West Nile Virus (WNV) is a member of the genus Flavivirus, in the family Flaviviridae, transmitted mainly by mosquito bites [1Murgue B, Murri S, Triki H, Deubel V, Zeller HG. West Nile in the Mediterranean basin: 1950-2000 Ann N Y Acad Sci 2001; 951: 117-26.].

After its first isolation in 1937 from the blood of a febrile patient in the West Nile Province in Uganda [2Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus isolated from the blood of a native of Uganda Am J Trop Med Hyg 1940; 20: 471-92.], WNV has not been regarded as a significant pathogen, because most of the cases were asymptomatic, and humans and animals were considered as accidental dead-end hosts. In the following decades, sporadic outbreaks of WNV infection associated with encephalitis and death have been reported in Israel (1950s) [3Weinberger M, Pitlik SD, Gandacu D, et al. West Nile fever outbreak, Israel, 2000: epidemiologic aspects Emerg Infect Dis 2001; 7: 686-91.], France (1962-1963) [1Murgue B, Murri S, Triki H, Deubel V, Zeller HG. West Nile in the Mediterranean basin: 1950-2000 Ann N Y Acad Sci 2001; 951: 117-26.], South Africa (1974) and India (1980/1981) [4George S, Gourie-Devi M, Rao JA, Prasad SR, Pavri KM. Isolation of West Nile Fever from the brains of children who had died of encephalitis Bull Word Health Org 1984; 62: 879-2.], but only after the severe human outbreak occurring in Romania in 1996 [5Hubálek Z, Halouzka J. West Nile fever - a reemerging mosquito-borne viral disease in Europe Emerg Infect Dis 1999; 5: 643-50.], WNV infection has become a major public health and veterinarian concern in Europe and in the Mediterranean basin.

In contrast with the picture observed in Europe, with occasional incursions and localized activity in areas with favorable conditions, a very different pattern was seen in the Western Hemisphere, where WNV unexpectedly emerged in 1999 in the district of New York, with encephalitis reported in humans and horses [6Jia XY, Briese T, Jordan I, et al. Genetic analysis of West Nile New York 1999 encephalitis virus Lancet 1999; 354: 1971-2., 7Lanciotti RS, Roehrig JT, Deubel V, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999; 286: 2333-7.]. In the following years, the virus was actor of a considerable spread across the United States, Canada, Central and South America. Until now, WNV activity has been reported in humans, birds, animals or mosquitoes from all states except Hawaii, Alaska, and Oregon, so that WNV can be considered one of the most widely distributed of the flaviviruses.

The WNV capability to cause either limited outbreaks or wide epidemics seems to be related to the genetic characteristics of different viruses. All of the major outbreaks of human encephalitis have been associated with WNV strains genetically classified as lineage 1, establishing an important correlation between viral genetics and disease phenotype. The WNV transmission and maintenance are strictly related to the presence of competent vectors (ornithophilic mosquito species) and avian hosts that maintain sufficient viremia for the infection of subsequent mosquitoes. Also other animal species show the capability to develop sufficient viremia for infection of mosquito vectors, but the role that these hosts play in the transmission dynamics of WNV is still unclear, albeit of significant interest. Humans and equines normally fail to generate sufficiently high viremic titers for the infection of mosquito hosts and are considered ‘‘dead end’’ hosts.

The aim of this review is to discuss virus characteristics, virus interaction with vectors and hosts, including the immune response, at the light of the potential spread and endemicity of WNV in EU.

West Nile virus is a mosquito-transmitted Flavivirus. The genus Flavivirus consists of nearly 80 different viruses, many of which are arthropod-borne human pathogens [8Lindenbach BD, Rice CM, Knipe DM, Howley PM, Eds. Flaviviridae: the viruses and their replication Fields Virology. 4th. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins 2001; 1: pp. 991-1041.]. WNV is a member of the Japanese encephalitis (JE) complex that includes St. Louis encephalitis (SLE), Murray Valley encephalitis (MVE), and the Kunjin (KUN) viruses [9Deubel V, Fiette L, Gounon P, et al. Variations in biological features of West Nile viruses Ann N Y Acad Sci 2001; 951: 195-206.], in the family Flaviviridae.

