{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T02:55:34Z","timestamp":1788404134641,"version":"build-2803163510"},"reference-count":39,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,1,3]],"date-time":"2020-01-03T00:00:00Z","timestamp":1578009600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.<\/jats:p>","DOI":"10.3390\/v12010061","type":"journal-article","created":{"date-parts":[[2020,1,3]],"date-time":"2020-01-03T11:55:07Z","timestamp":1578052507000},"page":"61","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3564-3423","authenticated-orcid":false,"given":"Evgeniya","family":"Volkova","sequence":"first","affiliation":[{"name":"Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Konstantin A.","family":"Tsetsarkin","sequence":"additional","affiliation":[{"name":"Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6341-3879","authenticated-orcid":false,"given":"Emilia","family":"Sippert","sequence":"additional","affiliation":[{"name":"Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felipe","family":"Assis","sequence":"additional","affiliation":[{"name":"Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangping","family":"Liu","sequence":"additional","affiliation":[{"name":"Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Rios","sequence":"additional","affiliation":[{"name":"Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander G.","family":"Pletnev","sequence":"additional","affiliation":[{"name":"Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1038\/nature22401","article-title":"Establishment and cryptic transmission of Zika virus in Brazil and the Americas","volume":"546","author":"Faria","year":"2017","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1016\/j.ncl.2018.06.003","article-title":"Zika Virus and Neurologic Disease","volume":"36","author":"Reid","year":"2018","journal-title":"Neurol. Clin."},{"key":"ref_3","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_4","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1038\/s41586-018-0446-y","article-title":"The emergence of Zika virus and its new clinical syndromes","volume":"560","author":"Pierson","year":"2018","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"S857","DOI":"10.1093\/infdis\/jix383","article-title":"Pandemic Zika: A Formidable Challenge to Medicine and Public Health","volume":"216","author":"Morens","year":"2017","journal-title":"J. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1056\/NEJMp1600297","article-title":"Zika Virus in the Americas--Yet Another Arbovirus Threat","volume":"374","author":"Fauci","year":"2016","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/BF00301989","article-title":"RNA viruses as gene expression vectors","volume":"3","author":"Huang","year":"1989","journal-title":"Virus Genes"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"13","DOI":"10.3390\/v1010013","article-title":"Alphaviruses in gene therapy","volume":"1","author":"Lundstrom","year":"2009","journal-title":"Viruses"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9713","DOI":"10.1128\/jvi.71.12.9713-9721.1997","article-title":"Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1","volume":"71","author":"Smith","year":"1997","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.copbio.2014.11.009","article-title":"Updates in inducible transgene expression using viral vectors: From transient to stable expression","volume":"32","author":"Mortimer","year":"2015","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Li, Y., Li, L.F., Yu, S., Wang, X., Zhang, L., Yu, J., Xie, L., Li, W., Ali, R., and Qiu, H.J. (2016). Applications of Replicating-Competent Reporter-Expressing Viruses in Diagnostic and Molecular Virology. Viruses, 8.","DOI":"10.3390\/v8050127"},{"key":"ref_12","unstructured":"Knipe, D., and Howley, P. (2007). Flaviviridae: The Viruses and Their Replication in Fields Virology, Lippincott-Raven Publishers."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1089\/vbz.2009.0067","article-title":"Infection, dissemination, and transmission of a West Nile virus green fluorescent protein infectious clone by Culex pipiens quinquefasciatus mosquitoes","volume":"10","author":"McGee","year":"2010","journal-title":"Vector Borne Zoonotic Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.virol.2005.01.021","article-title":"An infectious West Nile virus that expresses a GFP reporter gene","volume":"334","author":"Pierson","year":"2005","journal-title":"Virology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"14610","DOI":"10.1073\/pnas.1212379109","article-title":"Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro","volume":"109","author":"Schoggins","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"912","DOI":"10.7150\/thno.16615","article-title":"Visualization of a neurotropic flavivirus infection in mouse reveals unique viscerotropism controlled by host type I interferon signaling","volume":"7","author":"Li","year":"2017","journal-title":"Theranostics"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2712","DOI":"10.1099\/jgv.0.000938","article-title":"Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate","volume":"98","author":"Mutso","year":"2017","journal-title":"J. Gen. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1016\/j.chom.2016.05.004","article-title":"An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors","volume":"19","author":"Shan","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"11737","DOI":"10.1128\/JVI.01112-07","article-title":"Production of pseudoinfectious yellow fever virus with a two-component genome","volume":"81","author":"Shustov","year":"2007","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1186\/1743-422X-4-115","article-title":"Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes","volume":"4","author":"Bonaldo","year":"2007","journal-title":"Virol. J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e01255-17","DOI":"10.1128\/mBio.01255-17","article-title":"Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control","volume":"8","author":"Grabowski","year":"2017","journal-title":"MBio"},{"key":"ref_22","unstructured":"Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e01114-16","DOI":"10.1128\/mBio.01114-16","article-title":"A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development","volume":"7","author":"Tsetsarkin","year":"2016","journal-title":"MBio"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5350","DOI":"10.1038\/s41467-018-07782-x","article-title":"Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice","volume":"9","author":"Tsetsarkin","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e02904-18","DOI":"10.1128\/mBio.02904-18","article-title":"Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus","volume":"10","author":"Tsetsarkin","year":"2019","journal-title":"MBio"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1016\/j.virol.2012.06.028","article-title":"A novel coding-region RNA element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in Aedes aegypti mosquitoes","volume":"432","author":"Orozco","year":"2012","journal-title":"Virology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3430","DOI":"10.1128\/JVI.02677-14","article-title":"Overlapping local and long-range RNA-RNA interactions modulate dengue virus genome cyclization and replication","volume":"89","author":"Villordo","year":"2015","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6804","DOI":"10.1128\/JVI.00243-13","article-title":"Novel cis-acting element within the capsid-coding region enhances flavivirus viral-RNA replication by regulating genome cyclization","volume":"87","author":"Liu","year":"2013","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1739","DOI":"10.3390\/v3091739","article-title":"Functional RNA elements in the dengue virus genome","volume":"3","author":"Gebhard","year":"2011","journal-title":"Viruses"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3406","DOI":"10.1093\/nar\/gkg595","article-title":"Mfold web server for nucleic acid folding and hybridization prediction","volume":"31","author":"Zuker","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3330","DOI":"10.1093\/nar\/gkw061","article-title":"Kissing-loop interaction between 5\u2032 and 3\u2032 ends of tick-borne Langat virus genome \u2018bridges the gap\u2019 between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: Applications for virus attenuation and vaccine development","volume":"44","author":"Tsetsarkin","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Samsa, M.M., Mondotte, J.A., Iglesias, N.G., Assuncao-Miranda, I., Barbosa-Lima, G., Da Poian, A.T., Bozza, P.T., and Gamarnik, A.V. (2009). Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog., 5.","DOI":"10.1371\/journal.ppat.1000632"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1401","DOI":"10.1016\/j.cell.2007.04.040","article-title":"A mammalian microRNA expression atlas based on small RNA library sequencing","volume":"129","author":"Landgraf","year":"2007","journal-title":"Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1021\/acs.bioconjchem.6b00112","article-title":"NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence","volume":"27","author":"England","year":"2016","journal-title":"Bioconjug. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.coviro.2014.09.011","article-title":"Regulation of Flavivirus RNA synthesis and replication","volume":"9","author":"Selisko","year":"2014","journal-title":"Curr. Opin. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"7034","DOI":"10.1093\/nar\/gkr237","article-title":"Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus","volume":"39","author":"Tuplin","year":"2011","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e02326-16","DOI":"10.1128\/mBio.02326-16","article-title":"Synergistic Internal Ribosome Entry Site\/MicroRNA-Based Approach for Flavivirus Attenuation and Live Vaccine Development","volume":"8","author":"Tsetsarkin","year":"2017","journal-title":"MBio"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2056","DOI":"10.1128\/JVI.03051-13","article-title":"Internal ribosome entry site-based attenuation of a flavivirus candidate vaccine and evaluation of the effect of beta interferon coexpression on vaccine properties","volume":"88","author":"Frese","year":"2014","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e01582-17","DOI":"10.1128\/JVI.01582-17","article-title":"Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag","volume":"92","author":"Tamura","year":"2018","journal-title":"J. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/12\/1\/61\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:18:49Z","timestamp":1760361529000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/12\/1\/61"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,3]]},"references-count":39,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,1]]}},"alternative-id":["v12010061"],"URL":"https:\/\/doi.org\/10.3390\/v12010061","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,1,3]]}}}