An Openly Available SARS-CoV-2 and Coronavirus Toolkit
Author: Dr Suzannah Rihn (26th February 2021)
(Images created by Elena Sugrue using BioRender)
In the midst of what has been a very difficult time, one strikingly positive feature of the COVID-19 pandemic has been the capacity of researchers to rapidly join forces and redirect the focus of their work in order to aid the efforts to better understand SARS-CoV-2 (the virus that causes COVID-19) and to identify possible therapeutic targets. Not only have these efforts obviously involved many virologists here at the CVR, but they have included researchers in incredibly diverse research disciplines around the world.
In January of 2020, when reports of a novel virus from Wuhan were just emerging, and the very idea of a possible pandemic was only starting to unfold, the CVR was already starting to mobilise plans to focus research on this new coronavirus. Yet even in an institute with such a large cohort of virologists with diverse interests, we quickly realised that we were not only largely unaccustomed to working with coronaviruses, but we also lacked many of the molecular tools that would help us better understand SARS-CoV-2.
After recognising that many virologists and other scientists might find themselves similarly unfamiliar with coronavirus research, we realised that an important way that the CVR, as a national virology asset, could contribute to the COVID-19 research response would be to use our combined molecular virology expertise to create a ‘SARS-CoV-2 and coronavirus molecular toolkit’. Our idea was to create an openly available, low-cost set of molecular tools and reagents that would be straightforward for researchers from a variety of scientific backgrounds to utilise, in order to allow them to contribute to SARS-CoV-2 research initiatives.
The results of our toolkit effort, which have involved the efforts of many researchers at the CVR, along with research partners at the University of Dundee, Griffiths University and University of Tartu, have now been published in PLOS Biology. These tools have already been utilised in a number of high-profile publications in Cell (here and here) and Science, and have been distributed to researchers around the globe (see image at bottom of page), but it is our hope that they will continue to be valuable resources for SARS-CoV-2 researchers around the world. Even as we can all celebrate incredible vaccine successes, the continual threat of new SARS-CoV-2 variants, and the prospect of SARS-CoV-2 becoming endemic in global populations, means that there will remain a need for continued SARS-CoV-2 and coronavirus research for years to come.
Although all of the tools we’ve generated are described and validated in greater detail in the paper, we thought it would be useful to summarise the available resources, and their uses, in the descriptions below.
Single plasmid reverse genetics system, with reporter of choice. When a new virus emerges, one of the earliest challenges researchers wanting to better understand the virus often face is the struggle to establish a system in which the virus can be easily genetically manipulated. In the case of SARS-CoV-2, which has an unusually large RNA genome that can be difficult to transcribe, several reverse genetics systems, which allow the creation of replication competent virus, were reported in the months following the release of the first sequence of the virus (in January 2020). However, these systems have typically involved additional in vitro transcription and/or expression plasmids, and have often been difficult for those unaccustomed to coronavirus research to utilise.
With our partners at Griffiths University and University of Tartu, we now report a single plasmid reverse genetics system, that can include an mCherry, ZsGreen, or NLuc reporter, that is not only very straightforward to use through transient transfection, but is also relatively simple to genetically manipulate. This will enable researchers, including those without virology expertise, to quickly examine SARS-CoV-2 variants as they emerge. In particular, it will facilitate the examination of the contribution of single vs. multiple amino acid changes to emerging variant phenotypes. Furthermore, the incorporation of diverse reporters of choice means that the resulting viruses, which are stable over several passages, can be utilised in a very wide range of assays.
All the reverse genetics plasmids, which are based on the Wuhan-Hu-1 sequence, are openly available at our toolkit reagent website.
SARS-CoV-2 and related coronavirus antibodies. Antibodies are another important tool researchers can use to both learn more about new viruses, and to validate existing resources and research. Together with researchers at the MRC Protein Phosphorylation and Ubiquitylation Unit (PPU) at the University of Dundee, we have generated polyclonal sheep antibodies for nearly every SARS-CoV-2 protein, along with antibodies to the structural proteins of SARS-CoV and MERS-CoV, and the nucleocapsid of seasonal human coronaviruses 229E and OC43.
While many SARS-CoV-2 antibodies are now commercially available, our distinctly comprehensive panel of antibodies has also been extensively validated via immunofluorescence, western blot and immunoprecipitation using SARS-CoV-2 infected cells. These antibodies can therefore serve as an important tool for revealing the abundance and location of SARS-CoV-2 proteins during replication.
Along with the associated cDNA clones and proteins, the antibodies are all openly available at the toolkit website.
SARS-CoV-2 clinical isolates. During the initial preparation of our toolkit, we realised that an important facet of COVID research involves the use of SARS-CoV-2 viruses that have been isolated from patient samples (as these viruses may be indicative of any phenotypic changes as variants arise). We therefore have sought to isolate SARS-CoV-2 virus cultures directly from patient samples (typically swabs).
Although the number of clinical isolates available at the CVR has continued to expand, within our toolkit publication we describe the isolation, sequencing, validation and characterisation of three primary patient isolates of SARS-CoV-2. Interestingly, two of these viruses contain the N439K substitution in spike, which has been a focus of CVR research. These viruses are now openly available from the NIBSC (here) and BEI Resources (here).
SARS-CoV-2 permissive cell lines. Another challenge researchers face when a new virus emerges is the capacity to establish effective assays that can probe viral replication and test and/or validate possible therapeutic treatments. Within this challenge, one of the largest hurdles often involves identifying biologically relevant cells that permit efficient virus replication.
Within our toolkit publication, we also describe the generation of A549 (human lung cells) and Vero E6 cells that express exogenous ACE2 (the primary SARS-CoV-2 receptor) and TMPRSS2 (a serine protease which cleaves the viral spike, priming it for infection). These modified cells not only allow us to make non-permissive cells permissive to SARS-CoV-2 (in the case of A549 cells), but as discussed in the publication, they also improve viral plaque phenotypes and can be utilised in medium to high throughput cytopathic effect (CPE) based assays, which is particularly advantageous for screening possible therapeutic compounds. These modified cell lines, which are also very effective for isolating primary clinical isolates as described above, are openly available at the NIBSC.
We hope you agree that the components of our toolkit will continue to be valuable resources as SARS-CoV-2 research continues. If you would like to further discuss any of our resources, please feel free to get in touch, read our publication or visit our toolkit website.
Good luck to everyone with their research!
Global requests for our toolkit reagents as of December 2020:
