Dániel Cadar
email: danielcadar@gmail.com

Yannick Simonin
email: yannick.simonin@umontpellier.fr

Human Usutu Virus Infections in Europe: A New Risk on Horizon?

Keywords: Usutu, flavivirus, neurological disorders

The Usutu virus (USUV), a neurotropic mosquito-borne flavivirus discovered in 1959 in South Africa, has spread over the last twenty years across the European continent. This virus follows an enzootic cycle involving mosquitoes and birds. This caused epizootics with significant bird mortality in Europe in 2016 and 2018. It can also occasionally infect humans and other mammals, including horses and bats, which act as incidental or dead-end hosts. The zoonotic risk associated with this succession of avian epizootics in Europe deserves attention, even if, to date, human cases remain exceptional. Human infection is most often asymptomatic or responsible for mild clinical symptoms. However, human Usutu infections have also been associated with neurological disorders, such as encephalitis and meningoencephalitis. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages which could co-circulate spatiotemporally. In this review we discuss several aspects of the circulation of Usutu virus in humans in Europe, the neurological disorders associated, involved viral lineages, and the issues and questions raised by their circulation.

Structure and Viral Cycle 1.Structure and Organization

Usutu virus is an emerging encephalitic arbovirus of the Flaviviridae family [START_REF] Williams | The isolation of west nile virus from man and of usutu virus from the bird-biting mosquito mansonia aurites (theobald) in the entebbe area of uganda[END_REF]. It belongs to the Japanese encephalitis virus (JEV) serocomplex and it is phylogenetically close to several human and animal pathogens, e.g., West Nile virus (WNV) and Murray Valley encephalitis virus (MVEV) [START_REF] Mcintosh | nouvel arbovirus du groupe B[END_REF][START_REF] Kuhn | Structure of dengue virus: Implications for flavivirus organization, maturation, and fusion[END_REF]. It was first identified in 1959 near the Usutu River in Swaziland, South Africa and was subsequently isolated from field-caught Culex neavei mosquitoes by intracerebral inoculation of newborn mice [START_REF] Williams | The isolation of west nile virus from man and of usutu virus from the bird-biting mosquito mansonia aurites (theobald) in the entebbe area of uganda[END_REF][START_REF] Mcintosh | nouvel arbovirus du groupe B[END_REF].

The USUV particle has a spherical structure with a 40-60 nm diameter and is comprised of an envelope and an icosahedral capsid containing the viral genome (Figure 1). Immature virions have envelope (E) and premembrane (prM) protein heterodimers on their surface, which are cleaved during maturation of the virus particle. Mature virions thus have an envelope composed of structural proteins E and M, which are integrated into a lipid bilayer derived from the endoplasmic reticulum membrane of the host cell, and a capsid composed of protein C [START_REF] Kuhn | Structure of dengue virus: Implications for flavivirus organization, maturation, and fusion[END_REF]. The USUV genome contains a single-stranded RNA molecule of positive polarity coding one long open reading frame (ORF) of approximately 11 kb in length. It is flanked by a type I cap (m7GpppAmN) at 5 , which allows for stabilization and initiation of translation, and lacks a polyadenylated tail (polyA) at 3 [START_REF] Bakonyi | Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: Comparison with the South African Strain SAAR-1776 and other flaviviruses[END_REF]. The genome has a single reading frame encoding a viral polyprotein of about 3400 amino acids, itself flanked by 5 and 3 untranslated regions (5 -UTR and 3 -UTR) [START_REF] Ng | The 5' and 3' Untranslated Regions of the Flaviviral Genome[END_REF]. This polyprotein is cleaved to release three structural proteins, C, E, and prM, and seven non-structural proteins (Figure 1): NS1 (associates with NS4A to allow genomic replication); NS2A (coordinates the passage of packaged RNA to replication); NS2B (membrane protein, allowing the formation of a protease complex with NS3); NS3 (enzyme with different activities: trypsin-like serine protease, helicase, and RNA triphosphatase, also involved in RNA replication); NS4A (associates with NS1 for genomic replication); NS4B (involved in antiviral resistance); and NS5 (RNA-dependent RNA polymerase (RdRp)) [START_REF] Lindenbach | The Viruses and Their Replication[END_REF].

Viruses 2023, 14, x FOR PEER REVIEW 2 of 15 structural proteins (Figure 1): NS1 (associates with NS4A to allow genomic replication); NS2A (coordinates the passage of packaged RNA to replication); NS2B (membrane protein, allowing the formation of a protease complex with NS3); NS3 (enzyme with different activities: trypsin-like serine protease, helicase, and RNA triphosphatase, also involved in RNA replication); NS4A (associates with NS1 for genomic replication); NS4B (involved in antiviral resistance); and NS5 (RNA-dependent RNA polymerase (RdRp)) [START_REF] Lindenbach | The Viruses and Their Replication[END_REF].

Viral Cycle

Knowledge of the viral cycle of USUV is mainly based on the previous study of other flaviviruses, such as WNV. The attachment of the virus E protein to a cellular receptor on the plasma membrane is the first step in the viral cycle of flaviviruses [START_REF] Laureti | Flavivirus Receptors: Diversity, Identity, and Cell Entry[END_REF]. Cell receptors are known to be frequently associated with entry of certain flaviviruses, for example, integrin αvβ3, C-type lectin receptors (mannose receptors or DC-SIGN), or sulfated glycosaminoglycans (heparin sulfate). However, the USUV receptor is not known [START_REF] Laureti | Flavivirus Receptors: Diversity, Identity, and Cell Entry[END_REF]. A recent study shows that the C-type lectin receptor (CLR), langerin, could be one of the virus receptors in Langerhans cells (LCs) [START_REF] Martin | Usutu Virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus[END_REF]. Endocytosis, mostly clathrin-dependent, allows the entry of virions, then an acidification of the endosomal environment leads to the fusion of the E protein with the endosomal membrane and the release of the capsid into the cytoplasm. This uncoating releases the viral genome and allows for a direct translation of the +RNA into polyprotein by the cellular ribosomes, as if the viral +RNA was cellular mRNA. This polyprotein is then cleaved by cellular proteases and by the NS2B/NS3 complex, leading to the formation of structural and non-structural proteins. Following this translation step, the NS proteins form the replication complex by associating with endoplasmic reticulum membranes and other cellular proteins of the host cell. Replication then takes place, starting with the synthesis of a single-stranded (ss) negative (-) RNA, which serves as a template for the synthesis of ss positive (+) RNAs that transiently form a double-stranded RNA (ds) called dsRNA; this is the replication intermediate [START_REF] Klema | Flaviviral Replication Complex: Coordination between RNA Synthesis and 5'-RNA Capping[END_REF]. Subsequently, the assembly of genomic RNA and structural proteins at the replicative niche of the endoplasmic reticulum results in the formation of immature virions that can undergo post-translational modifications, such as glycosylation of the viral envelope which plays an important role in the recognition of cellular receptors. Transport into the Golgi apparatus allows for the

