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An overview of Usutu virus
Accepted Manuscript An overview of Usutu virus Paolo Gaibani, Giada Rossini PII:
S1286-4579(17)30085-0
DOI:
10.1016/j.micinf.2017.05.003
Reference:
MICINF 4473
To appear in:
Microbes and Infection
Received Date: 18 April 2017 Revised Date:
26 May 2017
Accepted Date: 29 May 2017
Please cite this article as: P. Gaibani, G. Rossini, An overview of Usutu virus, Microbes and Infection (2017), doi: 10.1016/j.micinf.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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An overview of Usutu virus
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Paolo Gaibani* and Giada Rossini
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Operative Unit of Clinical Microbiology, Regional Reference Centre for Microbiological
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Emergencies (CRREM), S. Orsola-Malpighi University Hospital, Bologna, Italy
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*
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Paolo Gaibani, PhD
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Operative Unit of Microbiology, S.Orsola-Malpighi University Hospital, Regional Reference
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Centre for Microbiological Emergencies (CRREM), 9 via G. Massarenti – 40138 Bologna,
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ITALY.
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Telephone: +39 051 6364316
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Fax: +39 051 6363076
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e-mail: [email protected]
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Corresponding Author:
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Usutu virus (USUV) is a mosquito-borne flavivirus that emerged in Africa in the middle of the 16th
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century and currently widely circulates in several European countries. Herein, we summarize
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current knowledge about USUV from ecology, epidemiology, phylogeny to clinical manifestations
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and diagnosis and discuss the role as human pathogen.
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Keywords: Usutu virus; Structure; Epidemiology; Ecology; Phylogeny; Human infection
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1. Introduction Usutu virus (USUV) is an arthropod-borne virus (arbovirus) belonging to the genus Flavivirus
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within the Flaviviridae family. As a member of the Japanese encephalitis virus (JEV) antigenic
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complex, USUV is closely related to numerous human and animal pathogens including West Nile
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virus (WNV), Murray Valley Encephalitis virus (MVEV), and St Louis encephalitis virus (SLEV)
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[1]. USUV is maintained in the environment through a typical enzootic cycle involving mosquitoes
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and birds. Since first identification in South Africa in the middle of 20th century, widespread
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circulation of USUV was observed in several countries [2]. In Europe, USUV emerged in 1996
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causing high numbers of bird deaths [3]. Five years later, USUV was responsible of high mortality
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rate among Eurosian Blackbirds (Turdus merula) in the surrounding area of Vienna, Austria [4].
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Currently, USUV is endemic in several countries in Europe [5-8].
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The clinical relevance of USUV as human pathogen has been hypothesized since the first
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descriptions of USUV-related infection in humans [9]. The first report of USUV infection in
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humans was described in Africa at the beginning of 1980s [10]. Thirty years later, two cases of
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USUV-related neuro-invasive diseases were reported in immune-compromised patients in Europe
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(Italy) [11,12]. Although different human cases of USUV infection have been reported until today
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[13,14], the effective role of the USUV as a human pathogen has yet to be clarified.
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The present review summarizes the up to date knowledge on USUV with particular focus on the
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structure, ecology, epidemiology, genetic diversity, as well as current understanding about human
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infection.
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1.1 Virus genome and structure
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USUV is a small and spherical virus with a lipid envelope derived from host cell membrane. The
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virion is 40–60 nm in diameter and contains a positive-sense RNA genome of 11 Kb in length with
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no 3′ poly(A) tail. Genomic organization shows a similar structure comparable to others flavivirus
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[15]. The genome consists of a single-stranded RNA genome with a 5’ cap structure, a unique open
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reading frame (ORF) and two untranslated regions (UTRs). The 5’ and 3’ UTRs varied respectively
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between 95 to 96 nt and 631 to 664 nt in length among different strains [16]. The UTRs are
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involved for the translation and replication of the viral genome. The predicted ORF is translated in a
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unique polyprotein of 3434 amino acids that is post-translationally processed into three structural
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(capsid, envelope, and pre-membrane) and eight non-structural proteins (NS1, NS2A, NS2B, NS3,
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NS4A, 2K, NS4B, and NS5). Like other mosquito-borne flavivirus, genes encoding the structural
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proteins are located on the 5’ end of viral genome and form the virion particle [17,18]. The capsid
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protein (C) forms the central core of the virion and is associated to the viral RNA. The envelope
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glicoprotein (E) mediates binding to the host cells and promotes viral entry into the host cells. The
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pre-membrane protein (prM) are necessary for virion assembly and maturation by assisting
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envelope folding [19,20]. The nonstructural proteins serve to different functions during infection
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and their functions are deduced on the basis of the similarity with other flavivirus genomes [21].
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NS1 exists in distinct forms (i.e. cellular and secreted) and is necessary on the replication of viral
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genome and virion maturation [22]. The NS2A, NS2B, NS4A and NS4B are small, hydrophobic
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proteins that are required for virus assembly and play a role in the inhibition of the IFN response
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[2]. NS3 and NS5 are two proteins with different enzymatic activities: NS3 protein encodes for
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viral serine protease (active only with NS2B cofactor), helicase, nucleoside triphosphatase and
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RNA triphosphatase. NS5 protein encodes for a methyltransferase (MTase) at the N-terminal, while
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C-terminal encodes for the RNA-dependent RNA polymerase [2,23].
