Standardized quantitative RT-PCR assays for quantitation of yellow fever and chimeric yellow fever-dengue vaccines.
Yellow fever-dengue chimeras (CYDs) are being developed currently as live tetravalent dengue vaccine candidates. Specific quantitative assays are needed to evaluate the viral load of each serotype in vaccine batches and biological samples. A quantitative real-time RT-PCR (qRT-PCR) system was developed comprising five one-step qRT-PCRs targeting the E/NS1 junction of each chimera, or the NS5 gene in the yellow fever backbone. Each assay was standardized using in vitro transcribed RNA qualified according to its size and purity, and precisely quantified. A non RNA-extracted virus sample was introduced as external quality control (EQC), as well as 2 extraction controls consisting of 2 doses, 40 and 4,000 GEQ (genomic equivalents), of this EQC extracted in parallel to the samples. Between 6 and 10 GEQ/reaction were reproducibly measured with all assays and similar titers were obtained with the two methods when chimeric virus samples were quantified with the E/NS1- or the NS5-specific assays. Reproducibility of RNA extraction was ensured by automation of the process (yield>or=50%), and infectious virus was isolated in >or=80% of PCR-positive sera from immune monkeys.
Mantel
N
N
Microbiology Research Department, Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. nathalie.mantel@sanofipasteur.com
Aguirre
M
M
Gulia
S
S
Girerd-Chambaz
Y
Y
Colombani
S
S
Moste
C
C
Barban
V
V
D000818:Animals; D002522:Chlorocebus aethiops; D017931:DNA Primers; D003715:Dengue / Q000821:virology; D053059:Dengue Vaccines; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D008252:Macaca fascicularis; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014614:Vaccines, Synthetic; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics; D010948:Viral Plaque Assay; D015004:Yellow Fever / Q000821:virology; D022341:Yellow Fever Vaccine; D015005:Yellow fever virus / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D053059:Dengue Vaccines; C100315:NS1 protein, Flavivirus; C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D022341:Yellow Fever Vaccine