Viral Cycle

Knowledge of the viral cycle of USUV is mainly based on the previous study of other flaviviruses, such as WNV. The attachment of the virus E protein to a cellular receptor on the plasma membrane is the first step in the viral cycle of flaviviruses [START_REF] Laureti | Flavivirus Receptors: Diversity, Identity, and Cell Entry[END_REF]. Cell receptors are known to be frequently associated with entry of certain flaviviruses, for example, integrin αvβ3, C-type lectin receptors (mannose receptors or DC-SIGN), or sulfated glycosaminoglycans (heparin sulfate). However, the USUV receptor is not known [START_REF] Laureti | Flavivirus Receptors: Diversity, Identity, and Cell Entry[END_REF]. A recent study shows that the C-type lectin receptor (CLR), langerin, could be one of the virus receptors in Langerhans cells (LCs) [START_REF] Martin | Usutu Virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus[END_REF]. Endocytosis, mostly clathrin-dependent, allows the entry of virions, then an acidification of the endosomal environment leads to the fusion of the E protein with the endosomal membrane and the release of the capsid into the cytoplasm. This uncoating releases the viral genome and allows for a direct translation of the +RNA into polyprotein by the cellular ribosomes, as if the viral +RNA was cellular mRNA. This polyprotein is then cleaved by cellular proteases and by the NS2B/NS3 complex, leading to the formation of structural and non-structural proteins. Following this translation step, the NS proteins form the replication complex by associating with endoplasmic reticulum membranes and other cellular proteins of the host cell. Replication then takes place, starting with the synthesis of a single-stranded (ss) negative (-) RNA, which serves as a template for the synthesis of ss positive (+) RNAs that transiently form a double-stranded RNA (ds) called dsRNA; this is the replication intermediate [START_REF] Klema | Flaviviral Replication Complex: Coordination between RNA Synthesis and 5'-RNA Capping[END_REF]. Subsequently, the assembly of genomic RNA and structural proteins at the replicative niche of the endoplasmic reticulum results in the formation of immature virions that can undergo post-translational modifications, such as glycosylation of the viral envelope which plays an important role in the recognition of cellular receptors. Transport into the Golgi apparatus allows for the cleavage of prM to M proteins, by a cellular protease called furin, as well as other post-translational modifications. Finally, the infectious viral particles are released from the cell by exocytosis [START_REF] Klema | Flaviviral Replication Complex: Coordination between RNA Synthesis and 5'-RNA Capping[END_REF][START_REF] Pierson | Degrees of maturity: The complex structure and biology of flaviviruses[END_REF][START_REF] Apte-Sengupta | Coupling of replication and assembly in flaviviruses[END_REF].

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Transmission Cycle 2.1. Transmission Routes

The transmission cycle of USUV involves ornithophilic mosquitoes as vectors, mainly of the genus Culex, and birds as amplifying hosts and reservoirs of the virus. It shares many characteristics with WNV [START_REF] Calisher | Taxonomy of the virus family Flaviviridae[END_REF][START_REF] Beck | Flaviviruses in Europe: Complex circulation patterns and their consequences for the diagnosis and control of West Nile disease[END_REF]. Infected mosquitoes will therefore feed preferentially on birds, often species belonging to the genera Passeriformes (sparrows, magpies, blackbirds) and Strigiformes (owls) [START_REF] Farajollahi | Bird biting" mosquitoes and human disease: A review of the role of Culex pipiens complex mosquitoes in epidemiology[END_REF][START_REF] Clé | Usutu virus: A new threat?[END_REF]. Birds infected by the mosquito's blood meal will develop sufficient viremia to allow transmission of the virus to a new mosquito during a subsequent bite. However, these Culex spp. mosquitoes, or gateway vectors such as Aedes albopictus mosquitoes, may take their blood meal from a mammalian host, such as a horse, bat, or human, considered as incidental hosts.

Vectors

Culex pipiens, an ornithophilic species which can also feed on humans, is the main vector of USUV in Europe. In addition, the vector competence of Culex pipiens, Culex neavei, and Culex quinquefasciatus for USUV have been demonstrated under laboratory conditions [START_REF] Cook | North American Culex pipiens and Culex quinquefasciatus are competent vectors for Usutu virus[END_REF][START_REF] Nikolay | Vector competence of Culex neavei (Diptera: Culicidae) for Usutu virus[END_REF]. Gateway vectors that are rather mammalophilic, such as Aedes japonicas, or anthropophilic, such as Aedes albopictus [START_REF] Puggioli | Does Aedes albopictus (Diptera: Culicidae) play any role in Usutu virus transmission in Northern Italy? Experimental oral infection and field evidences[END_REF][START_REF] Mancini | Mosquito species involved in the circulation of West Nile and Usutu viruses in Italy[END_REF][START_REF] Mannasse | Usutu virus RNA in mosquitoes, Israel, 2014-2015[END_REF], can also be naturally infected, even if their vector competence remains relatively weak. However, these species have been repeatedly found infected in field-collected samples in the Emilia-Romagna region, Northern Italy [START_REF] Cook | North American Culex pipiens and Culex quinquefasciatus are competent vectors for Usutu virus[END_REF][START_REF] Puggioli | Does Aedes albopictus (Diptera: Culicidae) play any role in Usutu virus transmission in Northern Italy? Experimental oral infection and field evidences[END_REF][START_REF] Calzolari | Co-circulation of two Usutu virus strains in Northern Italy between 2009 and 2014[END_REF]. There are currently no data on USUV's ability to sustain itself in mosquito populations by vertical transmission, unlike WNV.

