Tissue samples from the cerebellum and spinal cord were selected from nine rhesus monkeys (Macaca mulatta; 2–3-year-old; seven males and two females) inoculated intrathalamically (bilaterally) with a dose of 5.0 log10 PFU of wild-type WNV strain NY99-35262 (hereafter WNV) that were used as a positive control in our prior study of the WNV vaccine safety (Maximova et al., 2014) and from the cerebellum and spinal cord of four rhesus monkeys (Macaca mulatta; 2–3-year-old; one male and three females) that were mock-inoculated intrathalamically (bilaterally) with an identical to virus inoculum volume (0.25 ml) (Maximova et al., 2014) of diluent without the virus (Leibovitz’s L-15 medium [Invitrogen], supplemented with SPG buffer stabilizer) (detailed procedure of the bilateral intrathalamic inoculation of NHPs is described previously [Maximova et al., 2008]). All animal experiments were approved by the NIAID DIR Animal Care and Use Committee (animal study proposal #LID 7E). The NIAID DIR Animal Care and Use Program, as part of the NIH Intramural Research Program, complies with all applicable provisions of the Animal Welfare Act (http://www.aphis.usda.gov/animal_welfare/downloads/awa/awa.pdf) and other Federal statutes and regulations relating to animals. The NIAID DIR Animal Care and Use Program is guided by the ‘U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training’ (http://oacu.od.nih.gov/regs/USGovtPrncpl.htm).

Three WNV-infected and one mock-inoculated monkeys were euthanized at 3, 7, and 9 dpi. Detailed clinical, virological, and histopathological information about these animals can be found in our prior publications (Maximova et al., 2014). Tissue samples analyzed in this study were collected immediately following euthanasia and cardiac perfusion with a sterile saline in the BSL-3 environment. After removal, brains and spinal cords were aseptically dissected to be freshly preserved for RNA extraction (see later) or fixed in 10% phosphate-buffered formalin for immunohistochemistry, following the protocols similar to described previously (Maximova et al., 2014; Maximova et al., 2008). A central cerebellar coronal slice (4 mm thick) and a transverse lumbar spinal cord slice (4 mm thick) from each animal were used for RNA extraction. Immediately after dissection, each sample was placed into RNAlater (Ambion, AM7021) at 4°C. After a maximum 3 days of storage, the RNAlater was removed and tissues were stored at −80°C. For RNA extraction, the samples were thawed on ice and core tissue samples (3 mm in diameter; 4 mm thick) were extracted using sterile Harris Uni-Cores (Ted Pella, Redding, CA). The cerebellar cores were extracted from the spinocerebellar and cerebrocerebellar areas of the cerebellar cortex (including the molecular layer, Purkinje cell layer, granule cell layer, and white matter), as well as from the deep cerebellar nuclei, and pooled for each animal. The spinal cores were extracted from the ventral horns of the spinal gray matter bilaterally and pooled for each animal.