Armored RNA Technology for Production of Ribonuclease-Resistant Viral RNA Controls and Standards

Abstract

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. “Armored RNA” is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at −20°C or for 60 days at 4°C. Additionally, Armored RNA preparations are homogeneous and noninfectious.

Detection and monitoring of virus infections by real-time PCR

“…In order to avoid the risk of crosscontamination and to include well defined internal controls with physical properties similar to those of the target of interest, it is recommended to spike the specimens with non-human, naturally occurring viruses (see below). Alternatively, the so-called ''armored RNA'' technology can be implemented as an internal control, where the samples investigated are spiked with a known concentration of a synthetic RNA standard protected from degradation by packaging in pseudoviral particles (Pasloske et al, 1998). In our laboratory, we use the phocine (=seal) herpes virus type 1 (PhHV) as a DNA virus control and the phocine distemper virus (PDV) as an RNA virus control (both were kindly provided to us by Niesters, Department of Virology, University Hospital Rotterdam, the Netherlands).…”

Development and Evaluation of a One-Step Quantitative RT-PCR Assay for Detection of Lassa Virus

“…Furthermore, the same cDNA region was used to prepare ARC + based on a previously described procedure for MS2-phage-based ARPs (Cheng et al, 2006, Pasloske et al, 1998 with certain minor modifications. In brief, the PCR fragment containing the target region and additional flanking nucleotides (see above) was ligated into a linearized in-house plasmid vector containing the MS2 coat protein gene.…”

A novel delivery platform based on Bacteriophage MS2 virus-like particles

“…Moreover, they are not pathogenic during the experiment because these particles cannot replicate by themselves. Armored RNAs based on MS2 VLPs are now used for the detection of various viruses, such as human immunodeficiency virus (Pasloske et al, 1998), severe acute respiratory syndrome coronavirus (Drosten et al, 2001), enteroviruses (Beld et al, 2004;Donia et al, 2005), Avian influenza virus (Das et al, 2006), measles virus (Zhang et al, 2015a,b), and ebola virus .…”