Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes

Figure 1. An established protocol for short RNA-oligoprobe labeling in FFPE tissue sections A. GAPDH oligonucleotides were tested in the cell line HEK 293. Single fluorescent spots, each representing one single RNA molecule, are clearly visible. B. Normal deparaffinization procedure shows high background noise in FFPE islet sections even without probes (No Probes) and does not allow clear distinction of probe signal and background, when GAPDH oligonucleotides were added C.; Standard Protocol. Modification of the deparaffinization and post-hybridisation protocol leads to background reduction and increased signal intensity of GAPDH oligonucleotides D.; Modified Protocol. RNA Probes are labeled with Quasar 570 (red) and nuclei were stained with DAPI (blue), scale bar depicts 10µm.

Figure 2. RNA-oligonucleotide probe design and specificity A. Scheme of custom-designed oligonucleotide (CVB_1) annealing throughout the viral genome. B. Viral RNA probes were tested against non-infected, CVB3 and CVB2 (100% and 79% similarity to consensus sequence, respectively; positive control), CMV, (DNA virus; negative control) and HAV (<45% similarity to consensus sequence) infected HEK 293 cells. Cells were infected with an MOI of 5 and harvested after 24h (control, CVB3, CVB2) or 4 days (CMV, HAV) post-infection. RNA Probes are labeled with Quasar 570 (red) and nuclei were stained with DAPI (blue), scale bar depicts 10µm.

Figure 3. RNA-oligonucleotide probe sensitivity A. CM 9 and HEK 293 were infected with a dilution series (MOI 102-10-8) of CVB3 and stained with custom-designed oligonucleotides (CVB_1). Representative images of CM9 and HEK293 cells infected with either the highest or lowest dilution of CVB3 of the series are shown. White arrows highlight single viral spots. B. Ten single images were acquired for each dilution and single fluorescent spots were manually counted. Results for HEK 293 (diamonds) and CM 9 (squares) are displayed as single spots per cell in logarithmic scale. In total, viral particles were counted in 4470 HEK293 cells and CM9 cells. C. Viral RNA from a parallel experiment was extracted and analyzed by PCR; MOI of 100 (102) was set as 100%. D. Viral RNA (red) and VP1 (1/2000; green) staining on a CVB1-dilution array of FFPE infected GMK cells mixed with uninfected cells. RNA Probes were labeled with Quasar 570 (red) and nuclei were stained with DAPI (blue), scale bar depicts 10µm. E. Summary of the viral RNA and VP1 signals obtained from a CVB1-dilution array of FFPE infected GMK cells.

Figure 4. Coupling RNA-FISH and Immunohistochemistry A. Theoretical scheme of viral RNA and VP1 co-staining. Initially, labeled oligonucleotides cannot bind viral RNA within the capsid, but only when the virus is released. On the other hand, VP1 antibody can bind to the capsid surface. Over time as more RNA is released, more probes can anneal to their target sequence. When a sufficient amount of oligonucleotides is bound, green (VP1) and red (RNA) signal are visible in close proximity. B. CM9 cells were infected with CVB3 (MOI 1000) for 1h at 16°C, fixed and probed for viral RNA and VP1. VP1 (green) was found in close proximity with viral RNA (red), when capsid and RNA were present in the cytosol area (B; middle panel). When VP1 staining was performed before cell permeabilization no colocalization of viral RNA/VP1 was detected (B; right panel). No cross-reactivity was found with cellular proteins or RNAs (B; left panel). C. Co-staining and localization of viral RNA (red) and insulin (green) in CVB3-infected INS-1E cells and HgCl2 fixed paraffin embedded CVB-infected neonatal pancreas. Samples were stained with RNA FISH probes first, analyzed and then stained for insulin. The non-granular…

Figure 5. Viral RNA within an islet of a T1D patient A. The original probe set CVB_1 was complemented with two additional sets (CVB_2 and CVB_3). Scheme of custom-designed probe sets annealing throughout the viral genome. Arrows show localization of CVB_1, lines show the positions of CVB_2 and CVB_3 probe sets. B., C. Representative images of donors E375 and E514 form the UK cohort and of donor 3626 from the nPOD cohort. Virus RNA was found within the endocrine area (B) and outside the islets (C) shown by the co-staining of viral RNA probes (combination of CVB_1, CVB_2 and CVB3) (red) and insulin (Green). Tissues were first probed for viral RNA, analyzed and then stained for insulin. Nuclei were visualized by DAPI staining (blue); scale bar depicts 10µm.