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

作者：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

来源：Journal of Virological Methods, 2015, 213: 26-37.

DOI：10.1016/j.jviromet.2014.11.015

• Canine distemper virus vaccine vector
• SIV gag
• Duplex real-time qPCR
• Ferrets

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 Sly 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.

• 出版日期2015-3-1