In situ ZIKV RNA detection in tissues

Samples covering a range of tissue compartments including main organs, lymphoid tissues, reproductive system, peripheral and central nervous system, including the brain and skin were collected at post-mortem, fixed in 10% formal saline (Sigma) and embedded in paraffin wax (VWR) using previously reported procedures^{51 (link)}. Four micron thick sections were mounted on poly-L lysine coated slides (VWR) and prior to treatment de-waxed in xylene and re-hydrated via graded ethanol:water solutions. In situ ZIKV RNA detection was performed using the RNAscope 2.5 HD manual DAB detection system (Advanced Cell Diagnostics, 322300) and a combination of two ZIKV-specific probes (463781 and 464531) in accordance with manufacturer’s instructions. Negative (DaPB 310043) and positive (Hs-PPIB 313901) control probes were used to assess technique efficiency. Equivalent tissue sections from infection naïve RM and tamarins were stained with ZIKV-specific, positive or negative control probes to determine NHP tissue cross-reactivities. Sections were manually counter stained with haematoxylin. RNAscope positive cells were quantified by counting all positive cells within each tissue section and conversion to mean number of positive cells/mm². Grading definitions were generated using 10 random fields of view (x10 lens and x10 eye-piece magnification; equivalent to an area of 2.2 mm²).