ARTICLES
West Nile encephalitis epidemic in southeastern Romania
T F Tsai, F Popovici, C Cernescu, G L Campbell, N I Nedelcu, for the Investigative Team*
Summary
Background West Nile fever (WNF) is a mosquito-borne
flavivirus infection endemic in Africa and Asia. In 1996, the
first major WNF epidemic in Europe occurred in Romania,
with a high rate of neurological infections. We investigated
the epidemic to characterise transmission patterns in this
novel setting and to determine its origin.
Methods Hospital-based surveillance identified patients
admitted with acute aseptic meningitis and encephalitis in
40 Romanian districts, including Bucharest. Infection was
confirmed with IgM capture and indirect IgG ELISAs. In
October, 1996, we surveyed outpatients in Bucharest and
seven other districts to estimate seroprevalence and to
detect infected patients not admitted to hospital. We also
measured the rates of infection and seropositivity in
mosquitoes and birds, respectively.
Results Between July 15 and Oct 12, we identified 393
patients with serologically confirmed or probable WNF
infection, of whom 352 had acute central-nervous-system
infections. 17 patients older than 50 years died.
Fatality/case ratio and disease incidence increased
with age. The outbreak was confined to 14 districts in the
lower Danube valley and Bucharest (attack rate
12·4/100 000 people) with a seroprevalence of 4·1%. The
number of mild cases could not be estimated. WN virus
was recovered from Culex pipiens mosquitoes, the most
likely vector, and antibodies to WN virus were found in 41%
of domestic fowl.
Interpretation The epidemic in Bucharest reflected
increased regional WNF transmission in 1996. Epidemics
of Cx pipiens-borne WNF could occur in other European
cities with conditions conducive to transmission.
Lancet 1998; 352: 767–71
See Commentary page xxx
Division of Vectorborne Infectious Diseases, Centers for Disease
Control and Prevention, Fort Collins, CO 80522, USA (T F Tsai MD,
G L Campbell MD); Bucharest Preventive Medicine Centre,
Bucharest, Romania (F Popovici MD); Institute of Virology,
Bucharest (Prof C Cernescu MD); and Ministry of Health, Bucharest
(N I Nedelcu MD)
Correspondence to: Dr T F Tsai
THE LANCET • Vol 352 • September 5, 1998
Introduction
Mosquito-borne West Nile fever (WNF) is an endemic
febrile illness in Africa, the Middle East, and
southwestern Asia. The flavivirus has also been isolated in
Australia and sporadically in Europe but fewer cases in
human beings have been recognised. Clinical features are
acute fever with severe myalgia, arthralgia, and headache,
conjunctivitis, prominent lymphadenopathy, and a
roseolar rash, complicated, occasionally, by meningitis or
encephalitis.1–5 Outbreaks with hundreds of cases were
reported from Israel in 1950–57. In 1974, an epidemic in
a 2500 km2 area of central South Africa produced tens of
thousands of infections.6,7 Except for a small outbreak in
southern France in 1962, WNF has not been a public-
health threat in Europe.8 Between July and October,
1996, an epidemic of WNF with mainly neurological
infections occurred in Bucharest and the lower Danube
valley of southeastern Europe. We described after a field
investigation from Sept 28 to Oct 11, 1996, the epidemic,
its transmission patterns, and its likely source.
Methods
Romania is divided by the Carpathian range into a region of
mountains and high plains to the northwest and a large plain to
the south, with the Danube river forming most of the southern
border with Bulgaria. Bucharest, the capital city, consists of six
administrative sectors and is surrounded by the Ilfov District
(figure 1). The national and Bucharest municipal populations
were about 23 million and 2·3 million, respectively, in 1996.
In mid-August, 1996, unusually high numbers of acute
central-nervous-system infections were reported by clinicians
from the two infectious-disease hospitals in Bucharest. In early
September, the Centre for Medico-Military Scientific Research,
Bucharest, found WN virus haemagglutination-inhibition
antibodies in the blood of several patients, and the diagnosis was
confirmed by the WHO Collaborating Centre for Haemorrhagic
Fevers and Arboviruses, Pasteur Institute, Paris, France.
