{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T13:02:44Z","timestamp":1783083764285,"version":"3.54.6"},"reference-count":43,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2023,5,18]],"date-time":"2023-05-18T00:00:00Z","timestamp":1684368000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Science)","award":["SKLPBS2120"],"award-info":[{"award-number":["SKLPBS2120"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Human adenovirus 55 (HAdV-55) has recently caused outbreaks of acute respiratory disease (ARD), posing a significant public threat to civilians and military trainees. Efforts to develop antiviral inhibitors and quantify neutralizing antibodies require an experimental system to rapidly monitor viral infections, which can be achieved through the use of a plasmid that can produce an infectious virus. Here, we used a bacteria-mediated recombination approach to construct a full-length infectious cDNA clone, pAd55-FL, containing the whole genome of HadV-55. Then, the green fluorescent protein expression cassette was assembled into pAd55-FL to replace the E3 region to obtain a recombinant plasmid of pAd55-dE3-EGFP. The rescued recombinant virus rAdv55-dE3-EGFP is genetically stable and replicates similarly to the wild-type virus in cell culture. The virus rAdv55-dE3-EGFP can be used to quantify neutralizing antibody activity in sera samples, producing results in concordance with the cytopathic effect (CPE)-based microneutralization assay. Using an rAdv55-dE3-EGFP infection of A549 cells, we showed that the assay could be used for antiviral screening. Our findings suggest that the rAdv55-dE3-EGFP-based high-throughput assay provides a reliable tool for rapid neutralization testing and antiviral screening for HAdV-55.<\/jats:p>","DOI":"10.3390\/v15051192","type":"journal-article","created":{"date-parts":[[2023,5,18]],"date-time":"2023-05-18T06:00:29Z","timestamp":1684389629000},"page":"1192","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP"],"prefix":"10.3390","volume":"15","author":[{"given":"Wei","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuehong","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ye","family":"Feng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6860-6135","authenticated-orcid":false,"given":"Jing","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoping","family":"Kang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1090-3193","authenticated-orcid":false,"given":"Sen","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuchang","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiyan","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenguang","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huiyao","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"School of Public Health, Mudanjiang Medical University, Mudanjiang 157011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1908-2926","authenticated-orcid":false,"given":"Tao","family":"Jiang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China"},{"name":"School of Public Health, Mudanjiang Medical University, Mudanjiang 157011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,5,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"570","DOI":"10.3181\/00379727-84-20714","article-title":"Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture","volume":"84","author":"Rowe","year":"1953","journal-title":"Proc. Soc. Exp. Biol. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1055\/s-0041-1733802","article-title":"Adenovirus: Epidemiology, global spread of novel types, and approach to treatment","volume":"42","author":"Lynch","year":"2021","journal-title":"Semin. Respir. Crit. Care Med."},{"key":"ref_3","first-page":"001721","article-title":"ICTV Virus Taxonomy Profile: Adenoviridae 2022","volume":"103","author":"Aoki","year":"2022","journal-title":"J. Gen. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e2189","DOI":"10.1002\/rmv.2189","article-title":"Epidemiology of human adenovirus associated with respiratory infection in southern Brazil","volume":"31","author":"Pscheidt","year":"2021","journal-title":"Rev. Med. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Wang, X., Wang, D., Umar, S., Qin, S., Ling, Q., Gray, G.C., and Liu, Y. (2021). Molecular typing of human adenoviruses among hospitalized patients with respiratory tract infections in a tertiary Hospital in Guangzhou, China between 2017 and 2019. BMC Infect. Dis., 21.","DOI":"10.1186\/s12879-021-06412-0"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"586","DOI":"10.1055\/s-0036-1584923","article-title":"Adenovirus: Epidemiology, Global Spread of Novel Serotypes, and Advances in Treatment and Prevention","volume":"37","author":"Lynch","year":"2016","journal-title":"Semin. Respir. Crit. Care Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1007\/s12519-022-00568-8","article-title":"Current status of human adenovirus infection in China","volume":"18","author":"Mao","year":"2022","journal-title":"World J. Pediatr."