Galleria mellonella as an infection model for viral pathogens

(Galleria mellonella) waxmoth larvae are now being used by laboratories to study diseases caused by fungal and bacterial pathogens. It has been one of the great success stories of the initiatives to replace experiments with mammals with alternative and more ethical alternatives. Last year there were 231 publications using this model, compared to 194 publications and 2017 and 141 publications in 2016. The waxmoth larvae model is now used to study the ways in which fungi and bacteria cause disease and to test new ways of treating the diseases with drugs. There are several reasons why waxmoth larvae have become so widely used. First, they have an immune system, which resembles parts of the mammalian immune system, so they mimic the disease process much more effectively than, for example, cell culture models. Second, unlike many invertebrates they can be kept at 37oC. Finally, they are large enough to be handled easily and to be injected with exact doses of the microorganism and drugs.

However, waxmoth larvae have not yet been reported to be of value for the study of diseases caused by viruses. In part, this reflects the knowledge that viruses are very host-specific. But we are also aware that some viruses can infect both insects and mammals. For example, many viruses are spread by mosquitos and ticks and cause disease in human and other mammals. Moreover, there is evidence that when these viruses infect insects they can have detrimental effects. For these viruses it may be feasible to use insects to study the infections. In this short and focussed project we will carry out a study to investigate whether a range of insect-borne viruses can cause disease in waxmoth larvae. We have already carried out a pilot study indicating that a virus called Venezuelan Equine Encephalomyelitis (VEE) virus causes disease in waxmoth larvae. We will extend our pilot study with VEE virus and also test whether related viruses can cause disease in waxmoth larvae. This would then open up opportunities to use this model to replace experiments with mammals. If successful, we estimate that 50% of experiments using mammals could be replaced with studies using G. mellonella larvae. In addition, by carrying out preliminary studies in waxmoth larvae the design of any subsequent studies in mammals would be refined and the value of these experiments improved. Our work will stimulate other researchers to investigate whether other viruses are able to cause disease in waxmoth larvae allowing for further application of the model and a further reduction of animal use in the study of viruses.

Galleria mellonella have been used as an infection model for bacterial pathogens of humans since the 1980's. The model is now being used widely for research into bacterial and fungal infections and for antibacterial and antifungal drug screening. G. mellonella possess an innate immune system comprising cellular and humoral responses. The cellular response involves the production and mobilisation of hemocytes. Hemocytes share structural and functional similarity with mammalian phagocytic cells. Since the initial interaction with phagocytes often determines the outcome of infection, the galleria model provides information beyond that which could be obtained using cell cultures.

Mammalian infection models are frequently used to study viral disease, and to evaluate therapeutics. We will work with viruses which are insect-vectored. These viruses can replicate in the insect vector species, and there are reports of pathology and morbidity in infected insects. These findings have led to the suggestion by Shah et al., (Cell, 175, 1931-1945. 2018) that the "advantage of focusing on interactions conserved across viruses and hosts is that these interactions could serve as a starting point for broad-spectrum antiviral development with limited risk of viral resistance".

In this 12 month project we will build on our preliminary data showing that G. mellonella larvae are susceptible to Venezuelan Equine Encephalomyelitis (VEE) virus infection. We will evaluate whether this infection model shows morbidity or mortality in response to challenge with related viral pathogens (Western Equine Encephalomyelitis Virus, Eastern Equine Encephalomyelitis Virus, Semliki Forest Virus and Sindbis Virus). Our work will open new opportunities by providing a non-mammalian model for studying viral disease and for antiviral drug screening. This would allow a reduction in the number of studies involving regulated vertebrate models by replacing them with alternative insect infection models.

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1. 3Rs impact
We already have a large body of evidence that the Galleria mellonella infection model has been widely adopted by University, industry and government laboratories who are working with bacterial and fungal pathogen. The proposed work aims to develop the G. mellonella model to reduce the number of mammals used in research associated with insect-vectored viral pathogens. We already have a large body of evidence that this infection model. In the UK there were 118 publications in 2018 which reported infection of mammals and we calculate that at least 5546 mice, at least 189 rabbits and 141 non-human primates were used in 2018.

This study will involve the University of Exeter, who have pioneered the development of G. mellonella larvae as an alternative infection model for bacterial pathogens, and the Defence Science and Technology Laboratory (Dstl) at Porton Down. Between 2016 and 2018 the number of mice and non-human primates used to study viral pathogenesis and to evaluate therapies at Dstl was 865 and 16, respectively. These procedures were categorised as severe. We envisage that 50% of these mice would be replaced by G. mellonella larvae. Additionally, studies carried out in G. mellonella larvae before progression into mammals will allow the mammalian studies to be better designed, providing refinement of experiments.


2. Exploitation of networks and building a 3Rs legacy
We will also stimulate wider interest in the model and promote uptake widely by publishing the results of our work and by attending and presenting the results of our work at a national meeting (e.g. Microbiology society Annual meeting). The PI is also organising an international workshop on G. mellonella (15th-17th May 2020, Frascati Italy). This workshop would allow a presentation on the use of G. mellonella as a virus infection model.

We will also visit research groups working with viral pathogens of humans and who are currently users of mammalian infection models, to engage them in testing the models we are developing. We will visit the Pirbright Institute, the Roslin Institute, Public Health England and the Glasgow Centre for Virus Research during months 4-8 of the project. We have already engaged with potential users of G. mellonella infection model for viral pathogens, including the University of Tartu (direct users of the VEEV infection model), Public Health England and The Pirbright Institute who have expressed an interest in the model. These organisations have provided letters of support for this project.

Biosystems Technology Ltd (BST) is a University of Exeter spin-out company and has established itself as the market leader in the supply of research grade G. mellonella larvae. With the approval of Dstl and the University of Exeter, BST will continue to disseminate information on the use of G. mellonella as a model for virus disease beyond the lifetime of this award. Dissemination will take place via the BST website (https://biosystemstechnology.com/), the twitter account and the dedicated LinkedIn page. Currently there are almost 1000 followers of the Twitter and Linked-In pages and this number continues to grow. Finally, BST attends major networking events including the European Congress of Clinical Microbiology & Infectious Diseases and the FEMS Congress of European Microbiologists with a trade stand and will promote the use of G. mellonella as an infection model for viral pathogens.