Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid

Abstract

Procedures for cloning entire dengue serotype 2 virus genome in the multiple cloning site of a commercially available high copy number plasmid are described. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript II KS, which contains the colE1 origin of replication. When propagated at room temperature (20-25°C) under low level of antibiotic selection, the full-length recombinant plasmid was stable upon serial passages in two common Escherichia coli strains employed. Under the same culture conditions the whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofectin-mediated transfection, capped RNA transcripts derived from the plasmid initiated virus replication in C6/36 mosquito cells and BHK-21 cells within 3-4 days of transfection. Upon subsequent expansion in C6/36 cells, dengue viruses derived from the first- and eighth-plasmid passages achieved similar titers as the parent virus. They were also indistinguishable from the parent virus by the criteria of replication kinetics in mosquito and mammalian cell lines, and size and reactivity of selected viral proteins as detected with polyclonal and monoclonal antibodies. The cloning scheme and resultant recombinant plasmids based on high copy number cloning vectors allows greater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts. © 2001 Elsevier Science B.V.