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In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk ( Accipiter gentilis ) and a kestrel ( Falco tinnunculus ). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008-2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.

Background: West Nile (WNV) and Usutu (USUV) are emerging vector-borne zoonotic flaviviruses. They are antigenically very similar, sharing the same life cycle with birds as amplification host, Culicidae as vector, and man/horse as dead-end host. They can co-circulate in an overlapping geographic range. In Europe, surveillance plans annually detect several outbreaks., Methods: In Italy, a WNV/USUV surveillance plan is in place through passive and active surveillance. After a 2018 WNV outbreak, a reinforced integrated risk-based surveillance was performed in four municipalities through clinical and serological surveillance in horses, Culicidae catches, and testing on human blood-based products for transfusion., Results: Eight WNV cases in eight equine holdings were detected. Twenty-three mosquitoe catches were performed and 2367 specimens of Culex pipiens caught; 17 pools were USUV positive. A total of 8889 human blood donations were tested, and two asymptomatic donors were USUV positive., Conclusions: Different surveillance components simultaneously detected WNV only in horses and USUV only in humans and mosquitoes. While in endemic areas (i.e. northern Italy) entomological surveillance is successfully used as an early detection warning, this method in central Italy seems ineffective. To achieve a high level of sensitivity, the entomological trapping effort should probably exceed a reasonable balance between cost and performance. Besides, WNV/USUV early detection can be addressed by horses and birds. Further research is needed to adapt the surveillance components in different epidemiological contexts.

Lineage 2 West Nile virus (WNV) caused a vast epidemic in Europe in 2018, with the highest incidence being recorded in Italy. To reconstruct the evolutionary dynamics and epidemiological history of the virus in Italy, 53 envelope gene and 26 complete genome sequences obtained from human and animal samples were characterised by means of next-generation sequencing. Phylogenetic analysis revealed two Italian strains originating between 2010 and 2012: clade A, which apparently became extinct in 2013-2014, and clade B, which was responsible for the 2018 epidemic. The mean genetic distances in clade B increased over time and with the distance between sampling locations. Bayesian birth-death and coalescent skyline plots of the clade B showed that the effective number of infections and the effective reproduction number (Re) increased between 2015 and 2018. Our data suggest that WNV-2 entered Italy in 2011 as a result of one or a few penetration events. Clade B differentiated mainly as a result of genetic drift and purifying selection, leading to the appearance of multiple locally circulating sub-clades for different times. Phylodynamic analysis showed a current expansion of the infection among reservoir birds and/or vectors.

West Nile virus (WNV) is continuously spreading in Eastern and Southern Europe. However, the extent of vector competence of Aedes japonicus (Theobald, 1901) is controversial. In this work, we elucidated the dynamics of virus growth in this invasive mosquito species. Females of Ae. japonicus were reared from eggs collected in the field in Switzerland and fed on bovine blood spiked with two WNV lineage 1 strains (FIN, Italy; NY99, USA). Fully engorged females were incubated for 14 days under a fluctuating temperature regime of 24 ± 7 °C (average 24 °C), 45-90% relative humidity, which is realistic for a Central European mid-summer day. Infection, dissemination, and transmission rates were assessed from individual mosquitoes by analyzing the abdomen, legs and wings, and saliva for the presence of viral RNA. Saliva was also investigated for the presence of infectious virus particles. Overall, 302 females were exposed to WNV strain FIN and 293 to strain NY99. A higher infection rate was observed for NY99 (57.4%) compared to FIN (30.4%) (p = 0.003). There was no statistical evidence that the dissemination rate (viral RNA in legs and wings) was different between females infected with FIN (57.1%) compared to NY99 (35.5%) (p = 0.16). Viral RNA load of FIN compared to NY99 was significantly higher in the hemocoel (p = 0.031) of exposed females but not at other sites (legs and wings, saliva). This is the first study describing the vector competence parameters for two WNV strains in a European population of Ae. japonicus. The high dissemination and transmission rates for WNV under a realistic temperature regime in Ae. japonicus together with recent findings on its opportunistic feeding behavior (mammals and birds) indicate its potential role in WNV transmission in Central Europe where it is highly abundant.

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: West Nile virus (WNV) is a Flavivirus transmitted to vertebrate hosts by mosquitoes, maintained in nature through an enzootic bird-mosquito cycle. In Europe the virus became of major public health and veterinary concern in the 1990s. In Italy, WNV re-emerged in 2008, ten years after the previous outbreak and is currently endemic in many areas of the country. In particular, the northeastern part of Italy experience continuous viral circulation, with human outbreaks caused by different genovariants of WNV lineage 1, Western-European and Mediterranean subcluster, and WNV lineage 2, Hungarian clade. Alongside the WNV National Surveillance Program that has been in place since 2002, regional surveillance plans were implemented after 2008 targeting mosquitoes, animals and humans., Findings: In July and September 2014, West Nile virus lineage 2 was detected in pools of Culex pipiens s.l. mosquitoes from northeastern Italy. Whole genome sequencing and phylogenetic analysis of two representative samples identified the presence of WNV lineage 2 related to the Volgograd 2007 strain (99.3 % nucleotide sequence identity), in addition to WNV lineage 2 Hungarian clade., Conclusions: This is the first evidence of the circulation of a WNV lineage 2 strain closely related to the Volgograd 2007 outside Eastern Europe, where it has caused large human outbreaks. This strain may pose a new threat to animal and human health in Italy.

