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.

Tick-borne diseases cause a significant health burden on both the human and domestic livestock populations within the United Kingdom (UK). This includes the recently detected tick-borne encephalitis virus, a common cause of encephalitis in humans across Europe, and the livestock disease caused by louping ill virus. Both are types of flaviviruses and are closely related, and both are now endemic within the UK. Many questions remain concerning the biology of these viruses and there are key gaps in understanding virus distribution within tick vector populations, fundamental questions on flavivirus virus pathogenesis and a clear lack of serological tests that can distinguish between antibodies to either virus in order to tell which is circulating in the different host species. To address these gaps, the TickTools project aims to conduct a series of studies, each coordinated by one of the project partners. The Animal and Plant Health Agency (APHA) will conduct field surveys for adult ticks from across the UK and determine the microbiological make-up present within each sample, which will identify all viruses and bacteria present. This approach will also capture the genome of each tick that can be used to assess the relationships between tick-populations within the country, which in turn could reveal the interactions between these populations and how ticks, and their pathogens disperse. APHA will support the University of Glasgow Centre for Virus Research (CVR) in establishing a virus infection model to determine the pathogenesis of tick-borne flaviviruses. This will be achieved by comparing the virulent virus with an attenuated virus. This approach will identify potential therapeutic targets for prevention and control of flavivirus infection with the aim of preventing the most severe manifestations of virus infection. From these studies, CVR will supply organ tissue (spleen) to the University of Nottingham (UoN) to support their development of scFv antibodies that can be used to further study both viruses but also have potential as treatments for people or pets that become infected. The UoN will also develop antigen (peptide) panels that will discriminate between serological responses to infection with either TBEV or LIV. Using assays developed by UoN, serological surveillance in both human and animal populations will be possible.

Marl lakes have been designated a priority habitat within the EU Habitats Directive for their nutrient-poor waters with benthic vegetation of charophytes (stoneworts). However, it is thought that the ecological status of several English marl lakes, including Semerwater and Malham Tarn, has been adversely affected by eutrophication including an increase in algae and a decline in the diversity of the submerged plant community. In the absence of long-term records the nature of these changes are poorly understood and hence the information needed to successfully conserve and manage these unique waters is lacking. This project seeks to combine contemporary limnological and biological investigation with palaeoecological analysis to elucidate the nature, timescales and magnitude of changes in ecological and limnological processes involved in the response of marl lakes to nutrient enrichment. The study will address three main hypotheses: 1. In the absence of enrichment, marl lakes exist in a stable, clear water state, dominated by a species-rich community of charophytes 2. In response to eutrophication, marl lakes exhibit a gradual (>100 years) transition from macrophyte to phytoplankton dominance. A decline in charophyte species richness leads to an encroachment of elodeid macrophytes, characterised by sub-decadal oscillations between charophyte and elodeid dominance. These changes occur in conjunction with a reduction in marl precipitation. Finally, submerged macrophytes are displaced by phytoplankton populations concomitant with the total cessation of carbonate precipitation. 3. On a shorter timescale, reductions in plant species richness result in a progressive decrease in the seasonal duration of plant cover with associated alterations in zooplankton, invertebrate and fish populations. It is expected that the main findings of the work will be incorporated into management plans for the three study lakes, specifically assisting Natural England in setting conservation objectives for marl lakes as required by the EU Habitats Directive, and providing information to the Environment Agency on reference conditions and ecological data as required by the EU Water Framework Directive.

Manufactured chemicals are essential for the maintenance of public health, food production and quality of life, including a diverse range of pharmaceuticals, pesticides, and personal care products. The use of these compounds throughout society has led to increasing concentrations and chemodiversity in the environment. Whilst there has been focus on understanding the impacts of chemicals on a subset of freshwater biodiversity (particularly invertebrates and fish), we understand less about how chemical pollution impacts freshwater microbes. These microbial communities (the 'microbiome') number in the millions to billions of cells per millilitre of water or gram of sediment and form the most biodiverse and functionally important component of freshwater ecosystems. The biogeochemical and ecological functions delivered by freshwater microbes are essential to wider freshwater ecosystem health. The PAthways of Chemicals Into Freshwaters and their ecological ImpaCts (PACIFIC) project will focus on understanding the link between sources of anthropogenic chemicals and their pathways, fate and ecological impacts in freshwater ecosystems, with an emphasis on freshwater microbial ecosystems and the functions they perform. We will investigate the relationship between predicted diffuse and point source chemical pathways and measured chemical concentrations in water and sediments at locations across the Thames and Bristol Avon catchments, chosen to represent gradients of diffuse pollution sources. These locations will be chosen to coincide with Wastewater Treatment Works (WwTWs) to understand how sewage effluent contributes to chemical burden across these gradients. Liquid chromatography coupled with (high resolution) tandem mass spectrometry and QTOF (quadrupole Time-of-Flight) mass spectrometry will be used to perform targeted and untargeted profiling of chemical groups proven and suspected to impact freshwater ecology. A range of microbial community ecosystem endpoints will also be measured at each location to identify the impact of chemical exposure, including bacterial and fungal community composition via DNA sequencing, the expression of nutrient cycling and chemical stress and resistance genes, the production of extracellular enzymes involved with biogeochemical cycling, and the functional gene repertoire of whole microbial communities. We will perform experimental microcosm exposures on freshwater microbial communities, with increasing complexity and realism, deploying high-throughput screening to identify novel chemical groups (and their structural features) with the capacity to restructure these communities. Exemplar microbial community modifying chemicals will be investigated in more detail by applying cutting-edge molecular techniques to determine ecological exposure thresholds that represent different taxonomic and functional aspects of freshwater microbial ecosystems. Novel field based mesocosms will be used to explore wastewater exposures in more realistic, but controlled settings, allowing us to explore how chemical pollution may interact with other ecological drivers such as nutrients and temperature, and how microbial responses scale-up to higher trophic levels and alter ecosystem functioning. Spatially and temporally up-scaled models of diffuse and point source chemical pollution pathways will be combined with novel thresholds developed from the lab and field exposures, to determine chemical threats to freshwater microbes, supporting the development of tools for the better management of the risks of chemical pollution to freshwater ecosystem health. These will be combined with future hydrological, climate and socio-economic scenarios, informed by responses in our experiments, and co-developed with project collaborators, the Environment Agency, to explore future threats to microbial freshwater ecosystems and wider ecosystem health.