Investigations on Aspergillus fumigatus double-stranded RNAs and their effects on the fungus
Author(s)
Bhatti, Muhammad Faraz
Type
Thesis
Abstract
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.
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.
Date Issued
2011
Date Awarded
2011-10
Advisor
Coutts, Robert
Sponsor
Higher Education Commission of Pakistan
Creator
Bhatti, Muhammad Faraz
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)