A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay

Figure 1. Rescue effort of rSA11 strains expressing GFP from five genome locations. (A) Schematic representation of plasmids used for the rescue of rSA11 viruses encoding GFP. (B–H) Representative flow cytometry analysis of CV-1 cells infected with rSA11 rescue products. GFP sequence was inserted at the indicated positions. Expressions levels of GFP and rotavirus protein VP6 were examined. Pseudocolor plots were shown using color to denote areas of high and low population density.

Figure 2. Generation of rSA11 strains expressing GFP at the C terminus of NSP1 or NSP5 without 3’ ORF repeats. (A) Schematic representation of plasmids used for the recovery of rSA11 viruses encoding GFP downstream of NSP1 or NSP5. (B) Representative flow cytometry analysis of CV-1 cells infected with rSA11 strains generated with plasmids shown in (A). Expression levels of GFP and rotavirus protein VP6 were examined. Pseudocolor plots were shown using color to denote areas of high and low population density.

Figure 3. Properties of rSA11-GFP. (A) Genetic stability of rSA11-GFP. rSA11 and rSA11-GFP were serially passaged ten times on MA104 cells and analyzed by RT-PCR using primers flanking the insertion site. The expected band sizes are indicated in parentheses. (B) Growth kinetics of rSA11 and rSA11-GFP. MA104 cells were infected with viruses at an MOI of 0.01 IU/cell and harvested at indicated time points. Virus titer was determined in a flow cytometry-based infectivity assay using CV1 cells. Data are expressed as the mean and range of duplicates. (C) Plaque formation on MA104 cells by rSA11and rSA11-GFP. Data representative of three independent experiments.

Figure 4. rSA11-GFP based microneutralization assay. (A) Representative serum neutralization curves of four rhesus monkeys. rSA11-GFP, preincubated with serial diluted serum samples, was used to infect CV-1 cells. After overnight incubation, numbers of GFP positive cells were numerated. Median is shown from triplicated wells. (B) The conversion of the neutralization assay to a high-throughput platform. rSA11-GFP, preincubated with serial diluted serum samples, was used to infect CV-1 cells. After overnight incubation GFP positive cells were numerated by Acumen. A representative review of a 384-well plate is shown. The plate map is provided in Supplemental Table S1. No serum samples were used in the first column and the last column contained mocked infected cells. Serum samples were diluted threefold with 11-point titration. Wells are colored based on the numbers of GFP positive cells. Average for no serum, infected controls was ~100 objects/well while average for cells only was ~3. (C) The correlation of neutralization titers and ELISA titers of serum samples from 12 African green monkeys and its statistical analysis. **** means p value less than 0.0001.

Figure 5. Pre-existing immunity in human and other animal species. (A) Serum neutralization titers of 20 human donors. (B) Serum neutralization titers of animal samples from indicated species. The bars indicate the median.