The objective of this project is to use reverse genetics to develop better ways of making vaccines that protect against more than one disease (multivalent vaccines). This technology allows us to mutate RNA virus genomes through DNA copies (cDNA) of the RNA genome. The new genome cDNA can then be used to obtain the mutated form of the virus. In these studies we will use an existing vaccine for peste des petits ruminants virus (PPRV), as a vector to deliver antigens from other economically important viruses. PPRV causes a devastating plague in small ruminants and has a severe impact on animal welfare and the economies of many countries in Africa and Asia. In previous studies using this technology with a related virus, rinderpest virus (RPV), we were able to express foreign proteins efficiently in infected cells and to produce effective marker vaccines for RPV as well as identify some of the molecular factors which determine differences in virulence between virus strains. Recently it has been shown that the genome of measles virus (MV), a closely related virus, can be artificially segmented and that cDNAs of these segments can be used in a similar way to the full-length nonsegmented cDNA to rescue viable virus. The segmentation and rescue of PPRV will provide a new way to deliver immunogens from other small ruminant viral pathogens. Work with other nonsegmented negative strand (NNS) viruses has shown that there is a limit to the amount of extra genetic material that can be added to NNS virus genomes before a reduction in virus viability is seen. Segmentation of NNS genome can effectively overcome this limit, as evidenced by the ability of the segmented MV to encode at up to six foreign proteins efficiently. If this is applicable to related viruses then it would increase their coding capacity and enable us to produce multivalent vaccines to simultaneously protect against several economically important diseases of ruminants and increase their cost-effectiveness. Whilst RPV has been virtually eliminated from the globe as a result of a concerted vaccination campaign over the past 20 years, PPRV is a disease emerging in new regions of the world and is now causing great economic losses across much of the developing world as well as on the borders of the European Union. The current live-attenuated vaccines developed for PPRV are safe and highly effective and are, therefore, ideal candidates for use as vaccine vectors that can be tagged to allow differentiation between infected and vaccinated animals. We wish to explore the segmented approach using PPRV as a vector delivery system for multiple antigens from other economically significant viruses such as bluetongue virus (BTV) and Rift Valley Fever virus (RVFV), insect borne pathogens which can infect cattle and sheep, the latter also being able to infect humans. BTV and RVFV were once considered exotic diseases although recently BTV has entered the European Union, having a devastating effect on agriculture. RVFV has the potential to also enter Europe as insect vectors that carry BTV may also competent for RVFV infection. Current use of the PPRV vaccine generates a sterilising immunity that gives lifelong protection against the virus and, for RVFV, a similar response is thought to be generated post vaccination. However, for BTV a number of distinct genetic variants exist which, although diverse, cluster across distinct geographical regions. We wish to develop vaccines that target viruses circulating within a specific areas. Both RVFV and BTV are endemic across much of Asia and Africa and effective vaccination strategies are integral to their control. The three viral diseases targeted in this proposal are in line with the BBSRC's combating diseases of the developing world strategy as well as DFID's long term commitment to improving the sustainability of agriculture in developing countries.

In order to develop new vaccines, or new ways of delivering vaccines, it is necessary to demonstrate that they provide protection from the disease of interest. One of the most frequently employed ways of doing this is by the use of vaccination challenge studies. In such studies laboratory animals are immunized with the vaccine and then exposed to the disease of interest: if the vaccine is effective then the animals are protected from illness (or at least more protected than animals which did not receive the vaccine). In the case of certain diseases, the number of laboratory animals which are used in these kinds of studies can be reduced by measuring the immune response of the animals following vaccination. In such cases, the immune response can be used to predict how well an animal will be protected from disease, without the need to actually expose it to the disease causing organism. However, in the case of tuberculosis, as well as a number of other important diseases, reliable immune response predictors of vaccination success have eluded identification because there is no single immune response (e.g. antibody production) associated with protection from disease. This project aims to assess whether a recently developed technique called ?RNA sequencing? can be used to address this problem. The immune response to tuberculosis is complicated, and involves the combined action of several different types of cells, which are co-ordinated by protein messenger molecules and protein molecules on the surfaces of cells. In turn, these are controlled by genes, which can be either up-regulated or down-regulated. RNA sequencing allows the degree to which these genes are being up or down-regulated to be measured. Hence it is possible to study the combined immune response of an individual following vaccination, rather than studying specific components. The samples which will be analysed in this study come from badgers vaccinated against tuberculosis. Badgers are known to be susceptible to infection with tuberculosis, and can transmit the disease to cattle. As badgers are legally protected in the United Kingdom, vaccinating them against tuberculosis provides a potentially valuable approach to controlling the disease in cattle. This project aims to identify immune responses in badgers following vaccination which predict how well protected they will be from tuberculosis, using RNA sequencing. Identifying such responses has the potential to reduce the number of animals which need to be used in this kind of research.

Bovine tuberculosis (bTB) is one of the most difficult animal health problems that the farming industry in Great Britain faces today. The number of cattle infected with bTB has been increasing year on year by 18%, which leads to serious losses for affected farms due to the slaughter of infected animals and the imposition of cattle movement restrictions. Government spending on disease surveillance and compensation to farmers has also been following this upward trend, with spending over 2004-2012 expected to top £1 billion. From these statistics it is clear that the current disease control strategy is not working, yet the reasons for this are not obvious. One possibility is that new forms of the causative agent of bTB, Mycobacterium bovis, have evolved in GB that are able to circumvent the current control measures. Research by the VLA has found that evidence for this latter scenario is supported by the presence of a range of different types of M. bovis circulating in GB that seem to be successful in spreading around the country from their original place of isolation. This proposal sets out to determine whether these diverse types of M. bovis interact with the immune system of cattle in different ways, and so explain their success. To achieve this we will take advantage of the recent availability of the complete DNA sequences of both M. bovis and the bovine host. This will allow us to explore how the host and pathogen interact with each other at the level of individual molecules, and to build up a more detailed picture of how M. bovis causes disease in cattle. The information coming from this project will help government policy makers to develop new control strategies based on the exploitation of epidemiological information, and offers the chance to stop the upward spiral of bTB disease burden and linked expenditure in GB.

The global spread of H5N1 highly pathogenic avian influenza viruses and their ability to infect not only birds but humans emphasises that human and animal health are unavoidably linked. At present avian influenza remains an animal disease problem under urgent need for control but control in birds will also reduce the potential for a human influenza pandemic. Our knowledge of the behaviour of avian influenza viruses in domestic fowl and wild birds is limited. This proposal poses some fundamental questions that address how the easily the virus can infect chickens, turkeys and ducks; how much, and for how long, virus is shed following infection in each species; and how avian influenza virus infection is controlled by the immune response of birds. Fundamental studies of this type will be critical to the design and implementation of control measures in the short term and the long term.

Vector-borne RADAR looks to enhance surveillance of mosquito-borne diseases of wild birds in the UK that have wider medical and veterinary importance by adopting a One Health approach. Our first work package will increase the scope of our successful, multi-disciplinary network, which was responsible for the detection of Usutu virus in the UK in 2020, to include additional routes for sampling both bird hosts and mosquito vectors. This will follow a similar and successful paradigm that has been used in the Netherlands to detect emerging mosquito borne viruses of wild birds (www.onehealthpact.org). In essence we will co-opt bird observatories and licensed ringers in areas deemed at high-risk of virus incursion to sample migrant birds (to identify potential routes of virus incursion) and resident birds (to elucidate geographic extent and virus prevalence). At each of our designated bird sampling sites we will also set up mosquito traps to provide information on vector community composition, virus prevalence and potential transmission networks. Our second work package will develop an early warning system for the detection of potential zoonotic viral diseases of wild birds in the UK. Two existing and independent citizen science data schemes (the British Trust for Ornithology's [BTO] Garden BirdWatch, and the Garden Wildlife Health [GWH] project) will be co-opted to identify clusters of disease incident reports which correlate with a reduction in wild bird reporting rates, as a proxy to indicate areas of virus circulation and assess near real-time impact. This approach has been shown to be successful, by our consortium, in retrospectively identifying patterns of Blackbird mortality and declines in reporting rates caused by the 2020 Usutu virus outbreak in the UK. Our third work package will be conducted in collaboration with citizen scientists who participate in the BTO Garden BirdWatch. We will undertake a survey aiming to understand fine-scale Blackbird movements and habitat use in UK gardens. Data generated will provide detail on specific habitat use and identify potential virus transmission risks in relation to habitat features of gardens that are also likely to encourage higher mosquito populations (e.g. areas of stagnant water). Alongside this we will establish Blackbird trapping sites in London and the south-east, where licensed ringers will aim to catch birds in mist nets and colour-ring (so they can be identified from a distance without the need for further trapping) individual Blackbirds in order to quantify survival rates through time. Ringers involved in the scheme will also be trained to deploy mosquito traps and identify mosquitoes to provide evidence to link vector abundance and disease prevalence directly to wild bird survival. The data generated from this research proposal will improve our understanding of how exotic mosquito-borne viruses emerge and persist in temperate areas while also feeding directly into government policy & risk assessments on zoonotic diseases, public health messages and mosquito control strategies. Through engagement with citizen scientists, wildlife rehabilitators, zoological collections, and animal welfare and conservation NGOs, we will also raise awareness of implications for animal health and impacts on biodiversity.