Taiwan Partnering Award: Development of Animal Virus Vaccines - the Utilities of Replicon Systems and Infectious Copies

Lead Research Organisation: University of St Andrews
Department Name: Biology

Abstract

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Description This award has enabled us to formulate a program of research into the expression of virus glycoproteins for use as vaccines against animal viruses.
Exploitation Route Our collaborators in Taiwan have won a significant research award from the Ministry of Science and Technology to further develop animal vaccines.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology
 
Title cDNA clones 
Description We have designed cDNA clones encoding the Bovine Ephemeral Fever Virus (BEFV) glycoprotein (G) and non-structural G (GNS) glycoproteins - with, and without, the C-terminal transmembrane domain. In all cases the V5 epitope tag was genetically fused to the C-terminus of these constructs. These G / GNS coding sequences were cloned downstream of the reporter protein GFP, but linked via the 2A peptide sequence ([GFP-2A-BEFVG] and GFP-2A-BEFVGNS]). This strategy allowed us to monitor G or GNS expression via fluorescence. The expressed proteins bearing the C-terminal transmembrane anchoring domain were localised within the ER, Golgi and plasma membrane (detected using the anti-V5 monoclonal antibody). Those expressed products lacking the C-terminal transmembrane anchoring domain were localised to the ER and Golgi, but in these cases were secreted from the cell into the cell media. These secreted, V5 epitope-tagged proteins could be purified from the tissue-culture media by a single step method: solid matrix antibody antigen complexes coated with an anti-V5 monoclonal antibody. We are exploring the use of these reagents for both diagnostic and vaccine purposes. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact BEFV is a highly significant animal pathogen in SE Asia. Indeed, mortality rates in cattle can approach 50%. The antigens we have expressed are being developed to control this disease by generating an effective diagnostic tool and, potentially, a vaccine. Although expression within mammalian tissue culture cells adds to the expense of a vaccine, using these systems produces the correct glycosylation - often vital in the production of an effective immunogen. BEFV is highly unusual in encoding an non-structural form of the surface glycoprotein (GNS). The function of this protein is not understood: antibodies raised against this protein will be used to investigate the role of GNS in the virus replication cycle. 
 
Description BBSRC SLoLa BB/K003801/1 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Ryan (PI; St Andrews) lead an application to the BBSRC and was awarded an SLoLa grant ("The Molecular Biology of FMDV Replication: Towards New Methods of FMDV Disease Control": BB/K003801/1) in partnership with the Pirbright Institute and the Universities of Leeds, Dundee and Edinburgh. St Andrews and Leeds are using synthetic biology and standard site-directed mutagenesis to manipulate the FMDV replication proteins and RNA secondary structures to study their effects upon RNA replication and to attenuate the genome. The Pirbright Institute are converting these FMDV replicons into infectious copies, rescuing the viruses and studying virus replication and growth / immune response in animals. Edinburgh are using a variety of techniques to study virus/virus and virus/host-cell protein interactions. Dundee are using mass spectrometry (SILAC labeling) to study changes in the proteomes of virus-infected and replicon-transfected cells.
Collaborator Contribution The BBSRC SLoLa grant funded a 5-site team to investigate FMDV replication using replicon systems (alongside corresponding FMDV infectious copies / rescued viruses) and to use various strategies to produce new vaccines against FMDV. The Pirbright team are also expanding the FMDV genome sequence database (predominantly SAT serotypes to date) to inform further molecular biological investigations.
Impact Tulloch,F., Atkinson, N.J., Evans, D.J., Ryan, M.D. & Simmonds, P. (2014). RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife. Dec 9;3:e04531. doi: 10.7554/eLife.04531. Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P.Forrest, S., Lear, Z., Herod, M.R., Ryan, M.D., Rowlands, D.J & Stonehouse, N.J. (2014). Inhibition of the FMDV sub-genomic replicon by RNA aptamers. J. Gen. Virol. 95, 2649-2657. Tulloch, F., Pathania, U., Luke, G.A., Nicholson, J., Stonehouse, N.J., Rowlands,, D.J., Jackson, T., Tuthill, T., Haas, J., Lamond, A.I. & Ryan, M.D. (2014). FMDV replicons encoding green fluorescent protein are replication competent. J. Virol. Methods 209, 35-40. Herod, M.R., Ferrer-Orta, C., Loundras, E-A., Ward, J.C., Verdaguer, N., Rowlands, D.J. & Stonehouse, N.J. (2016). Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication J Virol. 90(15): 6864-6883. Invited talks; Martin Ryan - Uni. Leeds, Uni. Edinburgh, Uni. Nottingham, IVRI (Bangalore), Roslin Institute, National Pingtung University of Science and Technology (Taiwan). Nicola Stonehouse - Philosophy Café (Ilkley), Jaharingarinar (Bangladesh), Khulna (Bangladesh), St Andrews, Roslin Institute, VacChina 2015 (Shanghai). Fiona Tulloch - Glasgow Virology Workshop, Uni. Glasgow "Development of FMDV Replicon Systems for RNA Replication Studies and as a Screen for Attenuated Genomes" Society for General Microbiology, Liverpool, UK Apr 2014 "Screening for Attenuated FMDV RNA Replication Using a Novel Fluorescent Replicon & Rescue of Corresponding Viruses." Society for General Microbiology - Birmingham, UK Mar 2015 "Attenuation of Novel Fluorescent FMDV Replicons through Increases in Dinucleotide Frequencies." Microbiology Society, Liverpool, UK Mar 2016 - "Detection of undefined RNA secondary structure within the non-structural region of the FMDV genome." The Roslin Institute, Edinburgh, UK Apr 2016 - "Studying foot-and-mouth disease virus out-with high-containment facilities using a fluorescent replicon system." EuFMD Meeting, Italy: Pugla 2018 - A New Generation of LIve, Attenuated, FMDV Vaccines. Multi-disciplinary: Dundee - proteomic studies of FMDV-infected / FMDV replicon transfected cells; Edinburgh - virus/virus and virus/host-cell protein interaction studies; St Andrews / Leeds / Pirbright Institute - molecular virology.
Start Year 2013
 