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Liu, J., Nian, Q.G., Zhang, Y., Xu, L.J., Hu, Y., Li, J., Deng, Y.Q., Zhu, S.Y., Wu, X.Y., and Qin, E.D. (2014). In vitro characterization of human adenovirus type 55 in comparison with its parental adenoviruses, types 11 and 14. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0100665"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6111","DOI":"10.1002\/jmv.28074","article-title":"An outbreak of acute respiratory disease caused by HAdV-55 in Beijing, China, 2020","volume":"94","author":"Liu","year":"2022","journal-title":"J. Med. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1378\/chest.13-1186","article-title":"Emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia","volume":"145","author":"Cao","year":"2014","journal-title":"Chest"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1128\/JCM.01694-09","article-title":"Computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen","volume":"48","author":"Walsh","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"793","DOI":"10.46234\/ccdcw2021.197","article-title":"Outbreaks of acute respiratory disease associated with human adenovirus infection in closed camps, China, December 2011-March 2014","volume":"3","author":"Du","year":"2021","journal-title":"China CDC Wkly."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1111\/irv.12232","article-title":"Epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in China, 2009\u20132012","volume":"8","author":"Lu","year":"2014","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Liu, M.-C., Xu, Q., Li, T.-T., Wang, T., Jiang, B.-G., Lv, C.-L., Zhang, X.-A., Liu, W., and Fang, L.-Q. (2023). Prevalence of human infection with respiratory adenovirus in China: A systematic review and meta-analysis. PLOS Negl. Trop. Dis., 17.","DOI":"10.1371\/journal.pntd.0011151"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.15585\/mmwr.mm6639a2","article-title":"Human adenovirus surveillance\u2014United States, 2003\u20132016","volume":"66","author":"Binder","year":"2017","journal-title":"MMWR. Morb. Mortal. Wkly. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1408","DOI":"10.1002\/jmv.23325","article-title":"Seroprevalence of neutralizing antibodies to human adenovirus type 5 in healthy adults in China","volume":"84","author":"Yu","year":"2012","journal-title":"J. Med. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2984","DOI":"10.1128\/JCM.39.8.2984-2986.2001","article-title":"Simplified microneutralization test for serotyping adenovirus isolates","volume":"39","author":"Malasig","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2331","DOI":"10.1128\/jcm.32.9.2331-2334.1994","article-title":"Quantitative colorimetric microneutralization assay for characterization of adenoviruses","volume":"32","author":"Schnurr","year":"1994","journal-title":"J. Clin. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1016\/0166-0934(88)90134-6","article-title":"Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds","volume":"20","author":"Pauwels","year":"1988","journal-title":"J. Virol. Methods"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1095343","DOI":"10.3389\/fpubh.2023.1095343","article-title":"A seroepidemiological survey of adenovirus type 7 circulation among healthy adults in China and in Sierra Leone, West Africa","volume":"11","author":"Wang","year":"2023","journal-title":"Front. Public Health"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1080\/14760584.2019.1588113","article-title":"A review of 65 years of human adenovirus seroprevalence","volume":"18","author":"Mennechet","year":"2019","journal-title":"Expert Rev. Vaccines"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"30","DOI":"10.3389\/fmicb.2018.03040","article-title":"Seroprevalence of neutralizing antibodies to human adenovirus type 4 and 7 in healthy populations from southern China","volume":"9","author":"Ye","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1038\/s41426-018-0206-y","article-title":"Seroepidemiological investigation of HAdV-4 infection among healthy adults in China and in Sierra Leone, West Africa","volume":"7","author":"Wang","year":"2018","journal-title":"Emerg. Microbes Infect."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e43","DOI":"10.1038\/emi.2017.29","article-title":"Seroprevalence of neutralizing antibodies against adenovirus type 14 and 55 in healthy adults in Southern China","volume":"6","author":"Zheng","year":"2017","journal-title":"Emerg. Microbes Infect."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1007\/s12250-020-00295-2","article-title":"A sensitive and high-throughput flow cytometry-based assay for measuring antibody neutralization of human adenovirus type 3","volume":"36","author":"Liu","year":"2021","journal-title":"Virol. Sin."