Dissecting the molecular pathways of MDV oncoprotein Meq for understanding pathogenesis and aid vaccine development
Lead Research Organisation:
The Pirbright Institute
Department Name: Avian Oncogenic Viruses
Abstract
Infectious diseases result in direct and indirect losses at various steps of poultry farming and amongst them neoplastic disease caused by viruses is a major economic problem faced by the poultry industry worldwide. The oncogenic viruses causing neoplastic infection in chickens are herpesviruses comprising of Marek's disease virus (MDV) and retroviruses comprising of reticuloendotheliosis virus (REV) and avian leucosis virus (ALV). Marek's disease (MD) is a common disease of chickens involving paralysis and commonly death from the growth of highly malignant T lymphomas (cancers of white blood cells). MD is caused by a transmissible agent MDV. MDV is very contagious and is a major threat to the poultry industry worldwide. The estimated total loss from this disease worldwide is up to $2 billion. Presently, it is controlled by vaccination, and nearly 22 billion vaccine doses a year are used in an attempt to control the disease. Despite widespread vaccination, the threat from this disease is on increase due to continued evolution of MDV towards greater virulence, and more fundamental studies to understand the mechanisms by which this virus causes cancer is needed to develop more effective control programmes. Meq is the major oncoprotein in MDV induced tumorigenesis. Previously, we have identified Meq targetome in cancer cells. In this new grant proposal, to be carried out jointly between the Pirbright Institute and Roslin Institute, we want to extend these studies to obtain detailed information on the role of Meq and its targets in MDV induced oncogenesis using a number of advanced approaches including CRISPR/Cas9 genome editing we have recently established in the lab and rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for Meq interactome identification. The study is very important to understand the mechanisms by which this virus induces cancer, some of which are valuable in understanding cancer in other species including humans. Finally, the findings from the project will be very valuable in developing new approaches for the control of cancers caused by oncogenic viruses.
Technical Summary
The objectives of this project are to dissect the molecular pathways of MDV oncoprotein Meq for understanding pathogenesis and aid vaccine development. Meq is the major oncoprotein in MDV induced tumorigenesis. Yet the underlying molecular mechanisms are not fully understood. Our overall aim is to dissect the molecular events during Meq-induced neoplastic transformation exploiting the recent technological advances such as the CRISPR/Cas9 genome editing and rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). Specifically, we will investigate the role of Meq and its cancer related targets in maintaining the transformed phenotype of MDV1 cell line by deleting/mutating Meq/Meq-targets using CRISPR/Cas9 genome editing tool in MDV transformed cell line and perform proliferation assay for cell growth and RNA-seq for gene expression and pathways involved. RIME will be carried out to identify Meq interactome. Finally, we will delete Meq from very virulent plus (vv+) MDV1 virus and also to replace vv+ Meq with vaccine strain CVI988 Meq using CRISPR/Cas9 system and ask the question "Are the mutant viruses could be used as vaccine?" The data obtained will allow us to understand the role of Meq in maintaining the transformed phenotype and molecular interactions and pathways involved during MDV-induced oncogenesis.
Planned Impact
Poultry industry is a rapidly growing sector crucial for the global food security, acting as a major source of protein for the growing world population. Marek's disease (MD) is one of the major disease of poultry which causes serious economic losses and the global estimate of losses from MD is approximately $2 billion annually. Detailed understanding of the molecular basis of MDV induced oncogenesis, as the current proposal aims to achieve, will benefit development of new strategies for control.The beneficiaries of this research will include academic scientists, the poultry breeding companies and vaccine production companies, the Pirbright Institute, the BBSRC and its stakeholders such as Defra and the UK farming industry.
The research will have general impact with the wider scientific community, veterinary and medical practitioners, students and general public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored web pages, press releases to the media and outreach events in schools.
In the longer term the research may lead to medical benefits by improving control of human virus pathogens which will benefit the UK MRC and UK department of Health, the pharmaceutical industry and international organization such as the World Health Organization.
If the proposed studies lead to new approaches for controlling MDV or other viral diseases, additional funding will be sought from relevant funding agencies and other sources for further development. There is extensive experience within the Pirbright Institute of patent applications and commercialisations, new opportunities will feed into an established system for technology development and knowledge transfer by the Pirbright Business Development group.
The research will have general impact with the wider scientific community, veterinary and medical practitioners, students and general public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored web pages, press releases to the media and outreach events in schools.
In the longer term the research may lead to medical benefits by improving control of human virus pathogens which will benefit the UK MRC and UK department of Health, the pharmaceutical industry and international organization such as the World Health Organization.
