**1. Introduction**

Louping ill is a vector-borne disease endemic of the British Isles and Ireland, which is caused by the louping ill virus (LIV) [1]. In 2011, the Spanish goa<sup>t</sup> encephalitis virus (SGEV), a tick-borne flavivirus subtype closely related to LIV [1], naturally induced non-purulent encephalomyelitis in goats in Spain [2]. SGEV also induced clinical signs (febrile illness and neurological signs such as muscular tremors—mainly located in the neck—ataxia, and/or incoordination) and histopathological lesions in the nervous system in experimentally infected goats and lambs [3,4]. In those studies, lambs developed more severe histological lesions in the central nervous system (CNS) than goats, suggesting greater susceptibility to SGEV [4]. This greater susceptibility has been attributed to species di fferences and interindividual di fferences in the immune response; in fact, an e ffective specific inflammatory response against LIV has been shown to neutralize the spread of the infection in the nervous system in lambs [5].

The pathogenesis of an acute viral infection by flavivirus in the CNS involves complex virus–host interactions in which the recruitment of immune cells into the CNS plays a fundamental role in the outcome of the disease. The response to SGEV in goa<sup>t</sup> CNS has been shown to comprise microglia, T lymphocytes, and, a to lesser extent, B lymphocytes [6]. This is similar to the responses to LIV in mice and lambs [7], to West Nile virus in humans and horses [8,9], to Japanese encephalitis virus in humans [10], and to tick-borne encephalitis virus in humans and non-human primates [11,12].

A detailed understanding of the immune response to SGEV in the CNS of di fferent animal species may help clarify the di fferences among flavivirus. Therefore, the present study examined the phenotype and distribution of microglia, T and B lymphocytes, and astrocytes in the CNS of lambs experimentally infected with SGEV, and the results were compared with the previously reported immune response in goats [6].

#### **2. Materials and Methods**

#### *2.1. Animals and Sampling*

Samples were obtained from eight female 3-month-old Assaf lambs (identified as 14, 18, 19, 21, 25, 26, 28, and 29) from a previous study [4]. In that work, the animals were challenged subcutaneously on the right thorax caudal to the elbow with 1 mL of a suspension containing 1.0 × 10<sup>7</sup> plaque-forming units per mL of SGEV grown in BHK-21 baby hamster kidney cells. The following postmortem CNS samples were analyzed by histopathology in that study: cerebral cortex, corpus callosum, thalamus, hypothalamus, hippocampus, midbrain, cerebellum, pons, medulla oblongata, and four sections of spinal cord (cervical, thoracic, lumbar, and sacral). Samples were placed in 10% neutral bu ffered formalin, processed routinely through graded alcohols, and embedded in para ffi n wax. The severity of microscopic lesions was rated using our previously reported scale [4]: grade I, only perivascular cu ffing; grade II, perivascular cu ffing and small foci of glial cells; grade III, moderate non-suppurative encephalomyelitis, perivascular cu ffing, di ffuse or focal proliferation of glial cells, neuronal degeneration, neuron necrosis, and neuronophagia, demyelination, vacuolation of the neuropil, meningitis, and microvascular changes consisting of reactive endothelium and perivascular edema; and grade IV, which involved the same characteristics as grade III but more severe. In the eight animals, lesions were more frequent and severe in the midbrain, cerebellum, medulla oblongata, and all sections of the spinal cord than in other tissues examined [4].
