Infection of cell lines with experimental and natural ovine scrapie agents

Abstract

Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.

FIG. 1.

Permissibility of Rov9 and MovS6 cell lines to infection with natural and experimental sheep scrapie isolates. The average number of de novo infected cells (spots) detected by SSCA in 4 duplicate wells of Rov9 (upper panel, black) and MovS6 (lower panel, gray) cell lines after exposure to a 10⁻³ dilution of each scrapie inoculum is shown, and the PrP genotype is given for each isolate. The New Zealand-derived negative control represents a scrapie-negative isolate used to calculate the background level of nonspecific spots. The dashed lines represent the average number of spots detected in the negative control plus 3 times the standard deviation; below this line, the samples are considered uninfected. Error bars show standard errors of the means. Note the difference in y axis scale for the two cell lines.

FIG. 2.

Permissibility of second-generation Rov9 subclones to a 10⁻⁴ dilution of SSBP/1 detected by SSCA. Each gray bar represents the average number of de novo infected cells (spots) detected by SSCA for an individual subclone; the white bar represents the Rov9-B4 first-generation subclone; and the black bar represents the parental Rov9 cells. The dashed black line represents the number of spots detected with the first-generation subclone Rov9-B4. *, Rov9-1G10 subclone; #, Rov9-2A3 subclone.

FIG. 3.

Permissibility of Rov9, Rov9-B4, and Rov9-2A3 cell lines to a range of ovine scrapie isolates (a 10⁻³ dilution). The number of infected cells (spots) was determined using the SSCA. The cutoff values for determining samples to be positive or negative for infection, calculated from the average number of spots detected in the New Zealand-derived negative controls plus 3 times the standard deviation, are 107, 223, and 105 spots per 20,000 cells plated in the Rov9, Rov9-B4, and Rov9-2A3 cells, respectively. In some cases, the number of infected cells detected for the most sensitive subclones is an underestimate that is due to the ELISPOT plate reader being unable to separate the large numbers of individual spots. Error bars show standard errors of the means.

FIG. 4.

Western blot analysis of PrP^res in cell lysates of scrapie-challenged Rov9-2A3 cells (a) and ovine inocula (b). Molecular masses of size markers are shown in kDa.

FIG. 5.

Comparison of parental Rov9 cells and the most SSBP/1-permissive first- and second-generation subclones. (a) Dose-response graph showing the average number of infected cells (spots) detected by the SSCA when cells were challenged with increasingly dilute SSBP/1. x axis is a logarithmic scale. The dashed red line represents 1,000 infected cells. Error bars show standard errors of the means. (b) Western blot of PrP^C expression levels in Rov9 cells and the Rov9-B4 and -2A3 subclones. Amounts of 10 μg of total protein from induced (+ doxycycline) and uninduced (− doxycycline) cells were used. Lysates were PNGase treated to deglycosylate the PrP^C so that the total amount of PrP^C could be compared without the interference of glycosylated PrP. Molecular masses of size markers are shown on the left in kDa. (c) Immunofluorescent staining of PrP^C in Rov9 and the two subclones Rov9-B4 and Rov9-2A3. Cells were either induced or uninduced for 48 h prior to staining for PrP^C, which is colored green. Nuclei are falsely colored red.