Analysis of Functional Virus-generated PAMP RNAs Using IFNα/β ELISA Assay

Margit Mutso; Xiang Liu; Andres Merits; Suresh Mahalingam

doi:10.21769/BioProtoc.3282

Analysis of Functional Virus-generated PAMP RNAs Using IFNα/β ELISA Assay

Bio Protoc. 2019 Jun 20;9(12):e3282. doi: 10.21769/BioProtoc.3282.

Authors

Margit Mutso^{1

2}, Xiang Liu², Andres Merits¹, Suresh Mahalingam²

Affiliations

¹ Institute of Technology, University of Tartu, Tartu, Estonia.
² Institute for Glycomics, Griffith University, Gold Coast Campus, Southport, Queensland, Australia.

Abstract

Virus-generated PAMP RNAs are key factors that activate host immune response. The PAMP RNAs are therefore usually closely related with viral disease pathogenesis. Quantitative real time PCR is a conventional method to assess RNA. However, it cannot be used for detecting short dsRNAs generated by viral replicase. This protocol was established to analyze the PAMP RNAs produced by viruses which are able to induce host immune response. Classical viral PAMP RNAs and non-classical viral PAMP RNAs are analyzed separately. Briefly, to access total viral PAMP RNAs, total RNA was extracted from the virus infected cells and then transfected into Cop5 cells. Whereas, to assess non-classical viral PAMP RNAs, the constructs expressing viral replicase are transfected into Cop5 cells. The amount of IFNα/β produced by Cop5 cells, determined by ELISA, is correlated with the total and non-classical viral PAMP RNAs. Since this method is based on type I IFN response, it is therefore suitable for measuring the functional virus-generated PAMP RNAs and also for assessing the efficiency of these PAMP RNAs.

Keywords: IFNα/β detection; Infection; PAMP RNAs; RNA isolation; Virus.