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.antiviral.2019.01.008","article-title":"In vitro comparison of currently available and investigational antiviral agents against pathogenic human double-stranded DNA viruses: A systematic literature review","volume":"163","author":"Chemaly","year":"2019","journal-title":"Antivir. Res"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1438","DOI":"10.1128\/JCM.01928-09","article-title":"Outbreak of febrile respiratory illness associated with adenovirus 11a infection in a Singapore military training cAMP","volume":"48","author":"Kajon","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.coviro.2018.12.004","article-title":"Emergence and re-emergence of respiratory adenoviruses in the United States","volume":"34","author":"Kajon","year":"2019","journal-title":"Curr. Opin. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1002\/jmv.20441","article-title":"Respiratory disease caused by a species B2 adenovirus in a military camp in Turkey","volume":"77","author":"Chmielewicz","year":"2005","journal-title":"J. Med. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Zhang, J., Kang, J., Dehghan, S., Sridhar, S., Lau, S.K.P., Ou, J., Woo, P.C.Y., Zhang, Q., and Seto, D. (2019). A survey of recent adenoviral respiratory pathogens in Hong Kong reveals emergent and recombinant human adenovirus type 4 (HAdV-E4) circulating in civilian populations. Viruses, 11.","DOI":"10.3390\/v11020129"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"297","DOI":"10.2144\/btn-2022-0051","article-title":"Adenovirus vector system: Construction, history and therapeutic applications","volume":"73","author":"Syyam","year":"2022","journal-title":"BioTechniques"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhang, W., Mese, K., Schellhorn, S., Bahlmann, N., Mach, N., Bunz, O., Dhingra, A., Hage, E., Lafon, M.E., and Wodrich, H. (2020). High-throughput cloning and characterization of emerging adenovirus yypes 70, 73, 74, and 75. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176370"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1089\/hgtb.2018.044","article-title":"Novel vector construction based on alternative adenovirus types via homologous recombination","volume":"29","author":"Zhang","year":"2018","journal-title":"Hum. Gene Ther. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1038\/nprot.2007.135","article-title":"A protocol for rapid generation of recombinant adenoviruses using the AdEasy system","volume":"2","author":"Luo","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1095198","DOI":"10.3389\/fmedt.2023.1095198","article-title":"Novel and efficient method for the reconstruction of adenoviruses through isothermal assembly and its potential applications","volume":"5","author":"Wen","year":"2023","journal-title":"Front. Med. Technol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Zou, X., Zhu, Y., Li, C., Duan, Y., Zhang, L., Guo, X., Hou, W., Xie, Z., and Lu, Z. (2022). Construction of adenoviral vectors using DNA assembly technology. J. Vis. Exp. JoVE, 184.","DOI":"10.3791\/64033"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.jbiotec.2019.11.009","article-title":"Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning","volume":"307","author":"Guo","year":"2020","journal-title":"J. Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1016\/j.omto.2021.11.011","article-title":"A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology","volume":"23","author":"Ni","year":"2021","journal-title":"Mol. Ther. Oncolytics"},{"key":"ref_39","first-page":"61","article-title":"Generation of recombinant adenovirus using the Escherichia coli BJ5183 recombination system","volume":"130","author":"Reddy","year":"2007","journal-title":"Methods Mol. Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1089\/hgtb.2016.044","article-title":"A partial E3 deletion in replication-defective adenoviral vectors allows for stable expression of potentially toxic transgene products","volume":"27","author":"Haut","year":"2016","journal-title":"Hum. Gene Ther. Methods"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/sj.cgt.7701093","article-title":"Comparison of the E3 and L3 regions for arming oncolytic adenoviruses to achieve a high level of tumor-specific transgene expression","volume":"15","author":"Robinson","year":"2008","journal-title":"Cancer Gene Ther."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.virusres.2004.06.005","article-title":"Sequence polymorphism in the E3 7.7K ORF of subspecies B1 human adenoviruses","volume":"107","author":"Kajon","year":"2005","journal-title":"Virus Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5046","DOI":"10.1128\/JCM.41.11.5046-5052.2003","article-title":"Quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: Addressing preexisting immunity to vaccine and gene therapy vectors","volume":"41","author":"Sprangers","year":"2003","journal-title":"J. Clin. Microbiol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/5\/1192\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:37:33Z","timestamp":1760125053000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/5\/1192"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,18]]},"references-count":43,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2023,5]]}},"alternative-id":["v15051192"],"URL":"https:\/\/doi.org\/10.3390\/v15051192","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,5,18]]}}}