{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T01:18:28Z","timestamp":1785460708584,"version":"3.56.0"},"reference-count":127,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,10]],"date-time":"2026-01-10T00:00:00Z","timestamp":1768003200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Program of Natural Science Foundation of Hunan Province of China","award":["2024JJ3025"],"award-info":[{"award-number":["2024JJ3025"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Arthropod-borne orthoflaviviruses, including dengue, Zika, Japanese encephalitis, yellow fever and West Nile viruses, pose a significant global public health threat, causing hundreds of millions of infections annually with severe clinical symptoms. However, the lack of effective vaccines and antiviral drugs, coupled with the biosafety risks associated with handling live highly pathogenic strains, hinders progress in antiviral research. Pseudovirus technology, which uses single-round infectious viral particles lacking replication competence, has thus gained prominence as a safe and versatile tool for antiviral research. This review systematically summarizes the construction, optimization, and applications of orthoflavivirus pseudoviruses in antiviral research. The primary construction strategies of orthoflavivirus pseudoviruses rely on multi-plasmid co-transfection of viral replicons and structural protein expression vectors, leveraging the host cell secretory pathway to mimic natural viral assembly and maturation. The core applications of pseudovirus technology are highlighted, including high-throughput screening and detection of neutralizing antibodies, identification of antiviral drugs targeting viral entry or replication, and evaluation of vaccine immunogenicity. Despite these strengths, the approach still faces limitations, such as incomplete simulation of native viral structures and batch-to-batch titer variability, which may affect the physiological relevance of findings. In summary, orthoflavivirus pseudovirus technology has become an essential platform in both basic virology research and translational medicine, providing critical insights and tools in the ongoing fight against arthropod-borne orthoflaviviruses diseases.<\/jats:p>","DOI":"10.3390\/ijms27020722","type":"journal-article","created":{"date-parts":[[2026,1,12]],"date-time":"2026-01-12T09:13:01Z","timestamp":1768209181000},"page":"722","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Application of Orthoflavivirus Pseudovirus Technology in Antiviral Research"],"prefix":"10.3390","volume":"27","author":[{"given":"Yalan","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaqi","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaojun","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanyuan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1050-0693","authenticated-orcid":false,"given":"Hao","family":"Yuan","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaodan","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4916-7368","authenticated-orcid":false,"given":"Yong","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4726-5095","authenticated-orcid":false,"given":"Xiaoling","family":"Pan","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Science, School of Medical Technology and Translational Medicine, Hunan Normal University Health Science Center, 371 Tongzipo Road, Changsha 410013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1890","DOI":"10.1016\/S0140-6736(20)31375-1","article-title":"The Lancet Commission on dengue and other Aedes-transmitted viral diseases","volume":"395","author":"Lindsay","year":"2020","journal-title":"Lancet"},{"key":"ref_2","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. 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