If the proposed studies lead to new approaches for controlling MDV or other viral diseases, additional funding will be sought from relevant funding agencies and other sources for further development. There is extensive experience within the Pirbright Institute of patent applications and commercialisations, new opportunities will feed into an established system for technology development and knowledge transfer by the Pirbright Business Development group.
People |
ORCID iD |
YONGXIU YAO (Principal Investigator) | |
Venugopal Nair (Co-Investigator) |
Publications
Zhu ZJ
(2021)
Virus-encoded miR-155 ortholog in Marek's disease virus promotes cell proliferation via suppressing apoptosis by targeting tumor suppressor WWOX.
in Veterinary microbiology
Zhang Y
(2018)
Application of CRISPR/Cas9 Gene Editing System on MDV-1 Genome for the Study of Gene Function.
in Viruses
Zai X
(2022)
Identification of a Novel Insertion Site HVT-005/006 for the Generation of Recombinant Turkey Herpesvirus Vector.
in Frontiers in microbiology
Yao Y
(2019)
Herpesvirus of Turkeys (Meleagridis Herpesvirus 1) Encodes a Functional MicroRNA-221 Homolog with High Sequence Conservation
in Advances in Microbiology
Yang F
(2020)
Antiviral effect of baicalin on Marek's disease virus in CEF cells.
in BMC veterinary research
Xu M
(2021)
Novel mutation of avian leukosis virus subgroup J from Tibetan chickens.
in Poultry science
Tang N
(2019)
Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing.
in Journal of visualized experiments : JoVE
Tang N
(2021)
Application of CRISPR-Cas9 Editing for Virus Engineering and the Development of Recombinant Viral Vaccines.
in The CRISPR journal
Nair V
(2020)
Non-Coding RNAs
Li T
(2019)
Co-infection of vvMDV with multiple subgroups of avian leukosis viruses in indigenous chicken flocks in China.
in BMC veterinary research
Description | One of the key findings of this project was the establishment of an efficient pipeline for in situ CRISPR editing of the Marek's disease virus (MDV) genome in lymphoma-derived cell lines. Using this approach, we have demonstrated viral genes such as pp38 and MDV-miR-M4 are not essential for maintaining the transformed phenotype. On the other hand, deletion of MDV-encoded oncogene Meq or mutations preventing its interactions resulted in cell death demonstrating the essential role of Meq in transformation. In addition, we have demonstrated that targeted deletion of the MDV-2 essential gene glycoprotein B (gB) from MSB-1 cells coinfected with oncogenic MDV-1 and non-pathogenic MDV-2 viruses induces total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens. Another key finding was the establishment of CRISPRa (CRISPR activation) system to activate the latent viral genes in MDV cell lines. Using this approach, we have demonstrated targeted activation of pp38/pp24 expression in LCLs triggers lytic replication of MDV in MDV-transformed cell lines. |
Exploitation Route | Establishment of the pipeline for in situ CRISPR editing of lymphoma-derived cell lines, gives the opportunity for the identification of critical viral and host genes involved in features such as virus-host interactions, neoplastic transformation, and virus latency. Determination of the global myc-binding profiles in transformed macrophages could provide insights into its role in transformation. Achieving rapid detection of ALV infection is imperative in effective control of the spread of ALVs. |
Sectors | Agriculture, Food and Drink |
Description | BBSRC IAA The Pirbright Institute |
Amount | £300,000 (GBP) |
Funding ID | BB/S506680/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2018 |
End | 03/2021 |
Description | CRISPR/Cas system-based molecular diagnostics for avian viral pathogens |
Amount | £21,263 (GBP) |
Organisation | The Pirbright Institute |
Sector | Academic/University |
Country | United Kingdom |
Start | 01/2020 |
End | 03/2020 |
Description | Collaborative partnership for establish of PhysioMimix™ OOC system, part of Pirbright Institute Flexible Talent Mobility Account |
Amount | £180,000 (GBP) |
Funding ID | BB/S507945/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2020 |
End | 12/2020 |
Description | Development of CVI-988 based recombinant vaccine |
Amount | £240,000 (GBP) |
Organisation | Eco Animal Health Ltd |
Sector | Private |
Country | United Kingdom |
Start | 08/2019 |
End | 03/2021 |
Description | Development of HVT vectored vaccine |
Amount | £100,000 (GBP) |
Organisation | MSD Animal Health |
Sector | Private |
Country | United Kingdom |
Start | 11/2022 |
End | 04/2023 |
Description | Development of HVT-ND using CRISPR/Cas9 system |
Amount | £188,317 (GBP) |
Organisation | HIPRA |
Sector | Private |
Country | Spain |
Start | 06/2022 |
End | 05/2023 |
Description | Development of avian herpesvirus vector vaccines for poultry |
Amount | £432,000 (GBP) |
Organisation | MSD Animal Health |
Sector | Private |
Country | United Kingdom |
Start | 01/2021 |
End | 12/2023 |
Description | Development of improved HVT based vaccines using CRISPR/Cas9 system |
Amount | £258,820 (GBP) |
Organisation | Bill and Melinda Gates Foundation |
Sector | Charity/Non Profit |
Country | United States |
Start | 01/2021 |
End | 12/2022 |
Description | Development of multivalent HVT vectored vaccines |
Amount | £15,169 (GBP) |
Organisation | Vaxxinova |
Sector | Private |
Country | Netherlands |
Start | 06/2022 |
End | 05/2023 |
Description | PhD studentship |
Amount | £110,000 (GBP) |
Organisation | The Pirbright Institute |
Sector | Academic/University |
Country | United Kingdom |
Start | 10/2022 |
End | 03/2026 |
Description | PhD studentship |
Amount | £130,000 (GBP) |
Organisation | University of Oxford |
Sector | Academic/University |
Country | United Kingdom |
Start | 10/2022 |
End | 09/2026 |
Description | PhD studentship |
Amount | £91,830 (GBP) |
Organisation | The Pirbright Institute |
Sector | Academic/University |
Country | United Kingdom |
Start | 10/2019 |
End | 03/2023 |
Description | Production of HVT vectored VhH construct |
Amount | £155,000 (GBP) |
Organisation | Eco Animal Health Ltd |
Sector | Private |
Country | United Kingdom |
Start | 01/2020 |
End | 06/2020 |
Description | Seeding Catalyst Award |
Amount | £29,683 (GBP) |
Funding ID | ISCF-TFPSA-Pirbright |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2018 |
End | 02/2019 |
Title | Establishment of an efficient CRISPR/Cas9-based screening pipeline for herpesviruses to study the gene function and streamline the vector platform for recombinant vaccine development |
Description | The development of both cosmid DNA and Bacterial artificial chromosome (BAC) technologies has greatly facilitated the introduction of mutations into the viral genomes of herpesviruses to study gene functions. However, cloning of viral genomes as BAC plasmid and subsequent mutagenesis is inefficient, time-consuming and may introduce mutations by repeated passages. Following our success in efficient CRISPR/Cas9 editing of the MDV genome in both replicating virus and MDV transformed cell lines and of HVT genome for the recombinant vaccine development, we have developed an efficient CRISPR/Cas9-based screening pipeline for herpesviruses to identify the essential/non-essential genes for study of the gene function and streamline the vector platform for recombinant vaccine development. |
Type Of Material | Technology assay or reagent |
Year Produced | 2022 |
Provided To Others? | No |
Impact | Identification of essential and non-essential genes of herpesviruses provided opportunities to study gene function in vitro and in vivo. Knocking out the non-essential genes in the HVT vector can enhance HVT replication in vivo enabling recombinant HVT-based vaccines to induce stronger immune responses. |
Title | The establishment of an efficient pipeline for in situ CRISPR editing of the Marek's disease virus (MDV) genome in lymphoma-derived cell lines |
Description | The lymphoblastoid cell lines (LCLs) derived from MD lymphomas have served as valuable resources to study virus-host molecular interactions in transformed cells. However, detailed investigations into the functional role of different viral and host determinants in these cells have been difficult due to the lack of tools for in situ manipulation of viral/host genomes in MDV-transformed cell lines. Our recent success in efficient CRISPR/Cas9 editing of the MDV genome in LCLs has demonstrated the potential for targeted editing to dissect the regulatory pathways involved in latency, transformation, reactivation and lytic switch. |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Using this approach, we have demonstrated viral genes such as pp38 and MDV-miR-M4 are not essential for maintaining the transformed phenotype. On the other hand, deletion of MDV-encoded oncogene Meq or mutations preventing its interactions resulted in cell death demonstrating the essential role of Meq in transformation. |
URL | https://www.mdpi.com/1999-4915/10/6/279 |
Description | Finn Frey |
Organisation | University of Edinburgh |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Worked together to identify the host factors involved in MDV latency |
Collaborator Contribution | Provided chicken gRNA library for screening of MDV cell line |
Impact | Not yet |