Reservoirs and Amplifier Hosts

Although considered reservoirs, some avian species are particularly susceptible to USUV infection with a high mortality rate when a naïve population is reached. This is the case for blackbirds (Turdus merula), grey owls (Strix nebulosi), and sparrows (Passer domesticus) [START_REF] Chvala | Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005[END_REF][START_REF] Weissenböck | Usutu virus activity in Austria, 2001-2002[END_REF]. The virus and associated necrotic lesions, including neurological lesions, have been identified in many organs of cadavers of these species. Massive mortality of birds through Europe is also indicative of the high pathogenicity of USUV in different avian populations [START_REF] Becker | Epizootic emergence of Usutu virus in wild and captive birds in Germany[END_REF][START_REF] Chvala | Pathology and viral distribution in fatal Usutu virus infections of birds from the 2001 and 2002 outbreaks in Austria[END_REF][START_REF] Lecollinet | Dual emergence of Usutu virus in common blackbirds, Eastern France[END_REF][START_REF] Rijks | Widespread Usutu virus outbreak in birds in The Netherlands[END_REF]. Several avian species, such as the kestrel (Falco tinnunculus) or the babbler (Sylvia curruca), are believed to be responsible for the introduction of USUV into Europe. Subsequently, blackbirds, magpies, or sparrows are believed to have disseminated the virus throughout the continent.

Incidental Hosts

Incidental hosts of USUV and WNV are considered hosts that are not involved in the transmission cycle. They correspond to epidemiological dead ends for viral propagation because the viremia in the infected mammals is not high enough to ensure transmission via mosquito bites. USUV has been identified in dogs, wild boar, and several wild ruminants, such as deer (Cervus elaphus), sheep (Ovis aries), and Roe Deer (Capreolus capreolus) as well as zoo species [START_REF] Buchebner | Low Usutu virus seroprevalence in four zoological gardens in central Europe[END_REF][START_REF] Steinmetz | Emergence and establishment of Usutu virus infection in wild and captive avian species in and around Zurich, Switzerland-Genomic and pathologic comparison to other central European outbreaks[END_REF][START_REF] Caballero-Gómez | Evidence of exposure to zoonotic flaviruses in zoo mammals in Spain and their potential role as sentinel species[END_REF][START_REF] Constant | Evidence of exposure to USUV and WNV in zoo animals in France[END_REF][START_REF] Constant | One Health surveillance of West Nile and Usutu viruses: A repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020[END_REF]. Rodents, such as bats, as can also be infected [START_REF] Cadar | Usutu virus in bats, Germany[END_REF][START_REF] Diagne | Usutu virus isolated from rodents in Senegal[END_REF]. As for WNV, humans, and to a lesser extent horses, are also considered incidental hosts of USUV [START_REF] Constant | One Health surveillance of West Nile and Usutu viruses: A repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020[END_REF].

Geographical Distribution

USUV was first discovered in Culex neavei mosquitoes in 1959 in South Africa, in Swaziland near the Usutu River [START_REF] Mcintosh | nouvel arbovirus du groupe B[END_REF]. Then, the first human infection was identified in the Central African Republic in 1981, followed by a second in Burkina Faso in 2004 [START_REF] Nikolay | Usutu virus in Africa[END_REF]. Subsequently, the virus was identified in several African countries with surveillance programs (Ivory Coast, Morocco, Nigeria, Uganda, and Senegal), suggesting a wider distribution on this continent, especially in countries without adequate surveillance programs [START_REF] Nikolay | Usutu virus in Africa[END_REF].

The virus likely reached the European continent for the first time at the end of the 1950s, in Spain, and was reintroduced between 1970 and 1980 (in Italy and Austria) [36]. Retrospective studies suggest that USUV was associated with the deaths of a significant number of birds, including blackbirds in Italy in 1996 [START_REF] Engel | Reconstruction of the evolutionary history and dispersal of Usutu virus, a neglected emerging arbovirus in Europe and Africa[END_REF]. However, it was only in 2001 that the emergence of USUV in Central Europe was confirmed after a significant mortality of blackbirds was reported in Austria [START_REF] Weissenböck | Emergence of Usutu virus, an African mosquito-borne Flavivirus of the Japanese encephalitis virus group, central Europe[END_REF]. Subsequently, USUV was discovered in an occasional recurrent manner in animals (birds, bats, and horses) and mosquito vectors in several European countries (Belgium, Czech Republic, France, Germany, Hungary, Italy, Spain, and Switzerland), suggesting its endemization in these countries [START_REF] Clé | Usutu virus: A new threat?[END_REF][START_REF] Constant | One Health surveillance of West Nile and Usutu viruses: A repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020[END_REF]. New epizootics were recorded in 2016 and 2018 which affected several European countries, including Belgium, France, Germany, and the Netherlands [START_REF] Clé | Usutu virus: A new threat?[END_REF]. USUV was likely dispersed to Europe via migrations of its avian hosts from Africa to Europe, and then further spread via resident wild birds [START_REF] Engel | Reconstruction of the evolutionary history and dispersal of Usutu virus, a neglected emerging arbovirus in Europe and Africa[END_REF]. A second introduction then took place from the Central African Republic to Austria, and more precisely Vienna, where the first avian epizootic was recorded in 2001 [START_REF] Weissenböck | Emergence of Usutu virus, an African mosquito-borne Flavivirus of the Japanese encephalitis virus group, central Europe[END_REF].

