Detection of Torque Teno Sus Virus1 by an RNA in situ hybridization assay

Torque Teno viruses (TTVs) are ubiquitous, small DNA viruses which are highly epidemiologically associated with respiratory infections, hepatitis, neurological disease and autoimmune disorders in humans and animals. Swine TTVs (TTSuVs) can be considered opportunistic pathogens as they exacerbate clinical signs due to coinfecting agents. While further understanding of how TTVs contribute to disease is crucial, there is a notable lack of animal models and tools to study the in vivo infection patterns of TTV. RNA in situ hybridization (RNA-ISH) with multiple probe amplification has recently gained popularity due to its high levels of specificity and sensitivity and ability to detect agent specific RNA or mRNA. Currently there are no commercial TTSuV1 antibodies that allow viral antigen detection by immunohistochemistry assay that can be used to advance the understanding of TTSuV1 pathogenicity. Therefore, the goal of this study was to develop an RNA-ISH assay for TTSuV1. To generate positive control, PK-15 cells grown in chamber slides were either infected with TTSuV1 or transfected with the TTSuV1 genome. A cocktail of TTSuV1 ORF1-specific RNA probes was hybridized to the cells, and specific binding was successfully visualized using a chromogenic reaction. Liver, kidney, heart, spleen and intestines were collected from mice infected with TTSuV1 at 15- and 30-days post infection. Finally, the RNA-ISH was optimized for TTSuV1 mRNA detection in tissues. TTSuV1-specific signal was detected in the hepatocytes and renal tubular epithelium of infected mice at a detection rate of 33 % 15- and 30-days post infection. In summary, the described RNA ISH assay is a useful tool to visualizeTTSuV1 viral replication in tissues and has potential application to clinical specimens in the future.