Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope®) and assessment of their performance in tissues from aborted equine fetuses

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope ® ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques. The distribution and cellular localization of EAV RNA and antigen were similar in tissues from aborted equine fetuses. Evaluation of 80 FFPE tissues collected from 16 aborted fetuses showed that the conventional RNA ISH assay had a significantly lower sensitivity than the RNAscope ® and IHC assays, whereas there was no difference between the latter two assays. The use of oligonucleotide probes along with a signal amplification system (RNAscope ® ) can enhance detection of EAV RNA in FFPE tissues, with sensitivity comparable to that of IHC. Most importantly, these assays provide important tools with which to investigate the mechanisms of EAV pathogenesis.