title: Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain.
abstract: The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV.
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Yan
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Xuesong
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Pixi
authors.initials: P
authors.lastname: Yan
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Xuyuan
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Yuee
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Chaochao
authors.initials: C
authors.lastname: Ren
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Guoxin
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Yan
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Qiaoyang
authors.initials: Q
authors.lastname: Teng
authors.affiliation: Department of Avian Infectious Disease, and Innovation Team for Pathogen Ecology Research on Animal Influenza Virus, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China.
authors.forename: Zejun
authors.initials: Z
authors.lastname: Li
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D004372:Ducks / Q000821:virology*; D005416:Flavivirus / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D059386:Reverse Genetics
chemical_list: D020413:3' Untranslated Regions; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins