Functional analysis of Epstein-Barr Virus genome variation in relation to cell growth and disease

Most people are infected by Epstein-Barr virus (EBV) as young children, and carry the virus for their whole life with no ill effects, but the virus can cause several human diseases. Glandular fever occurs when people who were not infected as children first become infected with EBV as teenagers or adults. Also, several specific types of cancer are caused by EBV; together these cancers account for 1.5% of all human cancers worldwide. EBV may also play a role in multiple sclerosis. These diseases occur at quite different rates in different parts of the world and we suspect that the different EBV strains present in those parts of the world may be part of the reason for that. We therefore need to understand what variation is actually present in EBV strains and find out whether it makes a difference to function of the virus, in ways that might be relevant to the human diseases associated with EBV.

We have recently published the first large scale DNA sequencing of EBV from different parts of the world, and from various EBV-cancers. This application will make use of this new information to understand how the variation in EBV sequence has an impact on EBV biology. In fact, we now know that most EBV research so far has used EBV lab strains that are poor representatives of the viruses that circulate in the human population. We shall therefore isolate - in a pure form suitable for research - a set of diverse EBV strains and assess their characteristics. The viruses will be tested by infection of human cells relevant to the diseases, both in the lab and in an animal model.

We shall also test two specific examples of the impact of virus diversity on biology and disease that have been highlighted by our preliminary work. First we shall investigate how two of the EBV genes (called EBNA2 and EBNA3) work together to determine the much greater ability of certain EBV strains to cause growth of white blood cells, likely relevant to some of the blood cancers associated with EBV. Second, we shall test whether specific differences we have identified in Chinese EBV may help to explain the very high incidence of EBV associated nasopharyngeal cancer that occurs in Hong Kong and nearby parts of China.

Epstein-Barr virus causes infectious mononucleosis and contributes to 1.5% of human cancers, yet the vast majority of humans carry EBV as an asymptomatic lifelong infection. Most EBV research has focussed on a small number of laboratory strains, many of them defective, and has neglected the diversity of circulating strains. We recently published the first major sequence analysis of worldwide EBV strains; we shall now investigate how this sequence diversity affects virus function.

Our first aim is to identify viruses from among the sequenced strains that best represent wild-type (circulating) viruses and clone them in bacterial artificial chromosome (BAC) vectors. This will allow us to generate virus and investigate their behaviour in vitro (activation, proliferation and transformation of B cells; infection of epithelial and T cells; EBV gene expression differences) and in vivo - using mice with human immune system components. We shall seek to produce an in vivo model for EBV infection that more closely reflects human infection. We expect these new EBV clones will become standard strains for EBV research worldwide.

Our second aim is to investigate the two major specific variations that we have previously identified through the sequencing project. First is the linkage between EBNA2 and EBNA3s in defining type1 and type 2 EBVs. Our preliminary data imply some functional compatibility of the EBNA2 and EBNA3 variants that affects B cell transformation and growth. We shall test this by generating viruses with additional combinations of EBNA2/3 types and assessing their transforming ability. Second, we shall assess the function of specific polymorphisms in EBV strains associated with nasopharyngeal carcinoma (NPC) in southern China. We shall test whether specific polymorphisms in NPC-associated EBV strains in the BART miRNA region of EBV result in a more profound suppression of p53, such that they increase susceptibility to NPC.

Benefit to the research area
The accurate identification of wild type EBV genomes representative of the virus occurring naturally in different parts of the world and construction of BAC clones of those genomes for production of virus will be of major benefit to those working on EBV research. Determining how EBV is involved in human disease involves understanding the role of viral genes and other environmental and biological factors in the predisposition to disease. Our results can be exploited in an experimental medicine approach for the prevention, treatment or early diagnosis of EBV associated diseases.

Benefit to the public - health and disease
EBV causes a wide range of diseases including glandular fever (infectious mononucleosis) and about 1.5% of all cases of cancer worldwide. There is also evidence that it contributes to multiple sclerosis.
All aspects of EBV research and clinical intervention rely on basic definition of the virus genome, understanding the global picture of what is wild type EBV, how it may vary and how that might be relevant to disease. That is at the centre of this research project. The only EBV vaccine tested in humans so far reduced the incidence of glandular fever but did not prevent infection. To make an effective vaccine it is important to ensure that its efficacy will include natural variants of the virus that may be present in different populations or parts of the world. Immunotherapy is under development for EBV associated diseases, again it is important to target the correct sequences and strains.

Biotech and Pharma, economic impact
Our work will enable biotech and pharma work on vaccines, early detection or therapeutics targeted towards EBV. A clinical trial currently ongoing in Hong Kong (lead by Dr Anthony Chan) suggests that it may be possible to identify NPC cases at a very early treatable stage by a PCR assay for persistent EBV DNA load in the blood plasma. It will be important to understand strain variation to ensure detection of the correct EBV in that assay. This type of EBV DNA screening is alreday being implemented in some corporate health programmes in Hong Kong and is likely to become widespread there.

A vaccine for EBV is the next logical target after the success of the HPV and Hepatitis B vaccines in cancer viruses. This could enable cancer prevention and also prevention of IM, a common disease in the UK. Our research on EBV strain variation will also be relevant to the successful cytotoxic T cell therapy of post-transplant lymphoma caused by EBV. The CTLs recognise epitopes in EBV EBNA and LMP genes and variation in them is likely to affect the success of the CTL therapy.

Society, education and skills
IM is frequent in adolescents and young adults when it can have a significant impact on schooling and University. It is a frequent disease in the UK. There already a strong scientific case for further trials of immunisation of EBV seronegative adolescents with the purpose of preventing infectious mononucleosis in this age group.

Our project involves high level sequence analysis, bioinformatics, molecular biology and virology research. It provides a strong training in scientific methods, problem solving computing, information technology, statistics and data handling. These are all transferable skills which help society in general. In addition, our research labs also provide an attractive environment for the more general training of undergraduate and postgraduate science and medical students.

We also regularly accept visiting university summer students and provide work experience in the lab each year for outstanding school 6th formers who may be interested in a scientific or medical career. In our Department we have hosted visits from local schools, eg Paddington College and have participated in the INSPIRE project - presenting research to secondary school students to inspire them to talk and think about science.