The role of 3'-Deoxy-3',4'-didehydro-cytidine in the host response to viral infections.

The COVID-19 pandemic has highlighted an urgent need for better methods of diagnosing viral infections, as well as an improved understanding of how our bodies fight them. I recently looked at all the small molecules in blood samples from a group of adult patients in hospital with different types of infections including COVID-19, other viruses and bacteria. I found that a specific molecule, called 'ddhC', was only produced by patients with viral infections, and was rarely present in those without. This has raised several interesting questions:

A. Can we use ddhC to help diagnose viral infections?
Understanding to what extent ddhC is produced in different conditions will help better understand what ddhC does in the body, and inform how we might be able to use it as a diagnostic test. A diagnostic test that can rapidly identify patients with viral infections would help in two ways:

1. In a viral pandemic, a test like this that was 'ready and waiting' would enable prompt identification and isolation of people with the viral infection - and could be used even before tests specific for the new virus were rolled out, helping to stop its spread immediately. The current tests we have for viruses are limited in terms of accuracy and how long they take to generate a result. They can also take several weeks to develop.

2. It is currently difficult for doctors to rapidly distinguish between patients with viral and bacterial infections, due to the similarities in symptoms. Consequently, antibiotics (which only work on bacterial infections) are overprescribed, which drives worsening bacterial resistance to antibiotics. A test that can quickly identify patients with viral and not bacterial infections would help reduce unnecessary antibiotic prescriptions and stem worsening antibiotic resistance.

I will investigate how the body's ddhC production differs in:
a. patients who are only mildly unwell
b. early versus late timing of infection
c. infections in children as opposed to adults
d. bacterial and viral infections together
e. different infections, including malaria
Overall, these investigations will inform our understanding of how ddhC is produced in a wide range of scenarios, and guide its potential use as a diagnostic tool.

The current method to detect ddhC requires a machine called a mass spectrometer, which needs a large laboratory and is expensive to run. Therefore, I will also investigate whether we can detect ddhC using aptamers, which are small pieces of DNA that can potentially bind to and capture small molecules like ddhC, leading to development of a more straightforward diagnostic test not reliant on complex machinery.

B. What is the biological role of ddhC in viral infections?
Although I have detected ddhC in blood samples of patients with viral infections, I do not know its biological role. Does it work to protect against viral infections? Does its role differ in different infections? Previous work in cells has shown that a form of ddhC can act to disable specific parts of a virus responsible for its replication. This suggests that ddhC is protective in viral infections, but to know more we require further data.

I will look at the levels of ddhC produced by human cells in different viral infections, including different variants of SARS-CoV-2, the virus that causes COVID-19. I will then look at what happens to viral growth when we introduce external ddhC, and when we switch certain genes responsible for ddhC production on and off. Through these experiments, we will gain a better understanding of why the body produces ddhC in response to viral infections, which may help identify new treatments for these infections in the future.

In summary, the research I am proposing will lead to a better understanding of ddhC's role in both the diagnosis of and biological response to viral infections, which will help in our fight against viral diseases and worsening antibiotic resistance.

There is an urgent need for both improved diagnostics and a better understanding of the human host response in viral infections. Rapid diagnostics will help direct appropriate antimicrobial therapies and guide infection control precautions. Elucidation of host physiological antiviral defences will help identify risk factors for deterioration and targets for new therapeutics.

I recently performed untargeted metabolic profiling of serum samples from patients with different infections, using liquid chromatography coupled with mass spectroscopy (LC-MS). I found, for the first time, that the antiviral small molecule 3'-Deoxy-3',4'-didehydro-cytidine (ddhC) was detectable in human serum and could accurately differentiate viral infections from bacteraemic infections and controls, generating an area under the receiver operator characteristic curve of 0.954. This preliminary data has driven two hypotheses that I plan to explore:

Hypothesis 1. ddhC is selectively produced as an acute response to viral infections in adults and children and can be used as a diagnostic tool. I will use LC-MS to characterise the host ddhC response in broader clinical contexts, including infections at different timepoints, paediatric cohorts and other non-viral infections, enabling exploration of its utility in viral diagnostics. I will develop an aptamer-based assay for ddhC by using the systematic evolution of ligands by exponential enrichment method to identify optimised nucleic acid aptamers that can specifically bind ddhC.

Hypothesis 2. Viral replication is inhibited by both exogenous ddhC and viperin-derived endogenous ddhC-triphosphate. I will explore the biological role of ddhC and its associated gene viperin in viral infections. I will use in vitro respiratory epithelial cell models to investigate the natural ddhC response to infection and effects of exogenous ddhC addition and viperin overexpression on viral replication, focussing on SARS-CoV-2.