Live, attenuated vaccines are best constructed by introducing stable genetic alterations using techniques of molecular biology. Here, dengue virus is our model system. Flavivirus genomic RNAs contain a conserved stem-loop structure within the 3'-noncoding region (3'-SL). We showed, by constructing a series of mutant viruses containing D2/West Nile [WN] chimeric 3'-SL nt sequences, that an 11-bp double-stranded segment of the D2 3'-SL was absolutely required for D2 replication. One cDNA-derived D2 virus that contained a 2-nt difference from the wt D2 3'-SL nt sequence was severely restricted for growth in mosquito cells but replicated like wt D2 virus in monkey kidney cells. This is a desireable property of a live, attenuated vaccine virus. (L Zeng, B Falgout, L Markoff [1998] J Virol 72:7510-22). The mosquito cell growth-restricted mutant (mutF) was additionally characterized: (i) Nt sequence analysis showed that it had mutated during growth in monkey cells; an A residue was deleted in position 3 from the 3'-terminus of the genome, within the 7-bp substituted segment. (ii) Using neonatal outbred mice, we showed that the cDNA-derived wt parent virus was as neurovirulent as its """"""""parent"""""""" mouse-brain adapted D2 NGC isolate and that mutF virus retained the mouse-brain adapted phenotype of the wt viruses. (iii) Replication of D2mutF virus in adult Aedes sp. mosquitoes was assessed, compared to wt cDNA-derived D2 NGC virus. In albopictus mosquitoes, mutF replication was severely restricted out to day 7 after trans-thoracic infection (>100,000-fold); in aeqypti mosquitoes, mutF virus titers were about 40-fold reduced compared to wt up to day 4 p.i., when titers of the mutant virus began to approach that of wt. (iv) The genome of mutF virus isolated after replication in monkey cells was further sequenced upstream from the 3'-SL, to the 5'-end of the NS5 (RDRP) gene, to detect additional spontaneous mutations related to replication competence. No difference from the wt parent was detected. (v) The 2-nt mutation that defined MutF was introduced into an infectious full-length cDNA derived from the genome of D1 strain WestPac virus [provided by BF and SP]. This """"""""D1mutF"""""""" virus grew to wt titers in LLCMK2 cells but did not replicate in cultured mosquito cells, recapitulating the phenotype of the original D2mutF virus. Thus evidence suggests that the genetic modifications that define mutF would also confer the mosquito-cell host range restriction phenotype in the context of D3 and D4 genomes, and work is underway to prove this contention. Since the nt sequence of the 3'-SL is well conserved among all 4 serotypes of dengue viruses, the result is not so surprising. However, it does suggest an approach to the design of a tetravalent dengue vaccine composed of identically mutated viruses. Currently, D1mutF, D2mutF, and the respective wt parent dengue viruses are being tested for their ability to induce viremia in monkeys. This will provide information on the potential for mutF viruses to be attenuated in humans.