On Aug 20, the Ministry of Health started active surveillance
in Bucharest. Suspected cases of WNF were defined as patients
admitted to hospital with acute aseptic meningitis,
meningoencephalitis, or encephalitis with pleiocytosis. Clinical
and epidemiological data were collected and serum and
cerebrospinal-fluid samples were obtained. Active hospital-based
surveillance was expanded nationwide on Sept 10.
Serosurveys were done between Oct 2 and Oct 4 in Bucharest
and on Oct 28 and Oct 29 in seven other affected districts to
estimate the incidence of acute symptomless and mild WN viral
infection. Samples were obtained from blood taken for other
medical purposes in general clinics. The numbers of samples
were proportional to the sizes of clinic catchment areas and the
age distributions in each of the Bucharest clinics, or to the
catchment areas of the non-Bucharest clinics. In the Bucharest
serosurvey, histories were taken of recent illnesses, including
fever, myalgia, headache, rash, joint pain, lymphadenopathy,
jaundice, respiratory, gastrointestinal, and central-nervous-
system symptoms and signs. Symptoms among recently infected
IgM-positive respondents were compared with those of
seronegative controls matched for age and sex.
To ascertain whether there had been previous WN viral
transmission in the region, serum samples collected in
Bucharest, Tulcea, and Dolj for influenza serology during
1993–96, before the WNF epidemic began, were tested for WN
viral antibodies.
1
ARTICLES
Ukraine
Hungary
Arges
Dimbovita
Bucuresti
Olt
Dolj
Teleorman
Danube
Figure 1: WN encephalitis incidence by district, 1996
Viral isolation in cell cultures, suckling mice, or both was
attempted with 106 serum, cerebrospinal fluid, or necropsy
samples from patients with acute infection; in addition, six
serum and 22 cerebrospinal fluid samples were assessed by PCR
for WN virus genome. Extracted RNA was reverse transcribed
and cDNA was amplified with primers directed against a
conserved flaviviral envelope sequence; PCR products were
identified by hybridisation with a probe specific for WN virus.9
Viral isolates were identified by PCR, and for a cerebrospinal
fluid isolate (96-1030) antigenically, by comparison of its
reactivity with that of prototype WN virus (B956) in plaque-
reduction neutralisation tests against reactivity with reference
polyclonal antibodies (dengue 1–4, WN, yellow fever, and
Russian spring-summer encephalitis viruses), monoclonal
antibodies (central European and Russian encephalitis viruses),
and 11 serum samples taken during convalescence after WNF.10
Serum and cerebrospinal fluid samples were tested for IgM
and IgG antibodies to WN virus by capture and indirect EIAs,
respectively.10 IgM in clinical samples was captured with an
antibody to µ-chain, WN virus and control antigens were added,
and bound antigen was detected with a peroxidase-labelled
antiflavivirus antibody. IgG was measured by the addition of
samples to plates coated with WN virus and control antigens in
alternate wells, and bound IgG was detected with a peroxidase-
labelled antibody to human IgG. Acute WN virus infection was
confirmed if IgM, IgG, or both classes of antibody to WN virus
were present in cerebrospinal fluid, or if there was a conversion
in paired serum samples from negative to positive in IgM or IgG
EIA. Patients with specific IgM in a single serum sample were
classified as having a probable recent infection, and patients with
(specific) IgG only in a single serum sample as having had a past
infection. Because tick-borne encephalitis is the principal
flavivirus infection transmitted in Europe, a subgroup of patients
was tested for cross-reactive antibodies to central European
encephalitis virus.
