title: A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.
abstract: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.
authors: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
authors: Sang-Im
authors: SI
authors: Yun
authors: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
authors: Byung-Hak
authors: BH
authors: Song
authors: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
authors: Jin-Kyoung
authors: JK
authors: Kim
authors: Department of Microbiology, College of Medicine, Chungbuk National University, Cheongju, Republic of Korea.
authors: Gil-Nam
authors: GN
authors: Yun
authors: Department of Anatomy, College of Medicine, Chungbuk National University, Cheongju, Republic of Korea.
authors: Eun-Young
authors: EY
authors: Lee
authors: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America.
authors: Long
authors: L
authors: Li
authors: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America.
authors: Richard J
authors: RJ
authors: Kuhn
authors: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America.
authors: Michael G
authors: MG
authors: Rossmann
authors: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
authors: John D
authors: JD
authors: Morrey
authors: Department of Animal, Dairy, and Veterinary Sciences; Utah Science Technology and Research, College of Agriculture and Applied Sciences, Utah State University, Logan, Utah, United States of America.
authors: Young-Min
authors: YM
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000276:immunology; D004672:Encephalitis, Japanese / Q000235:genetics / Q000276:immunology / Q000821:virology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D022321:Japanese Encephalitis Vaccines / Q000235:genetics* / Q000276:immunology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009420:Nervous System / Q000821:virology*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014774:Virulence / Q000235:genetics; D014779:Virus Replication
chemical_list: D022321:Japanese Encephalitis Vaccines; D008562:Membrane Glycoproteins; D014613:Vaccines, Attenuated; D014759:Viral Envelope Proteins; C080628:glycoprotein E, Japanese encephalitis virus