We report the detection and isolation of four almost identical strains of West Nile virus (WNV) lineage 2from Culex modestus mosquitoes collected at three fish ponds in South Moravia, Czech Republic, during August 2013. Phylogenetic analysis demonstrated that the Czech WNV strains isolated are closely related to Austrian, Italian and Serbian strains reported in 2008,2011 and 2012, respectively. Our findings show the current northernmost range of lineage 2 WNV in Europe.

Human cases of West Nile virus (WNV) disease have been reported in Italy since 2008. So far, most cases have been identified in north-eastern Italy, where, in 2012, the largest outbreak of WNV infection ever recorded in Italy occurred. Most cases of the 2012 outbreak were identified in the Veneto region, where a special surveillance plan for West Nile fever was in place. In this outbreak, 25 cases of West Nile neuroinvasive disease and 17 cases of fever were confirmed. In addition, 14 WNV RNA-positive blood donors were identified by screening of blood and organ donations and two cases of asymptomatic infection were diagnosed by active surveillance of subjects at risk of WNV exposure. Two cases of death due to WNND were reported. Molecular testing demonstrated the presence of WNV lineage 1 in all WNV RNA-positive patients and, in 15 cases, infection by the novel Livenza strain was ascertained. Surveillance in other Italian regions notified one case of neuroinvasive disease in the south of Italy and two cases in Sardinia. Integrated surveillance for WNV infection remains a public health priority in Italy and vector control activities have been strengthened in areas of WNV circulation.

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.

West Nile Virus lineage 1 (WNV lin1) emerged in North-Eastern Italy in 2008 and, since then, it has been detected in animals, humans and mosquitoes. Three years later, in the same area, a lineage 2 (lin2) strain of WNV was found in birds and vectors. On August the 21st, during the 2012 WNV entomological surveillance plan, a WNV lin2 strain was detected by RT-PCR in a pool of Culex pipiens mosquitoes captured in Veneto region. According to the alignment of the partial sequences of the NS5 and NS3 genes, no differences between this Italian lineage 2 strain and the Nea Santa-Greece-2010 WNV isolate (Gr-10) were observed. Similarly to the Gr-10 strain, the putative NS3 amino acid sequences of the Italian strain showed proline in position 249 instead of histidine (H249P). Although proline in position 249 has been suggested to increase the virulence of WNV strains, neither human nor veterinary cases associated to this strain have been reported in the region. A prompt mosquito disinfestation was organized to avoid the spread of this potential threatening virus. The simultaneous circulation of both WNV lineage 1 and 2 confirms North-Eastern Italy as a high risk area for WNV emergence and highlights the need for a continuous surveillance.

West Nile virus (WNV) is one of the most serious public health threats that Europe and the Mediterranean countries are currently facing. In Italy, WNV emerged in 1998 and has been circulating since 2008. To tackle its continuous incursions, Italian national and regional institutions set up a surveillance program, which includes the serological screening of sentinel horses, sentinel-chickens and backyard poultry flocks and the surveillance on all equine neurological cases, resident captured and wild dead birds, and vectors. This communication aims to assess the importance of the entomological surveillance program as an early warning system for WNV circulation. In the province of Modena, the circulation of WNV lineage 2 strains was first detected in pools of Culex pipiens on July the 3rd, 42 days prior to the onset of the first 2013 human WNV neuroinvasive case reported in the same province. Similarly in Veneto, WNV was first detected on July 3rd in a pool of Cx. pipiens collected in the province of Venezia. The first human neuroinvasive case in this region occurred in the Rovigo province on July the 24th, seven days after the detection of WNV lineage 2 in a mosquito pool collected in the same province. Up to the end of July 2013, WNV circulation was further detected in several other pools of Cx. pipiens mosquitoes collected in Emilia-Romagna, Veneto and Lombardia. According to the NS3 partial sequence alignments including all recent European and Italian Lineage 2 strains, the new circulating WNV lineage 2 strains share high nt homology with the Hungarian and with the previous lineage 2 strains isolated in Veneto and Sardegna in 2011 and 2012. These data provide a clear and practical demonstration of the relevance of a reliable entomological surveillance program to early detect WNV in Italy.