Mosquito and avian surveys were done between Oct 1 and
Oct 9 at 13 sites in Bucharest and the Ilfov District. Adult
mosquitoes, collected by aspiration from resting sites, were
identified and pooled, and ground suspensions were inoculated
2 THE LANCET • Vol 352 • September 5, 1998
Incidence/100 000
0
0·1–1·0
1·1–10·0
Bucharest 12·4
Moldavia
Galati Ukraine
Buzau
Prahova Braila
Tulcea
Ialomita
Calarasj Black Sea
Giurgi Constanta
r
ve
Ri
Bulgaria
on to Vero cells for viral isolation by standard methods.10
Because WNF virus is transmitted in a mosquito-avian cycle,
blood samples from wild and domestic birds were tested for the
presence of neutralising antibody to WN virus by plaque-
reduction assay.10 The Ministry of Agriculture was asked for
notifications of equine encephalitis cases.
Results
Of 835 patients admitted to hospital with suspected
central-nervous-system infection and reported under the
surveillance system, 767 (92%) met the definition of
WNF cases. Appropriate blood, cerebrospinal fluid, and
necropsy samples were available from 441 patients,
among whom WN encephalitis was serologically
confirmed in 352 (80%). In addition, among 68 patients
who did not meet the case definition, 41 (60%) had
laboratory-confirmed or probable infection. A total of
393 patients had laboratory-diagnosed WNF.
Among these 393 patients, the neurological diagnoses
were meningitis (40%), meningoencephalitis (44%), and
encephalitis (16%). The onset of illness was typically
abrupt, with fever (91% of patients), acute headache
(77%), neck stiffness (57%), vomiting (53%), chills
(45%), and confusion (34%). Disorientation, disturbed
consciousness, and generalised weakness were the
predominant signs in patients with encephalitis; some
patients had decreased motor power with hypotonia, and
others had increased tone, hyper-reflexia, and abnormal
reflexes. Ataxia and extrapyramidal signs were recorded
in 17% of patients, and cranial-nerve palsies or seizures in
smaller proportions. The illness progressed to coma in
13% of cases, and among patients with confirmed or
probable infection there were 17 deaths (fatality/case ratio
4·3%), all in patients older than 50 years. Age-specific
fatality/case ratios increased substantially with age, from

50 Clinical incidence
40
Incidence per 100 000
30
20
10
0 0
Seroprevalence
Seroprevalence (%)
10
5 laboratory-confirmed infections lived in urban areas. In
0 the rural Ilfov District (28/100 000) than in urban areas
9 9 9 9 9 9
0– –1 –2 –3 –4 –5
10 20 30 40 50
Age-group (years)
Figure 2: Age-specific incidence and viral-specific IgM
seroprevalence of WN encephalitis, Bucharest, 1996
zero in patients younger than 50 years, to 3·4% in those
aged 50–59 years, 4·3% in those aged 60–69 years, and
14·7% in those older than 70 years, without significant
differences between men and women. Age-specific attack
rates also increased with age (figure 2).
Among the 41 patients with WN-virus infections who
did not meet the case definition, the diagnoses were acute
respiratory infection (11), acute febrile illness (three),
monoparesis (one), cerebrovascular disease (one),
urinary-tract infection (one), various chronic illnesses
(four), and no diagnosis (20). The 326 patients who met
the case definition but lacked appropriate samples for
testing differed significantly from patients with
appropriate samples in age, clinical diagnosis, residence,
onset dates, and fatality/case ratio (11%), which was
consistent with logistical difficulties in obtaining
appropriate samples from patients who died soon after
admission to hospital. These differences suggest that
untested cases were heterogeneous, and analysed only the
393 patients with laboratory-confirmed infection.
The onset of illness in the earliest confirmed case was
July 15, 1996. The epidemic peaked in the first week of
September and the last confirmed case had onset on
Oct 12 (figure 3). The first confirmed cases were reported
from Bucharest, and in other districts 3 weeks later.
