 0.05% Tween-20), and incubated with primary antibodies at 4\u0026deg;C overnight. After PBST, the membranes were incubated with secondary antibodies. The blots were visualized using a SuperSignal West Femto Trial Kit (34094; Thermo Fisher Scientific) and imaged with a ChemiDoc MP Imaging System (Bio-Rad). A polyclonal antibody against EV71 was generated in-house from rabbits immunized with VP1 protein. Rabbit anti-GAPDH (5174S) was purchased from Cell Signaling Technology (Danvers, MA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eBBB model and transendothelial electrical resistance (TEER) measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003ehCMEC/D3 cells were seeded on the apical side of transwell inserts (3462; Corning, Inc., Corning, NY, USA) with hCMEC/D3 complete medium in the upper and lower chambers. Cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e until confluence and fresh media was replaced daily. The apical chamber was exposed to EV71 strains at MOI 6.6 for 1 h, then replaced with fresh medium. TEER was measured using the Millicell\u0026reg;-ER system (#MERS00002; Merck Millipore, Kenilworth, NJ, USA), as described previously [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Briefly, the electrode was disinfected with 70% ethanol and equilibrated in pre-warmed medium. The longer arm of the electrode was placed in the lower chamber, and the shorter in the upper chamber. Triplicate readings were recorded for each insert. TEER values were calculated by multiplying the average resistance (Ω) by the membrane area (1.12 cm\u003csup\u003e2\u003c/sup\u003e). The background TEER value was used as a mock control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed with at least three replicates, and data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad Prism software (version 8.0.3, GraphPad, Software Inc., San Diego, CA, USA) was used for statistical analysis. A \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The statistical methods are noted in the figure legends.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eViral genome and phylogenetic tree\u003c/h2\u003e\u003cp\u003eThe GD10 strain sequence consisted of 7415 bp and contained a single open reading frame (ORF) encoding a 2193-amino acid polypeptide, deposited in GenBank under accession number KJ004559.1. Phylogenetic analyses were performed to assess genetic relationships between this strain and 22 reference strains isolated worldwide and deposited in GenBank. A phylogenetic tree was constructed based on full-genome nucleotide sequences. Phylogenetic analysis assigned the GD10 strain to EV71 subgroup C4. Various EV71 groups have emerged in China, including genogroups A, B, and C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Groups A and B exhibited limited circulation, whereas Group C showed extensive dissemination. The GD10 strain shared 88% nucleotide identity with other EV71 strains from Beijing, indicating close genetic relatedness and high homology. These findings confirm that EV71 subgroup C4 remains the predominant subgroup circulating in mainland China.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eInfection and replication of the GD10 strain\u003c/h2\u003e\u003cp\u003eWe initially evaluated CPE in Vero and RD cells to assess the infectivity of the GD10 clinical isolate. Both cell types were infected with the GD10 strain at an MOI of 0.01; the BrCr strain served as a positive control. The BrCr strain induced evident CPE in both cell lines at 36\u0026ndash;48 h post-infection (hpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, GD10 infection induced CPE in RD cells beginning at 36 hpi, whereas no evident cell death occurred in Vero cells. To evaluate replication kinetics, viral RNA expression was measured over time. The BrCr strain replicated robustly in both cell lines (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B). In RD cells, GD10 replicated significantly but with lower viral RNA levels than those in BrCr (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, GD10 RNA was undetectable in Vero cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results suggest that RD cells more effectively support GD10 replication. We also detected VP1 expression at the protein level using western blotting. Consistent with the RT-qPCR data, VP1 protein expression was increased in both cell types after BrCr infection but was absent following GD10 infection (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;F). These results suggest that antigenic variation within the VP1 protein prevented binding by the polyclonal antibody. Collectively, these data show that GD10 infection induced CPE and support efficient replication in RD cells but not in Vero cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell viability and cytotoxicity following GD10 infection\u003c/h2\u003e\u003cp\u003eGiven the observed CPE and viral replication (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we next assessed the cell death response to GD10 in Vero and RD cells. CCK-8 assay revealed a marked decrease in Vero cell viability following BrCr infection, whereas GD10 did not induce such a decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In RD cells, both BrCr and GD10 caused significant reductions in viability, consistent with the observed CPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cytotoxicity was further assessed by measuring LDH release into the supernatant and ATP levels. In Vero cells, BrCr infection led to increased LDH release starting at 12 hpi, with progressive elevation through 48 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). ATP levels also declined significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In contrast, GD10 infection did not affect LDH or ATP levels in Vero cells. In RD cells, both BrCr and GD10 increased LDH release and decreased ATP levels. These results indicate that GD10 did not induce detectable cytotoxicity in Vero cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eAbility of the GD10 strain to cross the BBB\u003c/h2\u003e\u003cp\u003eTo assess the effects of GD10 on the integrity of the BBB, we infected hCMEC/D3 cells cultured in transwell inserts to establish an \u003cem\u003ein vitro\u003c/em\u003e BBB model. Both BrCr and GD10 infection significantly reduced TEER relative to that in the mock control from the initial infection stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). TEER values progressively declined, reaching a minimum at 12 hpi. Although BrCr-induced TEER reduction was more pronounced than that induced by GD10, the difference was not significant. Concordantly, inactivated EV71 did not affect TEER, indicating that only active infection compromised BBB function. Viral RNA in the bottom compartment was measured to evaluate BBB penetration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At 12 hpi, the BrCr viral titer were significantly higher than that of GD10; however, both strains showed comparable viral titers by 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results suggest that GD10 compromised BBB integrity and crossed the barrier early during infection \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we described the genetic diversity and biological characteristics of the clinical EV71 subgroup C4 strain GD10. Compared with the BrCr strain, GD10 infection induced severe CPE in RD cells but not in Vero cells. Additionally, RD cells supported GD10 replication, whereas Vero cells did not. EV71 reportedly infects various cell lines, including human neuroblastoma (SK-N-SH), colorectal (HT29), neural (SF268), and microvascular endothelial (HMEC-1) cells [\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A previous study showed that five C4 strains from different Chinese regions induce CPE and replicate in Vero cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, our findings indicate that Vero cells do not efficiently support GD10 infection. Although BrCr and GD10 exhibited similar replication patterns in RD cells, BrCr viral RNA levels were significantly higher, suggesting a more rapid replication rate. We did not detect VP1 protein expression in RD cells following GD10 infection, possibly because the anti-EV71 antibody could not bind to the GD10 strain owing to the different antigenic epitopes residing in the BrCr strain. We further assessed cell viability and cytotoxicity, demonstrating that GD10 induced cell damage comparable with that caused by BrCr, with increased LDH release and decreased ATP production. These data indicate that GD10 infection induces lytic cell death. Collectively, GD10 exhibits distinct host-cell susceptibility relative to previously reported subgroup strains.\u003c/p\u003e\u003cp\u003eThe prototype BrCr strain (genogroup A) was isolated from a patient with central nervous system (CNS) disease in the USA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Subgroup C4 was identified as a major public health concern in China in 2007 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. EV71-associated HFMD often causes severe neurological complications in children aged\u0026thinsp;\u0026lt;\u0026thinsp;5 years, indicating that EV71 can cross the BBB and invade the CNS [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Three subgroup C4 EV71 strains induced pathological lesions in neonatal mice and rhesus monkeys [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Previously, we showed that GD10 infection caused severe clinical symptoms, including substantial weight loss and hindlimb paralysis, in both BALB/c and C57 mice, confirming its neurovirulence [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Using an \u003cem\u003ein vitro\u003c/em\u003e model, we demonstrated that GD10 could cross the BBB, as indicated by reduced TEER values. Furthermore, BrCr infection produced greater barrier disruption than GD10, suggesting that BrCr possesses higher neurovirulence.\u003c/p\u003e\u003cp\u003eThis study had some limitations. First, we did not compare GD10 with other Chinese subgroup C4 isolates. Second, few cell types were employed to characterize viral biology. Nevertheless, we thoroughly evaluated GD10 infectivity and replication at the cellular level, along with BBB disruption \u003cem\u003ein vitro\u003c/em\u003e, facilitating future studies on virus\u0026ndash;host interactions and CNS pathogenesis.\u003c/p\u003e\u003cp\u003eIn conclusion, we analyzed the genetic and biological properties of a clinically isolated EV71 subgroup C4 strain. GD10 exhibited adaptation for infection, replication, and induction of cell death in RD cells, and demonstrated BBB penetration \u003cem\u003ein vitro\u003c/em\u003e. Our findings improve the understanding of EV71 pathogenesis and may inform the development of multivalent EV71 vaccines.