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 (D2mutF) that contained a 2-nt deletion and a 2-nt substitution mutation compared to 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). MutF virus was additionally characterized: (i) Complete nt sequencing of D2mutF virus genome was done this year and revealed some spontaneous mutations upstream from the 3'-SL, associated with replication in monkey cells. (ii) D2mutF virus was shown to retain the mouse-brain adapted phenotype of its wt parent virus. (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, and in aeqypti mosquitoes, mutF virus titers were moderately reduced at early times after trans-thoracic infection. (iv) D2mutF virus replicated normally in a third tissue culture cell line, BHK. In summary, these data suggested that the D2mutF virus had a unique host-range restriction for replication in mosquito cells and in adult mosquitoes. Previously. the mutational changes that defined MutF were introduced into an infectious full-length cDNA derived from the genome of D1 strain WestPac virus. 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. This year, D1mutF virus and its wt human virulent parent virus were tested in monkeys for their immunogenicity and ability to cause viremia. Monkeys are the best available animal model for prediction of attenuation of dengue viruses in humans. D1mutF virus had all properties of a suitable vaccine candidate; it was highly immunogenic, inducing similar titers of neutralizing antibodies in sera compared to wt, and severely impaired in its ability to cause viremia, as defined by peak titers of virus in sera and by the total number of days of viremia detectable by RT-PCR analysis, using a validated assay. The D1mutF virus is of particular interest to vaccine developers, since D1 virus is the most common cause of serious dengue illness in SE Asia. Plans are in progress for additional testing of D1mutF in monkeys and eventually in phase I clinical trials in humans, with the collaboration of our colleagues at WRAIR. West Nile virus is a known cause of fever/arthralgia/rash syndrome and encephalitis in N Africa and the middle East. However, no known case of WNV infection of humans had occurred in N America until the past few years, when isolated cases of WN encephalitis have been documented in NYC, NJ and MD. Since we had prior experience with creating dengue/WN chimeric viruses, we attempted to create and recently were successful in creating a full-length infectious cDNA copy of the genome of WNV strain Eg101. Strain Eg101 was isolated in Egypt but its nt sequence is known to be closely comparable to that of WNV isolates from NYC. The ~10,500-nt genome of Eg101 was also completely sequenced in our lab this year and compared to NYC isolates and to that of WNV strains from elsewhere. This success was only very recently achieved, and our further plan is to mutagenize DNA sequences encoding the 3'-SL in the WNV genome, to create viruses that are the converse of the WN/D2 chimeras originally created using the D2 virus genome. From this study, we hope to derive candidate live attenuated WNV mutants suitable for vaccine development.