The incidence of cases in the 15 districts in the
epidemic area was four per 100 000 people in the general
population, without significant differences between the
sexes. The epidemic was confined to the Danube plain,
southeast of the Carpathian range and, excluding
Bucharest, the rate of infection was highest in districts
bordering the Danube River (figure 1). The attack rate
for Bucharest, 12·4 per 100 000, was higher than that for
all other districts, in which the mean rate of infection was
1·5 per 100 000. 240 (61%) of 393 patients with
THE LANCET • Vol 352 • September 5, 1998
ARTICLES
100
80
60
Cases
40
20
8 5 2 9 5 2 9 6 2 9 6 3 0 7 4
ly 1 2 2 g 1 1 2 t t 1 2 3 t 1
Ju July July July Au Aug Aug Aug Sep Sep ept ept ept Oc Oct
S S S
Week of onset
Figure 3: WN encephalitis cases by week of onset, Romania
Bucharest, however, the rate was significantly higher in
of the region (ten/100 000). In other districts more
9 70 patients who lived in rural areas were infected, but rates
–6 ⭓
60 could not be calculated.
WN virus genomic sequences were not detected in any
serum or cerebrospinal-fluid samples. A cerebrospinal-
fluid viral isolate (96-1030) recovered in cell cultures was
identified as WN virus by PCR. The virus was
antigenically indistinguishable from prototype WN virus
in plaque-reduction neutralisation-test assays with
reference polyclonal and monoclonal flavivirus
antibodies. In 11 human serum samples taken during
convalescence, however, neutralisation antibody titres
were consistently higher for the local WN viral isolate,
with a geometric mean ratio of 3·1; therefore, the isolate
could be considered a variety of WN virus.
No serological cross-reactivities were detected against
central European encephalitis virus in 11 serum samples
with IgM antibodies to WN virus (geometric mean titre
10 500) or in eight serum samples with titres of IgG
antibody to WN virus of 1:400.
Serosurveys showed higher proportions of outpatients
with IgG antibodies in Bucharest (39 [4·1%] of 959) than
in the other seven districts (eight [0·9%] of 868). In
Bucharest, the proportions of outpatients with IgG and
IgM antibodies were similar in both sexes in all age-
groups and regions of the city (data not shown). This
finding suggests a uniform risk of infection among city
residents (figure 2). At the time of the serosurvey, 2
months after the epidemic peak, only 18 of the 39
Bucharest residents with IgG antibodies also had
detectable IgM (1·9% of the sample). By extrapolation of
IgM and IgG prevalence rates to the general population,
we calculated that 43 000–96 000 people were infected
during the epidemic in Bucharest and 31 000 in the other
surveyed districts. The estimated clinical-to-subclinical
infection ratio was one to 140–320 (Bucharest data). In
the Bucharest survey, 31 recently infected people and 37
seronegative controls reported similar frequencies of
minor illness symptoms.
Among serum samples collected before the epidemic
(151 from Bucharest, 31 from Tulcea, 40 from Dolj), IgG
antibodies to WN virus were found in four Dolj samples.
3
ARTICLES
Sylvatic cycle Urban cycle
?Cx Cx Human
modestus pipiens beings
Africa
Middle Birds
Birds Birds
East
?Cx Cx
modestus pipiens
Figure 4: Sylvatic and urban transmission cycles in lower
Danube valley and model by which epidemic occurred
Culex pipiens was the predominant mosquito species in
Bucharest. All investigated sites were heavily infested,
including houses, poultry sheds in the rural Ilfov District,
and especially apartment complexes, many of which had
leaking pipes and standing water in basements. 3689
adult mosquitoes of seven taxa were collected, 94% of
which were Cx pipiens. A virus recovered from a pool of
Cx pipiens collected in central Bucharest was identified
antigenically as WN virus (H Savage, N Karabatsos,
unpublished observation), which resulted in a minimum
infection rate of 0·3/1000.
Chickens and other domestic fowl were commonly kept
at private residences in Bucharest. Neutralising
antibodies to WN virus were detected in 30 (41%) of 73
domestic fowl and in one (8%) of 12 wild birds. No
increase in equine encephalitis cases was reported.
