RESEARCH NOTE
Circulation of West Nile virus lineage 1
and 2 during an outbreak in Italy
F. Magurano1, M. E. Remoli1, M. Baggieri1, C. Fortuna1,
A. Marchi1, C. Fiorentini1, P. Bucci1, E. Benedetti1,
M. G. Ciufolini1, C. Rizzo2, S. Piga3, P. Salcuni4, G. Rezza1
and L. Nicoletti1
1) Istituto Superiore di Sanitá, Department of Infectious, Parasitic and
Immunomediated Disease, Rome, Italy, 2) Istituto Superiore di Sanità,
National Center for Epidemiology, Surveillance and Health Promotion,
Roma, Italy, 3) Department of Malattie Infettive, Ospedale SS. Trinità,
Cagliari, Italy and 4) Department of Infectious Diseases and International
Prophylaxis Office, Ministry of health, Rome, Italy
Abstract
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.
Keywords: lineage 1, lineage 2, neurogical disease, outbreak,
urine, West Nile
Original Submission: 18 April 2012; Revised Submission: 8
August 2012; Accepted: 9 August 2012
Editor: T. A. Zupanc
Article published online: 1 September 2012
Clin Microbiol Infect 2012; 18: E545–E547
10.1111/1469-0691.12018
Corresponding author: F. Magurano, Istituto Superiore di Sanità,
Department of Infectious, Parasitic and Immunomediated Diseases,
v.le Regina Elena 299 00161, Rome, Italy.
E-mail: fabio.magurano@iss.it
West Nile virus (WNV) belongs to the Japanese encephalitis
virus group. This flavivirus is widespread in Africa, Europe,
the Middle East and parts of Asia, and it has been introduced
Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases
VIROLOGY
into North America in the late 1990s [1]. Its transmission
cycle involves birds as vertebrate hosts, and ornithophilic
mosquitoes as maintenance vectors. Less than 1% of WNV-
infected humans develop acute neuroinvasive disease, includ-
ing meningitis, encephalitis, and flaccid paralysis [2].
Evidence of persistent WNV infection was shown in
experimentally infected monkeys and hamsters, where the
virus may be detected in urine samples for up to 12 months
[3,4]. These experimental data raise the possibility that per-
sistent renal infection may occur in humans. To this purpose,
WNV presence was demonstrated by reverse transcriptase
polymerase chain reaction (RT-PCR) in the urine of a patient
with acute and persistent WNV infection [5].
Isolates of WNV fall into two major genetic lineages: the
lineage 1 identified in North America, North Africa, Europe
and Australia; and lineage 2, which is endemic in Southern
Africa and Madagascar [6,7]. In recent years, lineage 2 circu-
lation has been identified in central and eastern Europe, both
in animal and human outbreaks [8–10].
In Italy, the first outbreak of WNV infection was reported
among horses in 1998 in Tuscany [11]. Then, the virus
re-emerged in 2008 in the north-east of the country, causing
sporadic cases and/or clusters of West Nile neuroinvasive
disease (WNND) in humans and horses every year [12,13].
All Italian cases were due to lineage 1. During the summer
of 2011, WN virus lineage 2 has been detected in urine sam-
ples of a febrile patient in the Marche Region [14]. More-
over, in 2011, evidence of circulation of both WNV lineage
1 and 2 (very close to the Hungarian one) was demonstrated
in animals in the north-east of Italy, suggesting a probable
introduction of lineage 2 from central and/or eastern Euro-
pean countries, possibly through migratory bird routes [15].
Here, we report a cluster of human cases of WNND that
occurred in the Sardinia region at the end of the 2011 sum-
mer season.
Between 26 September and 16 October 2011, we
received biological samples of nine hospitalized patients with
suspected WNND. All patients were from Sardinia, eight of
them from the southwestern province of Oristano and one
from the northeastern province of Olbia. Six of them had
central nervous system manifestations and four died. WNV
infection was confirmed for four cases, while the other two
cases were classified as probable cases (Table 1). All these
individuals were males and their median age was 68 years
(range, 34–83). The cluster was characterized by a high case-
fatality rate (CFR) (three of the four confirmed WNND
cases (75%) died), compared with that observed in Italy from
2008 to 2011 (16%, 7/43 confirmed WNND cases) [16] One
death occurred in a 34-year-old man. Whether that high
fatality rate was due to a high virulence of the circulating
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E546 Clinical Microbiology and Infection, Volume 18 Number 12, December 2012 CMI

TABLE 1. Viral diagnostic findings, West Nile virus-infected humans from the Sardinia region (Italy)

Elisa IgM PCR

Patient Age Origin Symptoms Status serum CSF PRNT serum CSF urine Strain

S 58 76 Oristano F –M Confirmed Death + + + + – + L1

S 59 79 Oristano F –M Confirmed Death + NA + – NA NA
S 60 34 Oristano F –M Confirmed Death + + + – + + L1
S 67 66 Oristano F Possible – NA – – NA NA
S 68 66 Oristano F Possible – NA ND – NA NA
S 69 73 Oristano F Possible – NA ND – NA NA
S 70 66 Oristano F –P Probable + NA + – NA NA
S 73 71 Olbia F –M Confirmed + + – + + NA L2
S 74 83 Oristano F –M Confirmed Death + + ND + – NA L1