Description BBSRC SLoLa BB/K003801/1 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Ryan (PI; St Andrews) lead an application to the BBSRC and was awarded an SLoLa grant ("The Molecular Biology of FMDV Replication: Towards New Methods of FMDV Disease Control": BB/K003801/1) in partnership with the Pirbright Institute and the Universities of Leeds, Dundee and Edinburgh. St Andrews and Leeds are using synthetic biology and standard site-directed mutagenesis to manipulate the FMDV replication proteins and RNA secondary structures to study their effects upon RNA replication and to attenuate the genome. The Pirbright Institute are converting these FMDV replicons into infectious copies, rescuing the viruses and studying virus replication and growth / immune response in animals. Edinburgh are using a variety of techniques to study virus/virus and virus/host-cell protein interactions. Dundee are using mass spectrometry (SILAC labeling) to study changes in the proteomes of virus-infected and replicon-transfected cells.
Collaborator Contribution The BBSRC SLoLa grant funded a 5-site team to investigate FMDV replication using replicon systems (alongside corresponding FMDV infectious copies / rescued viruses) and to use various strategies to produce new vaccines against FMDV. The Pirbright team are also expanding the FMDV genome sequence database (predominantly SAT serotypes to date) to inform further molecular biological investigations.
Impact Tulloch,F., Atkinson, N.J., Evans, D.J., Ryan, M.D. & Simmonds, P. (2014). RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife. Dec 9;3:e04531. doi: 10.7554/eLife.04531. Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P.Forrest, S., Lear, Z., Herod, M.R., Ryan, M.D., Rowlands, D.J & Stonehouse, N.J. (2014). Inhibition of the FMDV sub-genomic replicon by RNA aptamers. J. Gen. Virol. 95, 2649-2657. Tulloch, F., Pathania, U., Luke, G.A., Nicholson, J., Stonehouse, N.J., Rowlands,, D.J., Jackson, T., Tuthill, T., Haas, J., Lamond, A.I. & Ryan, M.D. (2014). FMDV replicons encoding green fluorescent protein are replication competent. J. Virol. Methods 209, 35-40. Herod, M.R., Ferrer-Orta, C., Loundras, E-A., Ward, J.C., Verdaguer, N., Rowlands, D.J. & Stonehouse, N.J. (2016). Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication J Virol. 90(15): 6864-6883. Invited talks; Martin Ryan - Uni. Leeds, Uni. Edinburgh, Uni. Nottingham, IVRI (Bangalore), Roslin Institute, National Pingtung University of Science and Technology (Taiwan). Nicola Stonehouse - Philosophy Café (Ilkley), Jaharingarinar (Bangladesh), Khulna (Bangladesh), St Andrews, Roslin Institute, VacChina 2015 (Shanghai). Fiona Tulloch - Glasgow Virology Workshop, Uni. Glasgow "Development of FMDV Replicon Systems for RNA Replication Studies and as a Screen for Attenuated Genomes" Society for General Microbiology, Liverpool, UK Apr 2014 "Screening for Attenuated FMDV RNA Replication Using a Novel Fluorescent Replicon & Rescue of Corresponding Viruses." Society for General Microbiology - Birmingham, UK Mar 2015 "Attenuation of Novel Fluorescent FMDV Replicons through Increases in Dinucleotide Frequencies." Microbiology Society, Liverpool, UK Mar 2016 - "Detection of undefined RNA secondary structure within the non-structural region of the FMDV genome." The Roslin Institute, Edinburgh, UK Apr 2016 - "Studying foot-and-mouth disease virus out-with high-containment facilities using a fluorescent replicon system." EuFMD Meeting, Italy: Pugla 2018 - A New Generation of LIve, Attenuated, FMDV Vaccines. Multi-disciplinary: Dundee - proteomic studies of FMDV-infected / FMDV replicon transfected cells; Edinburgh - virus/virus and virus/host-cell protein interaction studies; St Andrews / Leeds / Pirbright Institute - molecular virology.
Start Year 2013
 
