Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana
| dc.contributor.author | Regnard, Guy L | |
| dc.contributor.author | Rybicki, Edward P | |
| dc.contributor.author | Hitzeroth, Inga I | |
| dc.date.accessioned | 2017-09-18T08:12:56Z | |
| dc.date.available | 2017-09-18T08:12:56Z | |
| dc.date.issued | 2017-09-11 | |
| dc.date.updated | 2017-09-17T03:22:19Z | |
| dc.description.abstract | Background: Beak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana. Methods: The BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy. Results: In this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector. Conclusions: This is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV. | |
| dc.identifier.apacitation | Regnard, G. L., Rybicki, E. P., & Hitzeroth, I. I. (2017). Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana. <i>Virology Journal</i>, http://hdl.handle.net/11427/25228 | en_ZA |
| dc.identifier.chicagocitation | Regnard, Guy L, Edward P Rybicki, and Inga I Hitzeroth "Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana." <i>Virology Journal</i> (2017) http://hdl.handle.net/11427/25228 | en_ZA |
| dc.identifier.citation | Regnard, G. L., Rybicki, E. P., & Hitzeroth, I. I. (2017). Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana. Virology Journal, 14(1), 174. DOI: 10.1186/s12985-017-0847-9. | |
| dc.identifier.ris | TY - Journal Article AU - Regnard, Guy L AU - Rybicki, Edward P AU - Hitzeroth, Inga I AB - Background: Beak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana. Methods: The BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy. Results: In this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector. Conclusions: This is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV. DA - 2017-09-11 DB - OpenUCT DO - 10.1186/s12985-017-0847-9 DP - University of Cape Town J1 - Virology Journal LK - https://open.uct.ac.za PB - University of Cape Town PY - 2017 T1 - Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana TI - Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana UR - http://hdl.handle.net/11427/25228 ER - | en_ZA |
| dc.identifier.uri | http://dx.doi.org/10.1186/s12985-017-0847-9 | |
| dc.identifier.uri | http://hdl.handle.net/11427/25228 | |
| dc.identifier.vancouvercitation | Regnard GL, Rybicki EP, Hitzeroth II. Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana. Virology Journal. 2017; http://hdl.handle.net/11427/25228. | en_ZA |
| dc.language.iso | en | |
| dc.publisher | BioMed Central | |
| dc.publisher.department | Department of Molecular and Cell Biology | en_ZA |
| dc.publisher.faculty | Faculty of Science | en_ZA |
| dc.publisher.institution | University of Cape Town | |
| dc.rights.holder | The Author(s). | |
| dc.rights.uri | https://creativecommons.org/licenses/by/4.0/ | |
| dc.source | Virology Journal | |
| dc.source.uri | https://virologyj.biomedcentral.com/ | |
| dc.subject.other | Plant-made pharmaceutical | |
| dc.subject.other | Beak and feather disease virus | |
| dc.subject.other | Virus-like particle | |
| dc.subject.other | Circovirus | |
| dc.subject.other | Subunit vaccine | |
| dc.title | Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana | |
| dc.type | Journal Article | |
| uct.type.filetype | Text | |
| uct.type.filetype | Image |