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.

Keywords: Canine distemper virus vaccine vector; Duplex real-time qPCR; Ferrets; SIV gag.

Fig. 1

The relative frequency of mRNA transcription, genome organization for each vector, the scheme for the mRNA specific RT-qPCR, and the binding sites for qPCR primers and probe for the SIV gag gRNA and mRNA assays are depicted. (A). Transcriptional attenuation resulting in a gradient of mRNA synthesis such that the gag message is more abundant than the N message in the rCDV-SIVgag1 vector; the rCDV vector is a Vero cell adapted, Onderstepoort live-attenuated CDV vaccine (Galaxy D, Schering-Plough Animal Health Corporation, Omaha, NE, USA) obtained through a commercial source; and the rCDV-SIVgag1 vector with the Gag gene in the first position of the genome and a unique UAUAAU sequence common to CDV N and SIV gag genomes. (B). Diagram of the RT step used in the singleplex or duplex assay to synthesize cDNA from mRNA. The CDV-specific mRNA RT-primer that binds to a unique UAUUA sequence common only to CDV N and SIV gag mRNA transcripts, has a 22-base adapter sequence at the 5′ end followed by a 20-base oligo-dT sequence, and a 3′-terminal 5-base anchor (ATAAT). (C) Tagged cDNA generated with this primer is then PCR amplified with CDV N and SIV-gag forward primers and labeled probes and the CDV sequence tag-reverse primer that corresponds to the adapter sequence, as described in Section 2. (E). The black box from 256–339 Nt indicates the binding site for the qPCR primers () and probe () for the Gag specific gRNA RT-qPCR assay and the 1500–1669 Nt indicates the amplicon and the binding site for qPCR forward primer () and probe () used in the Gag specific mRNA RT-qPCR assay.

Fig. 2

Viral loads of CDV N in tissues from ferrets using the 2-step singleplex RT-qPCR assay. Detection of (A) gRNA and (B) mRNA at the indicated times post IN inoculation with rCDV. Viral loads in each tissue are displayed as either average gRNA or mRNA copies/gram of tissue. The detection limit is 3.0 × 10³ copies/g of tissue (shown by dotted line).

Fig. 3

Viral loads at the indicated times in GALT tissues from ferrets after IN inoculation with rCDV-SIVgag1. Detection of (A) CDV N gRNA, (B) SIV gag gRNA, (C) CDV N mRNA and (D) SIV gag mRNA using 2-step duplex RT-qPCR assays. The detection limit is 3.0 × 10³ copies/g of tissue (shown by dotted line).

Fig. 4

Viral loads in GALT tissues from ferrets after IM injection with rCDV-SIVgag1. Detection of (A) CDV N gRNA, (B) SIV gag gRNA, (C) CDV N mRNA and (D) SIV gag mRNA using 2-step duplex RT-qPCR assays. The detection limit is 3.0 × 10³ copies/g of tissue (shown by dotted line).

Fig. 5

Confirmations of gag insert deletion during in vivo replication of the rCDV-SIVgag1 in ferret tissues by traditional RT-PCR and sequencing. (A) RNA extracted from ferret tissues was RT-PCR amplified and run on a 1% agarose gel. Lane 1, TrackIt™ 1 kb Plus DNA Ladder; lane 2, mesenteric lymph nodes 7 days after intramuscular injection; lane 3, mesenteric lymph nodes 7 days after intranasal inoculation; lane 4, large intestines high density Peyer's patches 28 days after intramuscular injection; lane 5, RNA positive control; lane 6, plasmid DNA positive control; lane 7, no template negative control. (B) The solid line indicates the 1.6 kb gag mRNA with the solid box showing the amplicon used in the SIV gag specific gRNA RT-qPCR (see Fig. 1D). The dotted line indicates the position of the 1416 Nt deletion as determined by sequencing.

Supplemental Fig. 1

The linearity, amplification efficiency, sensitivity, and specificity of singleplex RT-qPCR assays to detect CDV N and SIV-gag genomic RNA and of the 2-step duplex assay to detect CDV N and SIV-gag genomic RNA. (A) Ten-fold serial dilutions of a synthetic RNA oligonucleotide spanning the CDV N amplicon were made and 1 × 10⁸ to 1 × 10¹ copies were used to initiate the CDV N gRNA RT-qPCR assay. (B) Similarly, 10-fold serial dilutions of a SIV-gag specific synthetic RNA oligonucleotide were made and 1 × 10⁸ to 1 × 10¹ copies of the amplicon assayed. (C) Also, 10-fold serial dilutions from 1 × 10⁸ to 1 × 10¹ copies of synthetic RNA oligonucleotides of (A) and (B) were assayed in a duplex RT-qPCR to detect CDV N and SIV-gag simultaneously. Standard curves were generated by plotting the log of genome copies against the cycle threshold (C_T). For singleplex assays, gene specific primer-probe sets were used in separate wells and in the duplex assay, gene specific primer-probe sets for both templates were combined in one well. Positive C_T values are ≤40 while negative C_T values are >40. All C_T values are means of duplicates.

Supplemental Fig. 2

Response of the duplex RT-qPCR assay on the C_T values of different ratios of CDV N-to-gag RNA concentrations in a sample. (A) N oligoribonucleotide template held constant at 1 × 10⁷ copies per RT-qPCR while serially titrating the gag template 10-fold over a range from 1 × 10⁷ to 1 × 10³ copies. (B). N oligoribonucleotide template held at 1 × 10⁴ copies per RT-qPCR while serially titrating the gag template 10-fold from 1 × 10⁴ to 1 × 10¹ copies. C_T values (vertical axis) for each target RNA and ratios of CDV N: SIV copies (horizontal axis). Standard curves were generated by plotting the log of gag genome copies against the measured cycle threshold (C_T). All C_T values are means of duplicates.