Establishing A Reverse Genetic System For Type 1 Feline Coronavirus
Lead Research Organisation:
University of Bristol
Department Name: Cellular and Molecular Medicine
Abstract
Feline coronavirus (FCoV) leads to the fatal disease known as feline infectious peritonitis in a small proportion of infected cats. Research into FCoV has so far been hindered by our inability to culture the most common serotype, Type 1, in vitro.
This project aimed to establish a reverse genetic system for Type 1 FCoV. Such a system would enable a deeper understanding of the role of viral mutations in the pathogenesis of disease and could serve as a platform for rational vaccine design. To this end, cDNA infectious clone and replicon constructs based on Type 1 FCoV were developed and transcribed. During this project, infectious virus or replicon-expressing cells were not recovered following transfection of the constructs into mammalian cells, but a foundation was laid for this to be achieved in future.
Recovery of recombinant Type 1 FCoV would necessitate a cell line capable of supporting this serotype's growth in vitro, so a specific aim of this project was to identify a cell entry receptor (CER) for Type 1 FCoV in order to develop a cell line permissive to infection with the virus. To this end, 'bait proteins' bearing the spike proteins of Type 1 and 2 FCoV were produced, and Type 2 bait protein was shown to be capable of recognising its CER. Feline intestinal organoid cultures were established, and an interaction identified between Type 1 bait protein and HSPA1A suggested that HSPA1A is a receptor for Type 1 FCoV.
Finally, a feline IFN-g ELISpot assay was used to measure the cellular immune response to peptides representing an epitope of Type 1 FCoV with and without a substitution. No significant difference in the ability of the peptide variants to stimulate a response was identified, but the assay could be used in the future to guide rational vaccine design.
This project aimed to establish a reverse genetic system for Type 1 FCoV. Such a system would enable a deeper understanding of the role of viral mutations in the pathogenesis of disease and could serve as a platform for rational vaccine design. To this end, cDNA infectious clone and replicon constructs based on Type 1 FCoV were developed and transcribed. During this project, infectious virus or replicon-expressing cells were not recovered following transfection of the constructs into mammalian cells, but a foundation was laid for this to be achieved in future.
Recovery of recombinant Type 1 FCoV would necessitate a cell line capable of supporting this serotype's growth in vitro, so a specific aim of this project was to identify a cell entry receptor (CER) for Type 1 FCoV in order to develop a cell line permissive to infection with the virus. To this end, 'bait proteins' bearing the spike proteins of Type 1 and 2 FCoV were produced, and Type 2 bait protein was shown to be capable of recognising its CER. Feline intestinal organoid cultures were established, and an interaction identified between Type 1 bait protein and HSPA1A suggested that HSPA1A is a receptor for Type 1 FCoV.
Finally, a feline IFN-g ELISpot assay was used to measure the cellular immune response to peptides representing an epitope of Type 1 FCoV with and without a substitution. No significant difference in the ability of the peptide variants to stimulate a response was identified, but the assay could be used in the future to guide rational vaccine design.
People |
ORCID iD |
| Andrew Davidson (Primary Supervisor) | |
| Samantha Saunders (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009122/1 | 01/10/2015 | 31/03/2024 | |||
| 1647135 | Studentship | BB/M009122/1 | 01/10/2015 | 30/09/2019 | Samantha Saunders |
| Description | The feline protein HSPA1A was identified as an interactor with the part of feline coronavirus that determines host cell entry. This finding suggests that HSPA1A is an attachment factor (if not a cell entry receptor) for the strain of feline coronavirus that we were studying. |
| Exploitation Route | Identifying the steps taken by feline coronavirus to enter a host cell will facilitate the identification and/or development of a cell line in which feline coronavirus can be cultured. Applications for a cell line supporting the growth of feline coronavirus are diagnosis of disease, screening candidate drugs and developing a vaccine. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | Pathology |
| Organisation | University of Zurich |
| Country | Switzerland |
| Sector | Academic/University |
| PI Contribution | A previous student in the lab developed a 'bait protein', which is the spike protein of the virus I am researching tagged with human IgG. I optimised the conditions for bait protein production, and prepared an aliquot to give to the collaborator. |
| Collaborator Contribution | Our collaborator tried out the bait protein as an immunohistochemistry reagent and reported the results back to us. |
| Impact | Preliminary results show that the bait protein may be a useful immunohistochemistry reagent, but there seems to be some non-specific staining. Our collaborator plans to try techniques to overcome this is the future. The collaboration involves synthetic biology and pathology. |
| Start Year | 2016 |
| Description | Article for Cats Protection Clinic magazine |
| Form Of Engagement Activity | A magazine, newsletter or online publication |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | I wrote an article in a magazine about the disease I am researching. The magazine is circulated to veterinary professionals. Colleagues in the field reported that it was useful and would change the way they approach diagnosis of the disease. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Research without Borders Discussion Panel |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | I took part in a discussion panel, along with three other postgraduate students and an academic, on the theme of data security. The event was open to the general public. We delivered short talks then took questions from the audience, who seemed very interested and engaged. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.bristol.ac.uk/doctoral-college/current-research-students/events-and-opportunities/resear... |
| Description | Research without Borders Showcase |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | I participated in a showcase of postgraduate research, where I designed and presented an interactive exhibit about viral infection and the role of receptors. People were very engaged with the exhibit and reported a better understanding of viruses. I won a prize for 'most engaging exhibit'. |
| Year(s) Of Engagement Activity | 2017 |
| URL | http://www.bristol.ac.uk/doctoral-college/current-research-students/events-and-opportunities/researc... |
| Description | Science outreach at Green Man festival |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | I helped to run a BBSRC science outreach stand at Green Man festival, open to all festival attendees for three days. The theme of the stand was agriculture, and we talked to people about different aspects of sustainable agriculture as well as the BBSRC. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.swbio.ac.uk/2018/10/16/promoting-plant-power-green-man-festival/ |
| Description | Talk at Supreme Cat Show |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | I spoke about the disease I am researching at the Supreme Cat Show, an annual national cat show that attracts a lot of cat breeders and enthusiasts. The talk mostly covered the mechanism of the disease and updates on diagnosis, treatment and prevention. |
| Year(s) Of Engagement Activity | 2016 |
| URL | https://www.gccfcats.org/Show-Information/Supreme-Show |