Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

Abstract

The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood

“…Importantly, these findings were reaffirmed by extending this analysis to PV14 (5.76 × 10 3 GE per ml LLOD; % CV < 1.26; 107% efficiency; 93.0% probability of single curve fit; Fig. 3b ) and external surrogate standards for EBOV NAATs 36 (1.46 × 10 3 GE per ml LLOD; % CV < 2.81; 97.4% reaction efficiency; 94.9% probability of single curve fit Fig. 3c ).…”

The Future of Digital Polymerase Chain Reaction in Virology

“…the extent to which reference standards behave like patient samples and are consistent across different assays [52]. Recently, dPCR was used to validated standard reference materials for several viruses, such as CMV [53] or Ebola virus [54]. In this context, it will be imperative to carefully investigate the impact of different dPCR platforms and the effect of the conformation of the standard DNA, i.e.…”

Commonly setting biological standards in rare diseases

“…NIBSC has developed procedures to enable the safe production and distribution of International Standards that precludes the need for high containment laboratories. This was achieved through the use of lentiviral vectors to package the nucleic acid of the target genes for nucleic acid amplification-based technology (NAT) assays [25]. This is a completely synthetic approach and avoids the need for handling and culture of wild type viruses.…”