Evidence of West Nile virus lineage 2 circulation in Northern Italy.
ABSTRACT A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: Phylogenetic relationships were examined for 29 southern African West Nile virus (formal name West Nile virus [WNV]) isolates from various sources in four countries from 1958 to 2001. In addition, sequence data were retrieved from GenBank for another 23 WNV isolates and Kunjin and Japanese encephalitis viruses. All isolates belonged to two lineages. Lineage 1 isolates were from central and North Africa, Europe, Israel, and North America; lineage 2 isolates were from central and southern Africa and Madagascar. No strict correlation existed between grouping and source of virus isolate, pathogenicity, geographic distribution, or year of isolation. Some southern African isolates have been associated with encephalitis in a human, a horse, and a dog and with fatal hepatitis in a human and death of an ostrich chick.Emerging infectious diseases 09/2002; 8(8):820-6. · 5.99 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Two different West Nile virus (WNV) strains caused lethal encephalitis in a flock of geese and a goshawk in southeastern Hungary in 2003 and 2004, respectively. During the outbreak in geese, 14 confirmed human cases of WNV encephalitis and meningitis were reported in the same area. Sequencing of complete genomes of both WNV strains and phylogenetic analyses showed that the goose-derived strain exhibits closest genetic relationship to strains isolated in 1998 in Israel and to the strain that emerged in 1999 in the United States. WNV derived from the goshawk showed the highest identity to WNV strains of lineage 2 isolated in central Africa. The same strain reemerged in 2005 in the same location, which suggests that the virus may have overwintered in Europe. The emergence of an exotic WNV strain in Hungary emphasizes the role of migrating birds in introducing new viruses to Europe.Emerging infectious diseases 05/2006; 12(4):618-23. · 5.99 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the well-described epidemiological patterns of WNV in North America, the basis for the emergence of WNV-associated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.Nature Genetics 10/2007; 39(9):1162-6. · 35.21 Impact Factor
Evidence of West Nile virus lineage 2 circulation in Northern Italy
G. Savinia,*, G. Capellib, F. Monacoa, A. Polcia, F. Russoc, A. Di Gennaroa, V. Marinia,
L. Teodoria, F. Montarsib, C. Pinonia, M. Pisciellaa, C. Terreginob, S. Marangonb,
I. Capuab, R. Lellia
aIstituto G. Caporale Teramo, Via Campo Boario, 64100 Teramo, Italy
bIstituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy
cRegione Veneto, Venice, Italy
West Nile virus (WNV) is a RNA virus included in the
Japanese Encephalitis serogroup within the Flaviviridae
family. In its natural cycle birds normally act as amplifying
hosts whereas mosquitoes, mainly of the genera Culex,
Aedes and Ochlerotatus, play the vector role. In this cycle
humans, horses, and other mammals are regarded as
incidental or dead-end hosts. Based on phylogenetic
analyses eight distinct lineages have been recently
proposed for the strains of WNV (Mackenzie and Williams,
2009; Vazquez et al., 2010). Of these, isolates grouped in
lineages 1 and 2 are nowadays by far the most widespread.
Lineage 1 strains have been reported in North America,
North Africa, Europe, and Australia, whereas strains of
lineage 2 have only recently shown to be capable of
spreading outside of their historical geographic range
which has been for long time confined between Southern
Africa and Madagascar (Lanciotti et al., 1999; Burt et al.,
2002). In 2004, a lineage 2 strain has been firstly recorded
in Hungary (Bakonyi et al., 2006) where it became endemic
(Bakonyi et al., 2006; Erdelyi et al., 2007) before extending
in Austria in 2008 and 2009 (Wodak et al., 2011). It is now
two years that a strain of lineage 2 has been circulating in
Greece (Papa et al., 2011a,b; Valiakos et al., 2011;
Chaskopoulou et al., 2011) and, just few months ago, a
WNV RNA belonging to lineage 2 was detected in a human
patient in the Center of Italy (Bagnarelli et al., 2011).
Veterinary Microbiology 158 (2012) 267–273
A R T I C L E
I N F O
Received 15 December 2011
Received in revised form 2 February 2012
Accepted 9 February 2012
West Nile virus lineage 2
A B S T R A C T
A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two
pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove
(Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was
molecularly identified by group and WNV lineage specific RT-PCRs and characterized by
partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same
fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary
(2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity,
respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010
Greek isolate by one amino-acid located at 249 site which is the site involved in genetic
modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather
than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1
strain is still circulating, creates a new scenario with unpredictable consequences. In this
situation comprehensive investigations on the occurrence, ecology, and epidemiology of
these different WNV strains circulating in Italy become the highest priority.
