title: Genetic instability of Japanese encephalitis virus cDNA clones propagated in Escherichia coli.
abstract: The genetic instability of Flavivirus cDNA clones in transformed bacteria is a common phenomenon. Herein, a cDNA fragment of the nucleotide (nt) 1-2913 of the genome of a flavivirus, Japanese encephalitis virus (JEV), was used to investigate factors that caused the instability of cDNA clones. Several cDNA fragments with different 5'- or 3'-termini of the 2913-nt cDNA were obtained by PCR amplification or restriction enzyme digestion and cloned into a pCR-Blunt II-TOPO vector. All the cDNA fragments were stably propagated at 25 °C. However, the 5'-untranslated region and half of the 3'-E gene could cause the instability of the 2913-nt cDNA at 37 °C. The 5'-terminus sequences of the 2913-nt fragment were subjected to testing of the prokaryotic promoter activity by luciferase assay and Western blot. The sequences of 54-120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. These findings revealed that the high prokaryotic promoter activity of the 54-120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5'-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria.
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Xuchen
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Wu
authors.initials: W
authors.lastname: Tong
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liang
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.
authors.forename: Guoxin
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China.|Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Tong
authors.affiliation: Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Shanghai, 200241, China. haozheng@shvri.ac.cn.|Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China. haozheng@shvri.ac.cn.
authors.forename: Hao
authors.identifier: http://orcid.org/0000-0001-8206-6633
authors.initials: H
authors.lastname: Zheng
mesh_terms: D020121:5' Untranslated Regions; D018387:Codon, Initiator; D018076:DNA, Complementary; D004664:Encephalitis Virus, Japanese / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D042822:Genomic Instability; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D018387:Codon, Initiator; D018076:DNA, Complementary; D012367:RNA, Viral
keywords: Genetic instability; Japanese encephalitis virus; Prokaryotic promoter