title: Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.
abstract: Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States. Electronic address: jcoleman@IAVI.org.
authors.forename: John W
authors.initials: JW
authors.lastname: Coleman
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Wright
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Olivia L
authors.initials: OL
authors.lastname: Wallace
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Palka
authors.initials: P
authors.lastname: Sharma
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Heather
authors.initials: H
authors.lastname: Arendt
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Martinez
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Joanne
authors.initials: J
authors.lastname: DeStefano
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Zamb
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States; Program in Molecular and Cellular Biology, School of Graduate Studies, The State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States.
authors.forename: Xinsheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States; Program in Molecular and Cellular Biology, School of Graduate Studies, The State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Parks
mesh_terms: D000005:Abdomen / Q000821:virology; D000818:Animals; D001921:Brain / Q000821:virology; D004217:Distemper Virus, Canine / Q000235:genetics / Q000502:physiology*; D004337:Drug Carriers; D005289:Ferrets; D015870:Gene Expression; D015683:Gene Products, gag / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D042822:Genomic Instability; D008221:Lymphoid Tissue / Q000821:virology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D018449:SAIDS Vaccines / Q000008:administration & dosage / Q000235:genetics; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000235:genetics; D014779:Virus Replication
chemical_list: D004337:Drug Carriers; D015683:Gene Products, gag; D018449:SAIDS Vaccines; D014613:Vaccines, Attenuated
keywords: Canine distemper virus vaccine vector; Duplex real-time qPCR; Ferrets; SIV gag