? 2012 Elsevier B.V. All rights reserved.
* Corresponding author at: Deptartment of Virology, National Refer-
ence Center for West Nile Disease, OIE Reference Laboratory for
Bluetongue, Istituto G. Caporale Teramo, Via Campo Boario, 64100
Teramo, Italy. Tel.: +39 0861 332440; fax: +39 0861 332251.
E-mail address: firstname.lastname@example.org (G. Savini).
Contents lists available at SciVerse ScienceDirect
jo u rn al ho m epag e: ww w.els evier.c o m/lo cat e/vetmic
0378-1135/$ – see front matter ? 2012 Elsevier B.V. All rights reserved.
Strains of both lineages have been shown to be able to
cause severe disease in birds, horses and humans (Lanciotti
et al., 1999; Bakonyi et al., 2006; Venter and Swanepoel,
2010). Neuroinvasiveness and virulence of a WND strain
have been recently related to the genotype and/or the
immunological status of the infected population (Burt
et al., 2002; Venter and Swanepoel, 2010; Papa et al.,
2011a). The nervous form of WNV when occurring is
ataxia, weakness, recumbency, and muscle fasciculation.
This article reports the first detection of a lineage 2 strain in
local birds and indigenous mosquitoes in Northern Italy.
2. Materials and methods
The activities described in this report were part of the
extensive National surveillance plan for monitoring
Flavivirus which is in place since 2002. The program
involved horses, insects as well as wild and domestic birds
as described by Calistri et al. (2010). In 2009, the
entomological and serological surveillance was locally
expanded by the Veneto region.
2.1. Entomological survey
Sixty-one CDC-CO2 traps were placed in rural and
periurban sites of all the 11 provinces of the Veneto (49
traps) and Friuli Venezia Giulia (12 traps) regions. Traps
operated from May through October and were activated
every 15 days for one night, from sunset to the next
Collected mosquitoes were immediately refrigerated,
taken to the laboratory, counted, identified using standard
taxonomic keys (Romi et al., 1997; Severini et al., 2009)
and pooled in 50 specimens maximum, according to
species, site and date and then stored at ?80 8C.
2.2. Bird surveillance
Within the framework of WN virus surveillance
activities in wild birds, an episode of high mortality in
doves was investigated. At the end of September 2011,
inhabitants of rural areas near Treviso, a municipality in
the Veneto region, noticed an unusual mortality of wild
collared doves (Streptopelia decaocto) and birds of other
species (i.e., blackbirds). Unfortunately, no other data were
available to the local veterinary authorities on these
episodes of mortality. Only one dead bird was collected by
a private citizen and carried to the Institute of Padua for
A complete necropsy was performed and samples of
brain, liver, kidney, spleen, lung and intestine were taken,
homogenized and suspended in phosphate-buffered saline
with antibiotics for viral detection.
2.3. Virus isolation
From the homogenized dove tissues and the RNA
positive mosquito samples, virus isolation attempts were
carried out on different cell lines as described by Savini
et al. (2011). In brief, homogenized tissue and mosquito
samples suspended in phosphate-buffered saline with
antibiotics were centrifuged (1000 ? g, 10 min). After
centrifugation, the supernatants were inoculated onto
confluent chicken embryo fibroblast, chicken embryo liver
and Vero cell cultures. Cell cultures were incubated at 37 8C
for 7 days and were checked daily for the occurrence of
cytopathic effects (CPE). Three blind passages were made
in absence of cytopathic effect. At the end of the third
passage and in case of CPE, the presence of WNV in the
supernatants was confirmed or excluded either by electron
microscopy or by Immunofluorescence using a monoclonal
antibody (produced by ICT, Italy) against WNV.
Samples of brain, liver, kidney, spleen, lung and
intestine of the dove were also processed in 9–10 day
old SPF embryonated chicken eggs (OIE, 2008).
2.4. Molecular analysis
2.4.1. Real time RT-PCR and nested RT-PCR
Viral RNA was extracted from a pool of kidney, heart
and brain and was tested by real time RT-PCR (RRT-PCR)
for Avian Influenza (AI) (Spackman et al., 2002), Newcastle
Disease (ND) (Wise et al., 2004), Usutu and West Nile (WN)
viruses (Tang et al., 2006). Lung and intestine were also
tested for ND and AI by RRT-PCR.
