The new Eurosurveillance website is almost here.

Eurosurveillance is on the updated list of the Directory of Open Access Journals and in the SHERPA/RoMEO database. Read more here.

On 6 June 2017, the World Health Organization (WHO) published updates to its ‘Essential Medicines List’ (EML). Read more here.

Follow Eurosurveillance on Twitter: @Eurosurveillanc

In this issue

Home Eurosurveillance Edition  2017: Volume 22/ Issue 2 Article 2
Back to Table of Contents
Previous Download (pdf)

Eurosurveillance, Volume 22, Issue 2, 12 January 2017
Rapid communication
Heitmann, Jansen, Lühken, Leggewie, Badusche, Pluskota, Becker, Vapalahti, Schmidt-Chanasit, and Tannich: Experimental transmission of Zika virus by mosquitoes from central Europe

+ Author affiliations

Citation style for this article: Heitmann A, Jansen S, Lühken R, Leggewie M, Badusche M, Pluskota B, Becker N, Vapalahti O, Schmidt-Chanasit J, Tannich E. Experimental transmission of Zika virus by mosquitoes from central Europe . Euro Surveill. 2017;22(2):pii=30437. DOI:

Received:15 December 2016; Accepted:10 January 2017

In 2015, Zika virus (ZIKV) emerged in Columbia and Brazil and spread rapidly across the American continent and the Caribbean, causing an epidemic with notable numbers of associated clinical cases of microcephaly and Guillain–Barré syndrome [1]. Mosquitoes of the species Aedes aegypti and Ae. albopictus are considered the primary and secondary vectors of ZIKV [2]. However, with transmission rates below 50%, their vector competence for ZIKV in the laboratory is low [3]. The question therefore remains whether other common mosquito species such as Culex spp. play a role in the transmission cycle of ZIKV. The few studies performed so far have provided inconclusive results and suggested that at least Culex quinquefasciatus might be able to transmit ZIKV [4-9]. In addition, for an assessment of the risk of possible spread to regions with temperate climate such as central Europe, information is lacking on ZIKV vector competence of mosquitoes under reduced temperature conditions (< 20°C).

This study aimed to evaluate the vector competence of central European mosquito species for ZIKV. Therefore, German populations of Culex pipiens pipiens biotype pipiens (Cx. p. pipiens), Culex pipiens pipiens biotype molestus (Cx. p. molestus), Culex torrentium and Ae. albopictus (Ae. albopictus, GER) were experimentally infected with ZIKV, using Ae. aegypti and an Italian Ae. albopictus (Ae. albopictus, ITA) as positive controls.

Experimental infection of mosquitoes

Two long-established laboratory strains (Ae. aegypti (Bayer company) and Cx. p. molestus (in colony since 2011, collected in Heidelberg, Germany)) and four species collected in summer 2016 (Cx. p. pipiens F0 (collected in Hamburg, Germany), Culex torrentium F0 (collected in Hamburg, Germany), Ae. albopictus F7 (collected in Freiburg, Germany) and Ae. albopictus F7 (collected in Calabria, Italy)) were analysed and maintained as previously described [10,11]. All colonies tested negative in pan-flavivirus PCRs [12].

Between 150 and 200 female mosquitoes 4–14 days-old were starved for 24 h before application of infectious blood meals containing ZIKV (strain ZIKV_FB-GWUH-2016, GenBank KU870645, fifth passage) [13] at a final concentration of 107 plaque-forming units (PFU)/mL. Artificial feeding was performed using a Hemotek Feeder (Aedes spp.) or by cotton sticks (Culex spp.). Engorged females were incubated at 80% humidity at either 18 °C or 27 °C. Analyses for ZIKV were done 14 and 21 days post infection (dpi) for approximately 35 randomly selected females and twice the number for Ae. aegypti at 27°C. For salivation, mosquitoes were anaesthetised and the proboscises were inserted into cropped 10 µL filter tips containing 10 µL phosphate-buffered saline (PBS). After 30 min, tips were removed and saliva-containing PBS was analysed for the presence of infectious virus particles by measuring its cytopathic effect (CPE) on Vero cells within the following 8 days. ZIKV in the supernatant of cytopathic cells was confirmed by qRT-PCR using Real Star Zika Virus RT-PCR Kit (Altona diagnostics, Hamburg, Germany). In addition, bodies of all challenged mosquitoes, excluding legs and wings, were analysed for ZIKV RNA by qRT-PCR.


At 14 or 21 dpi, ZIKV RNA was detected in the bodies of all challenged mosquito taxa, with infection rates ranging between 3 and 72% in the species–temperature combinations with ZIKV-positive bodies. Infection rates and virus titres were substantially higher in Aedes species, with viral RNA copies ranging from 102 to 104 in Culex spp. and from 104 to 109 in Aedes spp. (Table).


Susceptibility and transmission rates of mosquitoes experimentally infected with Zika virus (n = 856)

14 days post infection 21 days post infection
Mosquito taxa T in °C IRa
Mean (SD) log10
RNA copies/specimenb
Mean (SD) log10
RNA copies/specimen
Aedes aegypti 18 17/31
0/17 18/33
27 31/63
Aedes albopictus, ITA 18 19/30
0/19 14/39
27 22/31
Aedes albopictus, GER 18 4/32
0/4 11/32
27 20/31
Culex p. molestus 18 12/41
0/12 2/32
27 7/29
0/7 12/38
Culex p. pipiens 18 16/34
0/16 3/32
27 3/37
0/3 0/35
Culex torrentium 18 11/35
0/11 1/38
27 4/36
0/4 0/34

GER: from Germany; IR: infection rate; ITA: from Italy; NA: not available; SD: standard deviation; T: temperature; TR: transmission rate.

a Infection rate: number of ZIKV-positive mosquito bodies per number of fed females.

b RNA copies were averaged over all ZIKV-positive mosquito bodies excluding the zeros of ZIKV-negative mosquito bodies.

c Transmission rate: number of mosquitoes with ZIKV-positive saliva per number of ZIKV-positive mosquito bodies.

d Not available: Mean viral RNA copies and transmission rate could not be calculated for the species–temperature combinations with no ZIKV-positive bodies.

Virus load was generally higher at elevated incubation temperature (27 °C vs 18 °C). However, transmission of infectious virus particles as measured by CPE of Vero cells incubated with mosquito saliva was not detected in any of the Culex taxa. In contrast, saliva was positive for infectious virus particles in all Aedes species, but only at 27 °C incubation temperature. Interestingly, transmission rates at 21 dpi were similar in Ae. aegypti and Ae. albopictus from Germany but were substantially lower in Ae. albopictus from southern Italy (30% vs 13%).


Culex species from central Europe are known as established vectors, able to transmit numerous viruses including West Nile, Sindbis and Usutu virus [14,15]. The results presented here indicate that the three most common Culex taxa in central Europe (Cx. p. pipiens, Cx. p. molestus and Cx. torrentium) do not have vector competence for ZIKV. This is in agreement with results from other parts of the world including Italy [4-7,9], which all showed a low degree of compentence of the Cx. pipiens complex for ZIKV transmission.