Classification and Phylogeny

The introductions of USUV into Europe follow the main migratory flows: the first introduction from Africa to Spain overlaps with the East Atlantic migratory flow, and that from Africa to Central Europe overlaps with the Black Sea/Mediterranean migratory flow [START_REF] Engel | Reconstruction of the evolutionary history and dispersal of Usutu virus, a neglected emerging arbovirus in Europe and Africa[END_REF]. Therefore, USUV has been classified into eight lineages: African (Africa 1/2/3) or European (Europe 1/2/3/4/5) (Figure 2). Although the majority of strains currently circulating in Europe belong to the European lineages of USUV, African lineages continue to be introduced into the continent, such as the African 2 and 3 lineages discovered in 2018 in Culex pipiens in the South of France [START_REF] Eiden | Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015[END_REF]. The Africa 2 lineage of USUV has also been identified in France in a human patient [START_REF] Simonin | Human Usutu virus infection with atypical neurologic presentation[END_REF] and in owls in Berlin Zoo in 2015 [START_REF] Ziegler | Evidence for an independent third Usutu virus introduction into Germany[END_REF]. The Europe 1 lineage appears to derive from a Senegalese strain that reached Spain and was implicated in the first avian epizootic in Austria in 2001 [START_REF] Weissenböck | Emergence of Usutu virus, an African mosquito-borne Flavivirus of the Japanese encephalitis virus group, central Europe[END_REF]. The Europe 2 lineage comes from an Austrian strain dating back to 1993. This lineage reappeared during the autochthonous Italian cases of 2009/2010 and the Austrian and Hungarian cases in 2016 [START_REF] Kemenesi | First genetic characterization of Usutu virus from Culex pipiens mosquitoes Serbia, 2014[END_REF]. An Italian strain that circulated in 2007 would be the origin of the Europe 3 lineage. This lineage is responsible for the massive bird die-off observed in France in 2015, in Germany in 2011/2013, and in Belgium in 2016 [START_REF] Lecollinet | Dual emergence of Usutu virus in common blackbirds, Eastern France[END_REF][START_REF] Ziegler | Evidence for an independent third Usutu virus introduction into Germany[END_REF]43]. The Europe 4 lineage comprises only a few strains, some of which were circulating in Italy in 2010 and 2015 [START_REF] Vilibic-Cavlek | Epidemiology of usutu virus: The european scenario[END_REF]. Finally, the Europe 5 lineage was isolated from infected birds in Germany in 2016 [START_REF] Vilibic-Cavlek | Epidemiology of usutu virus: The european scenario[END_REF]. These different lineages seem to vary in virulence in both humans and birds and deserve further investigation for a better understanding of their distribution and virulence in animals and humans [START_REF] Diagne | Usutu virus isolated from rodents in Senegal[END_REF][START_REF] Clé | Differential neurovirulence of Usutu virus lineages in mice and neuronal cells[END_REF][START_REF] Benzarti | New insights into the susceptibility of immunocompetent mice to Usutu virus[END_REF][START_REF] Benzarti | Usutu virus infection of embryonated chicken eggs and a chicken embryo-derived primary cell line[END_REF].

Human Infections

Clinical Manifestations

In humans, the incubation period of USUV is estimated at about 3 to 12 days after an infected mosquito bite. This estimation is based on WNV infection as a reference because of the limited clinical data available for USUV infection [START_REF] Lustig | Surveillance and diagnosis of west nile virus in the face of flavivirus crossreactivity[END_REF]. After incubation, a short viremic phase is triggered and the patient develops the first symptoms. The virus is then detectable in urine. Seroprevalence studies suggest that USUV is asymptomatic or associated with mild symptoms in most cases. However, the incidence rate of asymptomatic

Human Infections

Clinical Manifestations

In humans, the incubation period of USUV is estimated at about 3 to 12 days after an infected mosquito bite. This estimation is based on WNV infection as a reference because of the limited clinical data available for USUV infection [START_REF] Lustig | Surveillance and diagnosis of west nile virus in the face of flavivirus crossreactivity[END_REF]. After incubation, a short viremic phase is triggered and the patient develops the first symptoms. The virus is then detectable in urine. Seroprevalence studies suggest that USUV is asymptomatic or associated with mild symptoms in most cases. However, the incidence rate of asymptomatic patients is currently unknown. The symptomatic phase of the infection is usually characterized by moderate fever, sometimes associated with rash and febrile jaundice [START_REF] Sejvar | Clinical Manifestations and Outcomes of West Nile Virus Infection[END_REF]. The zoonotic potential of USUV infection was first described in the African continent. The first two human cases were reported in the Central African Republic in the 1980s and in Burkina Faso in 2004 [START_REF] Nikolay | Usutu virus in Africa[END_REF]. Fever and skin rash were reported for both cases. Since, all human cases of USUV infection have been reported in Europe only, although seroprevalence studies conducted in other countries highlight the active circulation of this virus in the African continent [START_REF] Tinto | Screening of Circulation of Usutu and West Nile Viruses: A One Health Approach in Humans, Domestic Animals and Mosquitoes in Burkina Faso, West Africa[END_REF][START_REF] Raulino | Multiplex detection of antibodies to Chikungunya, O'nyong-nyong, Zika, Dengue, West Nile and Usutu viruses in diverse non-human primate species from Cameroon and the Democratic Republic of Congo[END_REF] (Table 1). CAR: Central African Republic.

The first autochthonous human cases of USUV infection in Europe were reported in 2009 in Italy and manifested as two cases of meningoencephalitis in immunosuppressed patients: one, a woman treated for B-cell lymphoma, and another who had an orthotopic liver transplant [START_REF] Cavrini | Usutu virus infection in a patient who underwent orthotropic liver transplantation, Italy[END_REF][START_REF] Pecorari | Rapid communications First human case of usutu virus neuro invasive infection, italy, august-september 2009[END_REF]. Since, most human clinical cases have been identified in Italy likely due to an active circulation of this virus in this country, but also due to an elaborate USUV surveillance program coupled with WNV monitoring. Indeed, in Italy USUV infection is a notifiable disease and USUV surveillance has been included in the national plan since 2017 [START_REF]National Surveillance and Response Integrated Plan for West Nile and Usutu Viruses-2018[END_REF]. Therefore, between 2008 and 2009, 12 additional cases were identified in cerebrospinal fluid (CSF) or blood samples of Italian patients [START_REF] Cavrini | A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid[END_REF][START_REF] Grottola | Usutu virus infections in humans: A retrospective analysis in the municipality of Modena, Italy[END_REF]. More recently, in 2018, one patient with USUV-associated encephalitis and six with fever were detected during the surveillance period [START_REF] Pacenti | Clinical and virological findings in patients with Usutu virus infection, northern Italy[END_REF] and in 2022 two new cases of febrile USUV infections were detected, while four asymptomatic donors were also identified as positive for the virus [START_REF]Sorveglianza Integrata del West Nile e Usutu Virus[END_REF]. This detection of USUV in blood donors has already been demonstrated before in Italy. Indeed, 38 blood donors were detected as positive in 2017-2018 in several regions of Italy [START_REF] Pacenti | Clinical and virological findings in patients with Usutu virus infection, northern Italy[END_REF][START_REF] Percivalle | West Nile or Usutu Virus? A Three-Year Follow-Up of Humoral and Cellular Response in a Group of Asymptomatic Blood Donors[END_REF][START_REF] Caracciolo | Comprehensive response to usutu virus following first isolation in blood donors in the friuli venezia giulia region of italy: Development of recombinant ns1-based serology and sensitivity to antiviral drugs[END_REF][START_REF] Carletti | Expanding Usutu virus circulation in Italy: Detection in the Lazio region, central Italy, 2017 to 2018[END_REF]. The consequences for blood donor recipients who receive USUV-positive blood are not yet known. Blood donors in endemic areas who have recently been sick with a clinical illness compatible with an arboviral disease should consider delaying blood donation. Blood donation agencies may want to assess the risk for transmission in endemic areas and consider updating their testing protocols accordingly [START_REF] Domanović | West nile and usutu virus infections and challenges to blood safety in the European Union[END_REF]. Note that there have been reports of blood donors with USUV infections testing positive after screening by the West Nile virus nucleic acid tests (NAT), suggesting potential screening cross-reactivity between these two related viruses [START_REF] Domanović | West nile and usutu virus infections and challenges to blood safety in the European Union[END_REF].