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHFMD: Hand, foot, and mouth disease; EV71: Enterovirus 71; LDH: \u0026nbsp;lactate dehydrogenase; BBB: blood\u0026ndash;brain barrier; CPE: cytopathic effect; MOI: \u0026nbsp;multiplicity of infection; TCID\u003csub\u003e50\u003c/sub\u003e:\u003csub\u003e\u0026nbsp;\u003c/sub\u003etissue culture infective dose 50; TEER: transendothelial electrical resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research \u0026amp; Development\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProgram of China [grant number 2022YFC2603800].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJX, HS, and LJ conceived and designed the study. TM, HL, and YL performed the experiments. TM, WL, ZY, LL and WZ analyzed the data. JX wrote the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTee HK, Zainol MI, Sam I-C, Chan YF. Recent advances in the understanding of enterovirus A71 infection: a focus on neuropathogenesis. Expert Rev Anti Infect Ther. 2021;19:733\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/14787210.2021.1851194\u003c/span\u003e\u003cspan address=\"10.1080/14787210.2021.1851194\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOoi MH, Wong SC, Lewthwaite P, Cardosa MJ, Solomon T. 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Virus Res. 2023;324:199038. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.virusres.2022.199038\u003c/span\u003e\u003cspan address=\"10.1016/j.virusres.2022.199038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"subgroup C4, viral kinetics, cytotoxicity, blood–brain barrier","lastPublishedDoi":"10.21203/rs.3.rs-7479059/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7479059/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHand, foot, and mouth disease (HFMD) is a widespread infectious disease primarily affecting infants and young children. Enterovirus 71 (EV71) comprises seven genogroups, among which subgroup C4 is the dominant viral agent in China and is frequently associated with HFMD and central nervous system infections. We analyzed the genetic characteristics of an EV71 subgroup C4 strain through whole-genome sequencing and investigated its biological characteristics, including infectivity, replication, and cytotoxicity, in human rhabdomyosarcoma (RD) and African green monkey kidney (Vero) cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe examined the genetic and biological features of a clinical EV71 GD10 strain isolated in China. Its relationships with strains listed in GenBank were evaluated using phylogenetic analysis. Viral infectivity and replication were assessed in RD and Vero cells. Cytotoxicity was evaluated by measuring cell viability, lactate dehydrogenase (LDH) release, and ATP levels. Effects on blood\u0026ndash;brain barrier (BBB) integrity were investigated \u003cem\u003ein vitro\u003c/em\u003e by assessing transendothelial electrical resistance and viral load across the barrier.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSequence analysis confirmed that GD10 belonged to subgroup C4 and closely resembled strains from China. GD10 infection induced a pronounced cytopathic effect and elevated viral RNA levels in RD cells but not in Vero cells. The infection time-dependently increased LDH release and reduced ATP levels. GD10 compromised BBB integrity and crossed the cellular barrier \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe GD10 strain demonstrated strong adaptability to RD cells and impaired BBB function. Our results improve the understanding of virus\u0026ndash;host interactions and may support efforts towards EV71 vaccine development.\u003c/p\u003e","manuscriptTitle":"Biological characteristics of an Enterovirus 71 subgroup C4 strain isolated in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 01:33:34","doi":"10.21203/rs.3.rs-7479059/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T17:39:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T07:48:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T14:21:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141092170375393385467394918647034762222","date":"2025-09-18T01:24:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94597038429823884357322167352053107363","date":"2025-09-17T14:44:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144814740274251073243450891019976195709","date":"2025-09-16T13:07:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T10:10:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-03T09:59:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-01T11:35:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-01T11:35:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-08-28T10:05:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2a1cec2-c955-473d-9b10-85db9643cb10","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:04:52+00:00","versionOfRecord":{"articleIdentity":"rs-7479059","link":"https://doi.org/10.1186/s12879-025-12241-2","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2025-12-04 15:57:28","publishedOnDateReadable":"December 4th, 2025"},"versionCreatedAt":"2025-09-25 01:33:34","video":"","vorDoi":"10.1186/s12879-025-12241-2","vorDoiUrl":"https://doi.org/10.1186/s12879-025-12241-2","workflowStages":[]},"version":"v1","identity":"rs-7479059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7479059","identity":"rs-7479059","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}