The invasive mosquito Ae. albopictus is established in large parts around the Mediterranean Sea and is considered to be the main vector in Europe for autochthonous human infections with chikungunya and dengue virus [16]. Aedes albopictus are regularly introduced into Germany as accidental cargo via road traffic from southern Europe [17]. In the winter 2015/16, successful overwintering of the species was observed for the first time in southern Germany [18]. The results presented here indicate that specimens of this overwintering population have considerable susceptibility to ZIKV, although only at elevated temperature of 27 °C. Moreover, the transmission rate in this overwintering population was substantially higher than in Ae. albopictus from the Calabrian region in southern Italy. Whether the difference in virus susceptibility between German and Italian Ae. albopictus populations is due to an ongoing process of adaptation to a new environment or to experimental conditions remains to be determined. Nevertheless, the susceptibility of European Ae. albopictus to ZIKV demonstrates the risk of arbovirus transmission associated with the establishment and ongoing spread of this invasive mosquito species in Europe. Of note, none of the tested Aedes populations were susceptible to ZIKV at 18 °C, which may limit the spread of ZIKV in central Europe to short summer periods with high temperatures. However, for a comprehensive risk assessment of ZIKV transmission in central Europe, further infection studies are needed at intermediate temperatures (e.g. 21 °C and 24 °C) as well as with other common Aedes species such as Ae. vexans or the newly established Ae. japonicus [19].


We thank Ella Weinert and Michelle Helms for excellent technical assistance and Jessica Börstler for analyses of Culex mosquitoes. ML was supported by the Leibniz Association, grant number SAW-2014-SGN-3. This work was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for Agriculture and Food (BLE) with the grant number 28-1-91.048-15.

Conflict of interest

None declared.

Authors’ contributions

Conceived and designed the study: AH, SJ, RL, JSC, ET. Performed the data collection: AH, SJ, ML. Analysed the data: AH, SJ, RL, JSC, ET. Provided the ZIKA virus strain: OV. Provided mosquito specimens: MB, BP, NB. Wrote the paper: AH, SJ, RL, ET. Contributed to the manuscript drafting: ML. All authors read and approved the final version of the manuscript.


  1. Musso D, Gubler DJ. Zika Virus.Clin Microbiol Rev. 2016;29(3):487-524.DOI: 10.1128/CMR.00072-15 PMID: 27029595

  2. World Health Organization Regional Office for Europpe (WHO/Europe). Zika virus technical report. Interim risk assessment WHO European Region. Copenhagen: WHO/Europe; 2016. Available from:

  3. Chouin-Carneiro T, Vega-Rua A, Vazeille M, Yebakima A, Girod R, Goindin D,  et al.  Differential susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika virus. PLoS Negl Trop Dis. 2016;10(3):e0004543.

  4. Aliota MT, Peinado SA, Osorio JE, Bartholomay LC. Culex pipiens and Aedes triseriatus Mosquito susceptibility to Zika virus.Emerg Infect Dis. 2016;22(10):1857-9.DOI: 10.3201/eid2210.161082 PMID: 27434194

  5. Amraoui F, Atyame-Nten C, Vega-Rúa A, Lourenço-de-Oliveira R, Vazeille M, Failloux AB. Culex mosquitoes are experimentally unable to transmit Zika virus.Euro Surveill. 2016;21(35):30333.DOI: 10.2807/1560-7917.ES.2016.21.35.30333 PMID: 27605159

  6. Boccolini D, Toma L, Di Luca M, Severini F, Romi R, Remoli ME,  et al.  Experimental investigation of the susceptibility of Italian Culex pipiens mosquitoes to Zika virus infection. Euro Surveill. 2016;21(35):30328.DOI: 10.2807/1560-7917.ES.2016.21.35.30328 PMID: 27605056

  7. Fernandes RS, Campos SS, Ferreira-de-Brito A, de Miranda RM, Barbosa da Silva KA, de Castro MG,  et al.  Culex quinquefasciatus from Rio de Janeiro Is Not Competent to Transmit the Local Zika Virus. PLoS Negl Trop Dis. 2016;10(9):e0004993.