Discussion
Although WNF is recognised in Africa, Asia, and Europe,
encephalitis cases have been reported previously only
from Israel, India, and Pakistan.1–7,11,12 Transmission of
WN virus in at least 11 European countries (France,
Cyprus, former Czechoslovakia, Greece, Italy, Portugal,
Romania, Spain, Turkey, former Yugoslavia, and Russia)
has been surmised from animal and human serosurveys
and by the occasional isolation of the virus from human,
horse, wild bird, mosquito (Aedes cantans, Anopheles
maculipennis, and Cx modestus), and tick (Hyalomma
plumbeum and H asiaticum) sources.1–3 With the exception
of a small outbreak in the Rhone delta (La Camargue),
France, however, cases of WNF have not been recognised
in Europe.8 Cases may have escaped detection because of
the mild and non-specific symptoms. WN is only rarely
complicated by hepatitis, pancreatitis, or encephalitis.11–14
The epidemic in Romania in 1996 was the first
important outbreak of WNF recorded in Europe. The
true number of cases and deaths may have been higher
than recorded because national surveillance was delayed,
and because appropriate clinical samples were available
from few patients. The paucity of acute-phase samples
probably contributed to the poor yield of viral recoveries.
The predominance of neurological cases reflected
hospital-based surveillance and the lack of case-finding
efforts in outpatient clinics. Confirmed infections in
patients not meeting the case definition probably
represented a mixture of coincidental WN virus infection
and other manifestations of the illness, such as myelitis
and respiratory infection.15,16 We attempted to identify
milder infections retrospectively in the Bucharest survey.
With a 6-week delay after the epidemic peak and the non-
specific nature of symptoms, however, we found no
difference in minor symptoms between recently infected
4 THE LANCET • Vol 352 • September 5, 1998
and seronegative respondents. Furthermore, even if mild
illnesses had been ten times more frequent than
neurological cases (for an extrapolated incidence of
124/100 000 people in the city), even with 959
respondents, we lacked sufficient statistical power to
exclude a much larger epidemic of mild illness over and
above the outbreak of neurological infections.
The Bucharest serosurvey found nearly identically low
infection rates in every age-group, whereas rates of
neurological disease increased substantially with age
(figure 2). The clinical/subclinical infection ratio
(1:140–320) showed that higher rates of central-nervous-
system infection in the elderly were due to host
susceptibility rather than exposure. Closely related
neurotropic flavivirus infections, such as St Louis
encephalitis and Japanese encephalitis, have similar
patterns, but the pathogenesis of age-related susceptibility
has not been elucidated.17
The uniform seroprevalence rates in all age-groups also
suggest that the virus had not been present previously in
this population or, if infections had occurred before, that
they were rare. In the seven non-Bucharest districts
surveyed, low seroprevalence rates suggested the absence
of widespread endemic transmission. The absence of
antibodies to WN virus among Bucharest residents
sampled before the outbreak was further evidence of a
new epidemic in the city. In this susceptible population,
the epidemic affected an extensive area of southeastern
Romania and, from anecdotally reported cases in
Bulgaria, an even larger area of the Danube delta. Better
case-finding probably contributed to the reported high
attack rate in Bucharest, but viral transmission may also
have been more intense in the urban and periurban Cx
pipiens-borne cycles. The absence of cases of WNF north
of the Carpathian range suggests that these mountains
were a barrier against further spread and transmission.
Sporadic cases of encephalitis, serologically diagnosed
as WN virus infection, have been suspected previously in
Romania.18–20 In addition, surveys in the lower Danube
valley have shown haemagglutination-inhibition antibody
rates to be as high as 18% among certain human
populations and 53% among migratory birds, although
WN virus was never isolated.21–23
Field studies have not differentiated between the
existence of a local WN virus transmission cycle,
although this may be plausible, and repeated viral
introductions by migratory birds, especially from Africa.
Reference hyperimmune antibodies did not distinguish
the locally recovered (human) WN virus isolate from
prototype WN virus, although a slight but consistent
antigenic difference was seen in serum samples from
recovering patients, which shows that the local strain
could be a distinct variety of WN virus.
