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Eurosurveillance, Volume 18, Issue 38, 19 September 2013
Rapid communications
Whole genome sequencing and phylogenetic analysis of West Nile virus lineage 1 and lineage 2 from human cases of infection, Italy, August 2013
  1. Department of Molecular Medicine, University of Padova, Padova, Italy
  2. Regional Reference Laboratory, Microbiology and Virology Unit, Padova University Hospital, Padova, Italy
  3. Department of Public Health and Screening, Veneto Region, Venice, Italy

Citation style for this article: Barzon L, Pacenti M, Franchin E, Lavezzo E, Masi G, Squarzon L, Pagni S, Toppo S, Russo F, Cattai M, Cusinato R, Palù G. Whole genome sequencing and phylogenetic analysis of West Nile virus lineage 1 and lineage 2 from human cases of infection, Italy, August 2013. Euro Surveill. 2013;18(38):pii=20591. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20591
Date of submission: 31 August 2013

A human outbreak of West Nile virus (WNV) infection caused by WNV lineage 2 is ongoing in northern Italy. Analysis of six WNV genome sequences obtained from clinical specimens demonstrated similarities with strains circulating in central Europe and Greece and the presence of unique amino acid changes that identify a new viral strain. In addition, WNV lineage 1 Livenza, responsible for a large outbreak in north-eastern Italy in 2012, was fully sequenced from a blood donor during this 2013 outbreak.


A human outbreak of West Nile virus (WNV) infection is ongoing in northern Italy. We report here the clinical presentation, laboratory test results, and WNV full genome sequences from human cases of WNV infection which were diagnosed in August 2013 in the Veneto region. This region is one of the most affected by the outbreak. WNV genome sequences obtained from clinical specimens of infected individuals were subjected to phylogenetic analysis, in order to gain information on the origin and evolutionary history of the responsible viral strains.

Cases of West Nile virus infection from the Veneto region

Cases included five patients with laboratory-confirmed West Nile neuroinvasive disease (WNND) and four with West Nile fever (WNF), aged 51 to 88 years-old, who were resident in Rovigo, Padova, and Verona provinces (Figure 1). In addition, three WNV RNA-positive blood and organ donors were identified by screening in Padova, Verona, and Venice provinces and, besides the 12 confirmed cases, further possible cases are currently under investigation. Confirmed human cases of WNND have also been notified in regions neighbouring Veneto, namely Emilia Romagna and Lombardy [1].

Figure 1. Map showing the places of residence of human cases of West Nile virus (WNV) infection confirmed in the Veneto region, Italy, August 2013 (n=12)

Of the 12 confirmed cases reported in Veneto, WNV lineage 2 (lin2) RNA was identified in plasma and/or urine of seven patients with WNND or WNF, and in a blood donor, while WNV lin1 was respectively detected in an organ donor and in a blood donor. The sites where different WNV lineages were identified are indicated in Figure 1.

Clinical and laboratory findings

A summary of clinical and laboratory findings from confirmed cases is reported in Table 1. Clinical presentation of patients with WNND and WNF included arthralgia, fatigue, fever (≥38°C), headache, myalgia, while patients with WNND had neurological manifestations, such as encephalitis, meningitis and paralysis. Mild symptoms (i.e. arthralgia, headache, myalgia, but not fever) occurred also in a WNV-positive blood donor a few days before donation. No deaths due to WNV infection were reported.

Isolation of the virus in cell cultures was obtained from urine samples collected from three patients with WNND or WNF and from two blood donors. Laboratory methods were performed as previously described [2].

