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Journal of Virology, April 2000, p. 3011-3019, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Siritorn Butrapet,1,2 Claire Y.-H. Huang,2 Dennis J. Pierro,2 Natth Bhamarapravati,1 Duane J. Gubler,2 and Richard M. Kinney2,*

Center for Vaccine Development, Institute of Science and Technology for Development, Mahidol University at Salaya, Nakhonpathom 73170, Thailand,1 and Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Fort Collins, Colorado 805222

Received 26 July 1999/Accepted 27 December 1999

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-MK2 cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 107.3 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 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.

* Corresponding author. Mailing address: Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522. Phone: (970) 221-6494. Fax: (970) 221-6476. E-mail: rmk1{at}cdc.gov.

Journal of Virology, April 2000, p. 3011-3019, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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