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1.2 Hosts, vectors and life cycle
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The natural life cycle of USUV involves ornithophilic mosquitoes as vector and birds as main
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amplifying host. USUV cycle is similar to that of other flaviviruses belonging to the JEV
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serocomplex [18], where humans are considered as incidental or “dead-end” hosts. Birds of
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different species may be infected by USUV and some of these may develop symptoms of disease.
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USUV has been detected in 62 bird species in African and European countries [10,24]. Different
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migratory wild species (i.e. Falco tinnunculus, Acrocephalus scirpaceus, Sylvia curruca, Sylvia
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communis and Ficedula hypoleuca) are considered responsible for USUV introduction in Europe
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from Africa [10], while others (i.e. Pica pica, Passer domesticus, Gallus gallus and Turdus merula)
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are considered responsible of USUV dissemination through Europe [24]. Interestingly, USUV
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caused high mortality among several bird species (especially Turdus merula) in Europe [4], while in
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Africa and Spain was not associated to high death in birds [25]. Birds may show symptoms varying
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from mild to severe signs of infection [26]. Severe symptoms due to USUV infection in birds
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included encephalitis, myocardial degeneration, and necrosis of the liver and spleen [27,28]
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USUV has been isolated from different mosquito species in several countries. Up to now, USUV
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has been found in eight species belonging to the genus Aedes, Anopheles, Culex, Culiseta,
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Ochlerotatus, Coquillettidia and Mansonia [24]. Although USUV has been found in several
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species, Culex pipiens is the main common vector for USUV [5,29], while Cx. neavei has been
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hypothesized to be involved in sylvatic transmission of USUV in Africa [30].
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Previous studies demonstrated that USUV has been not found in tick species, [31]. USUV infection
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has been demonstrated in different mammal species. In particular, USUV infection has been
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documented in bats (Pipistrellus pipistrellus), horses, dogs and red deer (Cervus elaphus) [32-35].
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1.3 Origin and epidemiology
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A recent study by Engel et al. hypothesized that USUV emerged in Africa at the beginning of 16th
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century and until middle of 20th century, USUV spreading remained exclusively limited to African
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continent where it was detected in different bird and mosquito species in several countries [10,16].
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Introductions of USUV in Europe from Africa probably started in the last 50 years and continue to
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occur. It has been hypothesized that USUV was firstly introduced in Europe by birds between 1950
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and the 1960’s in Spain, while second and third introductions occurred between 1970-1980 (Italy,
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Austria) and 1984-2006 (Spain) [16].
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The earliest evidence of USUV in Europe emerged in 2001 in Austria, associated to deaths in
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several species of resident birds [4]. However, retrospective analysis of archived tissue samples
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from dead Eurasian blackbird in Tuscany, Italy [3] provided evidence that USUV emerged in a
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pathogenic form in Europe in 1996 or earlier. In the following years, USUV was found in
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mosquitoes, birds and bats in several other European countries: Hungary [36], Switzerland [37],
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Spain [38], Italy [27], Czech Republic [39], Germany [40], Belgium [41] and France (2015) [42].
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Moreover, USUV infection has been demonstrated only serologically in birds in England [43],
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Poland [44] and Greece [45] and in a horse in Serbia [46]. USUV was found recurrently over
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several years in Austria (2001–2006) [47], Hungary (2003–2006) [36], Italy (2009–2016) [27,48-
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51], Spain (2006, 2009, 2012) [9,38,52], and Germany (2010-2015) [53,54] suggesting a
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persistence of the transmission cycle in the affected areas through overwintering mosquitoes or
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multiple reintroduction of the virus.
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During summer 2016, a large USUV epizootic was registered in Europe with widespread activity in
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Belgium, Germany, France and for the first time in Netherlands [55,56] highlighting the continuous
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geographical spread of USUV, also to new ecological niches.
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Frequently, USUV co-circulates with West Nile virus (WNV) in many European countries, in terms
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of geographic range of transmission, host and vector species. Considering the immunological cross-
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reactions between these two viruses, it remains to be clarified if such overlapping can influence the
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spatio-temporal patterns of virus circulation in Europe [24].
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1.4 Genetic variation and phylogeny
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USUV is classified in eight lineages which are distinct in two major groups, African and European,
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based on geographical origin of isolation [16]. Phylogenetic analysis based on the NS5 gene
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demonstrated that USUV strains from Africa belong to three distinct lineages (Africa 1 to 3), while
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European group is composed by five different lineages (Europa 1 to 5) [55].
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The lineage Africa 1 includes only one strain (CAR-1969 strain [GenBank accession no.
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KC754958]) isolated in the Central African Republic in 1969 [17]. The lineage Africa 2, originated
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in South Africa in the middle of 1940s, comprises USUV strains isolated in Senegal, Spain,
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Germany and France. The lineage Africa 3 includes strains isolated in Senegal, Germany,
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Netherlands, Belgium and a human isolate from Central African Republic isolated in 1981 (CAR-
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1981 [KC754955]). Europe 1 lineage originated from an ancestor that existed in Senegal and
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consists of strains isolated from Austria, Hungary, Switzerland, Senegal and recently humans
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isolates from Italy [13,16]. Europe 2 lineage consists of strains from Italy and Czech Republic
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including Bologna-2009 strain isolated from patient with meningo-encephalitis [HM569263] [57].
