*4.6. Immunohistochemistry*

Tissue sections were mounted on Vectabond™ reagen<sup>t</sup> (Vector Laboratories, Inc., Burlingame, CA, USA)-pretreated slides. Immunohistochemical detection of the WNV antigen was performed using a rabbit polyclonal antibody (BioReliance, Product 81–015, Rockville, MD, USA) at a dilution of 1:1000, following the protocol described previously [59]. We also characterized the inflammatory cell population in the cerebrum and cerebellum. For that purpose, we used primary antibodies, reagents, and protocols detailed in Table 4. Endogenous peroxidase activity was inhibited with a peroxidase-blocking reagen<sup>t</sup> (Dako EnVision®+System-HRP (AEC), DakoCytomation, Carpinteria, CA, USA) (CD3, GFAP) or with 3% H202 diluted in methanol (RCA-1), rinses were performed using 0.1% Tris-buffered saline/Tween20 (TBS 0.05 M, pH 7.5), unspecific primary antibody labeling was blocked with 2% albumin from bovine serum (BSA) (Sigma-Aldrich Chemie, Steinheim, Germany) diluted in 0.1% TBS/Tween20, and sections were counterstained with Mayer's hematoxylin.

**Table 4.** Reagents and protocols used to characterize inflammatory cells in the brain of experimentally WNV-infected red-legged partridges.


a Primary antibody products: RCA-1 product No. B-1085 (Vector Laboratories); GFAP product No. Z0334 (DakoCytomation, Glostrup, Denmark); CD3 product No. A0452 (DakoCytomation); CD79a product No. RM-9118 (Thermo Fisher Scientific, Runcorn, UK). b Proteinase K (DakoCytomation); RT: room temperature (22–25 ◦C). c Antibodies were diluted in 2% BSA−0.1% TBS/Tween20. ON: overnight. d Goat anti-rabbit IgG (Vector Laboratories) was diluted 1:200 in 0.1% TBS/Tween20 and applied for 1 h at RT; Labelled polymer-HRP anti-rabbit (Dako EnVision®+System-HRP (AEC), DakoCytomation) was applied according to manufacturer's recommendation. e Avidin-biotinylated enzyme complex (ABC system, Vector Laboratories) was applied for 30 min according to the manufacturer's recommendation and 3,3- diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories) was applied for 30 s according to the manufacturer's recommendations. AEC+substrate chromogen (Dako EnVision®+System-HRP (AEC), DakoCytomation) was applied for 15 min (CD3, CD79) and 3 min (GFAP) according to the manufacturer's recommendations.

> Tissue sections of WNV RRT-PCR-positive red-legged partridges were used as positive controls. Controls of specificity included several sections with substitution of the primary antibody by 2% BSA−0.1% TBS/Tween20 and negative rabbit antibody (BioReliance, Product 81–015), and tissue sections of a non-infected (WNV RRT-PCR-negative) red-legged partridge (from sham-inoculated control group) [17]. For the detection of T

cells, the positive control included a section of spleen of non-infected red-legged partridges. Negative controls included substitution of the primary antibody by 2% BSA−0.1% TBS/Tween20 and a brain section of a WNV RRT-PCR-negative partridge. A brain section of a non-WNV-infected partridge of the same age (control group [16]) served as reference for RCA-1 and GFAP.

We scored virus antigen staining according to its distribution and abundance in the tissues. The distribution of inflammatory cells within the brain was evaluated at 200x magnification. To detect changes in the abundance of CD3+ T cells and RCA-1+ cells during the course of infection, we counted the number of stained cells in 30 randomly selected fields at 400x magnification (in each brain region: cerebrum and cerebellum). Changes in the morphology, abundance, and staining intensity of GFAP+ astrocytes were also evaluated.

**Author Contributions:** Conceptualization, M.Á.J.-C. and U.H.; Funding acquisition, U.H.; Investigation, V.G., E.P.-R., E.S., M.Á.J.-C., and U.H.; Methodology, V.G., A.V.G.-G., E.S., and F.L.; Writing— original draft, V.G. and E.P.-R.; Writing—review and editing, V.G., A.V.G.-G., E.S., M.Á.J.-C., and U.H. All authors have read and agreed to the published version of the manuscript. An earlier version of this manuscript constituted a chapter of the PhD thesis of the first author V.G.

**Funding:** This study has been supported by the project PAC08-0296-7771 (JCCM), and from INIA-MARM funds (INIA CC08-020). Elena Sotelo was a fellow from INIA. Elisa Perez Ramirez was a fellow of the National Research Council (CSIC) and Ana Valeria Gutierrez was a fellow of the JCCM.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics and Animal Welfare Committee of the National Institute for Agricultural investigation and technology, Instituto Nacional de Investigación y Tecnología Agraria INIA (permit no. CEEA2008/013).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the different tables of this article.

**Acknowledgments:** We thank the personnel of the experimental farm of the University of Castilla-La Mancha "La Galiana" for their effort in this study. We would like to acknowledge the Junta de Comunidades de Castilla-La Mancha (JCCM) for their support. We thank Francisca Talavera Benitez for help with the preparation of tissue slides for immunohistochemistry and histopathology.

**Conflicts of Interest:** The authors declare no conflict of interest.
