West Nile virus in overwintering mosquitoes, central Europe
Rudolf et al. Parasites & Vectors
West Nile virus in overwintering mosquitoes, central Europe
Ivo Rudolf 3 4
Lenka Betášová 4
Hana Blažejová 4
Kristýna Venclíková 2 4
Petra Straková 3 4
Oldřich Šebesta 4
Jan Mendel 4
Tamás Bakonyi 0 7
Francis Schaffner 5 6
Norbert Nowotny 0 1
Zdeněk Hubálek 3 4
0 Institute of Virology, University of Veterinary Medicine , Vienna , Austria
1 Department of Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences , Dubai Healthcare City, Dubai , United Arab Emirates
2 Institute of Macromolecular Chemistry, v.v.i., Czech Academy of Sciences , Praha , Czech Republic
3 Department of Experimental Biology, Masaryk University , Brno , Czech Republic
4 Institute of Vertebrate Biology, Czech Academy of Sciences , v.v.i, Brno , Czech Republic
5 National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zurich , Zurich , Switzerland
6 Francis Schaffner Consultancy , Riehen , Switzerland
7 Department of Microbiology and Infectious Diseases, University of Veterinary Medicine , Budapest , Hungary
Background: West Nile virus (WNV) is currently the most important mosquito-borne pathogen spreading in Europe. Data on overwintering of WNV in mosquitoes are crucial for understanding WNV circulation in Europe; nonetheless, such data were not available so far. Results: A total of 28,287 hibernating mosquitoes [27,872 Culex pipiens, 73 Anopheles maculipennis (sensu lato), and 342 Culiseta annulata], caught in February or March between 2011 and 2017 in a WNV-endemic region of South Moravia, Czech Republic, were screened for the presence of WNV RNA. No WNV positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in three pools of Culex pipens mosquitoes collected in 2017 at two study sites. Conclusions: To the best of our knowledge, this is the first record of WNV RNA in overwintering mosquitoes in Europe. The data support the hypothesis of WNV persistence in mosquitoes throughout the winter season in Europe.
West Nile fever; West Nile virus; Flavivirus; Hibernation; Overwintering; Culex pipiens; Anopheles maculipennis; Culiseta annulata; Czech Republic
West Nile virus (WNV) is a mosquito-borne virus
(genus Flavivirus; family Flaviviridae) with nearly
cosmopolitan distribution [
]. In nature, it circulates
between birds (as amplifying hosts) and bird-feeding
mosquitoes, in Europe predominantly Culex pipiens [
Humans and horses are considered accidental dead-end
hosts. In 2004 a neuroinvasive lineage 2 WNV (WNV-2)
was discovered for the first time in Europe, i.e. in
southeastern Hungary . Since 2008, an unexpected
explosive spread of this WNV-2 resulted in several hundreds
of human and animal neuroinvasive cases in Hungary
], Austria [
], Greece [
], Serbia [
], and Italy [
In the Czech Republic, four identical strains of WNV-2
were isolated from Cx. modestus mosquitoes collected in
reed beds at South-Moravian fishponds in 2013 [
Overwintering of the introduced WNV-2 in Europe
was assumed (e.g. [
]), yet larger systematic studies
which specifically focus on the persistence of WNV in
overwintering mosquitoes are lacking in a European
WNV endemic area. However, answering this question is
crucial for the understanding of long-term persistence of
WNV and the WNV epidemiology in general in Europe,
especially in northern endemic regions. In contrast, in the
United States, several such investigations were carried out,
and WNV was occasionally detected in diapausing Cx.
pipiens, albeit at very low rates [
A long-term (seven-year) study was therefore
undertaken to examine the prevalence of WNV in
overwintering populations of mosquitoes collected from hibernacula
located in a WNV endemic region in South Moravia. We
performed molecular screening of female Cx. pipiens,
Anopheles maculipennis (s.l.) and Culiseta annulata
mosquitoes (they overwinter as nulliparous females) for WNV
to evaluate the hypothesis of hibernation of WNV in
overwintering mosquitoes in central Europe.
Study sites, mosquito collection and identification
Overwintering female mosquitoes were collected by
battery-operated aspirators (Hausherr’s Machine Works,
Toms River, N.J. 08753, USA) from walls and ceilings of
cellars including wine cellars, pensions, a water tower and
a castle nearby WNV positive study sites [
collections were carried out at localities Sedlec, Lednice,
Hlohovec, Valtice and Břeclav in South Moravia always in
February or March 2011 through 2017 (Table 1). Captured
mosquitoes were transported in closed and chilled
containers to the laboratory where they were identified on
chilled tables under a stereomicroscope, using the
determination key of Becker et al. [
] and then stored for
further processing in freezers at -60 °C. Mosquitoes of the
species Cx. pipiens were not further analysed to
discriminate between f. pipiens and f. molestus.
