{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T06:58:45Z","timestamp":1791874725142,"version":"4.3.4"},"reference-count":33,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,3,29]],"date-time":"2022-03-29T00:00:00Z","timestamp":1648512000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Zhiyong Ma","award":["No. 22N41900400"],"award-info":[{"award-number":["No. 22N41900400"]}]},{"name":"Jianchao Wei","award":["No. Y2020PT40"],"award-info":[{"award-number":["No. Y2020PT40"]}]},{"name":"Jianchao Wei","award":["No. 19ZR1469000"],"award-info":[{"award-number":["No. 19ZR1469000"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Nonstructural protein 2A (NS2A) of the Japanese encephalitis virus (JEV) contributes to viral replication and pathogenesis; however, a lack of NS2A-specific antibodies restricts studies on the underlying mechanisms. In this study, we constructed a recombinant JEV with a hemagglutinin (HA)-tagged NS2A (JEV-HA\/NS2A\/\u2206NS1\u2019) to overcome this challenge. An HA-tag was fused to the N-terminus of NS2A (HA-NS2A) at the intergenic junction between NS1 and NS2A. A peptide linker, \u201cFNG\u201d, was added to the N-terminus of HA-tag to ensure correct cleavage between the C-terminus of NS1 and the N-terminus of HA-NS2A. To avoid the side effects of an unwanted NS1\u2019 tagged with HA (HA-NS1\u2019), an alanine-to-proline (A30P) substitution was introduced at residue 30 of NS2A to abolish HA-NS1\u2019 production. The HA-tag insertion and A30P substitution were stably present in JEV-HA\/NS2A\/\u2206NS1\u2019 after six passages and did not exhibit any significant effects on viral replication and plaque morphology. Taking advantage of HA-NS2A, we examined the activities of NS2A during JEV infection in vitro using anti-HA antibodies. NS2A was observed to be localized to the endoplasmic reticulum and interact with viral NS2B and NS3 during virus infection. These data suggest that JEV-HA\/NS2A\/\u2206NS1\u2019 can serve as a model for the analysis of the biological characteristics and functions of NS2A in vitro during JEV infection.<\/jats:p>","DOI":"10.3390\/v14040706","type":"journal-article","created":{"date-parts":[[2022,3,29]],"date-time":"2022-03-29T21:45:51Z","timestamp":1648590351000},"page":"706","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Construction of a Recombinant Japanese Encephalitis Virus with a Hemagglutinin-Tagged NS2A: A Model for an Analysis of Biological Characteristics and Functions of NS2A during Viral Infection"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2477-4874","authenticated-orcid":false,"given":"Xiaochun","family":"Ma","sequence":"first","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenxi","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiqi","family":"Xia","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2216-5749","authenticated-orcid":false,"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Yang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1919-3653","authenticated-orcid":false,"given":"Abdul","family":"Wahaab","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Liu","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zongjie","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beibei","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4211-0817","authenticated-orcid":false,"given":"Yafeng","family":"Qiu","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3246-6273","authenticated-orcid":false,"given":"Jianchao","family":"Wei","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiyong","family":"Ma","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S98","DOI":"10.1038\/nm1144","article-title":"Emerging flaviviruses: The spread and resurgence of Japanese encephalitis, West Nile and dengue viruses","volume":"10","author":"Mackenzie","year":"2004","journal-title":"Nat. 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