The stability of the altered phenotype of isolate A17 was investigated by serially passaging spores (serially diluted up to 10-4 on each occasion) onto ACM plates without cycloheximide which were incubated at 37°C for 5 days. The altered phenotype was only stable over three passages and sectoring returned in all of the serial dilutions on the fourth passage (Fig. 5.10). The dsRNA isolated from the fourth passage of A17 was used as template for RT- PCR and generated the complete CP amplicon, confirming infection with AfuCV. These results suggest that some cells in isolate A17 was always residually infected with AfuCV even though initial RT-PCR amplification suggested otherwise. Once the cycloheximide stress was removed during serial passage the titre of the viral dsRNAs increased to a level sufficient to act as a template and generate amplicons.
Figure 5.10 Colony morphologies of Aspergillus fumigatus isolate A-56; isolate A17 and the fourth passage of A17, serially diluted 10-1 to 10-4 on ACM plates 5 days after incubation. Photographs were taken from the back (A) and from the front (B) of the plates to emphasise similarities and differences.
The results of the previous experiments suggested that it might be possible to cure A. fumigatus isolate A-56 from AfuCV infection but that the concentration of cycloheximide
required to achieve this was in excess of 90 mM. Thus the concentration of cycloheximide used in curing was increased to 150 mM and compared with lower levels of the antibiotic viz. 50 mM, 75 mM, 100 mM, in anticipation of toxic effects and reduced growth of the fungus. Plates of ACM were amended with cycloheximide at the concentrations stated, dried for 24 h, inoculated with equal numbers of A. fumigatus isolate A-56 spores and incubated at 37°C for 10 days. Cultures grown in the presence of 150 mM cycloheximide expanded much more slowly with an altered pigmentation as compared to those growing in the presence of lower concentrations of the antibiotic (Fig. 5.11). These cultures were used as a source for further spores which were serially diluted and spread onto 360 mm Petri ACM plates in order to select single colonies. Ten single colonies were selected which were inoculated onto ACM plates to confirm the characteristic, non-sectored morphology. Of these ten colonies, four colonies viz 4 (1), 4 (P), 6 and 5 were selected to investigate potential curing from virus infection.
Figure 5.11 Colony morphologies of Aspergillus fumigatus isolate A-56 grown on ACM amended with different concentrations of cycloheximide as shown, 10 days after inoculation. Note slow growth and altered colony pigmentation in the culture amended with 150 mM cycloheximide.
Spores of the four selected sub-isolates were grown in 5 ml liquid cultures of ACM, at 37°C for 3 days. Mycelia were harvested and processed using the RNeasy Plant mini kit (section 2.7.7.4) to isolate total RNA. In order to confirm that the four sub-isolates were bona fide cultures of Aspergillus fumigatus. RT-PCR amplification, using oligonucleotide primers designed to produce a specific ribosomal RNA amplicon (section 2.17) was performed and products of the anticipated sizes were obtained for all four isolates (Fig. 5.12).
Figure 5.12 Confirmation of the identity of Aspergillus fumigatus isolates by RT-PCR amplification of a specific ribosomal RNA amplicon 424 bp in size. Agarose gel electrophoretic separation of amplicons generated from template RNA isolated from four sub-isolates of Aspergillus fumigatus isolate A-56 is shown. NC contains no RNA and Hyperladder 1 (M; 10 kbp; Bioline) was used as marker and the position of the 0.6 kbp band is shown.
Having confirmed the fungal identity of the four, potentially cured sub-isolates derived from A. fumigatus isolate A-56 the remainder of the RNA isolated from each was purified (section 2.7.6.5), precipitated (section 2.7.6.2) and resuspended in water. The potentially cured state of the four sub-isolates was investigated by RT-PCR using oligonucleotide primers designed to amplify fragments of and the complete AfuCV RdRP and CP genes encoded on respectively dsRNA 1 and dsRNA 2 of the virus genome. Analysis of the amplicons generated in these experiments and a comparison with positive controls and negative controls indicated that three of the sub-isolates 4 (1), 4 (P) and 6 were completely cured from virus-infection but that isolate 5 was still infected with AfuCV (Figs. 5.13; 5.14).
Figure 5.13 Comparison of the amplicons generated by RT-PCR using oligonucleotide primers designed to produce a fragment of the AfuCV CP gene (lanes 2-7) or the complete CP gene (lanes 10-15). Total RNA isolated from Aspergillus fumigatus isolate A-56 infected with AfuCV (PC) and potentially cured, single colony isolates 5, 4(1), 4 (P) and 6 were used as templates for RT-PCR amplification. Negative controls (NC) contain no RNA and the Hyperladder 1 (M; 10 kbp; Bioline) was used as a marker. The positions of the 0.6 and 3kbp bands are shown.
Figure 5.14 Comparison of the amplicons generated by RT-PCR using oligonucleotide primers designed to produce a fragment of the AfuCV RdRP gene (lanes 2-7) or the complete CP gene (lanes 10-15). Total RNA isolated from Aspergillus fumigatus isolate A- 56 infected with AfuCV (PC) and potentially cured, single colony isolates 5, 4(1), 4 (P) and 6 were used as templates for RT-PCR amplification. Negative controls (NC) contain no RNA and the Hyperladder 1 (M; 10 kbp; Bioline) was used as a marker. The positions of the 0.6 and 4 kbp bands are shown.
Spores isolated from cultures growing in the presence of 150 mM cycloheximide and the four single colony isolates were point inoculated onto ACM plates, incubated for 7 days at 37°C and observed for characteristic morphologies.
Plates inoculated with spores from the original A. fumigatus isolate A-56 treated with 150 mM cycloheximide showed two different morphologies, suggesting the incidence of two populations of the fungus (Fig. 5.15), whereas plates inoculated with the four suspectedly cured, single colony isolates all demonstrated a single morphology. The three real cured isolates 4 (1), 4 (P) and 6 showed a similar morphology to one of the fungal populations found in the original isolate treated with 150 mM cycloheximide (Fig. 5.16) while the uncured (5) isolate was similar to the other population which in turn appeared similar to the original, untreated A-56 isolate (Fig. 5.17).
Figure 5.15 ACM plate showing two distinct fungal morphologies (red and blue arrows) following point inoculation with spores harvested from A. fumigatus isolates A-56 treated with150 mM cycloheximide.
Figure 5.16 A comparison of the colony morphologies of A. fumigatus shown in Fig. 5.15 with colonies grown from point inoculated spores of the cured, single colonies 4 (1) and 6. Both 4 (1) and 6 have colony morphologies similar to the morphology of the sub-population found with the originally treated culture shown with a red arrow. The photograph shows the appearance of the fungal colonies from the front (A) and the back (B) of the plates.
Figure 5.17 Comparisons of the morphologies of A. fumigatus singly colony 4(1)-cured from infection with AfuCV, single colony 5-infected with AfuCV and the original AfuCV-infected isolate A-56.
Based on the results of RT-PCR amplification and growth morphology analysis of sub- isolates 4(1), 4(P) and 6, which were originally derived from isolate A. fumigatus A-56 following treatment with 150 mM cycloheximide, the three sub-isolates are cured of infection with AfuCV. The maintenance of a similar growth morphology of one selected cured isolate 4(1) and RT-PCR assay following six serial passage confirmed stable maintenance of the virus-free status (Fig. 5.18). The isogenic status of AfuCV-infected (A-56) and the virus-free line 4 (1) of the original A. fumigatus isolate was confirmed by AFLP analysis which were kindly carried out by Dr Chris Linton, Cellular and Molecular Medicine, University of Bristol.