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The lineage Europe 3 includes USUV strains from Germany, Belgium and France, while Europe 4
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consists of USUV strains from Italy, including human isolates [13,55,58]. The recently designated
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Europe 5 lineage includes different strains all isolated from Germany [55].
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The genome identity among isolates is higher than 94%, with exception of CAR-1969 isolate which
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exhibited a nucleotide identity of 78.3%% compared to other USUV strains [17,25]. Comparison of
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untranslated regions (UTR) showed that 5’ UTR conserves a similar size and secondary structure
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among different lineages, with the exception of Africa 1 lineage. At the same time, specific
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nucleotide mutations are seen in 5’ UTR between African and European lineages (A3T, T4C, C10T,
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and T14C). In 3’ UTR, highly variable size heterogeneity is observed among different lineages [16].
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Comparative analysis between USUV genomes revealed specific amino acidic mutations related to
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geographical source of isolation and hosts. Geographic-specific mutations commonly to all African
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lineages are found in Capsid (A120V) and NS4B (M16I) proteins [16,17]. Moreover, host-specific
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mutations were observed in birds (prM, Y120N) and mosquitoes (Envelope, G195R). In humans,
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unique amino acid mutations are found in CAR-1981 strain (NS2A, S154L; NS3 Y474H; NS5,
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H173Q), the USUV strain isolated from African patient with and rash [17], and Bologna/09 strain
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(Envelope, S302G; NS5, D896E) USUV strain isolated from patient with meningoencephalitis in
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Italy [57]. In particular, specific mutations (E-S302G and NS5-D896E) in Bologna/09 strains have
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been hypothesized to be related to altered tropism to human neurological cells and neuroinvasive
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capacity of USUV strains [57].
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Recently, host-specific amino acid mutations in Envelope glycoprotein (E55A, N103K, V206E)
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was found in a USUV strain detected in a healthy blood donor from Germany [59].
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Up to now, different cases of USUV infection in humans have been reported both in African and
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European countries. USUV infection in humans may be asymptomatic, with no signs or symptoms
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of infection, or may be associated to a wide range of symptoms [9,10,59-62]. A total of 21 human
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cases of USUV infection have been reported until today (Table 1). Symptoms vary from moderate
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(i.e. rash, fever, headache) to severe signs of infection (i.e. neurological disorders) [10-12]. The first
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case of USUV infection in humans has been reported in Central African Republic at the beginning
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of the 1980s. The second case was identified in Burkina Faso in 2004. These two patients presented
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moderate symptoms that included fever, rash and jaundice [10]. In 2009, the first cases of
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meningoencephalitis due to USUV were reported in two immune-compromised subjects in Italy.
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The first case occurred in a patient with diffuse large B cell lymphoma presenting fever and
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neurological symptoms, while second case occurred in a patient that underwent an orthotropic liver
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transplant (OLT) [11,12]. In this last case, USUV was detected from blood that was tested positive
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by nucleic acid amplification technology for WNV [63]. Subsequently, USUV was found in
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cerebrospinal fluid from three patients with acute meningoencephalitis collected between 2008 and
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2009 [64]. Recent retrospective study conducted in the municipality of Modena in the Emilia-
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Romagna region (Italy) documented past USUV infection in patients with suspected viral
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encephalitis or meningoencephalitis during the period comprised between 2008-2009 [13]. In 2013,
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USUV-related neuroinvasive infection has been documented in three patients in Croatia [14].
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Recently, USUV infection has been described in a healthy blood donor tested positive for WNV by
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nucleic acid amplification technique (NAT) in Germany [59].
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Seroepidemiological studies showed that USUV antibodies have been detected in human serum
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samples collected from Germany, Italy and Serbia [13,59,61,62,65]. Seroprevalence of USUV-
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specific antibodies in healthy blood donors varied from 0.02% to 1.1% among different countries
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(Table 1).
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2.1 Diagnosis of USUV infection in humans
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USUV infection can be diagnosed by detection of specific antibodies, viral RNA genome and/or by
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virus isolation in cell culture. So far, no validated commercial serological and molecular assays are
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available. Antibody detection relies on home-made enzyme-linked immunoassay (ELISA) and/or
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immunofluorescence tests based on USUV antigens [60]; any positive result identified using these
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methods must be confirmed by more specific tests, such as plaque reduction neutralization test
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(PRNT) or microneutralization assay (mNTA), to rule out cross-reactivity with antibodies against
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closely related flaviviruses (e.g. WNV). Seroconversion, fourfold or greater increase in virus-
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specific antibody titers in paired samples taken at an interval of 10-15 days should be demonstrated
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for human case confirmation.
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The direct diagnosis of acute USUV infection is based on the detection of USUV RNA in clinical
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specimens (blood and cerebrospinal fluid) by nucleic acid amplification methods. Several in-house
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methods are described that can either identify USUV by specific real-time RT-PCR assays [64,66]
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or broadly detect flaviviruses by nested RT-PCR methods that target highly conserved genome
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sequences among flaviviruses [67], followed by virus identification by sequencing of the
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amplicons; this approach permits subsequent phylogenetic analysis to classify circulating viral
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strains. Furthermore, molecular tests designed for WNV RNA screening in blood and organ donors,
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can amplify USUV genome due a lack of specificity of the assays [58,62].