WNV is a spherical particle of approximately 50 nm in diameter: the lipid bilayer membrane, derived from the host cell, surrounds a nucleocapsid core containing a single stranded RNA genome of about 11 kilobases in length flanked by 5’- and 3’-untranslated regions, which have secondary structures necessary for the initiation of translation and for replication [8Lindenbach BD, Rice CM, Knipe DM, Howley PM, Eds. Flaviviridae: the viruses and their replication Fields Virology. 4th. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins 2001; 1: pp. 991-1041.]. The genome is packaged with the viral capsid protein (C) into a host-derived lipid bilayer in which 180 copies of the envelope protein (E) are embedded [10Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspective of the flavivirus life cycle Nat Rev Microbiol 2005; 3: 13-22.]. The RNA genome carries a 5’ cap at the 5’ end, and lacks a polyadenylated tail at the 3’ end. The genomic RNA corresponds to the messenger RNA for the translation of a single long open reading frame into one large polyprotein that is processed co- and post-translationally, by a virally encoded serine protease and multiple host proteinases, into three viral structural proteins (C, pre-M and E) and seven non-structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Surrounding the ORF are 5’ and 3’ non coding regions (NCRs) of around 100 nucleotides (nt) and 400-700 nt respectively [8Lindenbach BD, Rice CM, Knipe DM, Howley PM, Eds. Flaviviridae: the viruses and their replication Fields Virology. 4th. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins 2001; 1: pp. 991-1041.]. Understanding of the viral replication cycle is still far from being complete. Virions bind and enter cells via receptor-mediated endocytosis specific for viral envelope proteins [8Lindenbach BD, Rice CM, Knipe DM, Howley PM, Eds. Flaviviridae: the viruses and their replication Fields Virology. 4th. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins 2001; 1: pp. 991-1041.]. A structural reorganization of the E protein delivers the nucleocapsid and viral RNA into the cytoplasm where the genome is translated [11Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery Antiviral Res 2009; 81: 6-15.]. Viral proteins are processed from the single polyprotein of approximately 3000 amino acids by cellular and viral proteases. Following RNA replication within the cytoplasmic replication complexes, progeny virions assemble by budding through intracellular membranes of the rough endoplasmic reticulum. Once transported through the host secretory pathway, the mature virions are released into the cytoplasm, fused with the plasmatic membrane and finally are released by exocytosis into the extracellular compartment [8Lindenbach BD, Rice CM, Knipe DM, Howley PM, Eds. Flaviviridae: the viruses and their replication Fields Virology. 4th. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins 2001; 1: pp. 991-1041., 11Sampath A, Padmanabhan R. Molecular targets for flavivirus drug discovery Antiviral Res 2009; 81: 6-15.].

Studies of the phylogenetic relatedness on nucleic acid sequence data corresponding to a 255-bp region of the E glycoprotein gene of WNV strains isolated in different geographic regions, demonstrated that WNV isolates fall into two major genetic lineages diverging by 25 to 30% nucleotide differences [12Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses J Gen Virol 1997; 78: 2293-7., 13Lanciotti RS, Ebel GD, Deubel V, et al. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe and the Middle East Virology 2002; 298: 96-105.] and several subclades or clusters [7Lanciotti RS, Roehrig JT, Deubel V, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999; 286: 2333-7., 12Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses J Gen Virol 1997; 78: 2293-7., 14Charrel RN, Brault AC, Gallian P, et al. Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe Virology 2003; 315: 381-8.-16Scherret JH, Poidinger M, Mackenzie JS, et al. The relationships between West Nile and Kunjin viruses Emerg Infect Dis 2001; 7: 697-705.]. Lineage 1 is composed of WNV strains with a broad geographical distribution ranging from West Africa to the Middle East, Eastern Europe, North America, and Australia. This latter lineage includes viral strains with great nucleotide sequence homology, which account for the recent outbreaks in human, horses and birds. Lineage 1 can be further subdivided into at least three more clades. Clade 1a contains strains from Europe, Middle East, Asia, Africa and America, and presents different clusters including: (i) the most recent Israeli/American virus strains collected between 1997 and 2000; (ii) the strains isolated in Europe and Russia between 1996 and 2000 (Romania/1996-7, Russia 1999, and France/2000) [14Charrel RN, Brault AC, Gallian P, et al. Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe Virology 2003; 315: 381-8.], and (iii) the two closely related Italian 1998 and 2008 WNV strains [17Monaco F, Lelli R, Teodori L, et al. Re-Emergence of West Nile Virus in Italy Zoonoses Public Health 2009. [Epub ahead of print]]. A human WNV strain isolated in Italy in 2009 from an asymptomatic individual resident in Rovigo province also belongs to lineage 1, clade 1a, and is closely related to the two WNV strains isolated from magpies in Italy in 2008 [18Barzon L, Squarzon L, Cattai M, et al. West Nile virus infection in Veneto region, Italy, 2008-2009 Euro Surveill 2009; 14: pii=19289. Available from: http://www.eurosurveillance.org/View Article. aspx?ArticleId=19289]. In addition to those mentioned above, two more clusters have been identified, one including the strains isolated in the 1980s in Central African Republic, the other including the Egyptian strain of 1951 and the Romanian-1996 human strain.

