Abstract
Due to the exceptionally high mutation rates of RNA-dependent RNA polymerases, infectious RNA viruses generate extensive sequence diversity, leading to some of the lowest barriers to the development of antiviral drug resistance in the microbial world. We have previously discovered that higher barriers to the development of drug resistance can be achieved through dominant suppression of drug-resistant viruses by their drug-susceptible parents. We have explored the existence of dominant drug targets in poliovirus, dengue virus and hepatitis C virus (HCV). The low replication capacity of HCV required the development of novel strategies for identifying cells co-infected with drug-susceptible and drug-resistant strains. To monitor co-infected cell populations, we generated codon-altered versions of the JFH1 strain of HCV. Then, we could differentiate the codon-altered and wild-type strains using a novel type of RNA fluorescent in situ hybridization (FISH) coupled with flow cytometry or confocal microscopy. Both of these techniques can be used in conjunction with standard antibody-protein detection methods. Here, we describe a detailed protocol for both RNA FISH flow cytometry and confocal microscopy.
Highlights
[Background] The barriers to development of antiviral drug resistance vary greatly depending on the compound used and the host or viral target chosen
We were early adopters of the branched DNA technology originally developed by Affymetrix ( Thermo Fisher Scientific)
This technology uses tiered DNA oligos to build a network of up to 8,000 fluorophores on each target RNA. This unique type of RNA fluorescent in situ hybridization (FISH) can be coupled with protein detection using standard antibody conjugation and detected using confocal microscopy (ViewRNA® Cell Plus Assay) and flow cytometry (PrimeFlowTM RNA Assay). These branched DNA (bDNA) FISH techniques first generate a series of target probes that bind the RNA of interest at adjacent sequences, but leave 3’ extensions of unique sequence to bind the pre-amplifier DNA that is complementary to two different probes
Summary
[Background] The barriers to development of antiviral drug resistance vary greatly depending on the compound used and the host or viral target chosen. 500 ml Rapid-Flow Filter Unit, 0.2 μm (Thermo Fisher Scientific, catalog number: 566-0020) 11. PrimeFlowTM RNA Assay Kit (Thermo Fisher Scientific, catalog number: 88-18005-210) contains: a. ViewRNA® Cell Plus Assay Kit (Thermo Fisher Scientific, catalog number: 88-19000) contains: a.
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