Genetic analysis of WN viral strains recovered from
France showed their relation to a viral lineage also
including certain strains from Africa and Kunjin virus, a
flavivirus transmitted mainly in Australia and Asia, which
also produces a febrile exanthem and encephalitis.24
Introduction of a virus from Africa to Europe by
migratory birds has precedence in the transfer of Sindbis
virus from South Africa to northern Europe and the
emergence of Sindbis fever epidemics in Scandinavia in
1982.25–26 By analogy, sporadic isolations of WN virus
from migratory birds and their ticks in Europe show that
this virus could also be transferred in spring
migrations.1–3,8

In Africa, WN virus is principally transmitted in an
enzootic cycle among Cx univittatus and birds.6 Many
northward migratory routes follow a flyway across the
Middle East, Turkey, and the Black Sea, along which the
Danube delta is a main avian refuge.27 Frequent
introductions of WN virus, with establishment of a local
enzootic transmission in the Danube basin seem possible.
Cx modestus, identified as an enzootic WN virus vector in
southern France, is present in the Danube delta and
could mediate local sylvatic transmissions, from which
the virus could be transferred to an urban cycle or could
infect humans directly (figure 4). Such interactions of
sylvatic and urban transmission cycles have been reported
previously in the USA for St Louis encephalitis virus.17
Few mosquitoes were collected because of the lateness
of the season and only one viral isolate was recovered
from mosquitoes. Although the minimum infection rate
in Cx pipiens was only 0·3 per 1000, this species was
probably the epidemic vector in Bucharest at least.
Cx pipiens is the predominant mosquito species in the
city; apartment blocks and houses were heavily infested,
as well as domestic environments in rural locations where
apparently the mosquitoes feed readily on human beings
and birds. WN virus has been isolated previously from
the species and its capacity to transmit the virus has been
shown.1–3 The mosquito potentially could have
transmitted WN virus from person to person within
apartment buildings where flooded basements provide
favourable breeding conditions for Cx pipiens.3,28 To
prevent future outbreaks we recommended actions to
control Cx pipiens in Bucharest.
The other circumstances that favoured an epidemic of
WNF in Romania in 1996 are unknown. WNF outbreaks
in North Africa in 1994 and 1996 suggest a regional
increase in enzootic viral transmission.29,30 In some areas
of Europe, urban conditions are suitable for the
introduction and amplification of WN virus, with the risk
of future epidemics that follow the pattern of infrequent
but recurrent Cx pipiens-borne St Louis encephalitis
epidemics in cities of the eastern USA.17,31
Members of the Investigative Team
Romania—V Laurentia, L Spantulescu, Bucharest Preventive Medicine
Center, Bucharest; V Chitu, S Ruta, G Tardei, Institute of Virology,
Bucharest; D Crăciun, D Nicolaiciuc, D Pit‚igoi, Ministry of Health,
Bucharest; C Ceianu, G Nicolescu, A Ungureanu, L A Vladimirescu,
Cantacuzino Institute, Bucharest.
France—V Deubel, B LeGuenno, Centre National de Reference pour les
fievres Hemorragiques et les Arbovirus, Paris, France.
USA—L Han, H Savage, L Tengelsen, Centers for Disease Control and
Prevention, Fort Collins; EA Henchal, F Knauert, JA Mangiafico,
C Rossi, United States Army Medical Reasearch Institute of Infectious
Diseases, Fort Detrick.
Contributors
T F Tsai was responsible for guiding the epidemic investigation, data
analysis, and preparation of the paper. Florin Popovici managed the
databases and data analysis. Costin Cernescu supervised the laboratory
investigation. Grant Campbell supervised the field team and assisted in
data analysis. Sergio Nedelcu coordinated surveillance and administered
overall operations.
Acknowledgments
We thank Terry Gay, Office of International and Refugee Health,
Department of Health and Human Services, USA Public Health Service
for administrative assistance, and Peter LaPera, Randall Thompson, and
Mary Ann Micka of US Agency for International Development for
support.
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