Cases of human USUV infection have also been detected in other European countries, although the surveillance networks in place are often not as efficient. Three neurological cases were reported in Croatia in 2013: one patient presented with meningitis and two with meningoencephalitis. [START_REF] Santini | First cases of human Usutu virus neuroinvasive infection in Croatia, August, September 2013: Clinical and laboratory features[END_REF]. In 2018, three other neurological diseases were identified in Croatia, as well as a case in the Czech Republic and another one in Hungary [START_REF] Vilibic-Cavlek | Prevalence and molecular epidemiology of West Nile and Usutu virus infections in Croatia in the "One health" context, 2018[END_REF][START_REF] Zelená | First Autochthonous West Nile Lineage 2 and Usutu Virus Infections in Humans, July to October 2018, Czech Republic[END_REF][START_REF] Nagy | Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary[END_REF]. In 2020, one case was identified in Switzerland by metagenomic next-generation sequencing [START_REF] Zanella | Unmasking viral sequences by metagenomic next-generation sequencing in adult human blood samples during steroid-refractory/dependent graft-versus-host disease[END_REF]. Two large studies conducted in Austria on blood donors have also identified 24 positive donors [START_REF] Aberle | Increase in human west nile and usutu virus infections, Austria[END_REF][START_REF] Bakonyi | Usutu virus infections among blood donors, Austria, july and august 2017-Raising awareness for diagnostic challenges[END_REF] and a patient with meningitis [START_REF] Graninger | The First Case of Usutu Virus Neuroinvasive Disease in Austria[END_REF]. In France, USUV infection was reported in 2016 in a patient with idiopathic facial paralysis, and one febrile case was detected in 2022 [START_REF] Simonin | Human Usutu virus infection with atypical neurologic presentation[END_REF][START_REF] De Aquitaine | Confirmation d'Une Infection Autochtone à Virus Usutu (Secteurs des Landes et de Gironde)[END_REF].

