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

• Published in: Journal of virological methods 2015-03, Vol.213, p.26-37
• Main Authors: Coleman, John W., Wright, Kevin J., Wallace, Olivia L., Sharma, Palka, Arendt, Heather, Martinez, Jennifer, DeStefano, Joanne, Zamb, Timothy P., Zhang, Xinsheng, Parks, Christopher L.
• Format: Article
• Language: English
• Subjects: Abdomen - virology; Animals; Brain - virology; Canine distemper virus; Canine distemper virus vaccine vector; Distemper Virus, Canine - genetics; Distemper Virus, Canine - physiology; Drug Carriers; Duplex real-time qPCR; Ferrets; Gene Expression; Gene Products, gag - biosynthesis; Gene Products, gag - genetics; Genetic Vectors; Genomic Instability; Lymphoid Tissue - virology; Multiplex Polymerase Chain Reaction - methods; Mustela; Real-Time Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - methods; SAIDS Vaccines - administration & dosage; SAIDS Vaccines - genetics; Simian immunodeficiency virus; Simian Immunodeficiency Virus - genetics; SIV gag; Swine influenza virus; Vaccines, Attenuated - administration & dosage; Vaccines, Attenuated - genetics; Virus Replication
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2014.11.015

•The duplex assay monitored replication, tissue distribution, and mRNA expression.•The duplex assay monitored insert genetic stability during in vivo replication.•Primary site of CDV replication in ferrets was abdominal cavity lymphoid tissue.•CDV gRNA or mRNA was undetectable in brain tissue.•Specific primers were used in the RT step to distinguish gRNA from mRNA. 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.