The goal of this project is to clone a cDNA copy of the 10.7 kb DEN type1genome from which an infectious copy of DEN1 RNA can be transcribed. Creation of such a clone will allow introduction of defined mutations into the genome, which will be useful in many types of studies, such as attempting to make a live-attenuated DEN1 vaccine. RT-PCR was used to amplify and clone the entire DEN1 genome in a yeast shuttle vector. DNA was prepared after growth in E. coli, and was shown to have the correct restriction pattern. However, RNA transcripts from these clones were not infectious. Limited sequencing of four clones revealed numerous errors, including a stop codon in two cases; errors are localized to cDNA derived from one large cDNA product. An attempt was made to replace these suspect sequences with any one of three smaller cDNA products, but this too failed. We have begun to completely sequence one noninfectious clone: so far, about 8kb is done. Once completed, this sequence will be compared to the DEN1 parent, which should allow us to repair all mistakes and render this clone infectious. A major portion of the regulatory responsibilities of LVBV includes the evaluation of INDs for the development of live virus vaccines to protect against diseases caused by flaviviruses (e.g., dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), alphaviruses (e.g., Chikungunya virus, Eastern and Venezuelan equine encephalitis viruses), and arenaviruses (e.g., Junin virus, Hantaviruses). These are all positive-stranded RNA viruses. Thus, a viable strategy for producing a vaccine against any of these viruses is to identify potential attenuating mutations and engineer them into the viral genome using an infectious cDNA clone. Moreover, the principles applied to the identification and testing of putative vaccine strains are identical, regardless of the technology employed to derive them. Thus, the work described in this project will be invaluable in helping us to critique methods used by others to derive vaccine viruses and to evaluate them. In addition, we seek to develop our own attenuated dengue viruses as candidates for vaccine development.
