Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome.
Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV.
Wang
Hanzhong
H
Joint-Laboratory of Invertebrate Virology and Key Laboratory of Molecular Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
Deng
Fei
F
Pijlman
Gorben P
GP
Chen
Xinwen
X
Sun
Xiulian
X
Vlak
Just M
JM
Hu
Zhihong
Z
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D005144:F Factor / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure; D000077183:Occlusion Body Matrix Proteins; D010572:Pest Control, Biological / Q000379:methods; D020161:Physical Chromosome Mapping; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012093:Replicon / Q000502:physiology; D015183:Restriction Mapping / Q000379:methods; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D015678:Viral Structural Proteins
D000077183:Occlusion Body Matrix Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus