Alphavirus infection and nociception: differential effects on primary mouse dorsal root ganglia

Katz, Benjamin J. G. (2021) Alphavirus infection and nociception: differential effects on primary mouse dorsal root ganglia. MRes thesis, University of Nottingham.

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Alphaviruses such as Chikungunya Virus (CHIKV) and O’Nyong-Nyong Virus (ONNV) present an increasing problem in the world, with an increased vector efficacy allowing infection in previously uninfected areas such as southern Europe and a total infection count of over 5 million in the last 20 years. Most infected patients experience acute pain and a significant proportion go on to develop chronic rheumatoid-like arthritis whose mechanisms remain unclear. One potential target and source of mechanistic insight are the sensory dorsal root ganglia (DRG) that detect peripheral events and propagate nociceptive action potentials for recognition as pain in the CNS. This study aimed to determine if alphaviruses can infect DRGs and if so, which neuronal subpopulations are preferentially infected.

DRGs were excised from C57BL/6J mice (6-8wo) and cultured with growth media and 4uM aphidicolin on coverslips for 24h. ONNV-coverslips were infected with an aliquot of the virus (6.25x105 TCID50/ml) which had mCherry tagged to nsp3. Mock infection coverslips (CTRL) were not infected and both groups were incubated for 24h, 48h or 72h. Two immunohistochemistry experiments were performed on the DRG cultures with the following primary antibody target combinations: myelinated neurons (NF200+) and peptidergic neurons (CGRP+), as well as non-peptidergic neurons (IB4+) and neurons (NeuN+). Coverslips were imaged using a fluorescent microscope with cell counts analysed using ImageJ and GraphPad Prism.

No significant differences were found between CTRL and ONNV DRGs when considering total cell count. A statistically significant decrease in the mean relative percentage of CGRP-NF200- cells was found between CTRL and ONNV DRGs at 48h, (-11.86%, p = 0.0332), and 72h (-17.63%, p = 0.0024), as well as IB4-NeuN- cells at 72h (-21.31%, p = 0.0220). This occurred in parallel with an increase in ONNV infection in these populations. The numbers and percentages of peptidergic neurons were significantly increased in ONNV infected coverslips compared to CTRL at 72h.

This study has shown that ONNV can infect DRG neurons which may explain the manifestation of pain. Using mCherry labelled ONNV it was shown that ONNV preferentially infects myelinated peptidergic neurons and non-neuronal cells between 24h and 72h post infection, resulting in a significant increase in the relative proportion of peptidergic neurons, and a decrease in non-neuronal cells.

Further research is required to categorise the mechanisms underlying this infection, including the mechanism of axon transport, DRG responses to infection, and viral products released after cell death.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Chapman, Victoria
Hathway, Gareth
Keywords: Alphaviruses, Nociception, Dorsal Root Ganglia, Immunohistochrmistry
Subjects: Q Science > QR Microbiology > QR355 Virology
R Medicine > RC Internal medicine > RC 321 Neuroscience. Biological psychiatry. Neuropsychiatry
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 67027
Depositing User: Katz, Ben
Date Deposited: 08 Dec 2021 04:40
Last Modified: 20 Dec 2021 15:08

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