{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T01:18:33Z","timestamp":1785460713363,"version":"3.56.0"},"reference-count":56,"publisher":"Wiley","issue":"2","license":[{"start":{"date-parts":[[2025,2,3]],"date-time":"2025-02-03T00:00:00Z","timestamp":1738540800000},"content-version":"vor","delay-in-days":2,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Medical Virology"],"published-print":{"date-parts":[[2025,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Classical methods for constructing infectious cDNA clones of flaviviruses are often hindered by instability and toxicity. The Infectious\u2010Subgenomic\u2010Amplicons (ISA) method is an advancement which utilizes overlapping DNA fragments representing viral genomic sequence and in\u2010cell recombination to bypass bacterial plasmid assembly. However, the ISA method has limitations due to the toxicity of some ISA DNA fragments in bacteria during synthetic production. We validated modified ISA strategies for producing toxic ISA Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments. Three approaches were explored, including subdividing toxic DNA fragments into two sub\u2010fragments for synthetic clonal production, using a low\u2010copy bacterial plasmid, and subdividing the toxic DNA fragments into four short overlapping sub\u2010fragments, each up to 1.8\u2009kb. The latter novel approach in ISA applications enabled the synthesis of entirely bacteria\u2010free ISA DNA fragments. Our results demonstrate that subdividing toxic fragments into sub\u2010fragments smaller than 1.8\u2009kb for synthesis is the efficient strategy, circumventing the need for bacterial plasmids and ensuring rapid production of synthetic flaviviruses. This method also shortens the production timeline. We also compared the efficacy of JEV and WNV ISA in zinc finger antiviral protein 1 (ZAP) wild\u2010type and knockout cells and found that knockout cells may be more effective for ISA rescue of flaviviruses, including CpG\u2010enriched strains for live attenuated vaccines. The validated modified ISA strategies provide an efficient approach for producing synthetic JEV and WNV. This will enable rapid research during outbreaks of emerging flaviviruses by facilitating the quick generation of new virus variants.<\/jats:p>","DOI":"10.1002\/jmv.70205","type":"journal-article","created":{"date-parts":[[2025,2,3]],"date-time":"2025-02-03T04:50:53Z","timestamp":1738558253000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Infectious Subgenomic Amplicon Strategies for Japanese Encephalitis and West Nile Viruses"],"prefix":"10.1002","volume":"97","author":[{"given":"Prince Pal","family":"Singh","sequence":"first","affiliation":[{"name":"Department of Veterinary Biosciences, College of Veterinary Medicine The Ohio State University Columbus Ohio USA"},{"name":"School of Public Health University of Saskatchewan Saskatoon Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nguyen Phuong Khanh","family":"Le","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, College of Veterinary Medicine The Ohio State University Columbus Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7802-9223","authenticated-orcid":false,"given":"Uladzimir","family":"Karniychuk","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, College of Veterinary Medicine The Ohio State University Columbus Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,2,3]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-020-0714-0"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1017\/S0022172400027066"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2018.02.010"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1053\/j.semdp.2019.04.009"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-662-06099-5_2"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.antiviral.2014.12.007"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3527(08)60348-6"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.28978"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1995.0072"},{"key":"e_1_2_10_11_1","first-page":"285","article-title":"Transcription of Infectious Yellow Fever RNA From Full\u2010Length cDNA Templates Produced by In Vitro Ligation","volume":"1","author":"Rice C. M.","year":"1989","journal-title":"New Biologist"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(95)00332-Z"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.66.9.5425-5431.1992"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.068023-0"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2024.2356140"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.3390\/v12050579"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.03077"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-1884-4_14"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.943481"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2021.114385"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-023-29075-0"},{"key":"e_1_2_10_22_1","article-title":"New Reverse Genetics and Transfection Methods to Rescue Arboviruses in Mosquito Cells","volume":"7","author":"Atieh T.","year":"2017","journal-title":"Nature Publishing Group"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.3390\/v11070666"},{"key":"e_1_2_10_24_1","article-title":"Simple Reverse Genetics Systems for Asian and African Zika Viruses","volume":"6","author":"Atieh T.","year":"2016","journal-title":"Nature Publishing Group"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41426-018-0161-7"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41426-018-0053-x"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2023.1324069"},{"key":"e_1_2_10_28_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0138703"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.00715-19"},{"key":"e_1_2_10_30_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1005252"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12985-024-02557-x"},{"key":"e_1_2_10_32_1","doi-asserted-by":"publisher","DOI":"10.3390\/v15040830"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1007166"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1080\/15476286.2024.2388911"},{"key":"e_1_2_10_35_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature24039"},{"key":"e_1_2_10_36_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.823825"},{"key":"e_1_2_10_37_1","doi-asserted-by":"publisher","DOI":"10.1093\/ve\/vev012"},{"key":"e_1_2_10_38_1","doi-asserted-by":"publisher","DOI":"10.15252\/emmm.202114108"},{"key":"e_1_2_10_39_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.0030106"},{"key":"e_1_2_10_40_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00918-08"},{"key":"e_1_2_10_41_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1074276"},{"key":"e_1_2_10_42_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.77.21.11555-11562.2003"},{"key":"e_1_2_10_43_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1509745112"},{"key":"e_1_2_10_44_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003494"},{"key":"e_1_2_10_45_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01601-06"},{"key":"e_1_2_10_46_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01909-16"},{"key":"e_1_2_10_47_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1101676108"},{"key":"e_1_2_10_48_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12977-019-0500-3"},{"key":"e_1_2_10_49_1","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02683-20"},{"key":"e_1_2_10_50_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00001-19"},{"key":"e_1_2_10_51_1","doi-asserted-by":"publisher","DOI":"10.4161\/rna.5.2.6044"},{"key":"e_1_2_10_52_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13238-010-0117-8"},{"key":"e_1_2_10_53_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0712276105"},{"key":"e_1_2_10_54_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1006145"},{"key":"e_1_2_10_55_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00088-17"},{"key":"e_1_2_10_56_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.46767"},{"key":"e_1_2_10_57_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12985-024-02557-x"}],"container-title":["Journal of Medical Virology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jmv.70205","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,4,12]],"date-time":"2025-04-12T03:20:17Z","timestamp":1744428017000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/jmv.70205"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2]]},"references-count":56,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2025,2]]}},"alternative-id":["10.1002\/jmv.70205"],"URL":"https:\/\/doi.org\/10.1002\/jmv.70205","archive":["Portico"],"relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2024.08.09.607374","asserted-by":"object"}]},"ISSN":["0146-6615","1096-9071"],"issn-type":[{"value":"0146-6615","type":"print"},{"value":"1096-9071","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2]]},"assertion":[{"value":"2024-09-11","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-01-23","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-02-03","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70205"}}