Seroprevalence studies indicate a non-negligible exposure of humans to USUV infection risk (Table 2). These studies have been carried out in Austria, Germany, Italy, France, Hungary, the Netherlands, Romania, and Serbia, reporting an USUV antibody prevalence between 0.02% and 3% among healthy blood donors [START_REF] Constant | One Health surveillance of West Nile and Usutu viruses: A repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020[END_REF][START_REF] Faggioni | Prevalence of Usutu and West Nile virus antibodies in human sera[END_REF][START_REF] Scaramozzino | West Nile and Usutu viruses co-circulation in central Italy: Outcomes of the 2018 integrated surveillance[END_REF][START_REF] Pierro | Detection of specific antibodies against West Nile and Usutu viruses in healthy blood donors in northern Italy, 2010-2011[END_REF][START_REF] Bakonyi | Usutu virus infections among blood donors, Austria, july and august 2017-Raising awareness for diagnostic challenges[END_REF][START_REF] Carletti | Expanding Usutu virus circulation in Italy: Detection in the Lazio region, central Italy, 2017 to 2018[END_REF][START_REF] Coroian | Seroprevalence Rates against West Nile, Usutu, and Tick-Borne Encephalitis Viruses in Blood-Donors from North-Western Romania[END_REF][START_REF] Hrnjaković Cvjetković | Seroprevalence of Mosquito-Born and Tick-Born Microorganisms in Human Population of South Backa District[END_REF][START_REF] Gaibani | Detection of Usutu-virus-specific IgG in blood donors from Northern Italy[END_REF][START_REF] Zaaijer | Usutu virus infection in Dutch blood donors[END_REF][START_REF] Allering | Detection of Usutu virus infection in a healthy blood donor from South-West Germany[END_REF][START_REF] Nagy | West Nile and Usutu virus seroprevalence in Hungary: A nationwide serosurvey among blood donors in 2019[END_REF]. Higher prevalence (6/7%) was found in more exposed populations, such as forestry workers [START_REF] Grottola | Usutu virus infections in humans: A retrospective analysis in the municipality of Modena, Italy[END_REF][START_REF] Hrnjaković Cvjetković | Seroprevalence of Mosquito-Born and Tick-Born Microorganisms in Human Population of South Backa District[END_REF][START_REF] Percivalle | Usutu Virus Antibodies in Blood Donors and Healthy Forestry Workers in the Lombardy Region, Northern Italy[END_REF]. Several of these studies show that USUV circulates more actively in humans than WNV in Europe, and prevalence studies of USUV infection in mosquito vectors in Europe also highlight a higher level of exposure to USUV than to WNV [START_REF] Puggioli | Does Aedes albopictus (Diptera: Culicidae) play any role in Usutu virus transmission in Northern Italy? Experimental oral infection and field evidences[END_REF][START_REF] Calzolari | Usutu Virus Persistence and West Nile Virus Inactivity in the Emilia-Romagna Region (Italy) in 2011[END_REF][START_REF] Barzon | The complex epidemiological scenario of West Nile virus in Italy[END_REF]. However, seroprevalence data are still very scarce for assessing the real incidence of USUV in humans, and to date, there are no efficient serological diagnostic tools for large-scale screening. The technical disadvantages of serology testing are indeed confronted. This is often the case for flaviviruses due to lack of specificity linked to the substantial antigenic cross-reactions with representative flaviviruses, meaning results often require confirmation by the more fastidious seroneutralization approach [START_REF] Beck | Flaviviruses in Europe: Complex circulation patterns and their consequences for the diagnosis and control of West Nile disease[END_REF]. Although the USUV lineages involved in most of the studies remain unknown, few studies showed that several lineages circulate in the human population (Figure 3). Although the USUV lineages involved in most of the studies remain unknown, few studies showed that several lineages circulate in the human population (Figure 3). We can nevertheless note that infections due to the Europe 2 lineage are over-represented in the human cases with neurological diseases (Table 1). Since the appearance of the EU2 lineage in 2014 in Germany, initially detected in a blackbird [START_REF] Weidinger | Different dynamics of Usutu virus infections in Austria and Hungary, 2017-2018[END_REF], this lineage has become the dominant circulating USUV in several countries, such as Austria and Hungary [START_REF] Gaibani | Comparative Genomic and Phylogenetic Analysis of the First Usutu Virus Isolate from a Human Patient Presenting with Neurological Symptoms[END_REF]. In addition, several studies have reported increased virulence of this lineage. In particular, a study using chicken embryos showed the presence of viral antigens in the brains of infected embryos. These chicken embryos subsequently died following infection with a USUV EU2 isolate [START_REF] Benzarti | Usutu virus infection of embryonated chicken eggs and a chicken embryo-derived primary cell line[END_REF]. The EU2 lineage has also shown elevated virulence in mouse models and human neuronal cells [START_REF] Clé | Differential neurovirulence of Usutu virus lineages in mice and neuronal cells[END_REF]. Point mutation at position 835 in NS5 has been found in several EU2 strains [START_REF] Clé | Differential neurovirulence of Usutu virus lineages in mice and neuronal cells[END_REF]. This mutation was also found in the first reported human case in Europe from 2009 (associated with neurological complication), and it has also been associated with neuroinvasive capacity in JEV, WNV, Kunjin virus (KUNV), and Murray Valley encephalitis virus (MVEV) [START_REF] Cavrini | Usutu virus infection in a patient who underwent orthotropic liver transplantation, Italy[END_REF][START_REF] Haviernik | An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus in Mice[END_REF]. Furthermore, mutation at position 835 in the viral polymerase is located in the RdRp domain, thus most likely increasing the viral fitness by replication efficiency, which could result, in vivo, in an exacerbated virulence phenotype [START_REF] Clé | Usutu virus: A new threat?[END_REF]. Finally, a point amino acid substitution of glutamic acid for aspartic acid (E460D) in NS5 has demonstrated a role in the virulence of tick-borne encephalitis We can nevertheless note that infections due to the Europe 2 lineage are over-represented in the human cases with neurological diseases (Table 1). Since the appearance of the EU2 lineage in 2014 in Germany, initially detected in a blackbird [START_REF] Cadar | Widespread activity of multiple lineages of Usutu virus, Western Europe[END_REF], this lineage has become the dominant circulating USUV in several countries, such as Austria and Hungary [START_REF] Weidinger | Different dynamics of Usutu virus infections in Austria and Hungary, 2017-2018[END_REF]. In addition, several studies have reported increased virulence of this lineage. In particular, a study using chicken embryos showed the presence of viral antigens in the brains of infected embryos. These chicken embryos subsequently died following infection with a USUV EU2 isolate [START_REF] Benzarti | Usutu virus infection of embryonated chicken eggs and a chicken embryo-derived primary cell line[END_REF]. The EU2 lineage has also shown elevated virulence in mouse models and human neuronal cells [START_REF] Clé | Differential neurovirulence of Usutu virus lineages in mice and neuronal cells[END_REF]. Point mutation at position 835 in NS5 has been found in several EU2 strains [START_REF] Clé | Differential neurovirulence of Usutu virus lineages in mice and neuronal cells[END_REF]. This mutation was also found in the first reported human case in Europe from 2009 (associated with neurological complication), and it has also been associated with neuroinvasive capacity in JEV, WNV, Kunjin virus (KUNV), and Murray Valley encephalitis virus (MVEV) [START_REF] Cavrini | Usutu virus infection in a patient who underwent orthotropic liver transplantation, Italy[END_REF][START_REF] Gaibani | Comparative Genomic and Phylogenetic Analysis of the First Usutu Virus Isolate from a Human Patient Presenting with Neurological Symptoms[END_REF]. Furthermore, mutation at position 835 in the viral polymerase is located in the RdRp domain, thus most likely increasing the viral fitness by replication efficiency, which could result, in vivo, in an exacerbated virulence phenotype [START_REF] Clé | Usutu virus: A new threat?[END_REF]. Finally, a point amino acid substitution of glutamic acid for aspartic acid (E460D) in NS5 has demonstrated a role in the virulence of tick-borne encephalitis virus (TBEV) in mice [START_REF] Haviernik | An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus in Mice[END_REF]. Further studies are needed to evaluate if the over-representation of the EU2 lineage in acute infections and in neurological diseases is related to a higher virulence of this lineage or other reasons (vector competence, multiplication in the reservoir host, etc.).

Overall, to date, over 100 cases of acute human infection have been described in Europe, including 30 patients with neurological symptoms. Concerning the neurological disorders for which we have detailed information, we can notice that most patients have had comorbidities of varying severity, but that there are also patients developing neurological forms without identified concomitant pathological conditions (Table 3). Nevertheless, the number of cases reported to date is not sufficient for determining age or gender criteria in the exposure to neurological risks associated with USUV. Furthermore, it is still unknown whether these human cases represent the tip of the iceberg or the incidence of acute USUV infection with mild symptomatology remains minimal since the diagnosis of USUV infection is rarely considered. The emergence of avian epizootics is synchronous with a greater exposure of humans to zoonotic risks. In this light, the human cases detected in Italy were concomitant with an epizootic outbreak of USUV showing that humans are exposed to a higher zoonotic pressure than in that of WNV [START_REF] Gaibani | Detection of Usutu-virus-specific IgG in blood donors from Northern Italy[END_REF]. Similarly, the human cases detected from 2016 to 2018 were simultaneous with the most epizootics observed in Europe [START_REF] Clé | Usutu virus: A new threat?[END_REF][START_REF] Cadar | Blood donor screening for west nile virus (WNV) revealed acute usutu virus (USUV) infection, Germany[END_REF][START_REF] Bakonyi | Usutu virus infections among blood donors, Austria, july and august 2017-Raising awareness for diagnostic challenges[END_REF]. Phylogenetic analysis of the strains detected in humans in Germany and Austria confirmed that they were strains of the Europe 2 and 3 lineages, that had indeed circulated among blackbird and passerine populations at that time. Similarly, a different strain from the Africa 2 lineage that was detected in a human case in the south of France in Montpellier [START_REF] Simonin | Human Usutu virus infection with atypical neurologic presentation[END_REF] was also simultaneously identified in pools of mosquitoes captured nearby, in the Camargue area [START_REF] Eiden | Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015[END_REF]. Hence, significant circulation of USUV in both reservoirs and vectors, at a given time and place, increases the probability of human cases.

