The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged in Wuhan, China, in late December 2019 is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Today, it has impacted more than 41 million lives and caused over 1.1 million deaths worldwide. Many countries across Europe, Asia, and North America are still fighting second waves of infection.
Nucleic acid tests such as the reverse transcription-polymerase chain reaction (RT-PCR) have become the mainstay for early detection of SARS-CoV-2 infection. Positive controls have been produced for the molecular assays to validate the tests and ensure accuracy. However, most of the existing positive controls cannot serve as full-process control and need cold-chain distribution.
In an attempt to overcome these issues, a team of researchers from various departments at the University of California San Diego recently proposed using biomimetic virus-like particles (VLPs) as positive controls for assays used in SARS-CoV-2 detection. Their study is published on the preprint server bioRxiv*.
The researchers developed a biomimetic nanotechnology solution in which they packaged the RNA transcripts with the nucleic acid regions that bind to the viral primers and probes into a nanoparticle carrier. In other words, they generated a biomimetic positive control by producing a VLP technology that exploits the unique features of SARS-CoV-2 but is at the same time harmless in diagnostic assays. While several nanotechnology platforms such as polymer and lipid nanoparticles that can carry nucleic acids are currently available, the viral capsids are natural nucleic acid envelops and hence make a better choice for positive controls.
SARS-CoV-2 Detection Module (SDM)
VLPs derived from bacteriophage and plant viruses mimic the natural environment of viral RNA
The researchers packaged synthetic replication-deficient, SARS-CoV-2 RNA target sequences into VLPs derived from the bacteriophage Qbeta (Qβ), and the plant virus cowpea chlorotic mottle virus (CCMV). The chimeric VLPs were developed either by reconstitution and co-expression of the target detection module and coat proteins in vivo or by the assembly of detection module RNA sequences and coat proteins in vitro.
These positive controls based on VLPs closely mimic SARS-CoV-2 packaged RNA while being non-infectious and safe to use in the diagnostic setting, unlike the use of RNA from infected patients. Moreover, the study showed that these VLPs could serve as efficient, positive controls that are stable, scalable, and versatile for use in various assays clinical settings.
"A particular advantage of the CCMV system is the straightforward in vitro reconstitution – hence offering a high degree of modularity."
Qβ and CCMV were promising and stable positive controls for RT-qPCR
Overall, both the VLPs developed as part of this study – the bacteriophage-derived Qβ and the plant virus-derived CCMV – provide promising platforms for encapsidation of RNA target sequences and can serve as stable positive controls for RT-qPCR or other assays for the detection of infectious pathogens such as SARS-CoV-2.
The research team developed in vivo expression systems and in vitro reconstitution protocols, yielding CCMV- and Qβ-based nanoparticles that can encapsidate SARS-CoV-2 detection modules compatible with the CDC primer/probe sets. As the RNA is stabilized within the VLP, the positive control mimics the natural conditions familiar to the SARS-CoV-2 RNA template in environmental or clinical samples.
Safer, more accessible positive controls may help alleviate disparities in testing
Both the Qβ and CCMV platforms are highly scalable using bacterial fermentation and plant molecular farming. The in vivo expression of Qβ VLPs allows for fewer processing steps while the in vitro dis-assembly and reassembly of the chimeric CCMV VLPs offers better control of target molecule encapsidation. However, both these VLP systems were shown to be robust in clinical assays.
The team believes that these VLPs will make the handling of SARS-CoV-2 positive controls more accessible and safe for health care professionals in a wide variety of settings, including airports and border crossings, and also help alleviate the disparities in testing across different countries in the world.
"The potential to make these SARS-CoV-2 positive controls widely available at ambient conditions could help to alleviate some of the disparities in testing that are contributing to the increased COVID-19-related deaths in underserved populations in the US and across the world."
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.