{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:08:25Z","timestamp":1785431305522,"version":"3.56.0"},"reference-count":51,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T00:00:00Z","timestamp":1777420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32470144"],"award-info":[{"award-number":["32470144"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32070184"],"award-info":[{"award-number":["32070184"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["32470144"],"award-info":[{"award-number":["32470144"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"award":["32070184"],"award-info":[{"award-number":["32070184"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Enteroviruses are positive-sense single-stranded RNA viruses and common pathogens that are responsible for diverse public health diseases. To facilitate the study of the virus biology and pathogenesis of enterovirus, we developed a rapid method for construction of the enteroviral cDNA clones including enterovirus A71 (EV-A71) and coxsackievirus B5 (CVB5). As described for EV-A71, the full-length cDNA of CVB5 was amplified by long-distance PCR and cloned into a T7 promoter-containing plasmid using directional seamless cloning technology. The virus was successfully rescued by single transfection into cells stably expressing T7 polymerase and exhibited characteristics similar to the parental virus. Next, through systematic construction and the optimization of the EV-A71 and CVB5 reporter viruses, we successfully generated two novel reporter virus panels with high virus titers, rapid replication, and relatively stable genetic inheritance across passages using the new fluorescence proteins mScarlet3-H and the smallest miRFP670nano3. Analysis of critical determinants for the reporter virus construction revealed that reporter gene sizes, genomic insertion sites, and the usage of protease recognition sites are crucial parameters. The EV-A71 and CVB5 reporter viruses enable antiviral drug evaluation, as demonstrated by our identification of gemcitabine as a broad-spectrum inhibitor of both viruses. These systems also facilitate the functional interrogation of host factors, exemplified by our discovery that METTL3 promotes EV-A71 and CVB5 replication. These reverse genetic tools, including infectious cDNA clones and reporter viruses, will advance basic enterovirus biology and accelerate antiviral drug discovery.<\/jats:p>","DOI":"10.3390\/v18050514","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T15:01:18Z","timestamp":1777474878000},"page":"514","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid Construction and Characterization of Infectious cDNA Clones and Reporter Viruses of Enteroviruses, Including Enterovirus A71 and Coxsackievirus B5, with Systematic Identification of Critical Determinants for Successful Reporter Virus Generation"],"prefix":"10.3390","volume":"18","author":[{"given":"Hao","family":"Zheng","sequence":"first","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tong","family":"Zhao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China"},{"name":"Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, 138 Yixueyuan R., Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meixian","family":"Fu","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, 138 Yixueyuan R., Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zirui","family":"Niu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifan","family":"Xing","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xia","family":"Cai","sequence":"additional","affiliation":[{"name":"Biosafety Level-3 Laboratory, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5631-8884","authenticated-orcid":false,"given":"Jian-Er","family":"Long","sequence":"additional","affiliation":[{"name":"Key Laboratory of Medical Molecular Virology (MOE\/NHC\/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China"},{"name":"Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, 138 Yixueyuan R., Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1038\/s41579-018-0005-4","article-title":"The life cycle of non-polio enteroviruses and how to target it","volume":"16","author":"Baggen","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1016\/S1474-4422(10)70209-X","article-title":"Clinical features, diagnosis, and management of enterovirus 71","volume":"9","author":"Ooi","year":"2010","journal-title":"Lancet Neurol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1016\/S1473-3099(13)70342-6","article-title":"Hand, foot, and mouth disease in China, 2008\u20132012: An epidemiological study","volume":"14","author":"Xing","year":"2014","journal-title":"Lancet Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"778","DOI":"10.1016\/S1473-3099(10)70194-8","article-title":"Virology, epidemiology, pathogenesis, and control of enterovirus 71","volume":"10","author":"Solomon","year":"2010","journal-title":"Lancet Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1016\/j.jmii.2020.06.008","article-title":"Clinical characteristics of echovirus 11 and coxsackievirus B5 infections in Taiwanese children requiring hospitalization","volume":"54","author":"Chen","year":"2020","journal-title":"J. Microbiol. Immunol. Infect."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/S0140-6736(80)90731-X","article-title":"Diabetes and Coxsackie virus B5 infection","volume":"1","author":"Champsaur","year":"1980","journal-title":"Lancet"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"996","DOI":"10.2337\/db15-0956","article-title":"Coxsackievirus B5 Infection Induces Dysregulation of microRNAs Predicted to Target Known Type 1 Diabetes Risk Genes in Human Pancreatic Islets","volume":"65","author":"Kim","year":"2016","journal-title":"Diabetes"},{"key":"ref_8","first-page":"38","article-title":"Coxsackie B5 virus responsible for acute myocarditis inucklings","volume":"14","author":"Abraham","year":"1970","journal-title":"Rom. Med. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1038\/s41426-018-0186-y","article-title":"A neonatal mouse model of central nervous system infections caused by Coxsackievirus B5","volume":"7","author":"Mao","year":"2018","journal-title":"Emerg. Microbes Infect."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"351","DOI":"10.3201\/eid1802.111524","article-title":"Recombination of human coxsackievirus B5 in hand, foot, and mouth disease patients, China","volume":"18","author":"Han","year":"2012","journal-title":"Emerg. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1002\/jmv.23478","article-title":"A coxsackievirus B5-associated aseptic meningitis outbreak in Shandong Province, China in 2009","volume":"85","author":"Chen","year":"2013","journal-title":"J. Med. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1080\/21645515.2017.1384107","article-title":"Seroepidemiology of coxsackievirus B5 in infants and children in Jiangsu province, China","volume":"14","author":"Gao","year":"2018","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.jinf.2012.03.021","article-title":"Coxsackievirus B5, associated with neurological hand, foot and mouth disease, China","volume":"65","author":"Hu","year":"2012","journal-title":"J. Infect."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1089\/vim.2006.0085","article-title":"Serologic prevalence of coxsackievirus group B in Greece","volume":"20","author":"Mavrouli","year":"2007","journal-title":"Viral Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1093\/infdis\/156.2.288","article-title":"Reemergence of an epidemic coxsackievirus B5 genotype","volume":"156","author":"Hamby","year":"1987","journal-title":"J. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1016\/j.virol.2015.06.006","article-title":"Recent progress in understanding coxsackievirus replication, dissemination, and pathogenesis","volume":"484","author":"Sin","year":"2015","journal-title":"Virology"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Liu, Q., and Long, J.-E. (2025). Insight into the Life Cycle of Enterovirus-A71. Viruses, 17.","DOI":"10.20944\/preprints202501.1014.v1"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/j.cell.2019.05.030","article-title":"Viral Generated Inter-Organelle Contacts Redirect Lipid Flux for Genome Replication","volume":"178","author":"Laufman","year":"2019","journal-title":"Cell"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Yuan, J., Shen, L., Wu, J., Zou, X., Gu, J., Chen, J., and Mao, L. (2018). Enterovirus A71 Proteins: Structure and Function. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.00286"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3138","DOI":"10.1002\/jmv.26096","article-title":"Seroprevalence of group B Coxsackieviruses: Retrospective study in an Italian population","volume":"92","author":"Sciandra","year":"2020","journal-title":"J. Med. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Royston, L., and Tapparel, C. (2016). Rhinoviruses and Respiratory Enteroviruses: Not as Simple as ABC. Viruses, 8.","DOI":"10.3390\/v8010016"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1007\/s11262-013-0955-6","article-title":"Construction and characterization of an infectious cDNA clone of enterovirus type 71 subgenotype C4","volume":"47","author":"Zhang","year":"2013","journal-title":"Virus Genes."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"627","DOI":"10.1007\/s00705-020-04940-9","article-title":"Construction and characterization of an infectious cDNA clone of enterovirus 71: A rapid method for rescuing infectious virus based on stable cells expressing T7 polymerase","volume":"166","author":"Fu","year":"2021","journal-title":"Arch. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.virusres.2015.08.002","article-title":"Construction and characterization of an infectious clone of coxsackievirus A6 that showed high virulence in neonatal mice","volume":"210","author":"Yang","year":"2015","journal-title":"Virus Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1186\/s12985-019-1201-1","article-title":"Construction and characterization of an infectious cDNA clone of coxsackievirus A 10","volume":"16","author":"Liu","year":"2019","journal-title":"Virol. J."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.virusres.2015.05.011","article-title":"Construction and characterization of an infectious cDNA clone of Echovirus 25","volume":"205","author":"Hou","year":"2015","journal-title":"Virus Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"945","DOI":"10.1002\/jmv.20922","article-title":"Genetic and phenotypic diversity of echovirus 30 strains and pathogenesis of type 1 diabetes","volume":"79","author":"Paananen","year":"2007","journal-title":"J. Med. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1016\/S0166-0934(97)02178-2","article-title":"Amplification and cloning of complete enterovirus genomes by long distance PCR","volume":"65","author":"Lindberg","year":"1997","journal-title":"J. Virol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/j.virs.2022.03.005","article-title":"Construction and verification of an infectious cDNA clone of coxsackievirus B5","volume":"37","author":"Song","year":"2022","journal-title":"Virol. Sin."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1016\/j.virs.2023.05.010","article-title":"Construction of coxsackievirus B5 viruses with luciferase reporters and their applications in vitro and in vivo","volume":"38","author":"Guo","year":"2023","journal-title":"Virol. Sin."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Martinez-Perez, M., Velandia-Alvarez, S., Vidal-Verdu, C., Alvarez-Rodriguez, B., and Geller, R. (2025). A Fluorescent Reporter Virus Toolkit for Interrogating Enterovirus Biology and Host Interactions. Viruses, 17.","DOI":"10.20944\/preprints202505.0091.v1"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e01759-16","DOI":"10.1128\/JVI.01759-16","article-title":"In Vivo Imaging with Bioluminescent Enterovirus 71 Allows for Real-Time Visualization of Tissue Tropism and Viral Spread","volume":"91","author":"Caine","year":"2017","journal-title":"J. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Jin, W.P., Wang, C., Wu, J., Guo, J., Meng, S.L., Wang, Z.J., Yu, D.G., and Shen, S. (2023). Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice. Viruses, 15.","DOI":"10.3390\/v15091868"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.virs.2024.02.006","article-title":"Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening","volume":"39","author":"Zhang","year":"2024","journal-title":"Virol. Sin."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1016\/j.antiviral.2012.12.010","article-title":"Development and characterization of a stable eGFP enterovirus 71 for antiviral screening","volume":"97","author":"Shang","year":"2013","journal-title":"Antivir. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1477","DOI":"10.1128\/JVI.01578-09","article-title":"Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags","volume":"84","author":"Teterina","year":"2010","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"E8731","DOI":"10.1073\/pnas.1714385114","article-title":"Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes","volume":"114","author":"Song","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1159\/000321351","article-title":"Destabilization of coxsackievirus b3 genome integrated with enhanced green fluorescent protein gene","volume":"54","author":"Tong","year":"2011","journal-title":"Intervirology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"297","DOI":"10.3109\/00365548.2011.634433","article-title":"Epidemic characteristics of hand, foot, and mouth disease in Shanghai from 2009 to 2010: Enterovirus 71 subgenotype C4 as the primary causative agent and a high incidence of mixed infections with coxsackievirus A16","volume":"44","author":"Yan","year":"2012","journal-title":"Scand. J. Infect. Dis."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e01841-16","DOI":"10.1128\/JVI.01841-16","article-title":"Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo","volume":"91","author":"Rodgers","year":"2017","journal-title":"J. Virol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3008","DOI":"10.1099\/jgv.0.000967","article-title":"Enterovirus 71 antagonizes the antiviral activity of host STAT3 and IL-6R with partial dependence on virus-induced miR-124","volume":"98","author":"Chang","year":"2017","journal-title":"J. Gen. Virol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e00204-20","DOI":"10.1128\/JVI.00204-20","article-title":"The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses","volume":"94","author":"Xu","year":"2020","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1229","DOI":"10.1093\/molbev\/mst012","article-title":"Building Phylogenetic Trees from Molecular Data with MEGA","volume":"30","author":"Hall","year":"2013","journal-title":"Mol. Biol. Evol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1128\/JVI.73.1.152-160.1999","article-title":"Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination","volume":"73","author":"Lole","year":"1999","journal-title":"J. Virol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.virusres.2019.03.017","article-title":"Characteristics of Enterovirus 71-induced cell death and genome scanning to identify viral genes involved in virus-induced cell apoptosis","volume":"265","author":"Bai","year":"2019","journal-title":"Virus Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"6406","DOI":"10.1074\/jbc.M111.301622","article-title":"Molecular determinants of enterovirus 71 viral entry: Cleft around GLN-172 on VP1 protein interacts with variable region on scavenge receptor B 2","volume":"287","author":"Chen","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1038\/s41590-018-0275-z","article-title":"m(6)A modification controls the innate immune response to infection by targeting type I interferons","volume":"20","author":"Winkler","year":"2019","journal-title":"Nat. Immunol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"666","DOI":"10.1016\/j.chom.2016.10.002","article-title":"Dynamics of Human and Viral RNA Methylation during Zika Virus Infection","volume":"20","author":"Lichinchi","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Zhao, H., Gao, Z., Sun, J., Qiao, H., Zhao, Y., Cui, Y., Zhao, B., Wang, W., Chiu, S., and Chuai, X. (2024). N6-Methyladenosine Positively Regulates Coxsackievirus B3 Replication. Viruses, 16.","DOI":"10.3390\/v16091448"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1119","DOI":"10.1039\/D1SC04839B","article-title":"The smallest near-infrared fluorescence complementation system for imaging protein-protein and RNA-protein interactions","volume":"13","author":"Chen","year":"2022","journal-title":"Chem. Sci."},{"key":"ref_51","first-page":"3171","article-title":"Enhanced efficacy of gemcitabine by indole-3-carbinol in pancreatic cell lines: The role of human equilibrative nucleoside transporter 1","volume":"31","author":"Wang","year":"2011","journal-title":"Anticancer Res."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/5\/514\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T04:13:09Z","timestamp":1777608789000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/5\/514"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,29]]},"references-count":51,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["v18050514"],"URL":"https:\/\/doi.org\/10.3390\/v18050514","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,29]]}}}