title: Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53.
abstract: We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2 cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2 cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 vaccine virus and the virus passaged once in LLC-MK2 cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replication in vitro, it was not a major determinant of attenuation for PDK-53 virus in suckling mice.
authors.affiliation: Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA. rmk1@cdc.gov
authors.forename: R M
authors.initials: RM
authors.lastname: Kinney
authors.forename: S
authors.initials: S
authors.lastname: Butrapet
authors.forename: G J
authors.initials: GJ
authors.lastname: Chang
authors.forename: K R
authors.initials: KR
authors.lastname: Tsuchiya
authors.forename: J T
authors.initials: JT
authors.lastname: Roehrig
authors.forename: N
authors.initials: N
authors.lastname: Bhamarapravati
authors.forename: D J
authors.initials: DJ
authors.lastname: Gubler
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D003715:Dengue / Q000401:mortality / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D004195:Disease Models, Animal; D016679:Genome, Viral; D008253:Macaca mulatta; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010641:Phenotype; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013696:Temperature; D014613:Vaccines, Attenuated / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000378:metabolism; D010948:Viral Plaque Assay; D014765:Viral Vaccines / Q000494:pharmacology; D014774:Virulence
chemical_list: D018076:DNA, Complementary; C051821:E-glycoprotein, Dengue virus type 2; D014613:Vaccines, Attenuated; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C074932:prM protein, Flavivirus