Summary: In a collaboration with Steve Whitehead and Brian Murphy at the NIH we had introduced a 30 nt deletion - which they had shown was attenuating for a DEN4 infectious clone - in the 3' non-coding region of our DEN1 WP infectious clone. The recovered DEN1 mutant virus grew more slowly than its parent, and made a smaller plaque. This virus has now been shown to be immunogenic and attenuated in monkeys. These results have been submitted for publication to the Journal of Virology. In collaboration with E Kelly, we had sequenced and made an infectious clone of a DEN2 PDK50 vaccine candidate and its virulent parent. Clone-derived viruses were comparable 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 was tested in monkeys for attenuation and immunogenicity; this study should be completed soon. We had also made an infectious clone of the Army's candidate live attenuated Japanese encephalitis virus (JEV) vaccine, a vero cell adapted version of a PDK cell derivative of the Chinese JEV live vaccine strain SA14 14-2. Clone-derived virus grew with the same kinetics as its parent in cell cultures. This virus was tested in mice and has been shown to be immunogenic. Finally, with help from Bangti Zhao I have been trying to make an infectious clone of DEN3 in the pRS424 yeast-E. coli shuttle vector. I was able to make a full-length DEN3 clone in yeast, but all attempts to recover this clone in E coli failed; the only plasmids which can be recovered have suffered large deletions of the DEN3 sequences. Subsequently, I introduced the mutF mutation into the 3' end of the DEN3 clone in yeast. Both the WT and mutF yeast clones were used in PCR to amplify the full 10.7 kb genome, using Expand polymerase. Full-length run-off RNA transcripts were made from these PCR products. Electroporation of these transcripts into cells resulted in the recovery of WT and mutF DEN3 viruses. Partial sequence analysis has shown that the mutF mutation has been retained, but that these viruses are not identical in the structural gene region, differing at two amino acids out of the approximately 1 kb sequenced. Preliminary analysis of the phenotype shows that DEN3 mutF makes a very small plaque, is severly growth restricted on LLCMK2 cells, and does not grow at all on mosquito C6/36 cells. This result is complicated to interpret, since there are an as yet unknown number of adventitious differences between the WT and mutF viruses, due to the use of full-length PCR in their derivation. Plans are underway to sequence both viral genomes, after repeating the phenotypic analysis. Also, work is underway to try to stabilize the DEN3 clone in E coli: I plan to introduce insertions at various locations in the left half of the genome; based on previous work in DEN2, I expect this to allow the clone to grow in E. coli. Subsequently, these insertions can be removed by restriction digestion prior to RNA transcription to make infectious RNA.