{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:46:00Z","timestamp":1790210760770,"version":"4.1.0"},"reference-count":37,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2017,1,21]],"date-time":"2017-01-21T00:00:00Z","timestamp":1484956800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National key projects of \u201c863\u201d High Technology of P. R. China","award":["2012AA02A401"],"award-info":[{"award-number":["2012AA02A401"]}]},{"name":"Chinese mega project of science research for major new drug innovation and developmental research for quality control of JE live attenuated vaccine and polio vaccine","award":["2014ZX09304316-003"],"award-info":[{"award-number":["2014ZX09304316-003"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The attenuated Japanese encephalitis virus (JEV) strain SA14-14-2 has been successfully utilized to prevent JEV infection; however, the attenuation determinants have not been fully elucidated. The envelope (E) protein of the attenuated JEV SA14-14-2 strain differs from that of the virulent parental SA14 strain at eight amino acid positions (E107, E138, E176, E177, E264, E279, E315, and E439). Here, we investigated the SA14-14-2-attenuation determinants by mutating E107, E138, E176, E177, and E279 in SA14-14-2 to their status in the parental virulent strain and tested the replication capacity, neurovirulence, neuroinvasiveness, and mortality associated with the mutated viruses in mice, as compared with those of JEV SA14-14-2 and SA14. Our findings indicated that revertant mutations at the E138 or E107 position significantly increased SA14-14-2 virulence, whereas other revertant mutations exhibited significant increases in neurovirulence only when combined with E138, E107, and other mutations. Revertant mutations at all eight positions in the E protein resulted in the highest degree of SA14-14-2 virulence, although this was still lower than that observed in SA14. These results demonstrated the critical role of the viral E protein in controlling JEV virulence and identified the amino acids at the E107 and E138 positions as the key determinants of SA14-14-2 neurovirulence.<\/jats:p>","DOI":"10.3390\/v9010020","type":"journal-article","created":{"date-parts":[[2017,1,23]],"date-time":"2017-01-23T10:40:33Z","timestamp":1485168033000},"page":"20","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":51,"title":["Envelope Protein Mutations L107F and E138K Are Important for Neurovirulence Attenuation for Japanese Encephalitis Virus SA14-14-2 Strain"],"prefix":"10.3390","volume":"9","author":[{"given":"Jian","family":"Yang","sequence":"first","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"},{"name":"Department of Microbiology and Immunology, North Sichuan Medical College, Nanchong 637007, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huiqiang","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhushi","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lina","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianzhi","family":"Ni","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyu","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Arbovirus Vaccine, National Institutes for Food and Drug Control, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xianwu","family":"Zeng","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yonglin","family":"Wu","sequence":"additional","affiliation":[{"name":"China National Biotech Group, Beijing 100029, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3806-8046","authenticated-orcid":false,"given":"Yuhua","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Viral Vaccine, Chengdu Institute of Biological Products Co., Ltd., China National Biotech Group, Chengdu 610023, China"},{"name":"Department of Arbovirus Vaccine, National Institutes for Food and Drug Control, Beijing 100050, China"},{"name":"State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu 610000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,1,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1016\/0264-410X(92)90051-K","article-title":"Antigenic characterization of the live attenuated Japanese encephalitis vaccine virus SA14-14-2: A comparison with isolates of the virus covering a wide geographic area","volume":"10","author":"Wills","year":"1992","journal-title":"Vaccine"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1366","DOI":"10.1086\/517323","article-title":"Short-term safety of live attenuated Japanese encephalitis vaccine (SA14-14-2): Results of a randomized trial with 26,239 subjects","volume":"176","author":"Liu","year":"1997","journal-title":"J. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1505","DOI":"10.1099\/0022-1317-75-6-1505","article-title":"Comparison of nucleotide and deduced amino acid sequence of the 5\u2019 non-coding region and structural protein genes of the wild-type Japanese encephalitis virus strain SA14 and its attenuated vaccine derivatives","volume":"75","author":"Ni","year":"1994","journal-title":"J. Gen. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1099\/0022-1317-76-2-409","article-title":"Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14","volume":"76","author":"Ni","year":"1995","journal-title":"J. Gen. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3635","DOI":"10.1016\/j.vaccine.2010.02.105","article-title":"Phenotypic and genotypic characteristics of Japanese encephalitis attenuated live vaccine virus SA14-14-2 and their stabilities","volume":"28","author":"Yu","year":"2010","journal-title":"Vaccine"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.virol.2007.04.016","article-title":"JE Nakayama\/JE SA14-14-2 virus structural region intertypic viruses: Biological properties in the mouse model of neuroinvasive disease","volume":"366","author":"Chambers","year":"2007","journal-title":"Virology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1093\/infdis\/169.3.512","article-title":"Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis","volume":"169","author":"Jennings","year":"1994","journal-title":"J. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1006\/viro.1995.1268","article-title":"Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus vaccine grown under different cell culture conditions","volume":"209","author":"Taffs","year":"1995","journal-title":"Virology"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3655","DOI":"10.1128\/JVI.77.6.3655-3668.2003","article-title":"Yellow fever virus\/dengue-2 virus and yellow fever virus\/dengue-4 virus chimeras: Biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model","volume":"77","author":"Chambers","year":"2003","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3095","DOI":"10.1128\/JVI.73.4.3095-3101.1999","article-title":"Yellow fever\/Japanese encephalitis chimeric viruses: Construction and biological properties","volume":"73","author":"Chambers","year":"1999","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3131","DOI":"10.1099\/vir.0.81909-0","article-title":"Chimeric Japanese encephalitis virus\/dengue 2 virus infectious clone: Biological properties, immunogenicity, and protection against dengue encephalitis in mice","volume":"87","author":"Chambers","year":"2006","journal-title":"J. Gen. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1006\/viro.1996.0406","article-title":"Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: Effects on virus replication and mouse neurovirulence","volume":"222","author":"Muylaert","year":"1996","journal-title":"Virology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/S0168-1702(01)00371-9","article-title":"A \u201cminimal\u201d approach in design of flavivirus infectious DNA","volume":"81","author":"Mishin","year":"2001","journal-title":"Virus Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/S0065-3527(08)60348-6","article-title":"Functional cDNA clones of the Flaviviridae: Strategies and applications","volume":"53","author":"Ruggli","year":"1999","journal-title":"Adv. Virus Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S0166-0934(01)00331-7","article-title":"Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector","volume":"96","author":"Zhang","year":"2001","journal-title":"J. Virol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5425","DOI":"10.1128\/jvi.66.9.5425-5431.1992","article-title":"Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates","volume":"66","author":"Sumiyoshi","year":"1992","journal-title":"J. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6450","DOI":"10.1128\/JVI.77.11.6450-6465.2003","article-title":"Development and application of a reverse genetics system for Japanese encephalitis virus","volume":"77","author":"Yun","year":"2003","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/S0065-3527(03)61013-4","article-title":"Chimeric flaviviruses: Novel vaccines against dengue fever, tick-borne encephalitis, and Japanese encephalitis","volume":"61","author":"Lai","year":"2003","journal-title":"Adv. Virus Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1016\/0042-6822(90)90519-W","article-title":"Nucleotide sequence of the virulent SA-14 strain of Japanese encephalitis virus and its attenuated vaccine derivative, SA-14-14-2","volume":"177","author":"Nitayaphan","year":"1990","journal-title":"Virology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1099\/vir.0.80638-0","article-title":"Characterization of the E-138 (Glu\/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone","volume":"86","author":"Zhao","year":"2005","journal-title":"J. Gen. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1590\/S0100-879X1997000200002","article-title":"The yellow fever 17D vaccine virus: Molecular basis of viral attenuation and its use as an expression vector","volume":"30","author":"Galler","year":"1997","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref_22","first-page":"19","article-title":"Genetic property of attenuated Japanese encephalitis virus strain SA14-14-2 after subculture in suckling mouse brain","volume":"20","author":"Wu","year":"2007","journal-title":"Chin. J. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1006\/viro.1999.9859","article-title":"Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus","volume":"261","author":"Kolaskar","year":"1999","journal-title":"Virology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/0042-6822(89)90509-6","article-title":"Synthetic peptides derived from the deduced amino acid sequence of the E-glycoprotein of Murray Valley encephalitis virus elicit antiviral antibody","volume":"171","author":"Roehrig","year":"1989","journal-title":"Virology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"668","DOI":"10.1016\/0042-6822(90)90532-V","article-title":"Antibodies to dengue 2 virus E-glycoprotein synthetic peptides identify antigenic conformation","volume":"177","author":"Roehrig","year":"1990","journal-title":"Virology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1006\/viro.1993.1469","article-title":"Differences between cell membrane fusion activities of two dengue type-1 isolates reflect modifications of viral structure","volume":"196","author":"Despres","year":"1993","journal-title":"Virology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1038\/375291a0","article-title":"The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution","volume":"375","author":"Rey","year":"1995","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1006\/viro.1996.0457","article-title":"Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro","volume":"223","author":"Chen","year":"1996","journal-title":"Virology"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1099\/0022-1317-79-3-437","article-title":"Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA","volume":"79","author":"Gualano","year":"1998","journal-title":"J. Gen. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/0042-6822(92)90177-Q","article-title":"Mutations in the envelope protein of Japanese encephalitis virus affect entry into cultured cells and virulence in mice","volume":"191","author":"Hasegawa","year":"1992","journal-title":"Virology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1099\/0022-1317-76-4-865","article-title":"Neurovirulence and neuroinvasiveness of Murray Valley encephalitis virus mutants selected by passage in a monkey kidney cell line","volume":"76","author":"McMinn","year":"1995","journal-title":"J. Gen. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.1093\/infdis\/171.5.1144","article-title":"Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA","volume":"171","author":"Sumiyoshi","year":"1995","journal-title":"J. Infect. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1932","DOI":"10.1128\/JVI.76.4.1932-1943.2002","article-title":"Single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: Relevance to development and safety testing of live, attenuated vaccines","volume":"76","author":"Monath","year":"2002","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1099\/0022-1317-77-9-2085","article-title":"A mouse-attenuated envelope protein variant of Murray Valley encephalitis virus with altered fusion activity","volume":"77","author":"McMinn","year":"1996","journal-title":"J. Gen. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"934","DOI":"10.1128\/JVI.75.2.934-942.2001","article-title":"Molecular basis for attenuation of neurovirulence of a yellow fever virus\/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE)","volume":"75","author":"Arroyo","year":"2001","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Yun, S.I., Song, B.H., Kim, J.K., Yun, G.N., Lee, E.Y., Li, L., Kuhn, R.J., Rossmann, M.G., Morrey, J.D., and Lee, Y.M. (2014). A molecularly cloned, live-attenuated Japanese encephalitis vaccine SA14\u201314\u20132 virus: A conserved single amino acid in the ij hairpin of the viral E glycoprotein determines neurovirulence in mice. PLoS Pathog., 10.","DOI":"10.1371\/journal.ppat.1004290"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2385","DOI":"10.1128\/JVI.03116-13","article-title":"A single-amino-acid polymorphism in Chikungunya virus E2 glycoprotein influences glycosaminoglycan utilization","volume":"88","author":"Silva","year":"2014","journal-title":"J. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/9\/1\/20\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:26:42Z","timestamp":1760207202000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/9\/1\/20"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,1,21]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2017,1]]}},"alternative-id":["v9010020"],"URL":"https:\/\/doi.org\/10.3390\/v9010020","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,1,21]]}}}