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Direct detection of viral RNA in infected cells

Co-infection by multiple virus particles can lead to increased pathology and morbidity as is the case of Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) co-infections seen in older AIDS patients or super-infection of Hepatitis B and Hepatitis C. Understanding the lack of virus interference in these cases on a per cell basis may have implications in the future design and delivery of vaccines. To date, there have been no robust methods to track and study individual cells which have been co-infected. PrimeFlow® RNA is a tool for the direct detection of multiple viral transcripts within a single cell by flow cytometry, thus facilitating detailed study of co-infected populations of cells.

Detection of Hepatitis C viral RNA in co-infected human hepatocytes

Method
Hepatocytes were co-infected with two strains of Hepatitis C, JFH1-wild type (WT) and JFH1-codon altered (CA). Probes were designed to differentiate the two strains of HCV to study dominance relationships in co-infected cells. These probes were then applied using PrimeFlow® RNA and ViewRNA™ assays.

Results
In the PrimeFlow® RNA assay, clearly defined JFH1-wild type single positive, JFH1-codon altered single positive and double positive populations were observed. The orthogonal ViewRNA assay was used to verify that the probe design was highly specific for the indicated strains of Hepatitis C.

Conclusions
PrimeFlow® RNA is the first commercial assay that can robustly track and study individual cells which have been co-infected.

Figure 1: Co-infected viral RNA by PrimeFlow® RNA proof of principle
Co-infected Viral RNA by FlowRNA Proof of Principle

Legend: Hepatocytes were infected with JFH1-WT strain (top left), JFH1-CA (top right), hepatocytes from top left and top right were mixed post infection (bottom left) and hepatocytes were co-infected with JFH1-WT and JFH1-CA (bottom right). Using PrimeFlow® RNA, hepatocytes were analyzed for strain specific viral RNA.

Figure 2: View Specificity
View Specificity

Figure 3: Probe Specificity
Probe Specificity

Legend: ViewRNA assay using the same JFH1-WT and JFH1-CA specific probes from the PrimeFlow® RNA assay in JFH1-WT infected hepatocytes (top row) and JFH1-CA infected hepatocytes (bottom row) (Figure 2). ViewRNA assay for negative probe set in JFH1-WT infected hepatocytes (top row) and JFH1-CA infected hepatocytes (bottom row) (Figure 3).