The Molecular Biology of FMDV Replication: Towards New Methods of FMDV Disease Control.

One of our biggest challenges will be to meet a growing demand for food, especially in the developing world and economies such as India and China where the demand for meat products is rising. Animal diseases have a major impact on the productivity of the livestock industry and safeguarding animal welfare will be a major component of maximising food production. Foot-and-mouth disease virus (FMDV) is an animal pathogen which infects domesticated animals (cattle, sheep, goats and pigs). It is probably the most contagious mammalian virus known and much feared as the virus can spread extremely rapidly. The virus can also cause long-term 'persistent' infections that are in apparent; they are difficult to diagnose in the field and complicate disease control. FMDV can also infect many species of wildlife, and in Africa persistent infections in wildlife provides an important reservoir that may infect domestic livestock.
FMDV causes disease around the globe and is a continual threat to UK agriculture via the import of contaminated animals and animal products. Infection is rarely fatal, but it can have a dramatic impact upon the productivity of farm animals. The 2001 UK outbreak caused massive economic damage (billions of pounds) due to lost trade and impact upon farming communities. In addition, control of the disease through the slaughter of infected and high-risk animals was highly controversial and unpopular and led to heightened interest in a "vaccinate to-live policy".

Vaccines are often of two types. The first are inactivated or 'killed' viruses which cannot infect the host but still prime the immune system to protect against later infection. The second are live viruses which are weakened or 'attenuated': they do infect the host thus triggering a superior immune response but are not strong enough to cause disease. Current vaccines for FMDV are only of the 'killed' type. In contrast, for the closely related human virus poliovirus, live 'attenuated' vaccines have been used to effectively control both disease and transmission such that global eradication of poliovirus is within sight. The effectiveness of current 'killed' vaccines for FMDV are limited by a number of factors and there is an urgent need to develop new control measures: we therefore wish to develop novel 'attenuated' vaccines, one of the goals of this research. To do this safely will require a detailed understanding of the complex interactions between FMDV and its host.

Our proposed research will give novel insight into how FMDV interacts with its host-cell to achieve rapid replication or establish persistent infections. We will use this information to improve the effectiveness and safety of vaccines. The first strategy is to use modern molecular biology to change the virus, to make new strains that can protect animals without causing the debilitating disease - so-called live-attenuated viruses. The second strategy is to use the knowledge of how the virus grows in cells to make a new type of virus that could only grow in special 'helper' cells we will also create. Such viruses will not be able to grow in an animal and cause disease. This would make conventional 'killed' vaccine production a much safer process. Success in either approach would stimulate the routine use of vaccine to control FMDV around the globe. In the longer term, this could make a difference by reducing the overall, global incidence of FMD with enormous economic and social value worldwide. We argue that better control of FMD is essential for food security and must be coupled with the development of new vaccines, or new methods of producing vaccines, to make this policy effective. This is the purpose of our research.

FMDV replicates very rapidly leading to cell necrosis within 4-3hrs, but can also establish persistent infections. Amongst picornaviruses, the FMDV genome has a number of unique genome features whose functions are largely unknown.

Full length cDNA copies of the RNA genome (ca 8,500nts) are 'infectious': cell-transfection with plasmids, or RNA transcribed in vitro, 'rescues' virus. Thus genomes can be manipulated easily and phenotypes studied - an immensely powerful technique. Deletion of sequences encoding capsid proteins (or replacement with a reporter like GFP) produces RNA genomes which can replicate, but cannot package themselves: 'replicons' which can be studied outwith high-containment. University laboratories can now study RNA replication (screen and quantify using GFP fluorescence) using replicons. They can fully collaborate with the IAH, Pirbright, where virus can be studied, or, by insertion of capsid protein sequences Pirbright can covert (manipulated) genomes back into virus.

Our strategy is to perform systematic studies of the functions of the unique genome features (mutagenesis/interactions), the molecular basis of how the genome establishes persistent infections, how the virus modifies the host-cell and how the cell responds to infection (Y2H/siRNA screens, protemics). We will use synthetic biology to create genomes amenable to manipulation (serotype O1K and the SAT3 serotype; casuses persistent infections in Buffalo) such that tracts can be interchanged.

In combination with sequencing studies of natural isolates (all serotypes, acute and persistently-infected animals) we will develop a knowledge-base that will enable us to (i) both 'design' new attenuated genomes, but also use semi-random codon destabilisation to create genetically stable attenuated (vaccine) strains, (ii) genetically modify virus and host-cell genomes, creating special dependencies, to provide biosecure production of killed vaccines.

Key Areas of Impact: Our single aim is to produce new methods of controlling FMDV. Our strategy to achieve this is by producing live, attenuated, strains of FMDV, or, to improve existing methods of vaccine production.

Economic & Societal Impact.

Attenuated Strains: In this work, we WILL produce attenuated strains. Large animal experiments (efficacy, challenge) are expensive, and cannot be justified at this early stage. However, we realise that impact on disease control can only be realised by animal testing. If, at any stage in the project, it appears we have produced candidate vaccines strains, strains that meet the criteria set out above, we will seek resources specifically for animal testing through partnership with industry via the BBSRC 'Follow-on Funding' scheme. Success at this final stage would impact upon (necessitate changes in) EU legislation with regards trade practices for the adoption of such a vaccine in Europe, but could have a more rapid, major, impact in the developing world.

Biosecure Killed Vaccine Production: A more involved route to the market place, but killed vaccines is a proven technology with established methods of production. We have alluded to the problems with these vaccines and the hazards associated with their production: here, we seek to gain knowledge that can be harnessed to improve this technology, to make it safer (a generic method of modifying - any - FMDV genomes to grow in modified producer cells) and appropriate for routine vaccination. Issues concerning animal experimentation and commercial exploitation are as described above.

Sucess in either line of research would bring huge economic and societal benefits to societies in developing countries, but also bring greater future security for UK food supplies. Parenthetically, the major enconomic impact of the 2001 UK outbreak was in the tourism, not agricultural, sector of the economy: effective control of FMDV would benefit the much greater number of workers employed in the UK tourism industry.

Potential Future Impact - Emerging Viruses: When new virus diseases appear, one looks to the existing knowledge base for related viruses. Enzoonosis of animal viruses, for example, is a known source of new human pathogens. FMDV, as we presently understand it, is a relatively old virus that has undergone relatively recent diversification. It was thought the genus aphthovirus solely comprised FMDV. We now know of other, closely related, bovine and equine viruses which are now also classified as aphthoviruses. FMDV RNA replicates very effectively in cells from many species, including human HeLa and HEK291 cells. This program of work addresses FMDV, but the aphthovirus knowledge base needs to be expanded to meet any future contingencies.

We are committed to engage the public on the science it funds and promote debate. We will improve the impact of our public engagement activities - including internet profile through our web pages, make information widely available to academics and the public through peer review publications, scientific meetings and consultations, workshops, training courses and exhibitions. Many aspects of the science surrounding FMDV is of popular interest, covered in the national / international press, radio and TV (involving PIs in this application): we will continue to engage with the media. We have toured the UK to address farming communities on the impact of FMDV and the tools to control virus spread. We are committed to and participate in the STEMNET programme which operates in schools to inspire young people in Science & Technology. The PDRAs employed on this grant will be strongly encouraged to participate in this scheme.

We have the agreement of 3 international experts to participate in the management of this project. With regards the impact we wish to achieve in the area of vaccines, Tim Doel has worked for many years in the FMDV vaccine industry and his guidance will be highly valuable to the project.