West Nile virus (WNV) is currently circulating in several European countries and, over recent decades, concomitantly with enhanced surveillance studies and improved diagnostic capabilities, an increase in the geographical distribution and in the number of cases in Europe has been documented. In Italy in 2011, 14 human cases of WNV neuroinvasive infections due to lineage 1 strains were registered in several Italian regions. Here we report WNV partial sequences obtained from serum samples of two patients living in different regions of Italy (Veneto and Sardinia). Phylogenetic analysis, performed on a fragment (566 nt) of the envelope gene, showed that WNV strains circulating in Italy in 2011 belong to lineage 1a, but are different from lineage 1a strains isolated in 2008-2009.The data reported here are consistent with the hypothesis of multiple recent introductions of WNV lineage 1a strains into Italy.

During 2008-2009, several human cases of WNV disease caused by an endemic lineage 1a strain were reported in areas surrounding the Po river in north-eastern Italy. Since 2010, cases have been recorded in nearby northern areas, where, in 2011, both lineage 1a and 2 were detected. We describe here two new WNV complete genome sequences from human cases of WNV infection occurring in 2011 in the Veneto Region. Phylogenetic analysis showed that both genome sequences belonged to lineage 1a and were related to WNV strains of the Western Mediterranean subtype. The novel WNV genomes had high nucleotide and amino acid sequence divergence from each other and from the WNV strain circulating in Italy in 2008-2009. The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease that requires further investigation., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

In 2011, from 26 September to 16 October, a small outbreak of West Nile virus (WNV) disease occurred on the island of Sardinia (Italy). According to the national case definition, six cases with acute neurological disease were confirmed in hospitalized patients, and four of them died; one of these was only 34 years old. In two case, WNV RNA was detected in urine, suggesting renal involvement. Sequence analysis showed lineage 1 and 2 circulation., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

In recent years, West Nile virus (WNV) has re-emerged in the Western Mediterranean region. As a result, the number of complete WNV genome sequences available from this region has increased, allowing more detailed phylogenetic analyses, which may help to understand the evolutionary history of WNV circulating in the Western Mediterranean. To this aim, the present work describes six new complete WNV sequences from recent outbreaks and surveillance in Italy in 2008-2009 and in Spain in 2008 and 2010. Comparison with other sequences from different WNV clusters within lineage 1 (clade 1a) confirmed that all Western Mediterranean WNV isolates obtained since 1996 (except one from Tunisia, collected in 1997) cluster in a single monophyletic group (here called 'WMed' subtype). The analysis differentiated two subgroups within this subtype, which appear to have evolved from earlier WMed strains, suggesting a single introduction in the area, and further dissemination and evolution. Close similarities between WNV variants circulating in consecutive years, one in Spain, between 2007 and 2008, and another in Italy between 2008 and 2009, suggest that the virus possibly overwinters in Western Mediterranean sites. The NS3(249)-proline genotype, recently proposed as a virulence determinant for WNV, has arisen independently at least twice in the area. Overall, these results indicate that the frequent recurrence of outbreaks caused by phylogenetically homogeneous WNV in the Western Mediterranean since 1996 is consistent with a single introduction followed by viral persistence in endemic foci in the area, rather than resulting from independent introductions from exogenous endemic foci.

To determine the lineage of West Nile virus that caused outbreaks in Italy in 2008 and 2009, several West Nile virus strains were isolated from human specimens and sequenced. On the basis of phylogenetic analyses, the strains isolated constitute a distinct group within the western Mediterranean cluster.

In August 2008, West Nile disease re-emerged in Italy. The infection is affecting the North Eastern regions and, as of November 2008, has caused 33 clinical cases and five fatalities in horses. Until now, no deaths have been reported in birds. Mosquitoes, blood, serum and tissue samples, from horses and birds, within and around the outbreak area, have been collected and tested by various methods both serologically and virologically. West Nile virus strains have been isolated from blood samples of one horse and one donkey and from pools of brain, kidneys, heart and spleen of a pigeon and three magpies. When compared to the strain isolated during the 1998 Tuscany outbreak, the 255 bp sequence of the genome region coding for the envelope (E) protein of the isolated WNV strains, exhibited a 98.8% and 100% similarity at nucleotide and amino-acid level respectively., (© 2009 Blackwell Verlag GmbH.)

In Italy the first occurrence of West Nile virus (WNV) infection was reported in Tuscany region during the late summer of 1998. In August 2008, the WNV infection re-emerged in Italy, in areas surrounding the Po river delta, and involving three regions Lombardy, Emilia Romagna and Veneto. WNV was isolated from blood and organs samples of one horse, one donkey, one pigeon (Columba livia) and three magpies (Pica pica). The phylogenetic analysis of the isolates, conducted on 255 bp in the region coding for the E protein, indicates that these isolates belong to the lineage I among the European strains. According to the analysis, both the 1998 and 2008 Italian strains as well as isolates from Romania, Russia, Senegal and Kenya fell in the same sub-cluster.