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3. Conclusion After the discovery of USUV in the 1959, only few studies have been performed to characterize and
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investigate the circulation of this pathogen. Since early 2000s, however, the first description of
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USUV-associated high mortality in several resident birds in Europe and the subsequent evidence of
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the zoonotic potential of USUV infection with the reports of the first cases of human infections in
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two immunocompromised patients in Italy in 2009, contributed to increase awareness and attention
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on this viral infection.
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Currently, USUV results endemic in several European countries and diffusion of this pathogen to
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new geographic areas is continuously observed. These observations highlight an increasing need to
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implement and reinforce veterinary and entomological surveillance plans, resembling those for
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West Nile virus, in European countries to gain a better understanding of the geographical
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distribution, ecology, epidemiology, and genetic diversity of this virus.
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Human infections are considered sporadic; the limited availability of diagnostic tests, the high
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serological cross-reactivity together with co-circulation with closely related flaviviruses (e.g. WNV,
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TBEV) may have contributed to an under-recognition of USUV infections in humans. However, in
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the last years, an increasing number of reports about USUV human infections, especially in those
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country with active surveillance plan for WNV, let hypothesize a potential role of USUV as human
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pathogen.
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In humans, USUV RNA was found in several types of samples, including cerebrospinal fluid and
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blood, from patients with severe neurological syndromes [11-14] who presented with an
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immunocompromised status and/or with underlying chronic diseases. However, in none of these
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patients the seroconversion was demonstrated nor was possible to perform a serologic follow-up
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because of the retrospective design of the studies. Furthermore, the finding of an acute and
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asymptomatic USUV infection in a blood donor in Germany suggests a potential risk of
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transfusion-associated transmission of this infection. Based on these evidences, in countries where
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USUV circulates, human infection may be suspected in patients with febrile illness and
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neurological diseases of unknown aetiologies.
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In the last years, different studies have been prompted to investigate the development of vaccine
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that could protect against USUV infections [68-69]. Although vaccination may be considered as an
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effective strategy to prevent USUV infection, the limited number of human cases suggest that there
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is no an urgent need for the development of a vaccine against USUV for humans.
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In conclusion, a more complete knowledge of the kinetics of USUV infection in humans, i.e.
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duration of viraemia and time for detection of specific host immune response, should be mandatory
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to understand the pathogenic role of USUV in humans.
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Conflict of interest
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There is no conflict of interest.
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Acknowledgements
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This work was partially supported by the Emilia-Romagna region
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West Nile Virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September
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2016. Euro Surveill 2017;22: pii:30501
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453
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454 455
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PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene
457
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458
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460
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Cell and T cell epitopes for potential vaccine development. Scand J Immunol 2017;85:350-
464
64.
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Table 1. Human cases of USUV infection. Country, year, comorbidities and clinical characteristics of human USUV infections (upper part) and
Human cases
Year
Samples
Clinical presentation
Central African Republic
1
1981
Blood
Fever and rash
Burkina Faso
1
2004
Blood
Italy
1
2009
Italy
1
Italy
3
Comorbidities
Diagnosis
Reference
-
Virus isolation
[10]
Fever and jaundice
-
Virus isolation
[10]
Cerebrospinal fluid
Meningoencephalitits
large B cell lymphoma
Molecular (PCR, Sequencing)
[12]
2009
Blood
Encephalitis
thrombotic thrombocytopenic purpura / fulminant hepatitis
Molecular (PCR, Sequencing, NAT for WNV); Virus isolation
[11,62]
20082009
Cerebrospinal fluid
-
Molecular (PCR, Sequencing)
[64]
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No. Cases
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seroprevalence in healthy blood donors and sick individuals (bottom part).
Meningoencephalitis
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8
20082009
Cerebrospinal fluid
Encephalitis/Meningoencephalitis
liver disease / diabetes/ hypertension / mitral insufficiency
Molecular (PCR, Sequencing)
[13]
Italy
2
2009
Blood
Unknown
Healthy/ oncology / respiratory and infectious diseases
[13]
Croatia
3
2013
Blood
Meningitis/Meningoencephalitis
arterial hypertension, hyperlipidemia, hypertensive cardiomyopathy, permanent atrial fibrillation, coronary heart disease, diabetes mellitus
Molecular (PCR, Sequencing) Serology (ELISA, MNTA)
Germany
1
2016
Blood
None
-
Molecular (PCR, Sequencing, NAT for WNV); Serology (IFA)
[14]
[58]
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Italy
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Seroprevalence
Country
No. Cases
Year
Population
Seroprevalence
Serology results
Reference
Italy
4
2009
359§
1.1%
ELISA, MNTA
[60]
Italy
40
2008-
609°
(6.5%)°
MNTA
[13]
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2011 20102011
6000§
0.23%
Germany
1
2012
4200§
0.02%
Serbia
7
2015
93*
(7.5%)*
ELISA, MNTA
RI PT
14
SC
Italy
[61]
ELISA, IFA, MNTA
[62]
ELISA
[65]
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Abbreviations: NAT, nucleic acid amplification technique; ELISA, Enzyme-Linked Immunosorbent Assay; MNTA, micro-neutralization titre assay; IFA, immunofluorescent assay. ° Healthy and sick subjects § Healthy blood donors
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* Healthy individuals with high-estimated risk of mosquito-borne or tick-borne infections
S1286-4579(17)30085-0
DOI:
10.1016/j.micinf.2017.05.003
Reference:
MICINF 4473
To appear in:
Microbes and Infection
Received Date: 18 April 2017 Revised Date:
26 May 2017
Accepted Date: 29 May 2017
Please cite this article as: P. Gaibani, G. Rossini, An overview of Usutu virus, Microbes and Infection (2017), doi: 10.1016/j.micinf.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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An overview of Usutu virus
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Paolo Gaibani* and Giada Rossini
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Operative Unit of Clinical Microbiology, Regional Reference Centre for Microbiological
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Emergencies (CRREM), S. Orsola-Malpighi University Hospital, Bologna, Italy
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*
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Paolo Gaibani, PhD
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Operative Unit of Microbiology, S.Orsola-Malpighi University Hospital, Regional Reference
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Centre for Microbiological Emergencies (CRREM), 9 via G. Massarenti – 40138 Bologna,
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ITALY.
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Telephone: +39 051 6364316
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Fax: +39 051 6363076
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e-mail: [email protected]
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Corresponding Author:
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Usutu virus (USUV) is a mosquito-borne flavivirus that emerged in Africa in the middle of the 16th
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century and currently widely circulates in several European countries. Herein, we summarize
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current knowledge about USUV from ecology, epidemiology, phylogeny to clinical manifestations
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and diagnosis and discuss the role as human pathogen.
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Keywords: Usutu virus; Structure; Epidemiology; Ecology; Phylogeny; Human infection
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1. Introduction Usutu virus (USUV) is an arthropod-borne virus (arbovirus) belonging to the genus Flavivirus
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within the Flaviviridae family. As a member of the Japanese encephalitis virus (JEV) antigenic
56
complex, USUV is closely related to numerous human and animal pathogens including West Nile
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virus (WNV), Murray Valley Encephalitis virus (MVEV), and St Louis encephalitis virus (SLEV)
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[1]. USUV is maintained in the environment through a typical enzootic cycle involving mosquitoes
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and birds. Since first identification in South Africa in the middle of 20th century, widespread
60
circulation of USUV was observed in several countries [2]. In Europe, USUV emerged in 1996
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causing high numbers of bird deaths [3]. Five years later, USUV was responsible of high mortality
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rate among Eurosian Blackbirds (Turdus merula) in the surrounding area of Vienna, Austria [4].
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Currently, USUV is endemic in several countries in Europe [5-8].
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The clinical relevance of USUV as human pathogen has been hypothesized since the first
65
descriptions of USUV-related infection in humans [9]. The first report of USUV infection in
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humans was described in Africa at the beginning of 1980s [10]. Thirty years later, two cases of
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USUV-related neuro-invasive diseases were reported in immune-compromised patients in Europe
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(Italy) [11,12]. Although different human cases of USUV infection have been reported until today
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[13,14], the effective role of the USUV as a human pathogen has yet to be clarified.
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The present review summarizes the up to date knowledge on USUV with particular focus on the
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structure, ecology, epidemiology, genetic diversity, as well as current understanding about human
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infection.
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1.1 Virus genome and structure
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USUV is a small and spherical virus with a lipid envelope derived from host cell membrane. The
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virion is 40–60 nm in diameter and contains a positive-sense RNA genome of 11 Kb in length with
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no 3′ poly(A) tail. Genomic organization shows a similar structure comparable to others flavivirus
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[15]. The genome consists of a single-stranded RNA genome with a 5’ cap structure, a unique open
79
reading frame (ORF) and two untranslated regions (UTRs). The 5’ and 3’ UTRs varied respectively
80
between 95 to 96 nt and 631 to 664 nt in length among different strains [16]. The UTRs are
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involved for the translation and replication of the viral genome. The predicted ORF is translated in a
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unique polyprotein of 3434 amino acids that is post-translationally processed into three structural
83
(capsid, envelope, and pre-membrane) and eight non-structural proteins (NS1, NS2A, NS2B, NS3,
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NS4A, 2K, NS4B, and NS5). Like other mosquito-borne flavivirus, genes encoding the structural
85
proteins are located on the 5’ end of viral genome and form the virion particle [17,18]. The capsid
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protein (C) forms the central core of the virion and is associated to the viral RNA. The envelope
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glicoprotein (E) mediates binding to the host cells and promotes viral entry into the host cells. The
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pre-membrane protein (prM) are necessary for virion assembly and maturation by assisting
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envelope folding [19,20]. The nonstructural proteins serve to different functions during infection
90
and their functions are deduced on the basis of the similarity with other flavivirus genomes [21].
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NS1 exists in distinct forms (i.e. cellular and secreted) and is necessary on the replication of viral
92
genome and virion maturation [22]. The NS2A, NS2B, NS4A and NS4B are small, hydrophobic
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proteins that are required for virus assembly and play a role in the inhibition of the IFN response
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[2]. NS3 and NS5 are two proteins with different enzymatic activities: NS3 protein encodes for
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viral serine protease (active only with NS2B cofactor), helicase, nucleoside triphosphatase and
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RNA triphosphatase. NS5 protein encodes for a methyltransferase (MTase) at the N-terminal, while
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C-terminal encodes for the RNA-dependent RNA polymerase [2,23].
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1.2 Hosts, vectors and life cycle
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The natural life cycle of USUV involves ornithophilic mosquitoes as vector and birds as main
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amplifying host. USUV cycle is similar to that of other flaviviruses belonging to the JEV
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serocomplex [18], where humans are considered as incidental or “dead-end” hosts. Birds of
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different species may be infected by USUV and some of these may develop symptoms of disease.
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USUV has been detected in 62 bird species in African and European countries [10,24]. Different
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migratory wild species (i.e. Falco tinnunculus, Acrocephalus scirpaceus, Sylvia curruca, Sylvia
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communis and Ficedula hypoleuca) are considered responsible for USUV introduction in Europe
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from Africa [10], while others (i.e. Pica pica, Passer domesticus, Gallus gallus and Turdus merula)
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are considered responsible of USUV dissemination through Europe [24]. Interestingly, USUV
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caused high mortality among several bird species (especially Turdus merula) in Europe [4], while in
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Africa and Spain was not associated to high death in birds [25]. Birds may show symptoms varying
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from mild to severe signs of infection [26]. Severe symptoms due to USUV infection in birds
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included encephalitis, myocardial degeneration, and necrosis of the liver and spleen [27,28]
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USUV has been isolated from different mosquito species in several countries. Up to now, USUV
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has been found in eight species belonging to the genus Aedes, Anopheles, Culex, Culiseta,
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Ochlerotatus, Coquillettidia and Mansonia [24]. Although USUV has been found in several
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species, Culex pipiens is the main common vector for USUV [5,29], while Cx. neavei has been
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hypothesized to be involved in sylvatic transmission of USUV in Africa [30].
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Previous studies demonstrated that USUV has been not found in tick species, [31]. USUV infection
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has been demonstrated in different mammal species. In particular, USUV infection has been
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documented in bats (Pipistrellus pipistrellus), horses, dogs and red deer (Cervus elaphus) [32-35].
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1.3 Origin and epidemiology
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A recent study by Engel et al. hypothesized that USUV emerged in Africa at the beginning of 16th
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century and until middle of 20th century, USUV spreading remained exclusively limited to African
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continent where it was detected in different bird and mosquito species in several countries [10,16].
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Introductions of USUV in Europe from Africa probably started in the last 50 years and continue to
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occur. It has been hypothesized that USUV was firstly introduced in Europe by birds between 1950
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and the 1960’s in Spain, while second and third introductions occurred between 1970-1980 (Italy,
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Austria) and 1984-2006 (Spain) [16].
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The earliest evidence of USUV in Europe emerged in 2001 in Austria, associated to deaths in
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several species of resident birds [4]. However, retrospective analysis of archived tissue samples
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from dead Eurasian blackbird in Tuscany, Italy [3] provided evidence that USUV emerged in a
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pathogenic form in Europe in 1996 or earlier. In the following years, USUV was found in
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mosquitoes, birds and bats in several other European countries: Hungary [36], Switzerland [37],
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Spain [38], Italy [27], Czech Republic [39], Germany [40], Belgium [41] and France (2015) [42].
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Moreover, USUV infection has been demonstrated only serologically in birds in England [43],
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Poland [44] and Greece [45] and in a horse in Serbia [46]. USUV was found recurrently over
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several years in Austria (2001–2006) [47], Hungary (2003–2006) [36], Italy (2009–2016) [27,48-
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51], Spain (2006, 2009, 2012) [9,38,52], and Germany (2010-2015) [53,54] suggesting a
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persistence of the transmission cycle in the affected areas through overwintering mosquitoes or
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multiple reintroduction of the virus.
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During summer 2016, a large USUV epizootic was registered in Europe with widespread activity in
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Belgium, Germany, France and for the first time in Netherlands [55,56] highlighting the continuous
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geographical spread of USUV, also to new ecological niches.
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Frequently, USUV co-circulates with West Nile virus (WNV) in many European countries, in terms
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of geographic range of transmission, host and vector species. Considering the immunological cross-
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reactions between these two viruses, it remains to be clarified if such overlapping can influence the
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spatio-temporal patterns of virus circulation in Europe [24].
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1.4 Genetic variation and phylogeny
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USUV is classified in eight lineages which are distinct in two major groups, African and European,
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based on geographical origin of isolation [16]. Phylogenetic analysis based on the NS5 gene
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demonstrated that USUV strains from Africa belong to three distinct lineages (Africa 1 to 3), while
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European group is composed by five different lineages (Europa 1 to 5) [55].
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The lineage Africa 1 includes only one strain (CAR-1969 strain [GenBank accession no.
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KC754958]) isolated in the Central African Republic in 1969 [17]. The lineage Africa 2, originated
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in South Africa in the middle of 1940s, comprises USUV strains isolated in Senegal, Spain,
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Germany and France. The lineage Africa 3 includes strains isolated in Senegal, Germany,
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Netherlands, Belgium and a human isolate from Central African Republic isolated in 1981 (CAR-
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1981 [KC754955]). Europe 1 lineage originated from an ancestor that existed in Senegal and
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consists of strains isolated from Austria, Hungary, Switzerland, Senegal and recently humans
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isolates from Italy [13,16]. Europe 2 lineage consists of strains from Italy and Czech Republic
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including Bologna-2009 strain isolated from patient with meningo-encephalitis [HM569263] [57].
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The lineage Europe 3 includes USUV strains from Germany, Belgium and France, while Europe 4
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consists of USUV strains from Italy, including human isolates [13,55,58]. The recently designated
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Europe 5 lineage includes different strains all isolated from Germany [55].
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The genome identity among isolates is higher than 94%, with exception of CAR-1969 isolate which
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exhibited a nucleotide identity of 78.3%% compared to other USUV strains [17,25]. Comparison of
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untranslated regions (UTR) showed that 5’ UTR conserves a similar size and secondary structure
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among different lineages, with the exception of Africa 1 lineage. At the same time, specific
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nucleotide mutations are seen in 5’ UTR between African and European lineages (A3T, T4C, C10T,
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and T14C). In 3’ UTR, highly variable size heterogeneity is observed among different lineages [16].
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Comparative analysis between USUV genomes revealed specific amino acidic mutations related to
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geographical source of isolation and hosts. Geographic-specific mutations commonly to all African
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lineages are found in Capsid (A120V) and NS4B (M16I) proteins [16,17]. Moreover, host-specific
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mutations were observed in birds (prM, Y120N) and mosquitoes (Envelope, G195R). In humans,
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unique amino acid mutations are found in CAR-1981 strain (NS2A, S154L; NS3 Y474H; NS5,
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H173Q), the USUV strain isolated from African patient with and rash [17], and Bologna/09 strain
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(Envelope, S302G; NS5, D896E) USUV strain isolated from patient with meningoencephalitis in
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Italy [57]. In particular, specific mutations (E-S302G and NS5-D896E) in Bologna/09 strains have
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been hypothesized to be related to altered tropism to human neurological cells and neuroinvasive
182
capacity of USUV strains [57].
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Recently, host-specific amino acid mutations in Envelope glycoprotein (E55A, N103K, V206E)
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was found in a USUV strain detected in a healthy blood donor from Germany [59].
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185 2. USUV infection in humans
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Up to now, different cases of USUV infection in humans have been reported both in African and
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European countries. USUV infection in humans may be asymptomatic, with no signs or symptoms
189
of infection, or may be associated to a wide range of symptoms [9,10,59-62]. A total of 21 human
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cases of USUV infection have been reported until today (Table 1). Symptoms vary from moderate
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(i.e. rash, fever, headache) to severe signs of infection (i.e. neurological disorders) [10-12]. The first
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case of USUV infection in humans has been reported in Central African Republic at the beginning
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of the 1980s. The second case was identified in Burkina Faso in 2004. These two patients presented
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moderate symptoms that included fever, rash and jaundice [10]. In 2009, the first cases of
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meningoencephalitis due to USUV were reported in two immune-compromised subjects in Italy.
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The first case occurred in a patient with diffuse large B cell lymphoma presenting fever and
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neurological symptoms, while second case occurred in a patient that underwent an orthotropic liver
198
transplant (OLT) [11,12]. In this last case, USUV was detected from blood that was tested positive
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by nucleic acid amplification technology for WNV [63]. Subsequently, USUV was found in
200
cerebrospinal fluid from three patients with acute meningoencephalitis collected between 2008 and
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2009 [64]. Recent retrospective study conducted in the municipality of Modena in the Emilia-
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Romagna region (Italy) documented past USUV infection in patients with suspected viral
203
encephalitis or meningoencephalitis during the period comprised between 2008-2009 [13]. In 2013,
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USUV-related neuroinvasive infection has been documented in three patients in Croatia [14].
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Recently, USUV infection has been described in a healthy blood donor tested positive for WNV by
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nucleic acid amplification technique (NAT) in Germany [59].
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Seroepidemiological studies showed that USUV antibodies have been detected in human serum
208
samples collected from Germany, Italy and Serbia [13,59,61,62,65]. Seroprevalence of USUV-
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specific antibodies in healthy blood donors varied from 0.02% to 1.1% among different countries
210
(Table 1).
211 212
2.1 Diagnosis of USUV infection in humans
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USUV infection can be diagnosed by detection of specific antibodies, viral RNA genome and/or by
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virus isolation in cell culture. So far, no validated commercial serological and molecular assays are
215
available. Antibody detection relies on home-made enzyme-linked immunoassay (ELISA) and/or
216
immunofluorescence tests based on USUV antigens [60]; any positive result identified using these
217
methods must be confirmed by more specific tests, such as plaque reduction neutralization test
218
(PRNT) or microneutralization assay (mNTA), to rule out cross-reactivity with antibodies against
219
closely related flaviviruses (e.g. WNV). Seroconversion, fourfold or greater increase in virus-
220
specific antibody titers in paired samples taken at an interval of 10-15 days should be demonstrated
221
for human case confirmation.
222
The direct diagnosis of acute USUV infection is based on the detection of USUV RNA in clinical
223
specimens (blood and cerebrospinal fluid) by nucleic acid amplification methods. Several in-house
224
methods are described that can either identify USUV by specific real-time RT-PCR assays [64,66]
225
or broadly detect flaviviruses by nested RT-PCR methods that target highly conserved genome
226
sequences among flaviviruses [67], followed by virus identification by sequencing of the
227
amplicons; this approach permits subsequent phylogenetic analysis to classify circulating viral
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strains. Furthermore, molecular tests designed for WNV RNA screening in blood and organ donors,
229
can amplify USUV genome due a lack of specificity of the assays [58,62].
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3. Conclusion After the discovery of USUV in the 1959, only few studies have been performed to characterize and
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investigate the circulation of this pathogen. Since early 2000s, however, the first description of
234
USUV-associated high mortality in several resident birds in Europe and the subsequent evidence of
235
the zoonotic potential of USUV infection with the reports of the first cases of human infections in
236
two immunocompromised patients in Italy in 2009, contributed to increase awareness and attention
237
on this viral infection.
238
Currently, USUV results endemic in several European countries and diffusion of this pathogen to
239
new geographic areas is continuously observed. These observations highlight an increasing need to
240
implement and reinforce veterinary and entomological surveillance plans, resembling those for
241
West Nile virus, in European countries to gain a better understanding of the geographical
242
distribution, ecology, epidemiology, and genetic diversity of this virus.
243
Human infections are considered sporadic; the limited availability of diagnostic tests, the high
244
serological cross-reactivity together with co-circulation with closely related flaviviruses (e.g. WNV,
245
TBEV) may have contributed to an under-recognition of USUV infections in humans. However, in
246
the last years, an increasing number of reports about USUV human infections, especially in those
247
country with active surveillance plan for WNV, let hypothesize a potential role of USUV as human
248
pathogen.
249
In humans, USUV RNA was found in several types of samples, including cerebrospinal fluid and
250
blood, from patients with severe neurological syndromes [11-14] who presented with an
251
immunocompromised status and/or with underlying chronic diseases. However, in none of these
252
patients the seroconversion was demonstrated nor was possible to perform a serologic follow-up
253
because of the retrospective design of the studies. Furthermore, the finding of an acute and
254
asymptomatic USUV infection in a blood donor in Germany suggests a potential risk of
255
transfusion-associated transmission of this infection. Based on these evidences, in countries where
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USUV circulates, human infection may be suspected in patients with febrile illness and
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neurological diseases of unknown aetiologies.
258
In the last years, different studies have been prompted to investigate the development of vaccine
259
that could protect against USUV infections [68-69]. Although vaccination may be considered as an
260
effective strategy to prevent USUV infection, the limited number of human cases suggest that there
261
is no an urgent need for the development of a vaccine against USUV for humans.
262
In conclusion, a more complete knowledge of the kinetics of USUV infection in humans, i.e.
263
duration of viraemia and time for detection of specific host immune response, should be mandatory
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to understand the pathogenic role of USUV in humans.
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Conflict of interest
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There is no conflict of interest.
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Acknowledgements
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This work was partially supported by the Emilia-Romagna region
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273 274 275 276 277 278
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Table 1. Human cases of USUV infection. Country, year, comorbidities and clinical characteristics of human USUV infections (upper part) and
Human cases
Year
Samples
Clinical presentation
Central African Republic
1
1981
Blood
Fever and rash
Burkina Faso
1
2004
Blood
Italy
1
2009
Italy
1
Italy
3
Comorbidities
Diagnosis
Reference
-
Virus isolation
[10]
Fever and jaundice
-
Virus isolation
[10]
Cerebrospinal fluid
Meningoencephalitits
large B cell lymphoma
Molecular (PCR, Sequencing)
[12]
2009
Blood
Encephalitis
thrombotic thrombocytopenic purpura / fulminant hepatitis
Molecular (PCR, Sequencing, NAT for WNV); Virus isolation
[11,62]
20082009
Cerebrospinal fluid
-
Molecular (PCR, Sequencing)
[64]
SC
No. Cases
AC C
EP
TE D
M AN U
Country
RI PT
seroprevalence in healthy blood donors and sick individuals (bottom part).
Meningoencephalitis
ACCEPTED MANUSCRIPT
8
20082009
Cerebrospinal fluid
Encephalitis/Meningoencephalitis
liver disease / diabetes/ hypertension / mitral insufficiency
Molecular (PCR, Sequencing)
[13]
Italy
2
2009
Blood
Unknown
Healthy/ oncology / respiratory and infectious diseases
[13]
Croatia
3
2013
Blood
Meningitis/Meningoencephalitis
arterial hypertension, hyperlipidemia, hypertensive cardiomyopathy, permanent atrial fibrillation, coronary heart disease, diabetes mellitus
Molecular (PCR, Sequencing) Serology (ELISA, MNTA)
Germany
1
2016
Blood
None
-
Molecular (PCR, Sequencing, NAT for WNV); Serology (IFA)
[14]
[58]
EP
TE D
M AN U
SC
RI PT
Italy
AC C
Seroprevalence
Country
No. Cases
Year
Population
Seroprevalence
Serology results
Reference
Italy
4
2009
359§
1.1%
ELISA, MNTA
[60]
Italy
40
2008-
609°
(6.5%)°
MNTA
[13]
ACCEPTED MANUSCRIPT
2011 20102011
6000§
0.23%
Germany
1
2012
4200§
0.02%
Serbia
7
2015
93*
(7.5%)*
ELISA, MNTA
RI PT
14
SC
Italy
[61]
ELISA, IFA, MNTA
[62]
ELISA
[65]
M AN U
Abbreviations: NAT, nucleic acid amplification technique; ELISA, Enzyme-Linked Immunosorbent Assay; MNTA, micro-neutralization titre assay; IFA, immunofluorescent assay. ° Healthy and sick subjects § Healthy blood donors
AC C
EP
TE D
* Healthy individuals with high-estimated risk of mosquito-borne or tick-borne infections