Exploiting African swine fever virus surface proteins to develop rapid diagnostic tests and understand virus host interactions

This project will investigate a virus disease of pigs, African swine fever (ASF), which causes major economic losses in affected countries. These are mainly in sub-Saharan Africa although ASF has recently been introduced to the Caucasus and Russia increasing the risk of further global spread. Most of the virus isolates cause an acutely fatal disease in pigs, although there are some less virulent isolates. ASF has potentially devastating effects on the commercial and subsistence pig production. In countries such as Cameroon or Madagascar, the introduction of ASF resulted in the loss of between 50 and 80% of the pig population. ASF has serious implications for food security. Pigs are often reared in areas where beef production is difficult. They are excellent converters of food waste and agricultural by-products into high quality protein and have a relatively short production cycle. ASF is difficult to control for several reasons. These include the presence of reservoirs of the virus, ASFV, in wild pigs and soft tick vectors, the lack of a vaccine and the stability of the virus in pork and in the environment. Most outbreaks of ASF are not reported and control measures are therefore not implemented. This is because samples have to be submitted to reference laboratories for diagnosis. In many African countries these are either not present or sending samples to them is costly and requires a long time. This project will aid control of ASF by development of penside diagnostic tests which can be used locally on farms. Similar penside tests have been used to great effect in the diagnosis and control of other animal diseases, such as in the campaign which has resulted in the global eradication of the disease known as cattle plague or Rinderpest. These penside diagnostic tests will be made available commercially in the short term. The approach taken will be to characterise the proteins that are exposed on the surface of the ASF virus particles and then to produce antibodies that recognise these proteins. ASFV is a large complex virus and it may be necessary to test antibodies against several proteins to identify those that are best to use in the diagnostic tests. The antibodies will be incorporated into simple devices, similar to pregnancy testing kits, which can be used on farms. These will be used to detect virus in samples from pigs that are suspected to be infected with ASFV thus ensuring rapid and accurate diagnosis. The virus surface proteins will be used for two other purposes. These proteins are expected to be important for the virus to bind and enter host cells to begin production of progeny virus particles. Activating the host's immune system to recognise these proteins may inhibit virus infection and protect pigs from ASF disease. The ability of combinations of these virus surface proteins to stimulate an immune response in pigs and protect them from ASF disease will be tested. Promising candidate proteins will be incorporated into programmes for vaccine development against ASFV. Finally, the reagents generated in the project will be used for basic scientific studies to improve our understanding of how ASFV virus particles bind to and enter host cells and how virus particles are assembled. This information will be of broad scientific interest in understanding the interaction of viruses with the host cell which in the longer term may lead to novel strategies to control virus infections.

African swine fever virus (ASFV) causes an often acutely fatal disease of pigs, which results in major economic losses in many African countries. There is no vaccine and frequently disease is not diagnosed because of difficulties in sending samples to reference laboratories. ASFV particles are multi-layered and include more than 50 proteins. The two infectious forms of the virus include an intracellular mature form produced in cytoplasmic virus factories. This consists of a nucleoprotein core and shell surrounded by an internal envelope onto which the icosahedral virus capsid is assembled. The other infectious form of the virus is the extracellular virus, which contains an additional external membrane layer. The proteins on the surface of both of these virus particles will be characterised by tagging the proteins with biotin followed by affinity purification of biotinylated proteins and their identification using mass spectrometry. First, antibodies which recognise these virus surface proteins will be produced and used to develop penside diagnostic tests. Secondly, we will investigate the localisation of these proteins in virus particles and in infected cells and begin to characterise their roles in virus binding and entry and morphogenesis. These studies will include investigating if the antibodies or recombinant proteins can inhibit virus infection of cells. Thirdly, we will investigate the immune response to these proteins. This will involve analysis of the cellular and humoral immune response to these proteins in pigs which recover from infection with attenuated ASFV isolates. We will also vaccinate pigs using a DNA prime recombinant vaccinia virus boost strategy using pools of constructs expressing individual ASFV genes. The immune response following vaccination will be monitored and the ability to withstand lethal ASFV challenge tested. This will identify candidate protective antigens which can be included in vaccine development programmes.

African swine fever (ASF) causes high mortality of pigs and major economic losses in many African countries. The lack of a vaccine and accurate diagnosis in most countries contributes to difficulties in controlling ASF. This project will characterise proteins on the surface of virus particles and develop antibodies against these for use in penside diagnostic tests. These will be available in the short term. We will also investigate if these surface virus proteins are targets for generating a protective immune response in pigs. This will identify candidate protective antigens which can be incorporated into vaccine development programmes. Vaccine development is likely in the medium term. Knowledge gained from the project will include information on the role of the surface proteins in virus binding and entry and their interactions with bystander cells which do not become infected. Beneficiaries from the research will include the commercial partners who will have the opportunity to develop and market penside diagnostic tests for ASFV. We expect to have developed prototype devices and produced these on a larger scale for validation to OIE standards during the course of the project. An initial training course in use of the devices will be held during the project for selected participants from several African countries. Development on a commercial scale is expected soon after completion of the project. Implementation of the use of these will involve more widespread training of staff in veterinary services from different countries. The development of these diagnostic tests will be disseminated through publication in peer reviewed open access journals and on IAH and OVI websites. The information will also be disseminated via presentations at meetings involving scientists, veterinary authorities and by press releases. Use of these devices will provide more and better information concerning the prevalence of ASF and enable diagnosis at the local level. This will improve the implementation of control measures both by providing more information rapidly and involving stakeholders at the local level more directly. Outputs from the research will also contribute to development of vaccines against ASF in the medium term. Availability of vaccines is essential for effective control particularly in areas where the tick vector and other wildlife reservoirs of the virus are present. Beneficiaries of the improved control will include governments which will gain from the reduced costs of disease control and increased food security. Farmers in the commercial, rural poor and peri-urban sectors will also benefit as will other stake-holders in the food supply chain. These benefits will impact in countries where ASF is endemic and will also be available to ASF free countries if outbreaks did occur. The research will also contribute to basic knowledge on the interaction of ASFV with host cells which will be of interest generally to those studying virus host interactions. The reagents generated, including antibodies, will be publicised in articles in peer reviewed Journals, on IAH and OVI websites and in presentations at meetings of scientists and veterinarians. The staff employed on the project will benefit from training in molecular and cell virology, diagnostic test development and immune assays. Through interaction with commercial partners they will gain experience of the Biotechnology Industry. The project team is multidisciplinary and involves experts in ASFV and other economically important animal viruses with experience ranging from the molecular and cellular study of virus host interactions to studies on immune responses and pathogenesis at the whole animal level. Several of the PIs have experience in the commercial exploitation of antibodies and in interacting with potential beneficiaries outside of the immediate academic community in both Europe and African countries. These include government bodies, farmers and veterinary authorities.