{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T13:37:13Z","timestamp":1786541833105,"version":"build-2736575974"},"reference-count":52,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000030","name":"Centers for Disease Control and Prevention","doi-asserted-by":"publisher","award":["5U01GH002401-03"],"award-info":[{"award-number":["5U01GH002401-03"]}],"id":[{"id":"10.13039\/100000030","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Clin Microbiol"],"published-print":{"date-parts":[[2026,8,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      The co-circulation and rapid expansion of the genus\n                      <jats:italic toggle=\"yes\">Orthoflavivirus<\/jats:italic>\n                      , including dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Yellow fever virus (YFV), and Japanese encephalitis virus (JEV), pose significant global health challenges. Developing inclusive pan-genus molecular diagnostics is hindered by high nucleotide divergence (&gt;25\u201330%) and the computational limitations of traditional multiple sequence alignment in detecting conserved motifs across large data sets. To overcome these limitations, we developed a systematic alignment-free design pipeline that uses rigorous\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      -mer analysis and compacted De Bruijn graphs. We analyzed 11,846 RefSeq viral genomes to identify phylogenetically conserved, functionally relevant signatures within the\n                      <jats:italic toggle=\"yes\">Orthoflavivirus<\/jats:italic>\n                      genus as a case study. The pipeline identified a conserved 600 bp region within the non-structural protein 5 gene, facilitating the design of a broad-spectrum TaqMan RT-qPCR assay. Analytical validation demonstrated limits of detection (LODs) of 1\u201310 copies\/\u00b5L for DENV1\u20134, ZIKV, JEV, and YFV, with no cross-reactivity against non-target pathogens; WNV was consistently detected at 1,000 copies\/\u00b5L in pilot experiments. In a clinical evaluation of archived samples, the assay achieved 97.33% overall accuracy. It demonstrated 100% sensitivity and specificity for DENV serotypes, yielding significantly earlier cycle threshold (Ct) values compared to a standard commercial kit, while ZIKV detection showed 100% specificity with 71.43% sensitivity. This study validates an alignment-free,\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      -mer-guided strategy for uncovering conserved diagnostic targets in highly variable viral genera, followed by local MSA-assisted primer-probe design. The resulting assay offers a robust tool for broad frontline surveillance, and the computational framework provides a scalable solution for future pandemic preparedness.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Mosquito-borne viruses like dengue, Zika, and West Nile pose a massive threat to global health. However, because these viruses are highly diverse and mutate rapidly, creating a single diagnostic test to detect all of them has been a challenge. Traditional software struggles to find shared genetic targets across such different viruses. To solve this, we developed a new computational approach. By analyzing over 11,000 viral genomes, we successfully identified a shared genetic signature of a group of mosquito-borne viruses. We used this discovery to create a single, highly accurate test capable of detecting multiple dangerous mosquito-borne viruses at once. This breakthrough provides a crucial, ready-to-use tool for frontline clinical diagnosis and global outbreak surveillance. More importantly, our strategy can be quickly adapted to design broad-spectrum tests for other rapidly mutating viruses, significantly strengthening our ability to prepare for and respond to future pandemics.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00388-26","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T14:05:50Z","timestamp":1785161150000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Alignment-free\n                    <i>k<\/i>\n                    -mer-guided design of a pan-\n                    <i>Orthoflavivirus<\/i>\n                    RT-qPCR assay"],"prefix":"10.1128","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-6859-0974","authenticated-orcid":false,"given":"Khanate","family":"Sayasit","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chutikarn","family":"Chaimayo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Warinya","family":"Nuwong","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tharathip","family":"Boondouylan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nattaya","family":"Tanlieng","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kamol","family":"Suwannakarn","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8901-1088","authenticated-orcid":true,"given":"Intawat","family":"Nookaew","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00xcryt71","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical Informatics, University of Arkansas for Medical Sciences","place":["Little Rock, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9109-1259","authenticated-orcid":false,"given":"Navin","family":"Horthongkham","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University","place":["Bangkok, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms13071590"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijid.2025.107940"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1186\/s40249-025-01365-x"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature12060"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-58609-5"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.51027"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.2012.11-0473"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.15537\/1658-3175.8483"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2022.975398"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens12050643"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1155\/2017\/4248756"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/s44298-025-00114-z"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3002834"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.3390\/v16111653"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1101\/gr.279449.124"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-024-03320-9"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1051\/bioconf\/202516301008"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1603\/me11287"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0239381"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biodatasci-080917-013431"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-016-0997-x"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.12688\/f1000research.19675.1"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv271"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-017-1319-7"},{"key":"e_1_3_4_26_2","unstructured":"NCfBI N. 2004. NCBI virus bethesda (MD): national library of medicine (US). https:\/\/www.ncbi.nlm.nih.gov\/labs\/virus\/vssi\/#\/."},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jgg.2021.03.006"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.3390\/v8030066"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2020.556413"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/srep40712"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btr011"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2024.04.035"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-020-02135-8"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2009-10-3-r25"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp352"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btq033"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/mst010"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkl175"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.2353\/jmoldx.2010.090035"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkn198"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-83-10-2443"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1099\/jgv.0.000746"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-83879-6"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-025-02134-0"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens15020160"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02506-18"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2020.00453"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02042-09"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.01215-24"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00343-20"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.estlett.0c00535"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciw589"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.19-0881"}],"container-title":["Journal of Clinical Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/jcm.00388-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/jcm.00388-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T13:01:57Z","timestamp":1786539717000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/jcm.00388-26"}},"subtitle":[],"editor":[{"given":"Romney M.","family":"Humphries","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,12]]},"references-count":52,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8,12]]}},"alternative-id":["10.1128\/jcm.00388-26"],"URL":"https:\/\/doi.org\/10.1128\/jcm.00388-26","relation":{},"ISSN":["0095-1137","1098-660X"],"issn-type":[{"value":"0095-1137","type":"print"},{"value":"1098-660X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,12]]},"assertion":[{"value":"2026-03-17","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-06-29","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-27","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00388-26"}}