CONSTRUCTION OF INFECTIOUS FULL LENGTH CDNA CLONE OF DEN

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We are in the process of making full length "infectious" DEN1 cDNAs from both a virulent DEN1 virus, the Western Pacific (WP) strain, and from a live-attenuated vaccine candidate (PDK-20). The construction of the DEN1 WP infectious clone closely followed the methods used to make the infectious DEN2 clone. Briefly, the left-hand and right-hand ends of DEN1 Western Pacific strain were amplified by RT-PCR and then were cloned in proper orientation into the polylinker of the yeast shuttle vector pRS424. This DNA was linearized at a site in the polylinker between the two DEN ends. Meanwhile, a 5 kb cDNA was made from the middle of DEN1 by RT-PCR. This ~stuffer fragment~ overlaps both the left- and right-hand cDNAs contained in the pRS clone. Double homologous recombination between the stuffer fragment and the cloned left-hand and right-hand cDNAs upon co-transfection of yeast created a circular pRS plasmid containing the full-length DEN1 cDNA. Yeast colonies were screened by PCR, and plasmids containing full-length DEN1 cDNA were transfected into E. coli, DNA was prepared, and the structure confirmed by restriction analysis. Transcripts made from a series of independent clones produced typical dengue virus infections after electroporation into cells. Transcript-derived virus behaved like the parent WP virus in growth curves. The complete nucleotide sequence of one of the clones was determined, and the sequence of the WP RNA is about 75% completed; so far the two sequences are identical save for 1 silent mutation in the cloned cDNA. Work has begun to replace the FL DEN1 sequences with cDNA segments from the attenuated PDK-20 virus. First, an RT-PCR product representing the right half of PDK-20 was used to replace the same region of WP in the full-length clone. Transcripts from this chimeric construct were proven infectious. Then, the chimeric construct was used similarly to introduce the left half of PDK-20. Candidate clones have been identified and are being tested for infectivity. In addition, PDK-20 RNA is being sequenced. Once an infectious clone of PDK-20 is isolated, transcripts will be made and introduced into production Vero cells under cGMP by K. Eckels and his group at WRAIR. This candidate vaccine will then be compared in humans to the parental PDK-20 vaccine virus by B. Innis and colleagues at WRAIR. This will test whether an infectious clone of the average sequence of a virus population will behave the same in people as the parent vaccine virus. Having an infectious clone would allow the introduction ofspecific mutations into the PDK-20 virus in an effort to achieve further attenuation. Also, the Army group also has vaccine candidates for the other 3 dengue serotypes, and it is probable that at least two of these are not properly attenuated for use in humans. Thus, in the future, infectious clones could be derived from these other vaccine candidates and further mutagenized to achieve proper attenuation.