**1. Introduction**

West Nile virus (WNV) is an emerging virus involving birds and Culex mosquitoes in its transmission cycle. Spillover events from this cycle involve mammalian hosts, in particular horses and humans, considered dead-end hosts [1]. In humans, although most infections (about 80%) are asymptomatic, those with a clinical manifestation present with a mild febrile illness, known as West Nile fever, are often underdiagnosed. In some cases, especially among the elderly, a more severe infection may develop as West Nile neuroinvasive disease (WNND), associated with significant morbidity and mortality [2]. As WNND is observed in less than 1% of infected subjects, the frequency of subclinical infections leads to an underestimation of the actual circulation of the virus.

In Italy, WNV is endemic in the Northern regions, where several cases of infection in humans are reported every year. However, in recent years the virus has expanded its distribution with cases also registered in Central and Southern Italy [3].

WNV was reported for the first time in Italy in 1998 among horses residing in wetland areas of Tuscany, Central Italy [4], while the first human case of WNND was detected

**Citation:** Marchi, S.; Montomoli, E.; Viviani, S.; Giannecchini, S.; Stincarelli, M.A.; Lanave, G.; Camero, M.; Alessio, C.; Coluccio, R.; Trombetta, C.M. West Nile Virus Seroprevalence in the Italian Tuscany Region from 2016 to 2019. *Pathogens* **2021**, *10*, 844. https://doi.org/ 10.3390/pathogens10070844

Academic Editor: Francisco Llorente

Received: 28 April 2021 Accepted: 2 July 2021 Published: 5 July 2021

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ten years later, in 2008, in the Emilia-Romagna region, Northern Italy [5]. However, a retrospective study showed that in 2007 a woman living in Tuscany was infected with WNV [6], demonstrating that the virus was already circulating among humans in Central Italy before its isolation in Emilia-Romagna causing unrecognized human disease.

Following the identification of the first human cases of WNV infection, specific WNND surveillance systems were set up in the Emilia-Romagna and Veneto regions [7], followed by the implementation of national veterinary and human surveillance plans [8]. Surveillance activities include entomological, veterinary and human surveillance to be carried out from June to November, identified as the high-risk transmission period. Since 2008, WNV circulation has been reported in 10 Italian regions (Emilia-Romagna, Veneto, Lombardy, Sardinia, Sicily, Friuli Venezia Giulia, Piedmont, Tuscany, Basilicata, Apulia). From 2008 to 2017 a total of 231 cases of human WNND were reported [9].

The presence of WNV in Tuscany was reported in horses in 1998 [4], in 2009 [10], and in 2016 [11]. Although Tuscany was the first Italian region where the presence of WNV human infections was identified already in 2007 [6], since then only one imported case in 2011 in the province of Pisa [12] and two WNND cases in 2017 in the province of Livorno, a coastal area of the region, were reported [13] (Figure 1).

**Figure 1.** West Nile disease equine and autochthonous and imported human reported cases retrieved in Tuscany since 1998. The territories affected with West Nile virus (WNV) are colored in grey and those that are WNV-free are colorless. The maps were generated with Esri ArcGis Desktop 10.6.1 (www.esri.com, accessed on 2 June 2021). Red triangles, equine cases; Red circles, autochthonous human cases; Blue circle, imported human cases.

In Italy, human cases of WNV infection are usually detected starting from July and peaking in August–September. However, in 2018 the transmission season started earlier with the first detection of WNV from a pool of Culex mosquitoes in the Veneto region on the 7th of June [14], and the first confirmed human case was reported just 9 days later in the same province [15]. As of December 2018, a total of 577 confirmed cases of human infection were reported in the Veneto, Emilia-Romagna, Lombardy, Piedmont, Sardinia, and Friuli Venezia Giulia regions. In that very year, veterinary surveillance reported an increase in the circulation of WNV in mosquitoes, birds and horses in nine Italian regions (Emilia-Romagna, Veneto, Lombardy, Sardinia, Friuli Venezia Giulia, Piedmont, Lazio, Basilicata, and Apulia) [16].

Since 2018, the coastal provinces of Tuscany have been considered endemic areas [17], and since 2019, the province of Siena has been included among areas at high risk of transmission by national surveillance plans [18,19]. However, to date no cases of infection have been reported by routine surveillance activities.

Reports of epidemics in equine holdings sugges<sup>t</sup> the circulation of WNV in the Tuscany region. However, human cases reported by routine surveillance are few. To date, limited data are available on the prevalence of WNV in the region.

The primary aim of this study was to evaluate the prevalence of WNV antibodies in the Siena Province area, in the Tuscany region, to estimate the recent circulation of WNV in an area where no infection cases have ever been reported by surveillance activities. The second aim was to investigate any variation in WNV prevalence in the province after the increase in transmission observed during the 2018 season.