Mosquito homogenization, RNA extraction, PCR analysis
Mosquito pools of usually 50 (1 to 100) females sorted
by species, year and locality were prepared and
homogenised in 1.5 to 2.0 ml of cooled phosphate-buffered
saline pH 7.4 with 0.4% bovine serum albumin (fraction V)
and antibiotics. RNA was extracted from 150 μl of
cooled mosquito homogenates by using the QIAamp
Viral RNA Mini Kit (Qiagen, Hilden, Germany), according
to the manufacturer’s instructions. The mosquito
homogenates were tested by conventional reverse
transcriptionpolymerase chain reaction (RT-PCR) for flaviviral RNA by
using the protocol published by Scaramozzino et al. [
and the Qiagen OneStep RT-PCR Kit (Qiagen) [
Sequencing of PCR products and bioinformatic analyses
were performed according to a previous study .
A total of 27,872 female overwintering Cx. pipiens in
573 pools, 73 An. maculipennis (s.l.) in 15 pools, and
342 Cs. annulata in 28 pools collected between 2011
and 2017 were tested for the presence of flavivirus RNA.
All mosquito pools from 2011 to 2016 were negative,
while in three Cx. pipiens pools from 2017 WNV nucleic
acid was demonstrated: in specimen 17-06 from
Hlohovec collected on 27th February and in two samples
from Lednice collected on 21st February (nos. 17-12
and 17-18). Sequencing of partial NS5 fragments
(265 nt) of all three strains revealed the closest
similarity to WNV strain 13-104 circulating in the area (97%
nucleotide identity) [
]. All three sequenced strains
were identical to each other. A representative sequence
was deposited in the GenBank database under the
accession number MF162729. None of the pools was
positive for Usutu virus, another important flavivirus
which circulates in the region since 2012 [
causes epizootics in wild and domestic birds in Europe.
WNV surveillance studies carried out periodically from
2006 through 2017 within the framework of European
and national research projects comprising serological
surveys of bird, horse and human populations for WNV
neutralising antibodies as well as attempts to detect
WNV in mosquitoes, horses and humans demonstrated
limited WNV activity in the region [
]. The only
exception was repeated detection of WNV-2 in Cx. modestus
and Cx. pipiens mosquitoes in 2013 (minimum prevalence
rate 0.012%), 2015 (0.099%) and 2016 (0.056%), respectively
], unpublished data).
Regarding overwintering mosquitoes, one strain of
WNV (strain 99-222, type Rabensburg) was isolated
from Cx. pipiens females collected in a mini fortress at
Lanžhot on 13 October 1999, i.e. from mosquitoes
commencing to overwinter [
]. Similarly in Lower Austria,
close to places with diagnosed WNV-2 associated bird
mortalities, three pools of adult female Cx. pipiens
mosquitoes caught during October 2008 in cellars proved positive
for WNV-2 nucleic acid [
]. Vertical transmission of WNV
was confirmed by detecting WNV-2 nucleic acid in one
pool of 15 Cx. pipiens pupae as well as in a pool of two Cx.
pipiens egg rafts during a small-scale entomological survey
carried out close to the home of a WNV-positive blood
donor in Vienna [
Abbreviations: C.p., Culex pipiens, A.m., Anopheles maculipennis (sensu lato), C.a., Culiseta annulata, nd not done
Given the low WNV-2 endemicity in the Czech
Republic until 2013 it is understandable that, at least during the
winter periods 2011 to 2013, no WNV-positive
hibernating mosquitoes were found. Persistent WNV infections
in certain bird species (e.g. as recently demonstrated in an
exotic avian species in human care [
]) may to a lesser
extent also contribute to the circulation of the virus, either
through mosquitoes or oral transmission to predatory
birds. Nonetheless, the classical bird-mosquito cycle
including vertical transmission in mosquitoes is likely to be
the predominant biological cycle of WNV maintenance
and transmission. It is worth mentioning that some
individuals of the examined pools of Cx. pipiens mosquitoes
might belong to f. molestus, which is characterized by
autogeny, stenogamy, anthropophily and facultative diapause
]. Several publications (e.g. [
]) provide clear-cut
genetic evidence that WNV strains, once introduced to
central Europe, are maintained there and disperse to
For the first time, WNV was detected in overwintering
mosquitoes in Europe. This finding supports the
hypothesis of WNV persistence in mosquitoes throughout the
winter season in Europe. As a consequence, it can be
presumed that lineage 2 WNV infection in Europe is
sustained by the long-term persistence of the virus in
mosquitoes followed by vertical transmission and by the
maintenance of the mosquito-bird transmission cycle,
without the necessity of virus re-introductions. However,
further studies are needed to analyse in-depth mode of
circulation, overwintering and vertical transmission of
WNV in central Europe by monitoring overwintering
mosquito species, resident bird and horse populations as
well as humans.
This study was funded by the EU grant FP7–261504 EDENext. The contents
of this paper are the sole responsibility of the authors and do not necessarily
reflect the views of the European Commission. We also thank the Czech
Science Foundation (No. 16-20054S) for financial support. The funding bodies
had no role in study design, collection, analysis, and interpretation of data
and preparation of the manuscript.
Availability of data and materials
All data generated or analysed during this study are included in the article. A
representative sequence generated during the study was deposited in the
GenBank database under the accession number MF162729.
IR and ZH designed the study. LB, HB, KV, PS and OS participated in the
mosquito sampling and determination, LB, HB, KV, PS, OS carried out the
laboratory analyses, TB and JM participated in sequence analysis and the
acquisition of data, analysis and interpretation of results. IR, NN, ZH and FS
drafted the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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