Mosquito samples were also screened for the presence
of WNV by using one-step SYBR Green-Based RRT-PCR
(Ravagnan et al., 2011). This method which uses specific
primers (MAMD and cFD2; Scaramozzino et al., 2001)
designed on the conserved region of the non-structural
NS5 gene of the WNV, is capable of detecting several
Flaviviruses. Positive results were then confirmed either
by using the commercial Kit TaqvetTMWest Nile WNV (LSI,
Lissieu, France) which is able to detect WNV RNA of the
lineage 1 and 2 strains and the RT-PCR as described by
Bakonyi et al. (2006) targeting NS5 gene. To ascertain
which lineage the WNV RNA found in the samples
belonged to, lineage specific RT-PCRs were used. For
determining the presence of lineage 1 strains, the
Lanciotti et al. (2000) method modified as described by
Monaco et al. (2009) was used, whereas for discriminating
lineage 2 strains, the method as recently described by
Chaskopoulou et al. (2011), which targets the NS3 gene of
the WNV genome, was used. Briefly, total RNA was reverse
transcribed by the kit ‘‘TaqMan1Reverse Transcription
Reagents’’ (Applied Biosystem, USA) using random hex-
amers. The reverse transcription was carried out in 20 ml
of mix containing 0.5 ml of Multiscribe RNA enzyme (50 U/
ml), 2 ml of 10? RT buffer, 4.4 ml of the 25 mM MgCl2
buffer, 4 ml of dNTPs 2.5 mM, 1 ml of random hexamer
(50 mM) primer, 0.4 ml of Rnase inhibitor (20 U/ml) and
5 ml of RNA. The reaction was incubated 10 min at 25 8C
then at 48 8C for 30 min followed by final incubation at
95 8C for 5 min to inactivate the residual activity of the
reverse transcriptase. Viral RNA was amplified by a nested
RT-PCR. In brief, the first amplification generated a 778 bp
amplicon by using the external primer pair WN-NS3up1
(50-GCTGGCTTCGAACCTGAAATGTTG-30) and WN-NS3do1
(50-CAATGATGGTGGGTTTCACGCT-30). The second ampli-
fication product used the internal primer pair WN-
NS3up2 (50-GCAAGATACTTCCCCAAATCATCAAGG-30) and
G. Savini et al. / Veterinary Microbiology 158 (2012) 267–273
WN-NS3do2 (50-TGTCTGGGATCTCTGTTTGCATGTC-30) to
amplify a 423 bp region of the NS3 coding region of the
WNV genome. Both reactions were carried out in 50 ml of
final volume containing 5 ml of buffer 10?, 3 ml of MgCl2
(25 mM), 1 ml of dNTPs (10 mM), 0.3 ml of TaqGold and
1 ml of each primer (50 mM). The reaction was incubated
at 95 8C for 10 min followed by 30 cycles of denaturation
for 30 s at 94 8C, annealing at 60 8C for 30 s, extension at
72 8C for 1 min and final extension at 72 8C for 7 min.
Amplified products were visualized on 1% agarose gel
stained with Syber Safe (Invitrogen, USA) and the bands
were observed under UV light.
2.4.2. Amplification and sequencing
PCR products were purified with the Qiaquick PCR
Purification kit (Qiagen, Germany) and used for direct
sequencing in both directions using the internal primers
The sequencing reaction was set up using the Big Dye
Terminator kit (Applied Biosystems, USA), the excess of
dyes was removed using Cleanseq (Beckman Coulter, USA)
and the nucleotide sequences were determined using the
DNA sequencer ABI PRISM 3100 (Applied Biosystems,
USA). Raw sequence data were assembled using Contig
Express (Vector NTI suite 9.1, Invitrogen, USA) and
translated into amino acid sequences using Vector NTI
suite 9.1 (Invitrogen, USA). Consensus sequences from the
virus detected in Italian mosquitoes and bird tissues were
aligned with the WNV lineage 2 isolated in 2004 in
Hungary (DQ116961), in 2011 in Greece (HQ537483) and
to the reference strain isolated in 1937 in Uganda
(M12294) with ClustalW (Thompson et al., 1994) (Table 1).
Overall, 85,398 mosquitoes were collected belonging
to 14 species. Culex pipiens was the most abundant
mosquito species (81%) and was present in all the 61 sites
monitored. Species composition and relative abundance
were similar in the two regions, confirming the area as
environmental homogenous and highly suitable for C.
Only female mosquito samples identified at the species
level (n = 78,791) were screened by the one step RRT-PCR
for the presence of Flavivirus. Males (n = 766; 0.0001%) or
mosquitoes not identified at the species level (n = 5400;
6.3%) were not tested. In total, 2732 pools were tested and
5 of them (0.18%) were found positive for WNV RNA from
late July until mid September (Table 2).
At necropsy, the dove appeared undernourished but
no significant gross
Influenza, Newcastle Disease and Usutu viruses were
not detected by either molecular methods or virus
isolation on target organs. The pool of kidney, heart
and brain resulted positive for WN virus by both RRT-PCR
and RT-PCR, conversely the attempt to isolate the virus
All positive reactions were confirmed by the lineage 1
and 2 commercial RT-PCR. When the specific RT-PCRs were
used, RNA of lineage 1 WNV was found in 3 C. pipiens pools
while RNA of lineage 2 strain was found either in 2 pools of
C. pipiens collected in the province of Udine and in the bird
tissues. The NS3 gene of the lineage 2 strains was then
partially sequenced and the sequence aligned. The
nucleotide sequences confirmed the presence of lineage
2 strains. Results of the alignment of the partial NS3 gene
sequence are shown in Fig. 2. No differences were observed
between the Italian strains. The sequences also showed
100% similarity with those of the Hungarian strain but
differed by 2 nucleotides in positions 80 and 142 from the
2010 Greek isolate. When the nucleotide sequences were
translated to putative amino acid sequences, they differed
by one amino acid only at position 249 of the NS3 protein.
At this site in the Italian and Hungarian strains histidine
Strains of West Nile viruses included in the NS3 partial sequencing.
Nea Santa Greece 2010
Details of the West Nile virus infected Culex pipiens collected during the 2011 entomological survey.
WN area code
VEN, Veneto region; FVG, Friuli Venezia Giulia region ACV, area with WN viral circulation; AS, WN surveillance area; outside, area outside the AS area. MIR,
minimum infection rate (positive pools/mosquitoes tested ? 100) for the site and date of collection.
G. Savini et al. / Veterinary Microbiology 158 (2012) 267–273
Fig. 1. Areas where lineage 2 strains of West Nile virus have been detected in Northern Italy.
G. Savini et al. / Veterinary Microbiology 158 (2012) 267–273
replaces proline. The blast of the NS5 nucleotide sequence
also showed a similarity of 99% with the West Nile
Hungarian 2004 isolate.
This report provided further evidence that a lineage 2
strain is circulating in Italy. It was found in two pools of C.
pipiens and in the tissues of a resident collared dove found
dead in North East of Italy. This is the first time that a
lineage 2 strain has been detected in mosquitoes and birds
in Italy. A human case caused by a lineage 2 strain infection
was recently described in Ancona (Bagnarelli et al., 2011).
It was a mild case characterized by a period of high fever
with no response to antibiotics. The strain which was
detected in the urine sample, when sequenced, showed
99% identity to the complete genome of isolate goshawk-
Hungary/04 and to the more recent Nea Santa-Greece-
2010 (Bakonyi et al., 2006; Papa et al., 2011b; Bagnarelli
et al., 2011). According to the NS3 partial sequences, the
strains detected in mosquitoes and bird tissues in this
study, were identical and also showed 100% similarity with
the same fragment sequences of the strain detected in Italy
and of the strains which circulated in Hungary in 2004.
Therefore, it appears that the same strain of lineage 2 WNV
was likely responsible for the dove and mosquito infec-
tions even if the areas where the bird and the mosquitoes
were collected were not contiguous (Fig. 1). In contrast to
the previous Italian lineage 2 reported case, this lineage 2
strain detection occurred in an higher risk area compared
to, where lineage 1 WNV has circulated and/or is
circulating. It is currently difficult to track down the exact
route of entrance of the virus. The phylogenetic findings
and the sample collection dates imply a southward
expansion of the virus from the Central European
countries. The areas where the infected mosquitoes were
collected are adjacent to Slovenia and/or are part of the
flying paths of the long and short migration routes.
