Direct detection of viral RNA in human or other animal cells by RNA in situ hybridization (ISH) is a powerful tool to establish the etiology and pathogenesis of viral diseases. Nucleic acid-based molecular detection methods have revolutionized viral detection, offering several essential advantages such as sensitivity, specificity and speed.  Beyond those stated advantages, RNAscope  ISH uniquely offers molecular detection coupled with morphological context enabling visualization of the virus in different infected tissues and cell types. 

Why use RNAscope™ assay for viral pathogen research?

Sensitivity—with single RNA molecule detection technology, RNAscope ISH routinely identify individual viral particles in infected cells despite low or undetectable viral loads. Its high sensitivity level enables detection even in early stages of infection.

Specificity—proprietary probe design strategy enables accurate detection among highly related viral species/strains, providing accurate detection even in the presence of contaminants or similar/related viruses.

Speed—single-day workflow coupled with rapid 2 weeks new probe design and manufacture process makes RNAscope ISH a responsive solution for detection of emerging or exotic viruses.

Localization—providing both accurate molecular detection and visualization of infected cell types and tissues enables elucidation of viral transmission, pathogen-host interactions, localization of hidden reservoirs, etc.

Coinfection detection—RNAscope  duplex and multiplex assays offer the possibility of detecting multiple viruses or co-infection.

Differential detection—flexible probe design targeting the sense or anti-sense strand enables detection and differentiation of virus during latent or active stages

•  e.g. Retroviruses—RNAscope probes can be designed for detection of viral RNA in a virus particle; viral genomic RNA or mRNA in an infected cell; pro-viral DNA integrated in the nucleus of an infected cell.
•  e.g.  (+)ssRNA virus—RNAscope probes designed to target the sense strand detects viral genomic RNA and those designed to target the antisense strand detects complementary RNA (template of transcription/replication) 

RNAscope ISH and other viral detection methods

Real-time PCR assay is a sensitive, rapid and cost effective viral detection method.  However, this grind and bind method does not preserve morphological context and provides expression level averaged across the cell population and not at the single cell level. Researchers use PCR methods to detect and quantify viral presence and apply RNAscope ISH to compliment PCR data with single-cell expression level and localization.

NGS is a powerful a priori method especially valued for viral discovery and detection.  In the case of viral discovery, researchers use this new sequence to obtain RNAscope probes for detection and visualization of the virus within is infected environment. In the case of viral detection, NGS, a grind and bind method, does not preserve morphological context.

IHC/ Immuno-based techniques rely on detection of host antibodies against the virus or capturing viral components using antibodies raised against them. Although these indirect viral detection techniques offer morphological context, they are are becoming less routine due to lack of sensitivity and cost of custom antibody production. Compared to IHC, RNAscope ISH is preferred due to its unrivaled sensitivity and specificity along with its probe design accommodating detection of virtually any gene in any tissue of any species. 

Additional information is available on RNAscope applications in HIV/SIV and Zika viral research.