title: Determining genetic stabilities of chimeric dengue vaccine candidates based on dengue 2 PDK-53 virus by sequencing and quantitative TaqMAMA.
abstract: The genetic stabilities of the three attenuation loci of the candidate dengue 2 (D2) PDK-53 vaccine virus were evaluated for the PDK-53 virus and PDK-53-vectored chimeric D2/1, D2/3, and D2/4 viruses following 10 sequential passages in Vero cells. Sequencing revealed that the dominant NS1-53-Asp and the NS3-250-Val attenuation loci were extremely stable, whereas reversion occurred at the 5'NCR-57-U locus in 10 of the 18 viral lineages tested. A more sensitive and quantitative assay, the TaqMan mismatch amplification mutation assay (TaqMAMA), was employed to more finely discriminate the level of reversion at the 5'NCR-57 locus. This rapid genetic assay permitted detection of <or=1% reversion of 5'NCR-57 U-to-C in viral populations. By TaqMAMA, various levels of reversion at 5'NCR-57 were detected in all 18 of the PDK-53-based viral lineages tested at Vero passage 10, but only 3 lineages had reversion levels>80% in the viral population. Chimeric viruses based on the PDK-53-V (all three mutations present) genetic background were more stable than those developed in the PDK-53-E (5'NCR and NS1 mutations present) background. The TaqMAMA can be applied in quality control analyses to ensure that attenuated vaccine seeds contain undetectable or minimal levels of reversion at a given attenuation locus.
authors.affiliation: Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, P.O. Box 2087, Fort Collins, CO 80522, USA.
authors.forename: Siritorn
authors.initials: S
authors.lastname: Butrapet
authors.forename: Richard M
authors.initials: RM
authors.lastname: Kinney
authors.forename: Claire Y-H
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000235:genetics*; D042822:Genomic Instability; D016133:Polymerase Chain Reaction; D020365:RNA Helicases / Q000235:genetics; D016865:Reassortant Viruses / Q000235:genetics*; D017421:Sequence Analysis; D012692:Serial Passage; D012697:Serine Endopeptidases / Q000235:genetics; D014613:Vaccines, Attenuated / Q000235:genetics; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000956:Antigens, Viral; D020365:RNA Helicases; D012697:Serine Endopeptidases; D014613:Vaccines, Attenuated; D017361:Viral Nonstructural Proteins; D014765:Viral Vaccines; C079340:NS1 protein, Dengue virus type 2; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases