Fly infectivity assay for prion disease
Dr Raymond Bujdoso was awarded funding to test whether PrP transgenic Drosophila are a suitable invertebrate host to measure mammalian prion infectivity.
Principal Investigator: Dr Raymond Bujdoso
Organisation: University of Cambridge
Award type: Project grant
Start date: 2013
Duration: 2 years
Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of fatal neurodegenerative disorders that affect both humans and animals. They include CreutzfeldtJakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. The infectious agents are misfolded versions of the PrP protein, termed prions. PrP is a cell surface glycoprotein, found throughout the brain. It is highly conserved across vertebrate species and, although relatively little is known about its normal function, evidence suggests it is involved in a diverse range of biological processes from cellular differentiation through to the maintenance of myelin.
Prion diseases can arise spontaneously or be transmitted between individuals of the same and different species. This zoonotic potential led to the BSE crisis in the UK in the 1980s and the emergence of variant CJD in humans. Much of the early research into prion infectivity, including disease transmissibility and the existence of different strains, has been dependent on the use of animals, primarily non-human primates, sheep and goats. The demonstration that scrapie could be experimentally transmissible to rodents has, however, led to the widespread use of wild type and genetically modified mice as the ‘gold standard’ model.
Prion infectivity is typically measured by intracerebral or peripheral inoculation of a test material such as brain homogenate into an experimental host. There is often a long incubation period and clinical signs such as weight loss, ataxia and paralysis can take months or years to appear depending on route and dose of inocula, or prion strain used. The studies require close monitoring of the animals and the judicious use of humane endpoints given the potential for animal suffering.
3Rs benefits (actual and potential)
Raymond and colleagues generated Drosophila transgenic for ovine PrP to establish an invertebrate model of scrapie. Un-inoculated PrP transgenic flies show no adverse phenotypic effects. However, by feeding PrP transgenic Drosophila at the larvae stage with scrapieinfected sheep brain homogenate, Raymond demonstrated that the adult flies subsequently had hallmark features of mammalian prion disease, including the accumulation of infectious prions, a neurotoxic phenotype as assessed by locomotor assay, and enhanced mortality rate compared to controls.
Raymond investigated other aspects of prion transmission and infectivity in the fly model. This included studies in which head homogenate from scrapie-exposed PrP transgenic flies was inoculated into ovine PrP transgenic mice. The mice subsequently developed prion disease indicating that the fly can effectively propagate mammalian prions, providing a new model for studying prion biology. The Bujdoso laboratory have replaced the use of the rodent bioassay with the PrP fly model, saving around 200 mice a year.
Scientific and technological benefits
To test the sensitivity of the PrP transgenic fly model compared to the equivalent mouse model, animals were exposed to serial dilutions of scrapie-infected sheep brain homogenate. The flies showed a significant neurotoxic response to dilutions from 10-2 to 10-10 of the brain homogenate while the mouse bioassay detects a 10-6 dilution – in other words the fly bioassay is 10,000 times more sensitive than the mouse. This increased sensitivity has important practical applications. For example, Raymond has shown that PrP transgenic Drosophila can detect prion-infected blood from asymptomatic scrapie-infected sheep, opening the possibility for a confirmatory blood test for prion diseased individuals, including humans. Importantly, assessing prion infectivity in the fly takes about six weeks to complete while testing the same inoculum in mice can take months or years.
Raymond has published seven papers arising from the project grant, including in Brain, the Journal of Virology and the Biochemical Journal. A composite summary of the research was published on the NC3Rs gateway in 2018 – the paper has been downloaded 58 times to date.
Based on the utility of the model, Raymond has established new collaborations with research groups in Europe and North America and has generated bovine and cervid PrP flies in order to support these and replace the use of the natural hosts of prion diseases. Raymond was awarded an NC3Rs skills and knowledge transfer grant in 2017 for the validation of a fly bioassay for classical and atypical BSE prion infectivity. Working with collaborators, at the UK’s Animal and Plant Health Agency, a major reference centre for TSEs, Raymond has shown that bovine PrP transgenic Drosophila provide a highly sensitive and rapid bioassay to assess BSE infectivity. Following further validation work, this new invertebrate prion model could replace the use of 750 mice a year at the agency.
This case study was published in our 2019 Research Review.