title: Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.
abstract: In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.
authors: Analytical Research and Development North America, Sanofi Pasteur, Toronto, Ontario, Canada. cheryl.birmingham@sanofipasteur.com
authors: Cheryl L
authors: CL
authors: Birmingham
authors: Dominique
authors: D
authors: Dupont
authors: Patrice
authors: P
authors: Riou
authors: Corinne
authors: C
authors: Armanet
authors: Kerrie Nichol
authors: KN
authors: Edamura
authors: Briolange
authors: B
authors: Martinho
authors: Aurelie
authors: A
authors: Serres
authors: Severine
authors: S
authors: Jacouton
authors: Valerie
authors: V
authors: Detrez
authors: Bryan
authors: B
authors: McNeil
authors: Martha
authors: M
authors: Schreiber
authors: David
authors: D
authors: Gaillac
authors: Thierry
authors: T
authors: Bonnevay
authors: Lucy
authors: L
authors: Gisonni-Lex
authors: Laurent
authors: L
authors: Mallet
mesh_terms: D000818:Animals; D001354:Avian Leukosis Virus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D002460:Cell Line; D002642:Chick Embryo; D004340:Drug Contamination; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014612:Vaccines; D014779:Virus Replication
chemical_list: D014612:Vaccines