  8. Guo XX, Li CX, Deng YQ, Xing D, Liu QM, Wu Q,  et al.  Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus. Emerg Microbes Infect. 2016;5(9):e102.DOI: 10.1038/emi.2016.102 PMID: 27599470

  9. Huang Y-JS, Ayers VB, Lyons AC, Unlu I, Alto BW, Cohnstaedt LW,  et al.  Culex species mosquitoes and Zika virus. Vector Borne Zoonotic Dis. 2016;16(10):673-6.DOI: 10.1089/vbz.2016.2058 PMID: 27556838

  10. Vogels CBF, van de Peppel LJJ, van Vliet AJH, Westenberg M, Ibañez-Justicia A, Stroo A,  et al.  Winter activity and aboveground hybridization between the two biotypes of the West Nile virus vector Culex pipiens. Vector Borne Zoonotic Dis. 2015;15(10):619-26.DOI: 10.1089/vbz.2015.1820 PMID: 26394124

  11. Leggewie M, Badusche M, Rudolf M, Jansen S, Börstler J, Krumkamp R,  et al.  Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection. One Health. 2016;2:88-94.DOI: 10.1016/j.onehlt.2016.04.001

  12. Chao D-Y, Davis BS, Chang G-JJ. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes.J Clin Microbiol. 2007;45(2):584-9.DOI: 10.1128/JCM.00842-06 PMID: 17108075

  13. Driggers RW, Ho C-Y, Korhonen EM, Kuivanen S, Jääskeläinen AJ, Smura T,  et al.  Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Engl J Med. 2016;374(22):2142-51.DOI: 10.1056/NEJMoa1601824 PMID: 27028667

  14. Jöst H, Bürck-Kammerer S, Hütter G, Lattwein E, Lederer S, Litzba N,  et al.  Medical importance of Sindbis virus in south-west Germany. J Clin Virol. 2011;52(3):278-9.DOI: 10.1016/j.jcv.2011.08.002 PMID: 21893428

  15. Nikolay B. A review of West Nile and Usutu virus co-circulation in Europe: how much do transmission cycles overlap?Trans R Soc Trop Med Hyg. 2015;109(10):609-18.DOI: 10.1093/trstmh/trv066 PMID: 26286946

  16. Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H,  et al.  A review of the invasive mosquitoes in Europe: ecology, public health risks, and control options. Vector Borne Zoonotic Dis. 2012;12(6):435-47.DOI: 10.1089/vbz.2011.0814 PMID: 22448724

  17. Becker N, Geier M, Balczun C, Bradersen U, Huber K, Kiel E,  et al.  Repeated introduction of Aedes albopictus into Germany, July to October 2012. Parasitol Res. 2013;112(4):1787-90.DOI: 10.1007/s00436-012-3230-1 PMID: 23242268

  18. Pluskota B, Jöst A, Augsten X, Stelzner L, Ferstl I, Becker N. Successful overwintering of Aedes albopictus in Germany.Parasitol Res. 2016;115(8):3245-7.DOI: 10.1007/s00436-016-5078-2 PMID: 27112761

  19. Huber K, Schuldt K, Rudolf M, Marklewitz M, Fonseca DM, Kaufmann C,  et al.  Distribution and genetic structure of Aedes japonicus japonicus populations (Diptera: Culicidae) in Germany. Parasitol Res. 2014;113(9):3201-10. 25056941DOI: 10.1007/s00436-014-4000-z

Back to Table of Contents
Previous Download (pdf)

The publisher’s policy on data collection and use of cookies.

Disclaimer: The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the editorial team or the institutions with which the authors are affiliated. Neither ECDC nor any person acting on behalf of ECDC is responsible for the use that might be made of the information in this journal. The information provided on the Eurosurveillance site is designed to support, not replace, the relationship that exists between a patient/site visitor and his/her physician. Our website does not host any form of commercial advertisement. Except where otherwise stated, all manuscripts published after 1 January 2016 will be published under the Creative Commons Attribution (CC BY) licence. You are free to share and adapt the material, but you must give appropriate credit, provide a link to the licence, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use.

Eurosurveillance [ISSN] - ©2007-2016. All rights reserved

This website is certified by Health On the Net Foundation. Click to verify. This site complies with the HONcode standard for trustworthy health information:
verify here.