Novel West Nile virus (WNV) lineage 1 and 2 strains characterized by high virulence and epidemic potential for humans have emerged in recent years in Central and Eastern European and Mediterranean countries, where epidemics have become increasingly frequent . In Italy, the first human cases of WNV disease were notified in 2008, in north-eastern areas surrounding the Po river delta [2,3], where several human and equine cases were also identified in the following year [4,5]. Genome sequence analysis of WNV strains isolated in Italy in 2008 and 2009 showed that they were closely related [4,6,7], suggesting that the virus had overwintered and established an endemic cycle in north-eastern Italy. In 2010, human cases of WNV disease were reported only in the Veneto Region, in areas located to the north of those affected in the previous years . In this Italian Region, special surveillance programmes for WN neuroinvasive disease (WNND) and WN fever and for imported and autochthonous dengue and chikungunya were activated in 2010, as previously described [8,9]. In this study, we report an update on human cases of WNV infection diagnosed in this region in 2011 and describe two novel WNV lineage 1a full-length genome sequences obtained from human biological samples.
Of the 410 possible cases investigated in the Veneto Region during 2011, eight cases of WNND (five with encephalitis, one with encephalitis and myocarditis, one with meningitis, and one with polyradicoloneuritis) and two cases of WN fever were laboratory confirmed (all cases were WNV IgM-positive or IgM/IgG-positive in serum or cerebrospinal fluid, confirmed by plaque-reduction neutralization test; one case was WNV RNA-positive in plasma). These human cases of WNV disease represented the majority of cases notified in Italy in 2011; in addition, two cases of WNND were notified in the nearby Udine province in the Friuli-Venezia Giulia Region, four WNND cases were recorded in the Sardinia Region, and one case of WN fever in the Marche Region [10,11]. Patients from the Veneto Region were resident in relatively small areas in the provinces of Treviso and Venice surrounding the Piave and Livenza rivers, where WNV circulation had also been reported in 2010  (Fig. 1).
Figure 1. Map showing areas in north-eastern Italy where human cases of West Nile virus (WNV) infection were detected in 2008–2011. The sites where fully sequenced WNV human genomes were collected in 2009 and 2011 are indicated with black dots. The site where a WNV lineage 2-positive wild collared dove was collected in 2011 is also indicated in the map with a white dot.
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In the same areas, in September 2011, WNV nucleic acid amplification test screening led to the identification of four viraemic blood donors and one tissue donor, but in mid-August 2011, it missed a multi-organ donor who transmitted infection to four of five recipients, two of whom (the kidney recipients) developed WNND. A detailed description of these cases has been reported elsewhere . In one of the kidney recipients, who was evaluated by our laboratory, WNV RNA was still detectable in plasma, cerebrospinal fluid, urine and saliva at 1 month after transplantation. Following this experience, we monitored WNV RNA in the urine of WNV nucleic acid amplification test-positive blood donors and demonstrated that the virus was detectable also in urine when patients were viraemic (Barzon L and Pacenti M, unpublished observation).
In all human cases of WNV infection in whom WNV RNA was detectable, the presence of WNV lineage 1 was demonstrated by a specific real-time reverse transcription-PCR assay, as reported elsewhere . In the kidney transplant recipient and in a seronegative blood donor, who had a relatively high viral load, the whole viral genome was successfully amplified and sequenced from plasma and urine specimens. The two WNV full genome sequences were named Piave and Livenza (GenBank accession nos JQ928175 and JQ928174, respectively), because the two patients were resident near these two rivers (Fig. 1). Phylogenetic tree analysis classified both WNV genome sequences as lineage 1, clade 1a, within the monophyletic Western Mediterranean subtype (Fig. 2). The two genome sequences belonged to the same branch of the reconstructed phylogenetic tree, despite having 1.79% nucleotide divergence from each other, higher than expected from the fact that the viruses were detected almost simultaneously in close geographic areas. The two WNV genomes also had high nucleotide divergence (1.99% and 1.88%, respectively) from the strain circulating in Italy in 2008–2009 (Fig. 2). Recent studies using whole genome sequences of WNV isolates from different countries suggested that WNV clade 1a originated from a common ancestor in sub-Saharan Africa in the early twentieth century, and than spread by multiple migrations to Western and Eastern European countries in the 1970s and 1980s [15,16]. The Mediterranean subtype probably originated in the Mediterranean area approximately 20 years ago . It is conceivable that the 2011 WNV genomes detected in Veneto originated in the Mediterranean area. Less likely, their origin could be in other unknown areas where these types of viruses could be circulating without notice and be brought to the Mediterranean by unknown mechanisms. The two genome sequences might have been introduced independently in the Veneto region or, alternatively, they could have arisen as the two sequences evolved and diverged locally from a common ancestor already present in Veneto or in neighbouring regions, in a situation of endemic transmission in rural cycles remaining silent, as recently suggested for other Mediterranean WNV strains . However, the presence of significant amino acid changes in the viral polyprotein of the two genomes, as shown in Fig. S1 (see Supporting information), argues against this hypothesis and indicates two lines of independent evolution that separated a long time ago, unless recombination events had occurred and generated a rapid evolution. In particular, the viral polyproteins had amino acid changes unique to these genomes that were not previously reported in other WNV isolates as well as other amino acid changes that are not generally found in Eastern European and Mediterranean isolates, as detailed in the Supporting information (Fig. S1).
