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-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze 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 the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) 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 sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. 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 characteristics 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 determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.
Butrapet
S
S
Center for Vaccine Development, Institute of Science and Technology for Development, Mahidol University at Salaya, Nakhonpathom 73170, Thailand.
Huang
C Y
CY
Pierro
D J
DJ
Bhamarapravati
N
N
Gubler
D J
DJ
Kinney
R M
RM
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D003716:Dengue Virus / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D019143:Evolution, Molecular; D005819:Genetic Markers; D051379:Mice; D008813:Mice, Inbred ICR; D009154:Mutation; D009420:Nervous System / Q000821:virology; D010641:Phenotype; D011995:Recombination, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D010948:Viral Plaque Assay; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology; D014774:Virulence
D005819:Genetic Markers; D017361:Viral Nonstructural Proteins; D014765:Viral Vaccines