Differential Detection of Encapsidated versus Unencapsidated Enterovirus RNA in Samples Containing Pancreatic Enzymes—Relevance for Diabetes Studies

Figure 1. Overview of the preparation of pancreas homogenate dilutions spiked with coxsackievirus B1 (CVB1) and coxsackievirus A6 (CVA6).

Figure 2. (A) The experimental procedure of diluting infected or cells transfected with viral RNA into a background of untreated cells and pancreas extract. (B) Comparison of recovery of three forms of viral RNA from in pancreas extract treatment. Intact CVB3 virions, CVB3-infected cells, or TD-CVB3 transfected cells across a range of dilutions of infected/transfected cells in a background of untreated cells were treated with pancreatic extract before RNA extraction and recovery. RT-PCR results are converted from Ct (threshold cycle) to genomes using a standard curve established with known quantities of viral RNA. RT-PCR assay with SYBR green has an effect on endpoint Ct, which, when converted on standard curves (dashed line), indicates an effective lowest level of detection in assay. Cell Cont represents assay results from untreated cells. The calculated input of virus genomes in each experiment is shown in white bars. The red arrows indicate expected loss of virus signal from virus not adsorbed by cells during the 1-h infection incubation and washing step.

Figure 3. Detection of viral RNA from cells infected with CVB3 or transfected with equivalent genome copies of wild-type CVB3 RNA or truncated TD-CVB3 RNA. After infection or transfection for 6 h in the presence (A) or absence (B) of guanidine–HCl, the cells were diluted into non-transfected cells (as described in Figure 2A), treated with pancreas extract for 15 min on ice before extraction. RT-PCR analysis was the same as described for Figure 2B. Dashed line indicates effective limit of detection from standard curves and negative controls.