{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T07:13:57Z","timestamp":1791530037992,"version":"4.3.4"},"reference-count":36,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2023,9,2]],"date-time":"2023-09-02T00:00:00Z","timestamp":1693612800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002855","name":"Ministry of Science and Technology of the People\u2019s Republic of China","doi-asserted-by":"publisher","award":["2015ZX09102021"],"award-info":[{"award-number":["2015ZX09102021"]}],"id":[{"id":"10.13039\/501100002855","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.<\/jats:p>","DOI":"10.3390\/v15091868","type":"journal-article","created":{"date-parts":[[2023,9,4]],"date-time":"2023-09-04T02:26:12Z","timestamp":1693794372000},"page":"1868","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice"],"prefix":"10.3390","volume":"15","author":[{"given":"Wei-Ping","family":"Jin","sequence":"first","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Wang","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Wu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Guo","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheng-Li","family":"Meng","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ze-Jun","family":"Wang","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dai-Guan","family":"Yu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0916-053X","authenticated-orcid":false,"given":"Shuo","family":"Shen","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"20909","DOI":"10.1038\/s41598-020-77768-7","article-title":"Epidemical and etiological study on hand, foot and mouth disease following EV-A71 vaccination in Xiangyang, China","volume":"10","author":"Meng","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2426","DOI":"10.1128\/JCM.00007-11","article-title":"Complete genome analysis of coxsackievirus A2, A4, A5, and A10 strains isolated from hand, foot, and mouth disease patients in China revealing frequent recombination of human enterovirus A","volume":"49","author":"Hu","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"390","DOI":"10.7883\/yoken.JJID.2005.390","article-title":"Prevalence of coxsackievirus A5, A6, and A10 in patients with herpangina in Aichi Prefecture, 2005","volume":"58","author":"Yamashita","year":"2005","journal-title":"Jpn. J. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1186\/1743-422X-8-297","article-title":"Epidemics of enterovirus infection in Chungnam Korea, 2008 and 2009","volume":"8","author":"Baek","year":"2011","journal-title":"Virol. J."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Puenpa, J., Mauleekoonphairoj, J., Linsuwanon, P., Suwannakarn, K., Chieochansin, T., Korkong, S., Theamboonlers, A., and Poovorawan, Y. (2014). Prevalence and characterization of enterovirus infections among pediatric patients with hand foot mouth disease, herpangina and influenza like illness in Thailand, 2012. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0098888"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1177\/0009922820910822","article-title":"The epidemiological and clinical characteristics of hand, foot, and mouth disease in Hangzhou, China, 2016 to 2018","volume":"59","author":"Wang","year":"2020","journal-title":"Clin. Pediatr"},{"key":"ref_7","first-page":"959","article-title":"Detection and characterization of enterovirus associated with herpangina and hand, foot, and mouth disease in Seoul, Korea","volume":"57","author":"Park","year":"2011","journal-title":"Clin. Lab."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"vey020","DOI":"10.1093\/ve\/vey020","article-title":"A complex mosaic of enteroviruses shapes community-acquired hand, foot and mouth disease transmission and evolution within a single hospital","volume":"4","author":"Cobbin","year":"2018","journal-title":"Virus. Evol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e244","DOI":"10.1542\/peds.2006-3331","article-title":"Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005","volume":"120","author":"Chen","year":"2007","journal-title":"Pediatrics"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e1076-81","DOI":"10.1016\/j.ijid.2010.07.006","article-title":"The largest outbreak of hand; foot and mouth disease in Singapore in 2008: The role of enterovirus 71 and coxsackievirus A strains","volume":"14","author":"Wu","year":"2010","journal-title":"Int. J. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Yu, Y., Luo, Z., Jin, W., Mai, J., Qian, S., Lu, J., Wei, Z., Meng, S., Wang, Z., and Guan, X. (2021). Emergence of a novel recombinant of CV-A5 in HFMD epidemics in Xiangyang, China. BMC. Med. Genom., 14.","DOI":"10.1186\/s12920-021-01107-6"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e01743-20","DOI":"10.1128\/JVI.01743-20","article-title":"Efficacy of coxsackievirus A5 vaccine candidates in an actively immunized mouse model","volume":"95","author":"Jin","year":"2021","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.jviromet.2015.03.020","article-title":"Development of a stable Gaussia luciferase enterovirus 71 reporter virus","volume":"219","author":"Xu","year":"2015","journal-title":"J. Virol. Methods"},{"key":"ref_14","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":"Antiviral. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.3390\/v6072778","article-title":"Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening","volume":"6","author":"Xu","year":"2014","journal-title":"Viruses"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1101850","DOI":"10.3389\/fmicb.2022.1101850","article-title":"The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene","volume":"13","author":"Yu","year":"2023","journal-title":"Front. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1007\/s12250-015-3610-7","article-title":"Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone","volume":"30","author":"Deng","year":"2015","journal-title":"Virol. Sin."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"20038","DOI":"10.1073\/pnas.0807551105","article-title":"The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection","volume":"105","author":"Chapman","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","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_20","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1111\/irv.12602","article-title":"Construction of a recombinant rhinovirus accommodating fluorescent marker expression","volume":"2","author":"Han","year":"2018","journal-title":"Influenza. Other Respir. Viruses"},{"key":"ref_21","first-page":"1125","article-title":"Adoption of the 2A ribosomal skip principle to tobacco mosaic virus for peptide display. Front","volume":"8","author":"Fischer","year":"2017","journal-title":"Plant. Sci."},{"key":"ref_22","unstructured":"Standardization Administration of China (2022, August 05). Laboratory Animal-Guideline for Ethical Review of Animal Welfare. Standardization Administration of China, Available online: https:\/\/openstd.samr.gov.cn\/bzgk\/gb\/newGbInfo?hcno=9BA619057D5C13103622A10FF4BA5D14."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"104603","DOI":"10.1016\/j.micpath.2020.104603","article-title":"Amino acid substitutions in VP2, VP1, and 2C attenuate a Coxsackievirus A16 in mice","volume":"150","author":"Zhang","year":"2021","journal-title":"Microb. Pathog."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Chandler-Bostock, R., Mata, C.P., Bingham, R.J., Dykeman, E.C., Meng, B., Tuthill, T.J., Rowlands, D.J., Ranson, N.A., Twarock, R., and Stockley, P.G. (2020). Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts. PLoS Pathog., 16.","DOI":"10.1371\/journal.ppat.1009146"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e00032-19","DOI":"10.1128\/JVI.00032-19","article-title":"A novel fluorescent and bioluminescent bireporter influenza A virus to evaluate viral infections","volume":"93","author":"Nogales","year":"2019","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1517","DOI":"10.1099\/vir.0.052308-0","article-title":"An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein","volume":"94","author":"Seago","year":"2013","journal-title":"J. Gen. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Liu, F., Huang, Y., Wang, Q., and Shan, H. (2020). Construction of eGFP-tagged Senecavirus A for facilitating virus neutralization test and antiviral assay. Viruses, 12.","DOI":"10.3390\/v12030283"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"114744","DOI":"10.1016\/j.jviromet.2023.114744","article-title":"Development of recombinant West Nile virus expressing mCherry reporter protein","volume":"317","author":"Kobayashi","year":"2023","journal-title":"J. Virol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.antiviral.2011.05.001","article-title":"Development and characterization of a stable luciferase dengue virus for high-throughput screening","volume":"91","author":"Zou","year":"2011","journal-title":"Antiviral. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1042735","DOI":"10.3389\/fcimb.2022.1042735","article-title":"Dual-fluorescent reporter for live-cell imaging of the ER during DENV infection","volume":"12","author":"Corliss","year":"2022","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.jviromet.2015.07.006","article-title":"JC virus-iLOV fluorescent strains enable the detection of early and late viral protein expression","volume":"223","author":"Dang","year":"2015","journal-title":"J. Virol. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.virs.2022.03.007","article-title":"Construction and characterization of a full-length infectious clone of Getah virus in vivo","volume":"37","author":"Ren","year":"2022","journal-title":"Virol. Sin."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1186\/s13007-017-0175-5","article-title":"A rapid and efficient method for uniform gene expression using the barley stripe mosaic virus","volume":"13","author":"Cheuk","year":"2017","journal-title":"Plant. Methods."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"109675","DOI":"10.1016\/j.vetmic.2023.109675","article-title":"Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus","volume":"280","author":"Liu","year":"2023","journal-title":"Vet. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1209","DOI":"10.1094\/MPMI-04-20-0094-R","article-title":"Development of a reverse genetic system for studying Rose Rosette Virus in whole plants","volume":"33","author":"Verchot","year":"2020","journal-title":"Mol. Plant. Microbe. Interact."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e0030923","DOI":"10.1128\/jvi.00309-23","article-title":"Engineering oncolytic coxsackievirus A21 with small transgenes and enabling cell-mediated virus delivery by integrating viral cDNA into the genome","volume":"97","author":"Sam","year":"2023","journal-title":"J. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/9\/1868\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:45:28Z","timestamp":1760129128000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/9\/1868"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,2]]},"references-count":36,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["v15091868"],"URL":"https:\/\/doi.org\/10.3390\/v15091868","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,9,2]]}}}