Diagnosis

Diagnosis of USUV infection is classically made by detection of specific antibodies, by demonstration of the viral genome, genomic fragments, or by cell culture. Direct diagnosis is based on the detection of viral RNA in blood and CSF by reverse polymerase chain reaction (RT-PCR). Indirect or incidental detection of the virus occurs in most of the blood donor cases which cross-react with WNV in the COBAS systems used in several national surveillance programs for WNV screening. It can also be performed in some cases by cell culture. Many cell types tolerate viral replication. The cells mainly used are C636 insect cells or Vero cells, in which the virus develops a cytopathic effect, but many alternatives are possible [START_REF] Barr | Working with Zika and Usutu Viruses In Vitro[END_REF]. However, this direct detection method is not commonly used, as infection with these viruses usually results in a short-lived viremia as humans are an epidemiological dead-end for these viruses. Instead, serological tests are performed by ELISA or approaches using immunofluorescence. These tests suffer from a lack of specificity and must be systematically controlled by neutralization approaches to exclude systematic cross-reactions obtained with antibodies directed against other representatives of flaviviruses, particularly between WNV and USUV, which are phylogenetically closely related. To date, no commercial diagnostic test is available to detect USUV, unlike WNV.

Treatment and Prevention

There is no vaccine against USUV, although an experimental DNA vaccine appeared to be protective in mice [START_REF] Martín-Acebes | A recombinant DNA vaccine protects mice deficient in the alpha/beta interferon receptor against lethal challenge with Usutu virus[END_REF]. Given that USUV and WNV co-circulate in the same geographic regions, the question arises of cross-protection or an antibody-dependent enhancement (ADE) effect for USUV and WNV (or other flaviviruses). Studies in mice suggest partial post-flavivirus cross-protection in mice [START_REF] Blázquez | Limited susceptibility of mice to Usutu virus (USUV) infection and induction of flavivirus cross-protective immunity[END_REF].

To limit USUV infection, as for infection with other arboviruses, there is no specific treatment. Favipiravir, a viral RNA polymerase inhibitor, has been shown effective in mice [START_REF] Segura Guerrero | Favipiravir inhibits in vitro Usutu virus replication and delays disease progression in an infection model in mice[END_REF]. Supportive and symptomatic treatments may be used, such as paracetamol for pain and/or fever, hydration (orally or sometimes by injection), and antiemetics. Patients who develop meningoencephalitis or encephalitis may need to be monitored for elevated intracranial pressure and/or seizures. Although many efforts are being made to find specific or global treatments against arboviruses, the most widely used prevention methods are still based on vector control. Vector control is mainly based on the use of repellents and mosquito nets, or the use of insecticides which nevertheless have limitations, such as the resistance developed by arthropods against insecticides. To prevent mosquito breeding, stagnant water should be periodically removed from containers around homes.

Conclusions

The study of the circulation of USUV is not well documented. There are still many unknown aspects, especially concerning reservoirs and the real prevalence in humans. A better understanding of the circulation and risks associated with these viruses, and more globally with arboviruses, requires a review of our monitoring strategy, through the implementation of the One Health global approach. This approach consists of studying the interactions between animals, humans, and their various environmental factors (e.g., climatic condition, vector population dynamics) by setting up a transdisciplinary and multidisciplinary approach involving many actors. These include, for instance, human health professionals (e.g., physicians, pharmacists, virologists, epidemiologists), but also with the participation of professionals from the veterinary field and entomologists. The objective is to implement efficient surveillance programs and preparedness strategies for emerging diseases like USUV.

Figure 1 .

 1 Figure 1. Usutu virus virion structure and genomic organization. (A) Schematic representation of USUV virion structure: virion surface is enhanced with the envelope and membrane proteins, anchored in a lipid bilayer with an icosahedral symmetry. (B) Representation of the USUV genome. RNA of positive polarity composed of 11 genes. A single reading frame codes for a polyprotein which is cleaved co-and post-translationally by viral and cellular proteases releasing 3 structural proteins (C, prM, and E) and 8 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, 2K, NS4b, and NS5).

Figure 1 .

 1 Figure 1. Usutu virus virion structure and genomic organization. (A) Schematic representation of USUV virion structure: virion surface is enhanced with the envelope and membrane proteins, anchored in a lipid bilayer with an icosahedral symmetry. (B) Representation of the USUV genome. RNA of positive polarity composed of 11 genes. A single reading frame codes for a polyprotein which is cleaved co-and post-translationally by viral and cellular proteases releasing 3 structural proteins (C, prM, and E) and 8 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, 2K, NS4b, and NS5).

Figure 2 .

 2 Figure 2. Bayesian maximum clade credibility tree representing the phylogenetic placement of the human Usutu virus cases (in red) compared with some representative USUVs based on partial NS5 gene sequences. Phylogenetic analysis was performed by using Bayesian Markov chain Monte Carlo (MCMC) tree-sampling method implemented in BEAST v.1.8.0 (http://beast.bio.ed.ac.uk, accessed on 20 November 2022). Statistical supports of grouping from Bayesian posterior probabilities (clade credibility) are indicated at the nodes (colored circles). GenBank accession numbers, countries of origin, host, and years of detection of the sequences used to reconstruct the tree are indicated on the branches. Scale bar indicates mean number of nucleotide substitutions per site. Edited with Biorender.com.

Figure 2 .

 2 Figure 2. Bayesian maximum clade credibility tree representing the phylogenetic placement of the human Usutu virus cases (in red) compared with some representative USUVs based on partial NS5 gene sequences. Phylogenetic analysis was performed by using Bayesian Markov chain Monte Carlo (MCMC) tree-sampling method implemented in BEAST v.1.8.0 (http://beast.bio.ed.ac.uk, accessed on 20 November 2022). Statistical supports of grouping from Bayesian posterior probabilities (clade credibility) are indicated at the nodes (colored circles). GenBank accession numbers, countries of origin, host, and years of detection of the sequences used to reconstruct the tree are indicated on the branches. Scale bar indicates mean number of nucleotide substitutions per site. Edited with Biorender.com.

