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Title: Investigations on Aspergillus fumigatus double-stranded RNAs and their effects on the fungus
Author: Bhatti, Muhammad Faraz
ISNI:       0000 0004 2706 3685
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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The aim of this research was to assess the incidence of dsRNA mycoviruses in the opportunistic human pathogenic fungus Aspergillus fumigatus, where previously no dsRNA viruses had been reported and to investigate the effects of any dsRNAs on the growth and pathogenicity of the fungus. Thus far 366 isolates (clinical and environmental) have been screened, 24 of which posses dsRNA elements. Successful efforts were made to completely characterise the two dsRNA segments of the isolate 88, partitivirus to obtain novel sequence information. Fungal viruses or mycoviruses are widespread and they usually infect their hosts persistently without any detectable phenotypic effects. They have been however linked with both hypovirulence and hypervirulence but are normally cryptic. To obtain information on the effect of the dsRNAs on their respective hosts, efforts were made to ‘cure’ isolate 88 of its dsRNA infection by cycloheximide treatment. However, following cycloheximide treatment, a sensitive reverse transcription polymerase chain reaction (RT-PCR) amplification assay showed that the dsRNA elements, whilst being reduced in amount, were not eliminated completely and that high levels of cycloheximide also interfered with spore production, pigmentation and overall growth of the isolate. In further experiments attempts were made to mobilise the dsRNAs from 4 isolates viz. A-56, A-54, A-78 and isolate 88 into isolate Af-273y, which is hygromycin resistant and yellow in colour, by hyphal tip fusion, protoplast fusion and protoplast transfection with purified virus. Protoplast fusion and viral transfection experiments were successful for some isolates, as assessed by the RT-PCR assay and small scale extractions of nucleic acids. Subsequently comparative growth experiments by radial growth assay and mycelial weight measurements between isolate Af-273y and Af-273y transfected with isolate 88 partitivirus in essentially the same genetic background were performed. These experiments showed that the partitivirus infection resulted in a sectored phenotype and significantly lowered the growth of the fungus. All efforts to initiate the molecular characterisation of uncharacterised dsRNA elements found in isolates A-54, A-78 and isolate-66 have thus far proven unsuccessful but a new approach (cDNA library construction) is proposed for the characterisation of these dsRNAs.
Supervisor: Coutts, Robert Sponsor: Higher Education Commission of Pakistan
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral