U075833.pdf (37.31 MB)
An investigation into the presence of coxsackie viruses in idiopathic inflammatory muscle disease.
thesisposted on 2015-11-19, 08:49 authored by David Andrew. Hilton
Previous studies have claimed to have demonstrated the presence of coxsackie virus in skeletal muscle from cases of idiopathic inflammatory muscle disease (HMD) by either culture, the finding of paracrystalline arrays at electron microscopy or hybridisation techniques to demonstrate coxsackie virus RNA in muscle. Serological studies have also linked coxsackie virus exposure to HMD by the finding of elevated serum litres of antibodies to coxsackie viruses in cases of HMD and murine models of myositis can be induced by inoculation of neonatal mice with coxsackie virus. This study investigates the presence of coxsackie viruses in skeletal muscle from patients with HMD using three techniques: (i) In situ hybridisation to demonstrate the cellular localisation of virus RNA in muscle sections. (ii) Reverse transcription-polymerase chain reaction (PCR) to search for small amounts of virus RNA in extracted total RNA from muscle. (iii) Electron microscopy to search for the presence "virus-like" structures, in particular, paracrystalline arrays. Oligonucleotide probes, each of 30 bases in length, were designed to be homologous to conserved regions of the coxsackie B virus genome. A "cocktail" of 6 probes was used in order to detect as many different virus strains as possible and to increase the sensitivity of detection. The probes were synthesised on an oligonucleotide sythesiser then labelled at the 3' hydroxyl group with digoxigenin-ll-dUTP using terminal deoxynucleotidyl transferase. In addition control oligonucleotide probes to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA were designed and synthesised, in order to demonstrate RNA preservation within tissues. To demonstrate the efficacy of these probes for the detection of virus by in situ hybridisation, mice, infected with several different strains of coxsackie virus, were obtained. Formalin-fixed paraffin-embedded sections from these mice were hybridised with both coxsackie virus and control probes. Using the "cocktail" of coxsackie virus probes, all 11 strains of coxsackie virus were detected with no signal in the tissue from uninfected mice. Control probes demonstrated RNA preservation in all mice. The distribution of virus RNA largely matched that of the morphological changes. In the coxsackie A virus strains (A5, A7, A8, A10, A16 and A21), virus was distributed widely thorough out skeletal and cardiac muscle, with a focal distribution in brown fat. The coxsackie B virus strains (Bl-5) had a more limited distribution in skeletal muscle, predominantly involving the front limb girdle, head and neck muscles. In addition, with all coxsackie B virus strains, there was focal infection of the myocardium, salivary glands, pancreas, thyroid, liver, brown fat, hair roots and central nervous system. 26 cases of HMD were selected. The case notes and histology of all were reviewed to ensure that they fulfilled the published criteria for diagnosis of HMD. Sections from the cases (either paraffin embedded or cryostat) were used for in situ hybridisation with coxsaclde and control probes. All cases showed evidence of RNA preservation using control probes to mitochondrial rRNA (which was found to be more useful as control probes than the G3PDH probes). With the coxsaclde probes, 9 cases showed signal within interstitial mononuclear cells and 5 cases showed focal signal within muscle fibres. In both of these instances the signal did not disappear with RNase predigestion of sections and identical signal was seen when using a range of other oligonucleotide probes which had been designed to detect unrelated RNA species. The signal within interstitial mononuclear cells appeared to be within mast cells and disappeared following acetylation of tissue sections, suggesting that a charge related phenomenon was causing the oligonucleotide probes (negatively charged) to bind to basic substances within mast cell granules. (Abstract shortened by UMI.).
Date of award1994-01-01
Author affiliationCollege of Medicine, Biological Sciences and Psychology
Awarding institutionUniversity of Leicester