Table 1. Clinical and laboratory data of laboratory-confirmed cases of West Nile virus infection, Veneto region, Italy, August 2013 (n=12)

Epidemiological situation of West Nile virus infection in Europe and Italy

Since 2010, WNV, a mosquito-borne flavivirus, has become a public health concern in Europe, as it has been responsible for an increasing number of epidemic outbreaks in European countries and in neighbouring countries in the Mediterranean basin as well as in the Russian Federation [3]. In fact, after large human outbreaks with hundreds of cases of neuroinvasive disease (WNND) occurred in Romania in 1996 and in Russia in 1999, only small outbreaks were reported in European and Mediterranean countries, generally caused by WNV lin1 strains [4]. The epidemiological situation in Europe changed in 2010, when two large human outbreaks occurred in Greece [5] and in the Volgograd region, Russia [6,7]. During these two outbreaks, two unrelated WNV lin2 genotypes were respectively characterised. The genotype in the Greek outbreak (Greece-Nea Santa-2010) [8] was similar to that first found in a goshawk in Hungary in 2004 (Hungary04 strain) [9] while in the Volgograd outbreak the genotype was similar to that prior detected in human brain and blood samples in 2007 in Volgograd [6]. Recent epidemiological data indicate that WNV lin2 of the Greek/Hungarian cluster is spreading to other central and southern European countries [10,11], such as Serbia, where a large human outbreak occurred in 2012 [7] and is ongoing in 2013 [1].

The outbreak described in this report represents the first human outbreak of WNV lin2 infection reported in Italy. In fact, in Italy, most cases of human infection reported before 2013 were caused by WNV lin1. In particular, two unrelated WNV lin1 strains, both classified within the Mediterranean cluster by phylogenetic analysis, had been responsible for two different outbreaks in northern Italy. The first outbreak between 2008 and 2009 was caused by the WNV lin1 Italy 2008–2009 strain [12-14] and occurred in the same areas that are currently affected by WNV lin2 circulation, namely those surrounding the Po river in the Veneto, Emilia-Romagna, and Lombardy regions [12,15]; the second outbreak took place between 2011 and 2012 in the Venice and Treviso provinces of the Veneto region and was caused by the WNV lin1 Livenza strain [16,17].

Before 2013 only two unrelated human cases of WNV lin2 infection representing the Greek/Hungarian cluster were documented in the country. These had occurred in 2011 and included one case in Ancona (Marche region) and one in Olbia (Sardinia region), respectively [18,19]. In 2011 and 2012, however, WNV lin2 belonging to the Greek/Hungarian cluster was detected by entomological and veterinary surveillance in the island of Sardinia as well as in the Veneto and Friuli-Venezia Giulia regions in north-eastern Italy in areas where WNV lin1 was also circulating [20-22].

The fact that most human cases from Veneto in August 2013 are affected by WNV lin2 could suggest that this lineage has now become more widespread in north-eastern Italy and that it is playing an important part in the current human outbreak. To gain more insight into the origin of the WNV lin2 and WNV lin1 involved in this ongoing outbreak, respective genome sequences were sought.

Genome sequences derived from the 2013 West Nile virus outbreak cases in Veneto

A total of seven WNV lin2 and one WNV lin1 genome sequences were derived from blood or urine samples of cases, including two full genome sequences of WNV lin2 sequenced from samples collected at three days-interval from the same patient. Six WNV complete genome sequences and one almost complete were submitted to GenBank with accession numbers KF588365 and KF647248–KF647253.

Analysis of the West Nile virus lineage 1 sequence
Sequencing of the full genome of WNV lin1 detected in a blood donor from Venice province (i.e. Italy/2013/Livenza/35.1, GenBank accession number: KF647253) demonstrated over 99.9% nucleotide sequence identity with the Livenza strains fully sequenced in 2011 and 2012 and responsible for the large human outbreak that occurred in Venice and Treviso provinces in 2012 [16,17]. This finding demonstrates that the Livenza strain is still circulating in the affected area.