Figure 3 .

 3 Figure 3. Human cases of Usutu virus infection and the lineages involved.

Figure 3 .

 3 Figure 3. Human cases of Usutu virus infection and the lineages involved.

Table 1 .

 1 Description of human Usutu cases worldwide and associated viral lineages. In red: human USUV cases associated with neurological disorders.

	Acute Infections (n = 112)
	30 Neuroinvasive Diseases
	Country	Year	Cases	Sample Type	Symptomatology	Studied Population	USUV Lineages	Ref
	Burkina Faso	2004	1	Blood	Fever and jaundice	Clinical case	ND	[35]
	CAR	1981	1	Blood	Eruptive fever	Clinical case	AF3	[35]
	Austria	2017	6/12047	Blood	Healthy	Blood donors	EU2	[52]
		2018	18/31598	Blood	Healthy	Blood donors	EU2/AF3	[53]
		2021	1	CSF	Meningitis	Clinical case	EU2	[54]
	Croatia	2013	3/95	Blood	Meningoencephalitis	Meningoencephalitis patients	ND	[55]
					Neuroinvasive disease
		2018	3/178	Blood /Urine	included 1 fatal	Neuroinvasive cohort	EU2	[56]
					meningoencephalitis
	Italy	2009	1	CSF	Meningoencephalitis	Clinical case	EU1	[57]
		2009	1	Blood	Encephalitis	Clinical case	EU2	[58]
		2008-2009	3/44	CSF	Meningoencephalitis	Meningoencephalitis patients	ND	[59]
						Meningoencephalitis
		2008-2011	8/306 + 2/609	CSF/blood	Meningoencephalitis /healthy	patients + various healthy and sick	ND	[60]
						subjects
		2016-2018	25/73964	Blood	Healthy	Blood donors	ND	[61]
		2017-2018	9	Blood	Healthy	Blood donors	EU2/EU3/EU4	[62]
					1 encephalitis, 6with	Suspected autochthonous
		2017-2018	8	Blood/Urine	fever and 1 viremic	arboviral infection and	EU2	[63]
					blood donor	positive blood donors
		2018	2/8889	Blood	Healthy	Blood donors	EU2	[64]
		2018	1/44	Blood	Healthy	Blood donors	EU1	[65]
		2022	6	Blood	4 healthy, 2 fever	Clinical case	ND	[66]
	Czech Republic	2018	1	Blood	Meningoencephalitis	Clinical case	ND	[67]
	Germany	2016	1	Blood	Healthy	Blood donors	EU3	[68]
	France	2016	1/666	CSF	Idiopathic facial paralysis	Patients with infectious and/or neurological signs	AF2	[40]
		2022	1	Blood	Fever	Clinical case	ND	[69]
	Hungary	2018	1	Blood	Aseptic meningitis	Clinical case	EU2	[70]
	Netherlands	2018	7/12,040	Blood	Healthy	Blood donors	EU3	[71]
	Switzerland	2020	1/25	Blood	Disseminated fungal infection (fatal case)	Clinical case	AF3	[72]

Table 2 .

 2 Seroprevalence studies of USUV in the European populations.

	n = 127
	Country	Year	Number	Prevalence	Studied Population	Ref
	France	2019-2020	15/500	3%	Outpatients	[32]
	Germany	2012	1/4200	0.02%	Blood donors	[80]
	Hungary	2019	5/3005	0.17%	Blood donors	[81]
	Italy	2009	4/359	1.1%	Blood donors	[77]
		2008-2011	40/609	6.5%	Healthy/various sick subjects	[60]
		2010-2011	14/6000	0.23%	Blood donors	[78]
		2012	24/3069	0.78%	Blood donors	[79]
		2014-2015	6/33	18.1%	Healthy forestry worker [82]
		2014-2015	2/200	1%	Blood donors	[78]
		2018	2/8889	0.02%	Blood donors	[64]
	Netherlands	2018	7/12,040	0.06%	Blood donors	[71]
	Romania	2018	0/1200	0.00%	Blood donors	[82]
	Serbia	2015	7/93	7.5%	Healthy exposed subjects	[83]

Table 3 .

 3 Description of clinical case with identified neurological damage. NC: Not Communicated.

	Country	Clinic	Age (Years)	Gender	Health Condition/Comorbidity	Outcome
	Austria	Meningitis	81	Man	Hypertension, left-sided hemifacial spasms	Constant monitoring and hospital care (7 months after hospitalisation)
	Croatia	Meningoencephalitis	29	Woman	Healthy	NC
			61	Man	Healthy	NC
			56	Man	Arterial hypertension, coronary heart disease, hyperlipidemia, and diabetes	NC
		Neuroinvasive disease	25	NC	Healthy	NC
			84	NC	Healthy	NC
			60	NC	Immunocompromised. Chronic lymphocytic leukemia	Fatal
	Czech Republic	Meningoencephalitis	46	Woman	Healthy	Patient discharged after 12th day of hospitalisation.
	France	Idiopathic facial paralysis	39	Man	Healthy	Patient discharged 3 days after hospitalisation. Symptoms disappeared within a few weeks.
	Hungary	Meningitis	40s	Man	Healthy	Duration of hospitalisation was 7 days
	Italy	Meningoencephalitis	60s	Woman	Immunocompromised. Large B cell lymphoma.	NC
		Encephalitis	40s	Woman	Immunocompromised. Orthotropic liver transplantantation	Fulminant hepatitis, coma. The patient slowly regained motor function
		Meningoencephalitis	40	Man	Chronic liver disease	NC
			73	Man	Chronic obstructive pulmonary disease, diabetes	NC
		Encephalitis	54	Woman	Polyneuritis, hypertension	NC
		Encephalitis	60s	Man	Hypertension, diabetis	NC
		Encephalitis	67	Woman	Aortic and mitral insufficiency	NC

Funding: This research received no external funding.

Conflicts of Interest:

The authors declare no conflict of interest.

Author Contributions: D.C. and Y.S. contributed to the conception, drafting, writing and critical revision of the manuscript. All authors have read and agreed to the published version of the manuscript.