Description BBSRC SLoLa BB/K003801/1 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Ryan (PI; St Andrews) lead an application to the BBSRC and was awarded an SLoLa grant ("The Molecular Biology of FMDV Replication: Towards New Methods of FMDV Disease Control": BB/K003801/1) in partnership with the Pirbright Institute and the Universities of Leeds, Dundee and Edinburgh. St Andrews and Leeds are using synthetic biology and standard site-directed mutagenesis to manipulate the FMDV replication proteins and RNA secondary structures to study their effects upon RNA replication and to attenuate the genome. The Pirbright Institute are converting these FMDV replicons into infectious copies, rescuing the viruses and studying virus replication and growth / immune response in animals. Edinburgh are using a variety of techniques to study virus/virus and virus/host-cell protein interactions. Dundee are using mass spectrometry (SILAC labeling) to study changes in the proteomes of virus-infected and replicon-transfected cells.
Collaborator Contribution The BBSRC SLoLa grant funded a 5-site team to investigate FMDV replication using replicon systems (alongside corresponding FMDV infectious copies / rescued viruses) and to use various strategies to produce new vaccines against FMDV. The Pirbright team are also expanding the FMDV genome sequence database (predominantly SAT serotypes to date) to inform further molecular biological investigations.
Impact Tulloch,F., Atkinson, N.J., Evans, D.J., Ryan, M.D. & Simmonds, P. (2014). RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife. Dec 9;3:e04531. doi: 10.7554/eLife.04531. Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P.Forrest, S., Lear, Z., Herod, M.R., Ryan, M.D., Rowlands, D.J & Stonehouse, N.J. (2014). Inhibition of the FMDV sub-genomic replicon by RNA aptamers. J. Gen. Virol. 95, 2649-2657. Tulloch, F., Pathania, U., Luke, G.A., Nicholson, J., Stonehouse, N.J., Rowlands,, D.J., Jackson, T., Tuthill, T., Haas, J., Lamond, A.I. & Ryan, M.D. (2014). FMDV replicons encoding green fluorescent protein are replication competent. J. Virol. Methods 209, 35-40. Herod, M.R., Ferrer-Orta, C., Loundras, E-A., Ward, J.C., Verdaguer, N., Rowlands, D.J. & Stonehouse, N.J. (2016). Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication J Virol. 90(15): 6864-6883. Invited talks; Martin Ryan - Uni. Leeds, Uni. Edinburgh, Uni. Nottingham, IVRI (Bangalore), Roslin Institute, National Pingtung University of Science and Technology (Taiwan). Nicola Stonehouse - Philosophy Café (Ilkley), Jaharingarinar (Bangladesh), Khulna (Bangladesh), St Andrews, Roslin Institute, VacChina 2015 (Shanghai). Fiona Tulloch - Glasgow Virology Workshop, Uni. Glasgow "Development of FMDV Replicon Systems for RNA Replication Studies and as a Screen for Attenuated Genomes" Society for General Microbiology, Liverpool, UK Apr 2014 "Screening for Attenuated FMDV RNA Replication Using a Novel Fluorescent Replicon & Rescue of Corresponding Viruses." Society for General Microbiology - Birmingham, UK Mar 2015 "Attenuation of Novel Fluorescent FMDV Replicons through Increases in Dinucleotide Frequencies." Microbiology Society, Liverpool, UK Mar 2016 - "Detection of undefined RNA secondary structure within the non-structural region of the FMDV genome." The Roslin Institute, Edinburgh, UK Apr 2016 - "Studying foot-and-mouth disease virus out-with high-containment facilities using a fluorescent replicon system." EuFMD Meeting, Italy: Pugla 2018 - A New Generation of LIve, Attenuated, FMDV Vaccines. Multi-disciplinary: Dundee - proteomic studies of FMDV-infected / FMDV replicon transfected cells; Edinburgh - virus/virus and virus/host-cell protein interaction studies; St Andrews / Leeds / Pirbright Institute - molecular virology.
Start Year 2013
 
Description BBSRC SLoLa BB/K003801/1 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Ryan (PI; St Andrews) lead an application to the BBSRC and was awarded an SLoLa grant ("The Molecular Biology of FMDV Replication: Towards New Methods of FMDV Disease Control": BB/K003801/1) in partnership with the Pirbright Institute and the Universities of Leeds, Dundee and Edinburgh. St Andrews and Leeds are using synthetic biology and standard site-directed mutagenesis to manipulate the FMDV replication proteins and RNA secondary structures to study their effects upon RNA replication and to attenuate the genome. The Pirbright Institute are converting these FMDV replicons into infectious copies, rescuing the viruses and studying virus replication and growth / immune response in animals. Edinburgh are using a variety of techniques to study virus/virus and virus/host-cell protein interactions. Dundee are using mass spectrometry (SILAC labeling) to study changes in the proteomes of virus-infected and replicon-transfected cells.
Collaborator Contribution The BBSRC SLoLa grant funded a 5-site team to investigate FMDV replication using replicon systems (alongside corresponding FMDV infectious copies / rescued viruses) and to use various strategies to produce new vaccines against FMDV. The Pirbright team are also expanding the FMDV genome sequence database (predominantly SAT serotypes to date) to inform further molecular biological investigations.
Impact Tulloch,F., Atkinson, N.J., Evans, D.J., Ryan, M.D. & Simmonds, P. (2014). RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife. Dec 9;3:e04531. doi: 10.7554/eLife.04531. Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P.Forrest, S., Lear, Z., Herod, M.R., Ryan, M.D., Rowlands, D.J & Stonehouse, N.J. (2014). Inhibition of the FMDV sub-genomic replicon by RNA aptamers. J. Gen. Virol. 95, 2649-2657. Tulloch, F., Pathania, U., Luke, G.A., Nicholson, J., Stonehouse, N.J., Rowlands,, D.J., Jackson, T., Tuthill, T., Haas, J., Lamond, A.I. & Ryan, M.D. (2014). FMDV replicons encoding green fluorescent protein are replication competent. J. Virol. Methods 209, 35-40. Herod, M.R., Ferrer-Orta, C., Loundras, E-A., Ward, J.C., Verdaguer, N., Rowlands, D.J. & Stonehouse, N.J. (2016). Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication J Virol. 90(15): 6864-6883. Invited talks; Martin Ryan - Uni. Leeds, Uni. Edinburgh, Uni. Nottingham, IVRI (Bangalore), Roslin Institute, National Pingtung University of Science and Technology (Taiwan). Nicola Stonehouse - Philosophy Café (Ilkley), Jaharingarinar (Bangladesh), Khulna (Bangladesh), St Andrews, Roslin Institute, VacChina 2015 (Shanghai). Fiona Tulloch - Glasgow Virology Workshop, Uni. Glasgow "Development of FMDV Replicon Systems for RNA Replication Studies and as a Screen for Attenuated Genomes" Society for General Microbiology, Liverpool, UK Apr 2014 "Screening for Attenuated FMDV RNA Replication Using a Novel Fluorescent Replicon & Rescue of Corresponding Viruses." Society for General Microbiology - Birmingham, UK Mar 2015 "Attenuation of Novel Fluorescent FMDV Replicons through Increases in Dinucleotide Frequencies." Microbiology Society, Liverpool, UK Mar 2016 - "Detection of undefined RNA secondary structure within the non-structural region of the FMDV genome." The Roslin Institute, Edinburgh, UK Apr 2016 - "Studying foot-and-mouth disease virus out-with high-containment facilities using a fluorescent replicon system." EuFMD Meeting, Italy: Pugla 2018 - A New Generation of LIve, Attenuated, FMDV Vaccines. Multi-disciplinary: Dundee - proteomic studies of FMDV-infected / FMDV replicon transfected cells; Edinburgh - virus/virus and virus/host-cell protein interaction studies; St Andrews / Leeds / Pirbright Institute - molecular virology.
Start Year 2013