**1. Introduction**

West Nile virus (WNV) is an arthropod-borne flavivirus whose natural cycle involves bird hosts and mosquito vectors, with horses and humans as accidental or dead-end hosts [1]. Currently, it is considered one of the most widely distributed arboviruses in the world, causing numerous human, equine, and bird outbreaks and mortalities, both in the Old and New World [2,3]. In the Mediterranean basin, WNV activity is continuously increasing, and has been associated with several outbreaks affecting mainly humans and horses [4,5]. In this area, avian mortality due to WNV lineage 1 (L1) has been sporadic, while lineage 2 (L2) WNV has been responsible for significant outbreaks in central Europe and in magpies in Italy and Greece [6–13]. Nevertheless, under laboratory conditions in experimental infections, at least some Mediterranean L1 WNV strains have proven to be pathogenic for European wild bird species [14–18].

**Citation:** Gamino, V.; Pérez-Ramírez, E.; Gutiérrez-Guzmán, A.V.; Sotelo, E.; Llorente, F.; Jiménez-Clavero, M.Á.; Höfle, U. Pathogenesis of Two Western Mediterranean West Nile Virus Lineage 1 Isolates in Experimentally Infected Red-Legged Partridges (*Alectoris rufa*). *Pathogens* **2021**, *10*, 748. https://doi.org/ 10.3390/pathogens10060748

Academic Editor: El-Sayed Mohammed Abdel-Whab

Received: 8 May 2021 Accepted: 10 June 2021 Published: 13 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In an experimental study we demonstrated that the red-legged partridge (*Alectoris rufa*), a Mediterranean endemic gallinaceous bird species [19], is susceptible to the infection with two lineage 1 Western Mediterranean WNV strains, Morocco/2003 [20] and Spain/2007 [17]. However, the virulence of both strains differed, with 70% mortality in Morocco/2003-infected partridges as compared to 30% mortality in Spain/2007-infected birds, corroborating the results observed in a previous study in a mouse model [21].

The objective of the present work was to study the pathogenesis of WNV in the partridges in the mentioned experimental infection in detail to explain the different infection course and mortality observed between the two WNV strains. For this purpose, we compared the viral load, dynamics of virus appearance, and distribution and severity of microscopic lesions in different tissues. Additionally, we studied the dynamics of inflammatory cell activation and recruitment into the central nervous system (CNS) of the infected partridges. The objective was twofold: on one hand, to determine the evolution of the immune response in the CNS of the avian host, as this information is scarce for birds; and on the other hand, to determine differences in the response induced by the two WNV strains, as this could be considered a virulence marker [22].
