Understanding host interactions with high consequence pathogens using super resolution light microscopy in biocontainment.

This research proposal is for a piece of laboratory equipment called a Cell Discoverer 7. This is a microscope that allows us to see within cells. This is vital because infectious diseases important to animal and human health, such as viruses, work inside cells, and we need to see how they work in real time in order to better develop mechanisms to stop them. This equipment is important for us as we want to use it to study infectious diseases that impact on human and animal health at Containment Level (CL3). Currently, to our knowledge, there is no such facility in government supported research institutes or Universities. CL3 is a way of working safely with dangerous pathogens that pose a greater threat to human and/or animal health. Examples include, SARS-CoV-2, certain influenza viruses, many infectious diseases that we get from mosquitoes and bacteria like tuberculosis (TB). Working at CL3 is not easy and the experiments you can do are limited by safety considerations. Currently the state of the art is that any images of infection are from inactivated specimens - this leads to distortion of images/pictures and no live work. The Cell Discoverer 7 has some unique features that allow a real step change and allow us to really advance our research on high consequence infectious diseases of humans and animals. This advanced microscope will allow us to follow the proteins and effects of infectious diseases in real time e.g., in living things. This latter feature is the really important one - as this has not been done before at high containment. We will be to determine whether therapeutic compounds are effective in stopping disease progression as well as identify fundamental parts of the infectious disease lifecycle that we could target in the future. Because of the unique features of this microscope and our ability to use it at CL3, we have many letters of support from Research Council/Government supported Institutes and Universities where we would act as a research hotel and training facility. This would really drive forward the competitiveness of UK science and more importantly allow us to deal with the infectious disease challenges that are on the horizon. These include zoonotic infections (diseases that we get from animals), vector borne diseases due to climate change (diseases that we get from things like mosquitoes) and studying anti-microbial resistance.

We propose to install a state-of-the-art real time live cell imaging system in the internationally recognised Containment Level 3/outbreak response laboratories at the University of Liverpool, enhancing capabilities for live cell imaging on dangerous pathogens at the human/animal interface. We will use this equipment to:

(i). Study the trafficking of proteins from infectious diseases in the cell in real time and identify the dynamic interactions with host protein complexes.

(ii). Follow the effects of pathogens in real time on their host by our further development of organ slices, organoids, 3D cultures such as organ on a chip. This includes changes in morphology, the influx and efflux of different cell types in response to infection, including inflammation that is responsible for much of a disease phenotype. This focus will also allow us to contribute to the refinement, reduction and replacement in animal research.

The possible gains in sensitivity (limits of detection) combined with live cell imaging at CL3, are thus unique with respect to the instrumentation, both within the University of Liverpool, and across the UK. This advanced instrumentation is essential if we are to maintain UK infectious disease research at the forefront of this rapidly developing field, which underpins mainstream biological and veterinary and biomedical research. Our exemplar projects demonstrate the breadth and broad applicability of how this advanced live cell imaging instrumentation will expand the breadth and detail of information that can be elucidated from biological samples. Specifically, we will establish the first platform in the UK for live cell imaging at high containment and improve the characterisation of the dynamic interactions that occur within a cell, tissue, and organism in response to infection.