It may seem self-evident that a crucial reagent in the study of coronavirus is the virus itself. But producing live coronavirus in large quantities is actually easier said than done. An obvious approach is to obtain virus-containing biological samples such as sputum from infected individuals, and use them to infect susceptible cell lines where the virus can proliferate to large quantities. However, it is not always easy to obtain and ship samples containing viable virus, and it is also not always known what cell lines can support the proliferation of a given type of virus. Additionally, biological samples such as sputum can carry many bacterial, fungal, viral and mycoplasma contaminants besides the virus of interest, which necessitates stringent culture and validation procedures, including full sequencing of the cultured virus to ensure that it is the proper strain and has the correct sequence. Importantly, even if live virus can be produced from cell culture and fully validated, it is difficult to further genetically engineer the viral genome as is often needed for various studies.
The best solution for these problems is to reconstitute live virus from recombinant vectors using reverse genetics approaches. More specifically, vectors in the form of double-stranded DNA that carry the coronavirus genome of interest can be constructed, then transfected into packaging cells to produce large quantities of live virus. Such vectors can be further modified easily with desired changes such as adding reporter genes (e.g. EGFP) or introducing specific mutations. Additionally, given that coronavirus has a positive-sense, single-stranded RNA genome, the DNA-based viral vectors are not infectious themselves and are therefore very safe to handle and ship with minimal biosafety requirements.
VectorBuilder’s coronavirus vector system allows users to reconstitute live virus of any coronavirus species – wildtype or mutated version – by simply transfecting our vector into the proper packaging cells. The resulting virus can be further propagated and amplified to any quantity in the appropriate cell line.
Structure of coronavirus vectorThe structure of our coronavirus vector is depicted below:
CMV promoter: Human cytomegalovirus immediate early promoter. It can drive high-level transcription of the downstream viral RNA genome in packaging cells.
T7 promoter: Promoter from T7 bacteriophage. The T7 RNA polymerase from the phage recognizes this promoter to drive high-level transcription. It can be used for in vitro transcription of the downstream viral RNA genome.
Coronavirus genome sequence: The complete coronavirus genome sequence (~30 kb, not draw to scale) is placed here.
Poly(A): String of 25 A residues. Transcription of the upstream viral RNA genome through this region will result in the addition of a poly(A) tail to the transcript, which is required for the proper functioning of the viral genome.
HDV antigenome ribozyme: Hepatitis delta virus antigenome ribozyme. This ribozyme, when present in the transcript, catalyzes the self-cleavage of the transcript at the position between the poly(A) and the ribozyme, thus creating a transcript ending with the poly(A) tail.
T7 terminator: Transcriptional termination signal from T7 bacteriophage. The T7 RNA polymerase terminates transcription within this sequence.
BGH pA: Bovine growth hormone polyadenylation signal. Transcription driven by the CMV promoter is terminated within this sequence.
In our vector, the coronavirus genome sequence of interest is inserted downstream of the CMV and T7 promoters. When the vector is transfected into a packaging cell line, the CMV promoter drives transcription of the viral genome. The Poly(A) sequence immediately downstream of the viral genome sequence adds a string of A residues to the transcript. Transcription continues all the way to BGH pA where it is terminated by the polyadenylation signal there. The HDV antigenome ribozyme sequence present in the transcript catalyzes the self-cleavage of the transcript at the position between the poly(A) and the ribozyme, thus creating a transcript ending in a poly(A) tail that resembles the natural virus. This coronavirus genomic transcript can then go on to produce appropriate RNAs and proteins that ultimately lead to the production of live viral particles.
As an alternative virus packaging approach, the vector can be transcribed by T7 polymerase in vitro, which initiates transcription at the T7 promoter and ends in the T7 terminator. The HDV antigenome ribozyme would again self-cleave the transcript right after the poly(A) sequence. The resulting RNA can then be transfected into packaging cells, where it can produce appropriate RNAs and proteins that ultimately result in the production of live viruses.
Types of coronavirus vectors offered
Coronavirus constitutes a vast group of viruses that are extremely widespread in nature, infecting virtually all mammals and birds examined. Hundreds of coronavirus species have been characterized thus far. Of these, a few dozen can be deemed important because they infect humans, livestock, pets, or model animals, or they are evolutionarily closely related to them. The phylogenetic tree of these important coronavirus species is shown below:
Phylogenetic Tree of Important Coronavirus Species
More detailed information of these important coronavirus species is listed in the table below:
List of important coronavirus species
VectorBuilder offers viral vectors for all of the above coronavirus species. Vectors for other species or modified vectors carrying reporter genes or specific mutations can also be requested.
How do I order coronavirus vectors?
You can inquire about our coronavirus vectors by following the link below:Click here to send us a vector request
Live viruses generated from our coronavirus vectors are potentially infectious to humans and/or animals. Working with them therefore requires institutional approval and laboratory facilities meeting appropriate biosafety standards. Guidelines on what biosafety levels are required for various coronavirus species can be found in the Risk Group Database published by the American Biological Safety Association (ABSA).
Depending on the vectors requested, VectorBuilder reserves the right to evaluate the user's institutional approval and their technical qualifications to handle live viruses produced with the vectors. VectorBuilder may also require a material transfer agreement (MTA) that limits the use of the vectors to research activities aimed at improving public health.