Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5 ' noncoding region and nonstructural proteins 1 and 3

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-5 3, differs from its DEN-2 16681 parent by nine nucleotides. Using infectiou s cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyz e four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C -to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in th e structural proteins), nonstructural protein 1 (NS1)-53 Gly to-Asp, and NS 3-250 Glu-to-Val, The viruses were studied for plaque size, growth rate, an d temperature sensitivity in LLC-MK2 cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak tite rs of 10(7.3) PFU/ml or greater in LLC-MK2 cells, The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensi tivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mut ations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small- plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristic s of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype, Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determina nts of attenuation markers to nonstructural genomic regions makes the PDK-5 3 virus genotype attractive for the development of chimeric DEN virus vacci ne candidates.