Migratory birds from Africa have often been implicated
in the emergence of the WNV outbreaks in Europe
(Hubalek and Halouzka, 1999; Rappole et al., 2000;
Murgue et al., 2001; Malkinson et al., 1998; Rappole and
Hubalek, 2003). In this case, considering the high genetic
similarity with the Hungarian isolate, it is more likely that
birds migrating either along the south-eastern migration
route from Europe and western Asia to Africa, or along
short migration routes from Central to Southern Europe,
have introduced the virus. A similar infection pathway was
also proposed for the spread of Usutu virus in Italy (Savini
et al., 2011). Since in a month period the same strain virus
was detected in local birds and indigenous mosquitoes in
two different areas situated 50 km apart, it looks as if the
Italian lineage 2 strain established itself with the tendency
to spread westward. Many prerequisites are required to
maintain a WNV strain in an environment. Vector
competency and presence of susceptible vertebrate host
capable of transmitting the infection to vectors, are indeed
key parameters for the maintenance and spread of WNV in
a given environment. The areas where the lineage 2 strains
have been detected, have already proven to have these
prerequisites as lineage 1 WNV and also Usutu virus, which
share the same life cycle, have circulated and/or are
circulating. Although further studies carried out in non-
epidemic periods are needed to test this hypothesis, it is
then more than likely that this lineage 2 strain will
establish and further spread in the neighboring areas. As a
consequence, in the next season it is highly probable that
both lineages will contemporaneously circulate in the
same areas. Whether this will result in an enhancement or
reduction of their virulence is hard to say. Similarly, it is
difficult to predict the occurrence of possible genetic
recombination phenomena (Pickett and Lefkowitz, 2009).
Because cross protection between both lineages has been
observed in horses (Minke et al., 2011), the circulation of
both lineages could eventually result in a rapid endemiza-
tion of the area with no severe clinical consequences.
According to the phylogenetic analysis, the Italian as the
Hungarian lineage 2 strains differed from that isolated in
Greece as they have histidine rather than proline at
location 249 of the NS3 protein. This site has been involved
Fig. 2. Phylogenetic analysis of the Italian sequences. Sequence dataset
was analyzed using BioEdit version 7.0.9 and nucleotide alignment was
performed with Clustal-W (Thompson et al., 1994). Phylogenetic
analysis was conducted by using distance (NJ) method in PHYLIP (the
PHYlogeny Inference Package) program version 3.67. The reliability of
the phylogenetic trees was confirmed by performing bootstrap analysis
with 1000 replicate values. The tree obtained was rooted with ITA09
strain. The asterisk indicates significant bootstrapping values (>60%).
ITA09: West Nile virus (WNV) strain isolated in Italy in 2009; UGA37:
B956 lineage 2 prototype strain; GRE10: Greek WNV strain isolated in
2010; HUN04: Hungarian isolate detected in 2004; HumITA11: Italian
WNV strain detected in a human patient in 2011; SdeITA11: Italian WNV
strain detected in a wild collared dove in 2011; Cp1ITA11: WNV strain
detected in a pool of C. pipiens caught in Bagnara Arsa (UD); Cp2ITA11:
WNV strain detected in a pool of C. pipiens caught in Palazzolo della
G. Savini et al. / Veterinary Microbiology 158 (2012) 267–273
in genetic modulation of WNV pathogenicity, using
American crows (Corvus brachyrhynchos) as avian model
of WNV disease (Brault et al., 2007). The influence of
histidine in position 249 of the NS3 on the virulence of a
strain is still unknown. In line with those which circulated
in Hungary and Austria which were able to cause
encephalitis and death in a sparrow hawks (Accipiter
nisus), goshawks (Accipiter gentilis) (Bakonyi et al., 2006;
Erdelyi et al., 2007) and gyrfalcon (Falco rusticolus), the
Italian lineage 2 strain found in the tissues of a collared
dove was likely the cause of the bird death. In addition to
the dove, the lineage 2 strain was also detected in pools of
C. pipiens. Considering that C. pipiens has been demon-
strated to serve both as an enzootic and a bridge vector for
humans (Hamer et al., 2008), it is highly probable that
lineage 2 WNV will infect humans again. However is not
possible to predict what will be the impact of this infection
even if the clinical expression of the lineage 2 human case
reported in Italy was relatively mild.
This study demonstrated that a WNV strain belonging
to lineage 2 is circulating in North-East of Italy. As it was
found either in local collared dove and in indigenous
mosquito (C. pipiens), it seems to have developed
strategies of adaptation and become established. Its
NS3 partial sequence is identical to the other Italian
isolate detected in a human patient as well as to
Hungarian isolate with which it seems to share similar
features including the pathogenicity for birds. Compre-
hensive investigations on the occurrence, ecology, and
epidemiology of the different WNV strains circulating
in Italy should be of highest priority. Because of its
zoonotic potential, veterinarians, physicians, and public
health officials need to work more closely together to
control, prevent, and better estimate the epidemiological
impact and possible threat to animal and public health of
WNV infections. Greater communication and collabora-
tion between these different professional figures at both,
central and local level would, ultimately, improve
prevention and control strategies with great benefit for
human and veterinary public health.
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