Within clade 1b, highly homologous strains isolated in Australia from 1960 to 1994 [16Scherret JH, Poidinger M, Mackenzie JS, et al. The relationships between West Nile and Kunjin viruses Emerg Infect Dis 2001; 7: 697-705., 19Zeller HG, Schuffenecker I. West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas Eur J Clin Microbiol Infect Dis 2004; 23: 147-56.] can be grouped. This clade also includes the Australian (Kunjin) strains isolated from 1960 to 1994 [16Scherret JH, Poidinger M, Mackenzie JS, et al. The relationships between West Nile and Kunjin viruses Emerg Infect Dis 2001; 7: 697-705.], that can be distinguished from other lineage 1 WNV viruses by monoclonal antibody binding and cross-neutralization analysis [7Lanciotti RS, Roehrig JT, Deubel V, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999; 286: 2333-7., 15Savage HM, Ceianu C, Nicolescu G, et al. Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serological and molecular characterization of a virus isolate from mosquitoes Am J Trop Med Hyg 1999; 61: 600-11.]. According to Lanciotti et al. [13Lanciotti RS, Ebel GD, Deubel V, et al. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe and the Middle East Virology 2002; 298: 96-105.], clade 1c contains Indian WNV isolates collected between 1955 and 1980, but this finding was not confirmed in following studies [14Charrel RN, Brault AC, Gallian P, et al. Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa, the Middle East, and Europe Virology 2003; 315: 381-8.]. These viruses can also be distinguished from other lineage 1 viruses by serological assays [20Umrigar MD, Pavri KM. Comparative biological studies on Indian strains of West Nile virus isolated from different sources Indian J Med Res 1977; 65: 596-602.].

WNV strains from the northeastern United States and Israel are closely related, sharing 99.7% nucleotide sequence homology and grouping in a unique subclade within clade 1a. This high degree of sequence identity between US and Israel WNV strains support the hypothesis that the US WNV strains originated from the Middle East [7Lanciotti RS, Roehrig JT, Deubel V, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999; 286: 2333-7.].

Lineage 2 contains the B 956 prototype strain and other strains isolated exclusively in the sub-Saharan Africa and Madagascar [7Lanciotti RS, Roehrig JT, Deubel V, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999; 286: 2333-7., 12Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses J Gen Virol 1997; 78: 2293-7., 15Savage HM, Ceianu C, Nicolescu G, et al. Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serological and molecular characterization of a virus isolate from mosquitoes Am J Trop Med Hyg 1999; 61: 600-11.].

Recently, additional lineages have been proposed for viruses that exhibit considerable genetic differences from the two already existing [21Lvov DK, Butenko AM, Gromashevsky VL, et al. West Nile virus and other zoonotic viruses in Russia: examples of emerging-reemerging situations Arch Virol Suppl 2004; 18: 85-96.]: lineage 3 including a virus strain (Rabensburg virus) isolated in central Europe (Czech Republic), lineage 4 enclosing a unique virus isolated in the Caucasus [22Bakonyi T, Hubalek Z, Rudolf I, Nowotny N. Novel flavivirus or new lineage of West Nile virus, central Europe Emerg Infect Dis 2005; 11: 225-31.]. These last two viruses, however, may also be considered independent flaviviruses within the Japanese encephalitis group [22Bakonyi T, Hubalek Z, Rudolf I, Nowotny N. Novel flavivirus or new lineage of West Nile virus, central Europe Emerg Infect Dis 2005; 11: 225-31.]. Finally, a fifth lineage was suggested for a West Nile virus strain isolated in India [23Bondre VP, Jadi RS, Mishra AC, Yergolkar PN, Arankalle VA. West Nile virus isolates from India: evidence for a distinct genetic lineage J Gen Virol 2007; 88: 875-4.]. Grouping of isolates based on phylogenetic analysis does not correlate with the geographical distribution of the virus, demonstrating the fundamental importance of migrating birds in viral spread dynamics [12Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses J Gen Virol 1997; 78: 2293-7.].

The recent outbreaks associated with severe humans and avian diseases have all been caused by a subset of strains in lineage 1. In contrast, lineage 2 strains appear to be less virulent for humans, as they have been isolated from asymptomatic or mild cases or even during search for other pathogens [13Lanciotti RS, Ebel GD, Deubel V, et al. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe and the Middle East Virology 2002; 298: 96-105.].

The large epidemics and epizootics occurred in the US have considerably contributed to the improvement in WNV diagnosis in both humans and animals, focusing in the detection of either viruses or anti-WNV antibodies (Ab). Owing to the following enhanced surveillance programs, large collections of human and animal sera have been accumulated. The main difficulties in WN disease diagnosis are the requirement for level 3 safety labs, the necessity for a multi-species testing and for specific diagnosis that can overcome the cross-reactivity with other flaviviruses [24Dauphin G, Zientara S. West Nile virus: recent trends in diagnosis and vaccine development Vaccine 2007; 25: 5563-76.].