F, febrile symptoms; M, meningoencephalitis; P, polyradiculoneuritis; NA, not available; ND, not done.

strains, increased host susceptibility due to unknown factors

or an under-detection of milder cases remains undefined.
West Nile virus IgM-capture ELISA assay (FOCUS Diag-
nostics) was performed for the detection of IgM antibodies
against WNV in sera, plasma and CSF. Six sera and four CSF
samples were IgM positive by ELISA. IgM positive diagnosis
was confirmed by plaque reduction neutralization test
(PRNT) conducted against lineage 1 virus, or by genome
detection. In one case, PRNT was not performed due to the
small volume of serum received; however, this case was con-
firmed by positive PCR results both in serum and CSF. In
one case PRNT was found negative: in this case lineage 2
virus could be detected in serum (Table 1).
RT and hemi-nested PCR were performed to detect viral
RNA in serum, plasma, urine and CSF samples [17].
Positive PCR results could be obtained in three out of
nine serum samples, two out of four CSF samples and two
out of two urine samples (Table 1).
The PCR products were sequenced and aligned with the
FIG. 1. Maximum-likelihood (ML) phylogenetic tree for partial NS5
sequences that showed higher percentage of identity (99–
genomic segment nucleotide sequences (658 bp for L1 and 366 bp
97%) after Blast analysis. For phylogenetic analysis the
for L2) of West Nile virus (WNV) strains identified in Sardinia
HKY+G evolutionary model was chosen as the best-fitting
(Italy). Accession numbers of the sequence strains included in the
nucleotide-substitution model, according to the hierarchical
analysis: JF719069; HM152780; DQ786572; JF719067; JF719066;
likelihood-ratio test (LRT) implemented in the modeltest v.
DQ118127; HQ596519; DQ116961; JN858070; EF116943;
3.7 software [18]. Modeltest utilizes the PAUP software [19],
HQ537483; JF460774; AY453411; JF706286.
under a strict maximum likelihood (ML) approach, to test 56
different models of nucleotide substitution on a given 2008 and 2009 and to the strains circulating in Europe and
sequences alignment. Israel from late 2004 to 2011. A sequence from the WNV-
The ML tree (Fig. 1) showed a circulation of both WNV positive patient coming from Olbia (north-eastern Sardinia)
lineages, with the main cluster due to lineage 1, and one iso- showed a virus strain belonging to the genetic lineage 2
lated case in the eastern province of Olbia due to lineage 2. (Accession JX122764). This strain was closely related to
The spread of these lineages was apparently restricted to those responsible for the outbreaks that occurred in Greece
two geographically separated areas of the island. In particu- and Hungary [6] in 2010 and 2005, respectively, and to the
lar, sequences from three WNV-positive patients from Or- first autochthonous Italian human case found in 2011 in Anc-
istano (southwestern Sardinia) revealed a virus strain ona [14].
belonging to the genetic lineage 1 (Accession JX122763), In conclusion, WNV infection appears to be established in
related to the WNV strains circulating in Italy in the years the Mediterranean basin and every year tends to invade new

ª2012 The Authors

Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, E545–E547
CMI
areas. Both lineage 1 and lineage 2 strains circulate in
Europe, including Italy. In the summer of 2011, weather con-
ditions (frequent rainfalls, high temperatures and high relative
humidity) were favourable to mosquito population increase.
Culex pipiens, which was already identified as the vector of
WNV during the 1998 outbreak in Tuscany [20], was proba-
bly the main vector of WNV infection in the Sardinia out-
break. This hypothesis is supported by the WNV isolation
from Cx. pipiens pools collected in the province of Oristano
during last summer [21].
Further studies are needed to clarify the pathogenic
impact of different strains. Thus, integrated human and ani-
mal surveillance is important to monitor the viral spread.
Enhanced vector mosquito control programmes, together
with the implementation of blood transfusion measures, are
necessary to prevent infection transmission.
Of the two cases for which urine samples were available,
WNV could be detected in one case (S58) in urine and
serum (9 days after onset) but not in CSF (4 days after
onset), and in one case (S60) only in urine and CSF but not
in serum 11 days after the onset of the symptoms. Recent
studies detected WNV RNA in the urine of five out of 25
people (20%) tested several years after their initial acute
WNV disease [5]. Moreover, urine samples from the Marche
Region WNV case were positive by RT PCR up to 25 days
after the onset of the disease [14].
Collection and testing of urine samples might be consid-
ered in the national surveillance and control protocols, as
well as for transplant donors. Thus, these data suggest that
tests based on the detection of WNV RNA in serum and/or
CSF may give false-negative results, due to the short dura-
tion of viraemia. The addition of WNV RNA testing in urine,
as well as in plasma and CSF, in the work-up of patients with
suspected WNV infection, may improve the sensitivity of
molecular testing for the diagnosis of WNV infection.
Acknowledgements
The authors thank the staff at the Sardinia Sanitary Italy
agencies for providing clinical specimens. We thank Dr Mon-
ica Meola for technical support.
This research was partially funded by EU grant
HEALTH.2010.2.3.3-3 Project 261391 EuroWestNile.
Transparency declaration
The authors declare that they have no competing interests.
Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, E545–E547
Research Note E547
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