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J. Mol. Sci.","IJMS"],"abstract":"<jats:p>The Zika virus (ZIKV) remains an important public health concern owing to its association with neurological disorders and congenital abnormalities. Genetically, ZIKV is classified into two major lineages\u2014African and Asian\u2014and continues to evolve, with evidence of genetic diversification following its introduction into the Americas. The study of the envelope (E) gene of ZIKV is a key tool for understanding viral biology, phylogenetic analysis, and developing antiviral drugs and vaccine candidates. However, few studies have focused on designing primers capable of efficiently detecting and amplifying this essential gene. In this study, we developed universal primer sets capable of amplifying and sequencing the full-length ZIKV E gene across genetically diverse lineages and strains. Primers were designed based on representative ZIKV sequences available in public databases and validated using viral isolates and in vitro-spiked plasma samples. The assay demonstrated high specificity and analytical sensitivity, with the analytical detection limit established using serially diluted DNA amplicons from the target region. Furthermore, ZIKV was successfully detected in plasma samples spiked at 1 \u00d7 103 PFU\/mL, which enabled complete sequencing of the E gene. These findings highlight the utility of our primers for molecular epidemiological studies, strengthening arbovirus surveillance and improving diagnostic capacity in regions where multiple arboviruses co-circulate, using a simple and reliable assay.<\/jats:p>","DOI":"10.3390\/ijms27188017","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T10:19:16Z","timestamp":1788949156000},"page":"8017","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of Universal Primer Sets for Zika Virus Envelope Gene Amplification and Sequencing"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8971-2204","authenticated-orcid":false,"given":"L\u00e9o Shigueki","family":"Sato","sequence":"first","affiliation":[{"name":"Department of Virology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki 852-8523, Japan"},{"name":"Graduate School of 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Thailand","volume":"13","author":"Phumee","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Faye, O., Freire, C.C.M., Iamarino, A., Faye, O., de Oliveira, J.V.C., Diallo, M., Zanotto, P.M.A., and Sall, A.A. (2014). Molecular Evolution of Zika Virus during Its Emergence in the 20th Century. PLoS Negl. Trop. Dis., 8.","DOI":"10.1371\/journal.pntd.0002636"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1007\/s00705-023-05835-1","article-title":"Renaming of the Genus Flavivirus to Orthoflavivirus and Extension of Binomial Species Names within the Family Flaviviridae","volume":"168","author":"Postler","year":"2023","journal-title":"Arch. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1146\/annurev-pathmechdis-031521-034739","article-title":"Pathogenesis of Zika Virus Infection","volume":"18","author":"Giraldo","year":"2023","journal-title":"Annu. Rev. Pathol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1126\/science.aaf5316","article-title":"The 3.8 \u00c5 Resolution Cryo-EM Structure of Zika Virus","volume":"352","author":"Sirohi","year":"2016","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.jneuroim.2017.03.001","article-title":"Zika Virus: History, Epidemiology, Transmission, and Clinical Presentation","volume":"308","author":"Song","year":"2017","journal-title":"J. Neuroimmunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1552","DOI":"10.1056\/NEJMra1602113","article-title":"Zika Virus","volume":"374","author":"Petersen","year":"2016","journal-title":"N. Engl. J. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.antiviral.2016.03.010","article-title":"Zika Virus: History, Emergence, Biology, and Prospects for Control","volume":"130","author":"Weaver","year":"2016","journal-title":"Antivir. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"675","DOI":"10.2471\/BLT.16.171082","article-title":"Zika: The Origin and Spread of a Mosquito-Borne Virus","volume":"94","author":"Kindhauser","year":"2016","journal-title":"Bull. World Health Organ."},{"key":"ref_10","unstructured":"WHO (2025, April 15). Zika Virus Disease Outbreak 2015\u20132016. Available online: https:\/\/www.who.int\/emergencies\/situations\/zika-virus-outbreak."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2536","DOI":"10.1056\/NEJMoa0805715","article-title":"Zika Virus Outbreak on Yap Island, Federated States of Micronesia","volume":"360","author":"Duffy","year":"2009","journal-title":"N. Engl. J. Med."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Santana, L.M.B., de Moura, I.A., Mouzinho Ramos Tanaka, Y., and Fran\u00e7a, R.F.d.O. (2025). Zika Virus: A Review of Biology, Clinical Impacts, and Coinfections. Viruses, 17.","DOI":"10.3390\/v17050637"},{"key":"ref_13","first-page":"e3290","article-title":"A Comprehensive Review of the Manifestations and Pathogenesis of Zika Virus in Neonates and Adults","volume":"10","author":"Hussain","year":"2018","journal-title":"Cureus"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.meegid.2019.01.023","article-title":"Zika Virus Induces Abnormal Cranial Osteogenesis by Negatively Affecting Cranial Neural Crest Development","volume":"69","author":"Yan","year":"2019","journal-title":"Infect. Genet. Evol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1007\/s11910-020-01072-0","article-title":"Congenital Zika Virus Infection: A Review with Emphasis on the Spectrum of Brain Abnormalities","volume":"20","author":"Pinho","year":"2020","journal-title":"Curr. Neurol. Neurosci. Rep."},{"key":"ref_16","unstructured":"Wolford, R.W., and Schaefer, T.J. (2025, June 11). Zika Virus\u2014StatPearls\u2014NCBI Bookshelf, Available online: https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK430981\/."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1016\/j.chom.2016.04.006","article-title":"From Mosquitos to Humans: Genetic Evolution of Zika Virus","volume":"19","author":"Wang","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1126\/science.aaf5036","article-title":"Zika Virus in the Americas: Early Epidemiological and Genetic Findings","volume":"352","author":"Faria","year":"2016","journal-title":"Science"},{"key":"ref_19","first-page":"1","article-title":"Zika Virus Evolution on the Edges of the Pacific Ocean","volume":"6","author":"Diancourt","year":"2017","journal-title":"Emerg. Microbes Infect."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00292-17","DOI":"10.1128\/mSphere.00292-17","article-title":"Phenotypic Differences between Asian and African Lineage Zika Viruses in Human Neural Progenitor Cells","volume":"2","author":"Anfasa","year":"2017","journal-title":"MSphere"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1128\/genomeA.00800-16","article-title":"Complete Genome Sequences of Three Historically Important, Spatiotemporally Distinct, and Genetically Divergent Strains of Zika Virus: MR-766, P6-740, and PRVABC-59","volume":"4","author":"Yun","year":"2016","journal-title":"Genome Announc."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.meegid.2016.05.004","article-title":"Genomic Characterization and Phylogenetic Analysis of Zika Virus Circulating in the Americas","volume":"43","author":"Ye","year":"2016","journal-title":"Infect. Genet. Evol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1601","DOI":"10.1007\/s42770-020-00349-3","article-title":"Insights on Zika Virus Envelope Gene Conservation in American Outbreaks","volume":"51","author":"Thomazella","year":"2020","journal-title":"Braz. J. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"114459","DOI":"10.1016\/j.jviromet.2022.114459","article-title":"Differential Detection of Zika Virus Based on PCR","volume":"301","author":"Alzate","year":"2022","journal-title":"J. Virol. Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1590\/0074-02760170248","article-title":"Simple Protocol for Population (Sanger) Sequencing for Zika Virus Genomic Regions","volume":"113","author":"Cabral","year":"2017","journal-title":"Mem. Inst. Oswaldo Cruz"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1900606","DOI":"10.2807\/1560-7917.ES.2021.26.9.1900606","article-title":"Economic Evaluation of Whole Genome Sequencing for Pathogen Identification and Surveillance\u2014Results of Case Studies in Europe and the Americas 2016 to 2019","volume":"26","author":"Alleweldt","year":"2021","journal-title":"Eurosurveillance"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Pati\u00f1o, L., Ben\u00edtez, A.D., Carrazco-Montalvo, A., and Regato-Arrata, M. (2024). Genomics for Arbovirus Surveillance: Considerations for Routine Use in Public Health Laboratories. Viruses, 16.","DOI":"10.3390\/v16081242"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e7221","DOI":"10.1590\/1414-431x20187221","article-title":"Searching for the Best Real-Time RT-PCRs to Detect Zika Virus Infections: The Importance of Comparing Several Protocols","volume":"51","author":"Klein","year":"2018","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"880","DOI":"10.2471\/BLT.16.175950","article-title":"Assay Optimization for Molecular Detection of Zika Virus","volume":"94","author":"Corman","year":"2016","journal-title":"Bull. World Health Organ."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1658","DOI":"10.1093\/bioinformatics\/btl158","article-title":"Cd-Hit: A Fast Program for Clustering and Comparing Large Sets of Protein or Nucleotide Sequences","volume":"22","author":"Li","year":"2006","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1093\/molbev\/mst010","article-title":"MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability","volume":"30","author":"Katoh","year":"2013","journal-title":"Mol. Biol. Evol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e01239-16","DOI":"10.1128\/mBio.01239-16","article-title":"How Did Zika Virus Emerge in the Pacific Islands and Latin America?","volume":"7","author":"Pettersson","year":"2016","journal-title":"mBio"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"622","DOI":"10.1016\/S2352-4642(25)00168-3","article-title":"Comparison of Dengue, Chikungunya, and Zika among Children in Nicaragua across 18 Years: A Single-Centre, Prospective Cohort Study","volume":"9","author":"Carrillo","year":"2025","journal-title":"Lancet Child Adolesc. Health"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Bhadra, S., Salda\u00f1a, M.A., Han, H.G., Hughes, G.L., and Ellington, A.D. (2018). Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay. Viruses, 10.","DOI":"10.3390\/v10120714"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1177\/1040638720905833","article-title":"Guidelines for Sanger Sequencing and Molecular Assay Monitoring","volume":"32","author":"Crossley","year":"2020","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Liang, D., Leung, R.K.K., Lee, S.S., and Kam, K.M. (2017). Insights into Intercontinental Spread of Zika Virus. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0176710"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.jviromet.2019.01.011","article-title":"Multiplex Real-Time RT-PCR for Detection and Distinction of Spondweni and Zika Virus","volume":"266","author":"Rademan","year":"2019","journal-title":"J. Virol. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"20761","DOI":"10.2807\/1560-7917.ES2014.19.14.20761","article-title":"Potential for Zika Virus Transmission through Blood Transfusion Demonstrated during an Outbreak in French Polynesia, November 2013 to February 2014","volume":"19","author":"Musso","year":"2014","journal-title":"Euro Surveill."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3410","DOI":"10.1038\/s41598-018-21224-0","article-title":"Direct DNA and RNA Detection from Large Volumes of Whole Human Blood","volume":"8","author":"Cai","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1128\/JCM.39.2.485-493.2001","article-title":"Purification and Characterization of PCR-Inhibitory Components in Blood Cells","volume":"39","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Latham, S., Hughes, E., Budgen, B., and Morley, A. (2023). Inhibition of the PCR by Genomic DNA. PLoS ONE, 18.","DOI":"10.1371\/journal.pone.0284538"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"eaag0538","DOI":"10.1126\/scitranslmed.aag0538","article-title":"Rapid and Specific Detection of Asian- and African-Lineage Zika Viruses","volume":"9","author":"Chotiwan","year":"2017","journal-title":"Sci. Transl. Med."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.jcv.2008.05.005","article-title":"One-Step RT-PCR for Detection of Zika Virus","volume":"43","author":"Faye","year":"2008","journal-title":"J. Clin. Virol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.1093\/bioinformatics\/btp033","article-title":"Jalview Version 2\u2014A Multiple Sequence Alignment Editor and Analysis Workbench","volume":"25","author":"Waterhouse","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"e115","DOI":"10.1093\/nar\/gks596","article-title":"Primer3\u2014New Capabilities and Interfaces","volume":"40","author":"Untergasser","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, T.L. (2012). Primer-BLAST: A Tool to Design Target-Specific Primers for Polymerase Chain Reaction. BMC Bioinform., 13.","DOI":"10.1186\/1471-2105-13-134"},{"key":"ref_47","unstructured":"Jullien, N. (2025, May 02). AmplifX: Manage, Test and Design Your Primers for PCR | Institute of NeuroPhysiopathology. Available online: https:\/\/inp.univ-amu.fr\/en\/amplifx-manage-test-and-design-your-primers-for-pcr."},{"key":"ref_48","unstructured":"(2025, October 16). National BioResource Project (NBRP) NBRP Human Pathogenic Viruses. Available online: https:\/\/www.tm.nagasaki-u.ac.jp\/nbrp-virus\/en\/."},{"key":"ref_49","unstructured":"European Virus Archive (EVA) (2026, July 02). Zika Virus 976 Uganda|EVA. Available online: https:\/\/www.european-virus-archive.com\/virus\/zika-virus-976-uganda."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1101\/gr.8.3.175","article-title":"Base-Calling of Automated Sequencer Traces Using Phred. I. Accuracy\u2009Assessment","volume":"8","author":"Ewing","year":"1998","journal-title":"Genome Res."},{"key":"ref_51","unstructured":"(2025, August 08). Phrap and Cross Match. Available online: http:\/\/www.phrap.org\/phredphrap\/phrap.html."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/18\/8017\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T04:11:51Z","timestamp":1789186311000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/18\/8017"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,9]]},"references-count":51,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ijms27188017"],"URL":"https:\/\/doi.org\/10.3390\/ijms27188017","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,9]]}}}