Start Year | 2018 |
Description | Mick Watson |
Organisation | University of Edinburgh |
Department | The Roslin Institute |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provide samples for host-virus interaction study of MDV |
Collaborator Contribution | Data analysis of RNA-seq on CRISPR/Cas9 edited MDV cell lines. |
Impact | One publication from this collaboration: Zhang Y, Tang N, Luo J, Teng M, Moffat K, Shen Z, Watson M, Nair V#, Yao Y#. Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines. J Virol. 2019 Jun 12. pii: JVI.00713-19. doi: 10.1128/JVI.00713-19. |
Start Year | 2018 |
Description | Professor Guozhong Zhuang |
Organisation | Henan Agricultural University |
Country | China |
Sector | Academic/University |
PI Contribution | Provided knowledge and expertise on MDV research |
Collaborator Contribution | Provided samples for MDV research |
Impact | One publication has been generated: Sun A, Yang S, Luo J,Teng M, Xu Y, Wang R, Zhu X, Zheng L, Wu Y, Yao Y, Nair V, Zhang G, Zhuang G. UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells. Veterinary Research. 2021, 52:20. 10.1186/s13567-021-00901-5 |
Start Year | 2019 |
Description | Professor Luo |
Organisation | Henan Academy of Agricultural Sciences |
Country | China |
Sector | Academic/University |
PI Contribution | Provided knowledge, reagent and information on MDV research |
Collaborator Contribution | Provided samples and reagent for MDV research |
Impact | 6 publications have been generated: 1. Sun A, Yang S, Luo J,Teng M, Xu Y, Wang R, Zhu X, Zheng L, Wu Y, Yao Y, Nair V, Zhang G, Zhuang G. UL28 and UL33 homologs of Marek's disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells. Veterinary Research. 2021, 52:20. 10.1186/s13567-021-00901-5 2. Zhu Z, Teng M, Li H, Zheng L, Liu J, Yao Y, Nair V, Zhang G, Luo J. Virus-encoded miR-155 ortholog in Marek's disease virus promotes cell proliferation via suppressing apoptosis by targeting tumor suppressor WWOX. Veterinary Microbiology. 7 November 2020, https://doi.org/10.1016/j.vetmic.2020.108919 3. Zhu Z, Teng M, Li H, Zheng L, Liu J, Chai S, Yao Y, Nair V, Zhang G, Luo J. Marek's disease virus (Gallid alphaherpesvirus 2, GaHV-2)-encoded miR-M2-5p simultaneously promotes cell proliferation and suppresses apoptosis through RBM24 and MYOD1-mediated signaling pathways. Frontiers in Microbiology. 03 November 2020 | https://doi.org/10.3389/fmicb.2020.596422 4. Luo J, Teng M, Zai X, Tang N, Zhang Y, Mandviwala A, Reddy VRAP, Baigent S, Yao Y, Nair V. Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System. Viruses. 2020 Apr 20;12(4): E466. doi: 10.3390/v12040466. 5. Zhang Y, Tang N, Luo J, Teng M, Moffat K, Shen Z, Watson M, Nair V#, Yao Y#. Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines. J Virol. 2019 Jun 12. pii: JVI.00713-19. doi: 10.1128/JVI.00713-19. 6. Zhang Y, Luo J, Tang N, Teng M, Reddy VRAP, Moffat K, Shen Z, Nair V#, Yao Y#. Targeted Editing of the pp38 Gene in Marek's Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System. Viruses. 2019 Apr 26;11(5). pii: E391. doi: 10.3390/v11050391. |
Start Year | 2018 |
Description | AN INTERVIEW WITH PROFESSOR VENUGOPAL NAIR |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Professor Venugopal Nair is a Research Group Leader at The Pirbright Institute, a visiting Professor of Avian Virology at the Department of Zoology, and a Jenner Investigator at the Jenner Institute, University of Oxford. He is also a member of the Microbiology Society, and in this interview, he tells us more about his research into viral diseases of livestock. |
Year(s) Of Engagement Activity | 2020 |
URL | https://microbiologysociety.org/membership/meet-our-members/focus-area-viruses/an-interview-with-pro... |
Description | Interview by CGTN |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Increased profile of both Institute and The UK-China Centre of Excellence for Research on Avian Diseases (CERAD) |
Year(s) Of Engagement Activity | 2019 |
URL | https://newseu.cgtn.com/news/2020-01-28/British-and-Chinese-scientists-join-forces-to-fight-avian-di... |
Description | School visit (Tilingborne) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Stimulating increased interest in science and research |
Year(s) Of Engagement Activity | 2019 |
Description | School visit (Woking) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Stimulating increased interest in science and research |
Year(s) Of Engagement Activity | 2019 |
Description | Science Festival (Cheltenham) |
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 | Stimulating increased interest in science and research |
Year(s) Of Engagement Activity | 2019 |