Phylogenetic and amino acid analyses of West Nile virus lineage 2 sequences
Sequence alignment demonstrated that all the genome sequences derived from the cases infected with WNV lin2 (WNV lin2 Italy/2013) shared over 99.9% nucleotide sequence identity, and the two WNV lin2 genome sequences derived from the same patient had 100% identity. At variance, the identity with other WNV lin2 genomes was lower, e.g. 99.5% vs the WNV lin2 Hungary04 strain [9] and 99.4% vs a WNV lin2 Greece-Nea Santa-2010 strain [8]. Likewise 99.7% and 99.1% nucleotide sequence identities were also respectively observed with the NS3 and NS5 regions of a WNV lin2 detected in a mosquito pool collected in 2012 in Rovigo province, in the same area of the current outbreak [21]. Finally the present outbreak sequences presented 99.5 % nucleotide sequence identity with the full genome of the WNV lin2 isolated from the patient in Ancona in 2011 [19]. Phylogenetic analysis showed that the WNV lin2 Italy/2013 genomes were included in the Greek/Hungarian cluster which contains the Hungarian (Hungary04) and Greek (Greece-Nea Santa-2010) strains, but generated a distinct branch in the phylogenetic tree, indicating that they represent a new strain (Figure 2). This finding suggests that a single monophyletic group of WNV lin2 is arising from the Greek/Hungarian cluster, which corresponds to a group of viruses that are evolving as they reach new territories in their spread from central Europe and areas in the Balkans.

Figure 2. Molecular phylogenetic analysis of six West Nile virus lineage 2 genome sequences detected in individuals from the Veneto region, Italy, August 2013

At protein level, the WNV lin2 Italy/2013 genomes encoded a set of unique amino acids compared to other fully sequenced WNV lin2 genomes of the Greek/ Hungarian cluster (Table 2). Most of the substitutions compared to the Hungary 04 reference apparently seem not to change dramatically the properties of referring proteins. Nonetheless, mutations observed on the surface of the non-structural protein 5 (NS5) protein (i.e. E638K* and D831G) in two individual genome sequences were predicted by in silico site-directed mutagenesis to cause a local altered electrostatic potential in the RNA-directed RNA polymerase domain (Figure 3). The relevance of these mutations will be assessed by further sequencing of WNV genome sequences and by experimental studies with viral isolates and site-directed mutagenesis of infectious clones. Of note, none of the WNV lin2 Italy/2013 genomes had the H249P substitution in non-structural protein 3 (NS3) that characterises the Greece-Nea Santa-2010 strain.

Table 2. Variant amino acids encoded by West Nile virus (WNV) lineage 2 genome sequences, including the reference Hungary 2004 sequence, other related sequences, and six genome sequences obtained during the WNV outbreak in Italy, August 2013

Figure 3. 3D images of mutations in the RNA-directed RNA polymerase domain of West Nile virus non-structural protein 5 (NS5) 

Conclusion

Overall, the results of this molecular epidemiology study shows that genetically different lineages of WNV are capable of establishing in Europe, remain circulating for several years in the same territory, and spread slowly to neighbouring areas, in agreement with other reports from Europe [13,23]. In this local transmission and spread dynamics in Europe, WNV overwintering in mosquitoes and amplification in local susceptible bird populations are key factors, while WNV re-introduction by migrating birds from long-distance Euro-African routes seems to be less relevant [24].

In conclusion, a novel WNV lin2 strain of the Greek/Hungarian cluster is responsible for a human outbreak of neuroinvasive disease that is ongoing in northern Italy. The virus is co-circulating with the WNV lin1 Livenza strain that caused a large human outbreak in 2012.


Acknowledgements
This study was supported by Veneto region and by the European Commission under FP7, Project 261426 (WINGS-West Nile Integrated Shield Project).

Conflict of interest
None declared.

Authors’ contributions
Luisa Barzon coordinated the study and wrote the manuscript; Monia Pacenti, Riccardo Cusinato, Silvana Pagni, Margerita Cattai, and Laura Squarzon performed surveillance activities and virological tests; Elisa Franchin and Giulia Masi performed WNV genome sequencing; Enrico Lavezzo and Stefano Toppo performed bioinformatics analyses of WNV genome sequences; Francesca Russo coordinated WNV surveillance activities; Giorgio Palù supervised the study and revised the manuscript.


*Authors' correction:
At the request of the authors, ‘E636K’ was replaced with ‘E638K’. This change was made on 27 September 2013.



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