The election method for WNV detection in vertebrate, mosquito pools and avian samples remains viral isolation which can be performed from cerebrospinal fluid (CSF), blood or tissues in infected cell cultures, even though this technique requires good quality samples [25Castillo-Olivares J, Wood J. West Nile virus infection of horses Vet Res 2004; 35: 467-83.].

Antibody testing in patients or animal sera is of large usefulness for diagnosing WNV infection, both in the course of large field studies or screening and when samples are taken in absence of or late after symptomatic phase. Serological testing is mainly based on detection of anti-E protein antibodies, although the cross-reactivity of the neutralizing antibodies response against flaviviruses limits the specificity of the tests. This is particularly true in areas where other Flaviviridae are concomitantly spread [26Innis BL, Nisalak A, Nimmannitya S, et al. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate Am J Trop Med Hyg 1989; 40: 418-27., 27Kuno G, Gubler DJ, Oliver A. Use of ‘original antigenic sin’ theory to determine the serotypes of previous dengue infections Trans R Soc Trop Med Hyg 1993; 87: 103-5.]. The plaque reduction neutralization test (PRNT) detecting WNV specific neutralizing antibodies in CSF and serum remains the assay required for confirmation of flavivirus infections and identification of the infecting agent [28Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay J Clin Microbiol 2000; 38: 4066-71.], although paired serum samples are needed to confirm seroconversion.

Infected cell culture supernatants or preparations from WNV infected suckling mouse brains (SMB) are antigens classically used for WNV serodiagnosis. Alternatively, recombinant antigens such as the envelope glycoprotein E [29Herrmann S, Leshem B, Landes S, Rager-Zisman B, Marks RS. Chemiluminescent optical fiber immunosensor for the detection of anti-West Nile virus IgG Talanta 2005; 66: 6-14.], virus-like particles (VLP) [30Holmes DA, Purdy DE, Chao DY, Noga AJ, Chang GJ. Comparative analysis of immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay using virus-like particles or virus-infected mouse brain antigens to detect IgM antibody in sera from patients with evident flaviviral infections J Clin Microbiol 2005; 43: 3227-6.], or the non-structural NS1 [31Hukkanen RR, Liggitt HD, Kelley ST, et al. Comparison of commercially available and novel West Nile virus immunoassays for detection of seroconversion in pig-tailed macaques (Macaca nemestrina) Comp Med 2006; 56: 46-54.], NS3 and NS5 [32Wong SJ, Boyle RH, Demarest VL, et al. Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination J Clin Microbiol 2003; 41: 4217-23.] proteins may presently be produced and purified in large scale for use in different assay formats in the absence of particular containment facilities. The IgM capture enzyme-linked immunosorbent assay (ELISA) on serum or CSF is widely used for diagnosis of WNV in humans in the United States [33Martin DA, Biggerstaff BJ, Allen B, Johnson AJ, Lanciotti RS, Roehrig JT. Use of immunoglobulin M cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States Clin Diagn Lab Immunol 2002; 9: 544-9.]. In addition, a series of immunological tests (from different ELISA formats to Western blotting to Solid-phase dot-ELISA technology) is in use in several laboratories for both human and animal diagnostics worldwide [34Niedrig M, Sonnenberg K, Steinhagen K, Paweska JT. Comparison of ELISA and immunoassays for measurement of IgG and IgM antibody to West Nile virus in human sera against virus neutralisation J Virol Methods 2007; 139: 103-5.-36Loroño-Pino MA, Farfan-Ale JA, Blitvich BJ, Beebe JL, Jarman RG, Beaty BJ. Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the diagnosis of West Nile virus infections in humans Clin Vaccine Immunol 2009; 16: 749-55.]. However, these tests are laborious, time-consuming, and sometimes need to be conducted in level 3 biosafety laboratories [24Dauphin G, Zientara S. West Nile virus: recent trends in diagnosis and vaccine development Vaccine 2007; 25: 5563-76.].

Further exploitation of sensitive and specific assays for prompt differential diagnosis of WNV infection in either humans and animals in the field is still needed and may take advantage of modern knowledge of WNV antigen determinants, recombinant DNA and protein technology, and methods for production of MAbs with different affinity [37Jibin Z, Xiumei L, Hongping W, et al. Production and characterization of monoclonal antibodies to nucleoprotein of Marburg virus Hybridoma 2008; 27: 423-9., 38Deyev SM, Lebedenko EN. Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design Bioessays 2008; 30: 904-18.]. As for other viruses, major expectations are laid in the field of genome testing approaches by conventional, nested or real-time RT-PCR, which are enormously faster than cultivation methods and can be adjusted for use in clinical and epidemiological applications [28Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay J Clin Microbiol 2000; 38: 4066-71., 39Phipps LP, Gough RE, Ceeraz V, Cox WJ, Brown IH. Detection of West Nile virus in the tissues of specific pathogen free chickens and serological response to laboratory infection: a comparative study Avian Pathol 2007; 36: 301-5.-41Moureau G, Temmam S, Gonzalez JP, Charrel RN, Grard G, de Lamballerie X. A real-time RT-PCR method for the universal detection and identification of flaviviruses Vector Borne Zoonotic Dis 2007; 7: 467-77.].

Single-tube real-time RT-PCR, which can be performed on RNA extracted from a wide variety of tissues in all infected animal species, shows many advantages compared to end-point RT-PCR because it is more rapid, often more sensitive, more specific, and minimizes contamination [28Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay J Clin Microbiol 2000; 38: 4066-71.]. In addition, together with an automated RNA extraction, real-time RT-PCR can be used in large-scale surveillance, and allows quantitative measurement of viral nucleic acids [42Ostlund EN, Crom RL, Pedersen DD, Johnson DJ, Williams WO, Schmitt BJ. Equine West Nile encephalitis, United States Emerg Infect Dis 2001; 7: 665-9.]. In several studies, molecular detection assays based on broadly reactive (degenerate) primers may be used for the simultaneous detection and quantification of distinct flaviviruses using species-specific and group-specific primers in a single reaction [40Dyer J, Chisenhall DM, Mores CN. A multiplexed TaqMan assay for the detection of arthropod-borne flaviviruses J Virol Methods 2007; 145: 9-13.] or targeting the flavivirus consensus amplimers located at the RNA-dependent RNA polymerase domain of the NS5 protein [43Chao DY, Davis BS, Chang GJ. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes J Clin Microbiol 2007; 45: 584-9.].

Various approaches for screening of WNV and viral antibodies in blood, body fluids and other tissues have been developed and used as routine screening tools for blood donation and cell tissues/organs transplantations over the last several years [44Zhang W, Wu J, Li Y, Li F, Njoo H. Rapid and accurate in vitro assays for detection of West Nile virus in blood and tissues Transfus Med Rev 2009; 23: 146-54.]. Despite implementation of nucleic acid testing, particularly by real-time RT-PCR, WNV transmission through blood transfusion and organ transplant still occurs, thus remarking sensitivity limits of the present screening assays, particularly on mini-pool samples [45Centers for Disease Control and Prevention. West Nile Virus infections in organ transplant recipients - New York and Pennsylvania, August-September, 2005 Morb Mortal Wkly Rep 2005; 54: 1021-3., 46Centers for Disease Control and Prevention. West Nile virus transmission through blood transfusion - South Dakota, 2006 Morb Mortal Wkly Rep 2007; 56: 76-9.]. Individual donation testing for WNV blood screening is more sensitive, but presents an unfavourable cost-effectiveness [47Stramer SL, Custer B, Busch MP, Dodd RY. Strategies for testing blood donors for West Nile virus Transfusion 2006; 46(12): 2036-7.].

Although more advanced than for other arboviruses, molecular methods for WNV detection still have wide margins for technical implementation to both adapt their use to the variety of clinical samples (tissue, blood or CSF) from humans and animals, and particularly vector tissue extracts [48Naze F, Le Roux K, Schuffenecker I, et al. Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting J Virol Methods 2009; 162: 1-7.-50Ulloa A, Ferguson HH, Méndez-Sánchez JD, et al. West Nile virus activity in mosquitoes and domestic animals in Chiapas, México Vector Borne Zoonotic Dis 2009; 9: 555-60.], and to take into account more recent knowledge from sequence analysis of strains circulating and evolving in various geographical areas and habitats. Problems are also related to the very low viral loads that can be expected especially in post-mortem tissues or while screening subjects in the absence of symptoms. Similar to antigen-antibody based detection assays, efforts are also needed for molecular approaches to overcome the problems posed by the occasionally close similarity of nucleotide sequences between different members of Flaviviridae when a genus-specific diagnosis is required [48Naze F, Le Roux K, Schuffenecker I, et al. Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting J Virol Methods 2009; 162: 1-7., 51Maher-Sturgess SL, Forrester NL, Wayper PJ, et al. Universal primers that amplify RNA from all three flavivirus subgroups Virol J 2008; 5: 16-0., 52Scaramozzino N, Crance J-M, Jouan A, DeBriel DA, Stoll F, Garin D. Comparison of Flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences J Clin Microbiol 2001; 39: 1922-7.]. Although suited for a rapid and sensitive diagnosis at the experienced lab level, molecular methods are hardly usable for local field investigations particularly on outdoor animals or farms, where development of user-friendly antigen-based field test would be desirable.

According to the existing literature, ornithophilic (bird-feeding species) mosquitoes are commonly considered as the principal vectors of West Nile virus [5Hubálek Z, Halouzka J. West Nile fever - a reemerging mosquito-borne viral disease in Europe Emerg Infect Dis 1999; 5: 643-50.]. In particular, the vector species mainly involved in WNV transmission to humans are Culex pipiens Linneus 1758, the widespread common mosquito in the northern hemisphere, and its vicariant species Cx. quinquefasciatus Say 1823, in the southern hemisphere.

Nevertheless, little is known about the factors that may influence the epidemiological cycle of the disease, that may assume different characteristic depending on the type of environment (rural, urban, etc.). WNV is taken up by a competent ornithophilic mosquito vector, during blood feeding on an infected bird [53Rappole JH, Derrickson SR, Hubálek Z. Migratory birds and spread of West Nile virus in the Western Hemisphere Emerg Infect Dis 2000; 6: 319-28.]. After a short period necessary to the replication of the virus, the infected mosquito will spread WNV biting the main hosts (migratory or indigenous birds, which also act as a reservoir), and/or horses or humans, which are “incidental dead-end” hosts [54Campbell GL, Marfin AA, Lanciotti RS, Gubler DJ. West Nile virus Lancet Infect Dis 2002; 2: 519-29.].

Overall, the epidemiological cycle of WNV in temperate areas may be divided in two different moments [55Higgs S, Snow K, Gould EA. The potential for West Nile virus to establish outside of its natural range: a consideration of potential mosquito vectors in the United Kingdom Trans R Soc Trop Med Hyg 2004; 98: 82-7., 56Crook PD, Crowcroft NS, Brown DW. West Nile virus and the threat to the UK Commun Dis Public Health 2002; 5: 138-43.]:

1. A rural/wild cycle, that involves migratory and indigenous birds (such as white storks and pigeons, respectively), and one or more ornithophilic mosquito species such as Culex univittatus (Africa, Middle East), Cx. tarsalis (East Europe), Cx. pipiens restuans (USA) Cx. Modestus (France, Russia), and that is known as “enzootic” (or exoanthropic) cycle. Characteristic environments where the wild cycle commonly occurs are wetlands, river deltas and flooded plains that migratory birds choose for nesting, coming in contact with the potential mosquito vectors breeding in the same area.
2. An urban, synanthropic cycle that involves domestic birds, such as chicken, house sparrows and pigeons, and a mosquito species that feed on both humans and birds, such as Culex pipiens/molestus. This cycle is named “epizootic” [57Campbell GL, Ceianu CS, Savage HM. Epidemic West Nile encephalitis in Romania: waiting for history to repeat itself Ann N Y Acad Sci 2001; 951: 94-101., 58Ceianu CS, Ungureanu A, Nicolescu G, et al. West nile virus surveillance in Romania: 1997-2000 Viral Immunol 2001; 14: 251-62.], and may occurs also in urban areas when human activities make available a number of breeding sites for mosquito populations, as happened in Bucharest, Romania, and in the US [5Hubálek Z, Halouzka J. West Nile fever - a reemerging mosquito-borne viral disease in Europe Emerg Infect Dis 1999; 5: 643-50.-7Lanciotti RS, Roehrig JT, Deubel V, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States Science 1999; 286: 2333-7.].

Depending on the kind of environment, the abundance of mosquito species and on their degree of competence for WNV transmission, two main hypotheses about competence of the vectors have been formulated [59Bernard K, Maffei G, Jones S, et al. West Nile virus infection in birds and mosquitoes, New York State, 2000 Emerg Infect Dis 2001; 7: 679-85.]: (i) in the former, the enzootic cycle is sustained by one or more mosquito species, mainly ornytophilic, which ensure WNV transfer from migratory to indigenous birds, allowing the dissemination and amplification of the virus. Different species, with a broader range of hosts and anthropophagic, will act as a “bridge” vector, between infected birds and horses/humans; (ii) alternatively, a single species able to bite birds as well as mammals may act as both enzootic and epizootic vector.

In both hypotheses, the species with a broad range of host could in fact be Cx. pipiens.

A number of factors are involved in WNV vector competence. Firstly the vector must be genetically susceptible to become infected with WNV and supportive of its replication, and secondly, the vector must be able to transmit it to the natural host. For these reasons, the virus isolation in a field-collected mosquito does not necessarily imply competence to transmit it [60Sardelis MR, Turell MJ, Dohm DJ, O'Guinn ML. Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus Emerg Infect Dis 2001; 7: 1018-22.-65Goddard LB, Roth AE, Reisen WK, Scott TW. Vector competence of California mosquitoes for West Nile virus Emerg Infect Dis 2002; 8: 1385-91.]. Under laboratory conditions, all of common species of the genus Culex may become infected, as well as other species belonging to different genera (i.e. Aedes, Ochlerotatus, Culiseta, Coquillettidia). Up to date, more than 70 species of mosquitoes have been found to be naturally infected with WNV worldwide, but their possible involvement in the epidemiology of the disease is probably negligible in most cases. Some concern is only related to the high competence showed by Ae. albopictus and Oc. japonicus, two species highly anthropophilic which may sustain and amplify the infection in the urban areas [59Bernard K, Maffei G, Jones S, et al. West Nile virus infection in birds and mosquitoes, New York State, 2000 Emerg Infect Dis 2001; 7: 679-85., 61Sardelis MR, Turell MJ, Ochlerotatus J. Japonicus in Frederick County, Maryland: discovery, distribution, and vector competence for West Nile virus J Am Mosq Control Assoc 2001; 17: 137-41.].

In the last years, WNV has been circulating in various European countries. Particularly, in Italy infection has quickly spread at least through five neighboring regions in 2008-2009 [17Monaco F, Lelli R, Teodori L, et al. Re-Emergence of West Nile Virus in Italy Zoonoses Public Health 2009. [Epub ahead of print], 66Rizzo C, Vescio F, Declich S, et al. West Nile virus transmission with human cases in Italy, August - September 2009 Euro Surveill 2009; 14: pii: 19353. Available from: http://www.eurosurveillance. org/ViewArticle.aspx?ArticleId=19353, 67Barzon L, Franchin E, Squarzon L, et al. Genome sequence analysis of the first human West Nile virus isolated in Italy in 2009 Euro Surveill 2009; 14: pii=19384. Available online: http://www. eurosurveillance.org/ViewArticle.aspx?ArticleId=19384]. This outbreak followed the earlier significant outbreak of 1998, occurred in Tuscany only among horses [68Autorino GL, Battisti A, Deubel V, et al. West Nile virus epidemic in horses, Tuscany region, Italy Emerg Infect Dis 2002; 8: 1372-8.].

The entomological inquiries carried out after these events, a 3-year longitudinal survey in Tuscany [69Romi R, Pontuale G, Ciufolini MG, et al. Potential vectors of West Nile Virus following an equine disease outbreak in Italy Med Vet Ent 2004; 18: 14-9.] and the 5-year national surveillance plan, that included 15 wetland sites across the country [70Toma L, Cipriani M, Romi R, Goffredo M, Lelli R. First report on entomological field activity for the surveillance of west Nile disease in Italy Vet Ital 2008; 44: 483-96.], gave univocal results indicating that Culex pipiens was the predominant species in all sites, representing more than 50% of all samples.

It is important to note that, whereas in the tropics WNV transmission may occur all year round, in temperate regions it appears to be limited to the season favorable to the massive development of the vector populations (usually in the late summer). In some cases, it is possible that the potential mosquito vectors become infected in springtime, after biting on migratory birds, and that a few months are necessary for the virus to amplify and spread in a limited area [68Autorino GL, Battisti A, Deubel V, et al. West Nile virus epidemic in horses, Tuscany region, Italy Emerg Infect Dis 2002; 8: 1372-8.]. However, this cannot explain the simultaneous appearance of distinct WNV foci scattered on different regions occurred in Italy in 2008 and 2009. Since infected mosquito may remain able to transmit WNV to other animals even for months after the infected blood meal, it should be considered the possibility that WNF may have overwintered in these temperate regions throughout vertical transmission. This phenomenon has been observed both in the field and in the laboratory, but its real relevance in the epidemiology of WNV is still to be assessed [71Baqar S, Hayes CG, Murphy JR, Watts DM. Vertical transmission of West Nile virus by Culex and Aedes species mosquitoes Am J Trop Med Hyg 1993; 48: 757-62.-75Goddard LB, Roth AE, Reisen WK, Scott TW. Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species J Med Entomol 2003; 40: 743-6.].

Altogether, the findings from Italian investigations indicate the involvement of Cx. pipiens as a major WNV vector both in enzootic and epizootic cycles. Since the late 1930’s [76La Face L, Osservazioni sul C. pipiens autogenicus Rendiconti Ist San Pub 1938; 1: 669-78., 77La Face L. Osservazioni sulla biologia del complesso “Culex pipiens” Parassitologia 1962; 4: 155-8.], the possibility that Cx. pipiens in the Paleartic region consists of a species complex or of different biological forms has been and is still debated. The existence of at least two forms with different characteristics related to different environment adaptation [78Vinogradova EB. Culex pipiens pipiens mosquitoes: taxonomy, distribution, ecology, physiology, genetics, applied importance and control Chp 1. Sofia, Bulgaria: PENSOFT Publishers 2000; pp. 4-45.], namely Cx. pipiens pipiens (the original rural, mainly ornithophilic) and Cx. molestus (the urban form, mainly antrhopophilic), is in fact currently accepted [78Vinogradova EB. Culex pipiens pipiens mosquitoes: taxonomy, distribution, ecology, physiology, genetics, applied importance and control Chp 1. Sofia, Bulgaria: PENSOFT Publishers 2000; pp. 4-45.-82Kent RJ, Harrington LC, Norris DE. Genetic differences between Culex pipiens f.molestus and Culex pipiens pipiens (Diptera: Culicidae) in New York Med Entomol 2007; 44: 50-9.]. Nevertheless the possible existence of further forms, and their role in the transmission of the WNV still need to be deeply investigated.

Experimental DNA vaccines have been developed expressing genes encoding the WNV membrane (M) and envelope (E) proteins in eukaryotic expression vectors, and successfully tested in animals and avian species [132Davis BS, Chang GJ, Cropp B, et al. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays J Virol 2001; 75: 4040-7.-134Chang GJJ, Davis BS, Stringfield C, Lutz C. Prospective immunization of the endangered California condors (Gymnogyps californianus) protects this species from lethal West Nile virus infection Vaccine 2007; 25: 2325-30.].

Recombinant WNV proteins have been used in immunization experiments, and recombinant E protein can be a candidate vaccine to prevent WN virus infection in animals and humans, as the positive result in mice [135Lieberman MM, Clements DE, Ogata S, et al. Preparation and immunogenic properties of a recombinant West Nile subunit vaccine Vaccine 2007; 25: 414-23.] and non human primates suggest [136Lieberman MM, Nerurkar VR, Luo H, et al. Immunogenicity and protective efficacy of a recombinant subunit West Nile virus vaccine in rhesus monkeys Clin Vaccine Immunol 2009; 16: 1332-7.].

Attenuated, inactivated and killed viruses are also protective against infection. Inactivated virus immunization can induce stronger humoral responses compared to the DNA plasmid vaccine, but they can present some limitations. In particular, the attenuated vaccines are economic to produce and potent in elicit immune response, but are not suitable for use in the immunocompromised, while the inactivated vaccines, although safer, are rather expensive to produce and less potent.

Recently, a novel class of vaccines against flaviviruses started are under evaluation. They are based on the production of defective viral particles that are unable to spread between normal cells, and are unable to cause disease in vaccinated animals. A proposed single-cycle, encapsidation defective flavivirus vaccine appears to be reasonably cheap to produce, potent, and safe in the immuno-compromised subjects [137Widman DG, Frolov I, Mason PW. Third-generation flavivirus vaccines based on single-cycle, encapsidation-defective viruses Adv Virus Res 2008; 72: 77-126.].

However, the suitability of vaccine implementation for either human or animal use remains questionable due to the relatively low number and severity of cases worldwide, and the extreme difficulties envisaged in controlling virus spread through the large animal reservoir represented by susceptible avian species.

As the other RNA viruses, WNV has a great potential to evolve and mutate in order to exploit environmental opportunities and become more efficient in spreading into new areas. Indeed, in the last decade, WNV has dramatically increased geographic range and has demonstrated a greater pathogenicity to birds and mammalian hosts, including humans. Virological factors have included genetic adaptation for both increased replication in avian hosts and wider range of competent mosquito vectors. Moreover, emergence of WNV strains capable to disseminate more rapidly and with greater efficiency at elevated temperatures pointed out the potential importance of temperature as a selective criteria for the emergence of WNV genotypes with increased vectorial capacity.

The pathogenicity in avian host of WNV has been related to the higher virus replication and capability of spreading, and mutations that can affect this character increase the fitness of the virus in a new environment.

The ongoing epidemic that since August 2008 is affecting Northern Italy is an event that can be considered as case study for the better understanding of the mechanism on WNV persistence in an environmental niche.

During this epidemic, some important evidences have been collected: i) virus spreading across a wide area and involvement of human and animal hosts; ii) overwintering of WNV and recrudescence of the outbreak in the following summer season and iii) mutation of the virus towards a higher pathogenicity in the avian species.

With regard to overwintering of the virus, this phenomenon is poorly known. WNV infection in overwintering mosquitoes is rare in nature, and the detection of viremic birds in winter season raises the question of the possibility of persistently infected hosts like bird species or other animals.

Many other questions on the potential persistence and spread of WNV in EU are still unsolved, but data so far support the idea that further knowledge on the immune response of the avian hosts, vector fitness and development of control strategies including vaccination can provide better understanding of the kinetics of WNV infection.