The nucleic acid sequences of the pre-membrane/ membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90 %. The similarity among deduced amino acids was between 95 % and 200 %, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.

The family Flaviviridae comprises the genus Flavivirus, which contains 65 related species and two possible members. They are small, enveloped RNA viruses (diameter 45 nm) with peplomers comprising a single glycoprotein E. Other structural proteins are designated C (core) and M (membrane-like). The single strand of RNA is infectious and has a molecular weight of about 4 × 10⁶ and an m⁷G ‘cap’ at the 5’ end but no poly(A) tract at the 3’ end; it functions as the sole messenger. The gene sequence commences 5’-C-M-E... The replication strategy and the mode of morphogenesis are distinct from those of the Togaviridae which are slightly larger and morphologically similar in some respects. Flaviviruses infect a wide range of vertebrates, and many are transmitted by arthropods.

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(14), Jamaica, and the Dominican Republic (13, 17). The rapid geographic expansion of the virus is attributed to movement by viremic birds during local and migratory flight behavior. To date, there is no effective drug treatment against WN virus infection and surveillance and mosquito control measures have not significantly influenced the number of human infections (27). A vaccine against WN virus represents an important approach to the prevention and control of this emerging disease.The ChimeriVax technology has been successfully used to develop a live vaccine against Japanese encephalitis (JE) virus that is now in phase II trials (23). JE virus is a close genetic relative of WN virus (31), a fact that expedited use of this technology to develop multiple WN virus vaccine candidates. The ChimeriVax technology employs the yellow fever (YF) 17D vaccine capsid and nonstructural genes to deliver the envelope genes (prM and E) of other flaviviruses. In the work presented here, the envelope genes of YF 17D were replaced with the corresponding genes of the wild-type WN virus NY99 strain previously described by Lanciotti et al. (19). The resulting YF/WN chimera lacked the mouse neuroinvasive property of WN virus and is less neurovirulent than YF 17D vaccine in both mouse and monkey models. Because WN virus, like other flaviviruses in the genus, is neurotropic for mammals (21, 29), attenuating point mutations were later introduced in the envelope of the YF/WN chimera to further reduce its virulence. Mutation sites were targeted only to regions of the envelope (E) protein gene and were based on previous observations by others (1,3,28,32) pertaining to attenuation phenotypes in related flaviviruses: specifically JE and tick-borne encephalitis viruses. Site-directed mutations in the WN virus E gene of the chimeric prototype vaccine, ChimeriVax-West Nile 01 , (ChimeriVax-WN 01 ) resulted in a significant reduction in virus neurovirulence. Here we discuss a vaccine in a YF vaccine backbone; the WN virus envelope (E) protein mutagenesis rationale; and the assessment of the safety, immunogenicity, efficacy, and genetic stability of these ChimeriVax-WN vaccine candidates in the mouse and macaque models. MATERIALS AND METHODSYF/WN chimeric clones and molecular procedures for virus assembly. Chimeric flaviviruses were constructed with the ChimeriVax two-plasmid technology previously described (9). Briefly, the two-plasmid system provides plasmid stability in Escherichia coli by dividing the cloned YF backbone into two plasmids. This provides smaller plasmids that are more stable to manipulate the YF sequences facilitating replacement of the prM and E genes of the flavivirus target vaccine. The WN virus prM and E genes used were cloned from the WN flamingo isolate 383-99 sequence (GenBank accession no. AF196835; kindly provided by John Roehrig, Centers for Disease Control and Prevention, Fort Collins, Colo.). Virus prME sequence cDNA was obtained by reverse transcription-PCR (RT-PCR) (XL-PCR kit; Applied Biosystems, Foster City, C...

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