title: The A66G back mutation in NS2A of JEV SA14-14-2 strain contributes to production of NS1' protein and the secreted NS1' can be used for diagnostic biomarker for virulent virus infection.
abstract: Japanese encephalitis virus (JEV) is the most common cause of the prevalent encephalitis in Asia-Pacific region and poses a serious risk to public health. Here, we developed a reliable reverse genetics system based on the JEV SA14-14-2 strain to further explore the mechanism for the synthesis of NS1' protein and to investigate the function of NS1' protein during virus infection. NS1' is an additional form of NS1 protein with 52 amino acid carboxy-terminal extension and is expressed by the members of the Japanese encephalitis (JE) serogroup due to the translation frameshift. A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1'. The NS1' protein has no significant effect on the virus replication properties in BHK-21 cells. Animal experiments demonstrated that the NS1' protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain. But the NS1'-expressing virus (rA66G) could induce a higher humoral immune response than the NS1'-non-expressing virus (rSA14-14-2). NS1' protein can be detected in the serum of JEV rA66G infected animal and in the cultural media of that infected mammalian cells. Interesting, only the dimer of NS1' can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1' was in agreement with that of the secreted virion. In comparison with the live-attenuated JE vaccine strain which is incapable of formation of NS1', most of the virulent JEV strains produce the NS1' protein. And the secreted NS1' may serve as an early surrogate biomarker for viremia to distinguish the field infection from the vaccine inoculation. In total, in the present study, we identified the nt 66 in the viral NS2A gene as one of the critical site for the -1 programmed ribosomal frameshift to produce the NS1' protein and demonstrated the secreted NS1' could be used for diagnostic biomarker during JEV infection.
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Jingman
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Jinyan
authors.initials: J
authors.lastname: Gu
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China. Electronic address: crb@njau.edu.cn.
authors.forename: Ruibing
authors.initials: R
authors.lastname: Cao
authors.affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
authors.forename: Puyan
authors.initials: P
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004195:Disease Models, Animal; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000276:immunology / Q000378:metabolism* / Q000472:pathogenicity; D004672:Encephalitis, Japanese / Q000175:diagnosis* / Q000276:immunology / Q000401:mortality / Q000821:virology*; D005814:Genes, Viral; D016679:Genome, Viral; D006801:Humans; D056724:Immunity, Humoral; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D014774:Virulence; D014779:Virus Replication
chemical_list: D015415:Biomarkers; D018076:DNA, Complementary; C100315:NS1 protein, Flavivirus; D017361:Viral Nonstructural Proteins
keywords: Diagnostic biomarker; Humoral immune response; Japanese encephalitis virus; NS1′ protein; Neurovirulence