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

Procedures for cloning entire dengue serotype 2 virus genome in the multipl e cloning site of a commercially available high copy number plasmid are des cribed. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript I I KS, which contains the colE1 origin of replication. When propagated at ro om temperature (20-25 degreesC) under low level of antibiotic selection, th e full-length recombinant plasmid was stable upon serial passages in two co mmon Escherichia coli strains employed. Under the same culture conditions t he whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofect in-mediated transfection, capped RNA transcripts derived from the plasmid i nitiated 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 simila r titers as the parent virus. They were also indistinguishable from the par ent 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 resultan t recombinant plasmids based on high copy number cloning vectors allows gre ater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts. (C) 2001 Elsevier Science B.V. All rights reserved.