Previously, in collaboration with B Puri we made full-length cDNA clones from a virulent dengue type 1 virus (DEN1 WP) and from a live-attenuated DEN1 vaccine candidate, which had been adapted to grow in dog kidney cells (DEN1 PDK20). Transfection of cells with RNA transcribed in vitro from these clones produces a dengue infection. Clone-derived viruses behave like the corresponding parent DEN1 in growth curves, and the recovered DEN1 PDK20 has the same small plaque phenotype as its parent. We made a series of recombinants between the WP and PDK20 infectious clones, and characterized the plaque size of the resulting chimeric viruses. Most chimeras had an intermediate plaque size, suggesting that multiple mutations are involved in the determination of this phenotype. In collaboration with S Whitehead and B Murphy at the NIH we introduced a 30 nt deletion in the 3' non-coding region, which they had shown was attenuating for a DEN4 infectious clone, into the DEN1 WP clone. The deletion mutant virus grows more slowly than its parent, and makes a smaller plaque size. This virus is to be tested for immunogenicity and attenuation in monkeys. Another collaboration with B Puri involved a DEN4 PDK20 vaccine candidate. So far, we have sequenced the PDK20 virus and its parent, and we made an infectious full-length cDNA clone of the vaccine. Further work on this is unlikely, as B Puri has taken a job at the patent office. In collaboration with E Kelly, we have sequenced and made infectious clone of a DEN2 PDK50 vaccine candidate and its virulent parent. Clone-derived viruses are being compared to their parents for growth kinetics in tissue culture cells. The cloned DEN2 PDK50 was manufactured under GMP by K. Eckels and his collaborators, and this virus is being tested in monkeys for attenuation and immunogenicity. Finally, we recently completed an infectious clone of the Army's candidate live attenuated Japanese encephalitis virus (JEV) vaccine, a vero cell adapted version of a PDK cells derivative of the Chinese JEV live vaccine strain SA14 14-2. Clone-derived virus grows with the same kinetics as its parent in cell cultures. This virus is being tested in mice for immunogenicity.
