title: Generation of a reliable full-length cDNA of infectiousTembusu virus using a PCR-based protocol.
abstract: Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV.
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Te
authors.initials: T
authors.lastname: Liang
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Shulin
authors.initials: S
authors.lastname: Cui
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Shenghua
authors.initials: S
authors.lastname: Qu
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Fumin
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Kang
authors.initials: K
authors.lastname: Ning
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China. Electronic address: Zhangdb@cau.edu.cn.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China. Electronic address: zdb@cau.edu.cn.
authors.forename: Dabing
authors.initials: D
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D005416:Flavivirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D005822:Genetic Vectors; D042822:Genomic Instability; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral
keywords: Duck; Full-length cDNA; Tembusu virus; Virus rescue