title: [Construction of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 via Rde/ET recombination system].
abstract: OBJECTIVE
  In recent years,manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). With these BAC clones in hand,we manipulated the genome of HVT by utilizing Red/ET recombination system, and developed a biologically safe live vaccine based on the HVT BACs.
  
  
  METHODS
  In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs,and added inducer L-arabinose into the cells. We prepared the cells into competent cells and electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells. So the functional cassette was inserted into the U(S)2 locus. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin (HA) gene of (HPAIV) A/Goose/ Guangdong/1/96(H5N1) flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates.
  
  
  RESULTS
  Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the U(S)2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts (CEFs).
  
  
  CONCLUSIONS
  We achieved one rescued recombinant virus which designated as rHVT-HA3. The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.
authors.affiliation: National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China.
authors.forename: Desong
authors.initials: D
authors.lastname: Lan
authors.forename: Xingming
authors.initials: X
authors.lastname: Shi
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Wang
authors.forename: Changjun
authors.initials: C
authors.lastname: Liu
authors.forename: Mei
authors.initials: M
authors.lastname: Wang
authors.forename: Hongyu
authors.initials: H
authors.lastname: Cui
authors.forename: Guobin
authors.initials: G
authors.lastname: Tian
authors.forename: Jisong
authors.initials: J
authors.lastname: Li
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Tong
mesh_terms: D000818:Animals; D001089:Arabinose / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000276:immunology*; D026002:Herpesvirus 1, Meleagrid / Q000235:genetics* / Q000502:physiology; D008381:Herpesvirus 2, Gallid / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology; D007252:Influenza Vaccines / Q000627:therapeutic use; D005585:Influenza in Birds / Q000517:prevention & control; D014422:Turkeys; D014613:Vaccines, Attenuated / Q000627:therapeutic use
chemical_list: D006389:Hemagglutinins, Viral; D007252:Influenza Vaccines; D014613:Vaccines, Attenuated; D001089:Arabinose