*2.4. Virus Antigen*

Virus antigen staining was mild to moderate in most cases. Immunopositivity was more widespread at 6 dpi and in the tissues of partridges infected with Morocco/2003 (Table 3), consistent with RRT-PCR results. At 3 dpi, WNV antigen was detected in macrophages in spleen and inflammatory cells and myofibers of the heart of both groups. In Morocco/2003-infected partridges there was also a mild immunostaining in tubular epithelial cells of the kidney, isolated acinar cells of the pancreas, and a small group of inflammatory cells in the cecal tonsils. At 6 dpi, the WNV antigen was stained in inflammatory cells in the lung, heart, spleen, kidney, and pancreas. It was also detected in cardiac myofibers (Figure 3A), glomerular mesangial cells (Figure 3B), and tubular epithelial cells of the kidney, acinar cells of the pancreas, as well as in cells of the crypts and myofibers of the muscularis externa of the large intestine. Only in birds infected with Morocco/2003 was the WNV antigen evidenced in smooth muscle cells of the splenic vessels, hepatocytes, and Kupffer cells of the liver, as well as smooth muscle cells of the vascular wall of one vessel and several myofibers of the muscularis externa of the duodenum. At 14 dpi, IHC was negative in all examined tissues (Table 3).

### *2.5. Inflammatory Cells in the Brain*

### 2.5.1. Microglia Activation/Macrophage Infiltration

At 3 dpi, a mild reaction of microglial cells was observed in Morocco/2003-infected partridges. Nevertheless, it was at 6 dpi when these cells were more active in both groups, especially in birds infected with Morocco/2003 (Figure 4A). At 14 dpi, while the activity of microglial cells decreased in the aforementioned group, these remained very active in Spain/2007-infected individuals (Figure 4A).

**Table 3.** WNV antigen detection in tissues of experimentally WNV-infected red-legged partridges. WNV antigen was detected by immunohistochemistry at different days post-inoculation (dpi) in both groups, Spain/2007 (SP07) and Morocco/2003 (MO03). Immunostaining was graded according to its distribution and percentage of stained cells. −: no staining, ±: focal single cells, +: focal or multifocal and <20% cells stained, ++: multifocal or diffuse and 20–50% cells stained, +++: multifocal or diffuse and >50% cells stained. NA: tissue sample not analyzed.


**Figure 3.** Detection of WNV antigen by immunohistochemistry in tissues of experimentally-infected red-legged partridges. (**A**) Heart; partridge inoculated with Spain/2007, 6 dpi. WNV antigen in the cytoplasm of cardiac myofibers. (**B**) Kidney; partridge inoculated with Morocco/2003, 6 dpi. WNV antigen in the cytoplasm of glomerular mesangial cells. Scale bars = 100 μm.

**Figure 4.** Characterization of inflammatory cell reaction in the brain of red-legged partridges experimentally infected with WNV Morocco/2003 (MO03) and Spain/2007 (SP07) strains. (**A**) Semi-quantitative analysis of the number of RCA-1+ microglial cells/macrophages in the cerebrum and cerebellum at different days post-inoculation (dpi). Bars represent the mean of stained cells in 30 randomly selected fields (at 400x magnification) ± standard deviation. The horizontal dashed line represents the mean of stained cells in the cerebrum and cerebellum of a non-WNV-infected partridge. (**B**) Cerebellum; partridge inoculated with Morocco/2003, 6 dpi. RCA-1 positive staining in the cytoplasm of phagocytic foamy macrophages in the molecular layer. Scale bar = 100 μm. (**C**) Semi-quantitative analysis of the number of CD3+ T cells in the cerebrum and cerebellum at different dpi. Bars represent the mean of stained cells in 30 randomly selected fields (at 400x magnification) ± standard deviation. (**D**) Cerebellum; partridge inoculated with Morocco/2003, 6 dpi. CD3 positive staining in T cells located near the Purkinje cell layer (arrowheads). Scale bar = 100 μm.

In the cerebrum, at 3 dpi, RCA-1+ ramified cells were diffusely distributed in the parenchyma. At that time, there was also the mild presence of amoeboid (large soma and short, thick cellular processes) and rounded cells (corresponding both to activated microglia and macrophages). From 6 dpi onwards, most microglia changed to an activated amoeboid morphology and increased the presence of microglia/macrophage nodules, which in many cases surrounded neurons and vessels. RCA-1+ cells were especially abundant in the peripheral pallium and in the region located near the lateral ventricle. In the cerebellum, microglial cell reaction was milder than in the cerebrum (Figure 4A), but the evolution of changes in cellular morphology was similar. These cells were more active in the molecular layer, some of them surrounding Purkinje cells (Figure 4B). At 6 dpi, cell activation was also moderate in the granular layer and white matter, especially in Morocco/2003-infected partridges.

### 2.5.2. Astrocyte Activation

GFAP+ cells were detected from 3 dpi on, with mild changes in their distribution and relative abundance during the infection course and between infected groups. Compared to a non-infected partridge, there were mild differences in staining distribution and the quantity of stained cells but none in astrocyte morphology or staining intensity.

In the cerebrum, moderate astrocytosis (i.e., increased number of astrocytes) was detected near the lateral ventricle in both groups and in the lamina medularis dorsalis in Morocco/2003-infected birds. In the cerebellum, there was mild astrocytosis in the granular layer, slightly more marked at 6 dpi and in Morocco/2003-infected partridges. In this group, and especially in one bird euthanized at 6 dpi, some GFAP+ fibers invaded the molecular layer and among Purkinje cells. Moderate astrocytosis was also observed in the white matter of Morocco/2003-infected birds.

### 2.5.3. T Cell Infiltration

CD3+ T cells infiltrated the brain parenchyma after 3 dpi, and at 6 dpi these cells were more numerous, especially in Morocco/2003-infected birds (Figure 4C). At 14 dpi, the number of T cells decreased, more sharply in the Morocco/2003-infected group (Figure 4C). In some cases, brain zones of T cell infiltration corresponded to brain zones of microglia activation and/or macrophage infiltration. Few T cells were detected in meningeal vessels, and only in Morocco/2003-infected partridges.

In the cerebrum, CD3+ T cells were found diffusely distributed, but also forming part of perivascular infiltrates and of inflammatory foci/nodules associated, in some cases, with neuronal necrosis. The peripheral pallium was especially infiltrated by these cells. In the cerebellum, the vast majority of CD3+ T cells were distributed in the molecular layer, in some cases forming part of inflammatory nodules surrounding Purkinje cells (Figure 4D). There was also moderate infiltration in the granular layer and mild infiltration in the white matter.
