title: [Sequencing the complete genome of rabies virus CVS-11 strain and constructing its full-length infectious cDNA clone].
abstract: OBJECTIVE
  To sequence the complete genome of CVS-11 strain and establish a reverse genetic system of CVS-11 to further study its pathogenic mechanism, virulence genes and antigenic sites.
  
  
  METHODS
  We amplified12 fragments covering the complete genome of the CVS-11 strain by RT-PCR, and then cloned to pEASY-Blunt vector for sequencing the complete genome of CVS-11. We analyzed single restriction enzyme sites of the full length cDNA of the CVS-11 strain by DNAMAN and designed 4 pairs of specific primers. We amplified the full-length cDNA of CVS-11 by RT-PCR. We obtained four fragments and cloned into pcDNA3. 1. We named the full-length cDNA plasmid pcDNA3. 1-CVS-11. We also cloned helper plasmids pcDNA3.1-N, P, L and G expressing N, P, L and G protein of CVS-11 strain, respectively. We co-transfected NA cells with the full-length plasmid and four helper plasmids. We identified the supernatant of the transfected and then passaged NA cells by immunofluorescence staining and RT-PCR and found the recombinant virus rCVS-11 rescued successfully.
  
  
  RESULTS
  Sequencing results showed that the complete genome of CVS-11 was composed of 11 927 nucleotides. The complete genome of CVS-11 encoded 5 structure proteins and gene array was the same as other reported rabies viruses. We successfully constructed a reverse genetic system of CVS-11, namely the full length plasmid pcDNA3. 1-CVS-11 and 4 help plasmids pcDNA3. 1-N, P, L, G and rescued the rCVS-11 from a full-length infectious cDNA clone.
  
  
  CONCLUSIONS
  The reverse genetic system of the CVS-11 strain laid the foundation for future studies on rabies virus.
authors.affiliation: College of Animal Science and Veterinary Medicine, Department of Prologue and Veterinary, Jilin University, Changchun 130062, China. red99.smile@163.com
authors.forename: Xianghong
authors.initials: X
authors.lastname: Xue
authors.forename: Xuexing
authors.initials: X
authors.lastname: Zheng
authors.forename: Weiwei
authors.initials: W
authors.lastname: Gai
authors.forename: Hongru
authors.initials: H
authors.lastname: Liang
authors.forename: Jinzhu
authors.initials: J
authors.lastname: Ma
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Tiecheng
authors.initials: T
authors.lastname: Wang
authors.forename: Na
authors.initials: N
authors.lastname: Feng
authors.forename: Geng
authors.initials: G
authors.lastname: Huang
authors.forename: Yongkun
authors.initials: Y
authors.lastname: Zhao
authors.forename: Songtao
authors.initials: S
authors.lastname: Yang
authors.forename: Xianzhu
authors.initials: X
authors.lastname: Xia
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D010957:Plasmids / Q000235:genetics; D011820:Rabies virus / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D014162:Transfection / Q000379:methods
chemical_list: D018076:DNA, Complementary