Figure 2. Phylogenetic trees based on the two West Nile virus full-length genomes sequenced from human specimens, Veneto Region, Italy, 2011. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura two-parameter model . The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the strains analysed. Branches corresponding to partitions reproduced in <50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated strains clustered together in the bootstrap test (1000 replicates) are shown next to the branches (only >80 values). Initial trees for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one-quarter of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with a Markov Cluster distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 10 425 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 .
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In September 2011, in the same area where the novel human WNV lineage 1a sequences were detected (Fig. 1), a dead wild collared dove was found to be positive for WNV lineage 2, with sequence similarity to the Hungary/04 strain, and the entomological surveillance led to the identification of mosquito pools positive for both WNV lineage 1 and 2 .
The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease, which requires enhanced surveillance and further investigation.
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Figure S1. Summary of amino acid changes detected in the polyprotein of West Nile virus (WNV) full genome sequences compared with the consensus sequence of Eastern European and Mediterranean WNV subtypes, as described by May et al. [May et al. J Virol 2011]. The WNV strains are included in the figure have the following accession numbers: Marocco 96-111-horse (AY701412); France PaAn001-horse (AY268132); Marocco WNV04.05-horse (AY701413); France 2004-magpie (DQ786573); Portugal 2004-mosquitoes (AJ965628); Italy1998-horse (AF404757); Italy 2008-magpie (FJ483548); Ita09-human (Po river) (GU011992); Italy 2011/Livenza-human (JQ928174); Italy 2011/Piave-human (JQ928175); Spain 2007-eagle (FJ766332); Kenya 1998-mosquitoes (AY262283); Romania-RO97-50-mosq (AF260969); Russia 1999-mosquitoes (AF317203); LEIV-Vlg00-human (AY278442). The polyproteins of the WNV Piave and Livenza genomes (GenBank accession no. JQ928175 and JQ928174, respectively) had unique amino acid changes that were not previously reported in other WNV isolates (i.e., NS4B-A123T in the WNV Livenza genome and NS4B-A50T and NS5-E824D in the WNV Piave genome), as well as other amino acid changes that are not generally found in Eastern European and Mediterranean isolates (supplementary figure). These mutations include, in the WNV Piave genome, the NS5-R287K amino acid change that is also present in WNV clade 1c and in lineages 2, 3, and 4, and, in the WNV Livenza genome, the C-I118V change that is also present in WNV clade 1c, the conservative NS2B-A103V amino acid substitution that is typically found in Eastern European isolates, in the NY99 strain and in WNV lineage 3 (Rabensburg), the NS3-N158S that is present in the Ast02-3-717 isolate from Russia and in WNV lineage 3, and the non-conservative NS3-T249P amino acid substitution associated with avian virulence [Brault et al Nat Genet 2007].
Brault AC, Huang CY, Langevin SA, et al. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet 2007; 39: 1162–6.
May FJ, Davis CT, Tesh RB, et al. Phylogeography of West Nile virus: from the cradle of evolution in Africa to Eurasia, Australia, and the Americas. J Virol 2011; 85: 2964-2974.
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