Announcements
Eurosurveillance remains in the updated list of the Directory of Open Access Journals (DOAJ). It was first added to the DOAJ on 9 September 2004. Eurosurveillance is also listed in the Securing a Hybrid Environment for Research Preservation and Access / Rights MEtadata for Open archiving (SHERPA/RoMEO) [2], a database which uses a colour‐coding scheme to classify publishers according to their self‐archiving policy and to show the copyright and open access self-archiving policies of academic journals. Eurosurveillance is listed there as a ‘green’ journal, which means that authors can archive pre-print (i.e. pre-refereeing), post-print (i.e. final draft post-refereeing) and archive the publisher's version/PDF.

Follow Eurosurveillance on Twitter: @Eurosurveillanc

Read our articles on Zika virus infection

Note of concern published for 'Epidemiological investigation of MERS-CoV spread in a single hospital in South Korea, May to June 2015', http://bit.ly/29QFXPp


In this issue


Home Eurosurveillance Edition  2016: Volume 21/ Issue 35 Article 1
Back to Table of Contents
Download (pdf)
Next

Eurosurveillance, Volume 21, Issue 35, 01 September 2016
Rapid communication
Amraoui, Atyame-Nten, Vega-Rúa, Lourenço-de-Oliveira, Vazeille, and Failloux: Culex mosquitoes are experimentally unable to transmit Zika virus

+ Author affiliations


Citation style for this article: 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):pii=30333. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.35.30333

Received:16 August 2016; Accepted:01 September 2016


Outbreaks due to Zika virus (ZIKV) are expanding and affecting most tropical regions [1]. The rapid spread may be related to the efficiency of human-biting Aedes aegypti and Aedes albopictus mosquitoes, which are ZIKV vectors. However, both mosquito species were unexpectedly poorly competent vectors for ZIKV as shown by our laboratory in a previous study [2]. Other factors have been suggested to explain the rapid spread of ZIKV across the Americas [2]: a human population immunologically naive for the newly introduced virus, higher densities of Ae. aegypti or the involvement of other anthropophilic vectors such as Culex mosquitoes. In light of this, we experimentally infected two laboratory colonies of Culex species, Cx. quinquefasciatus and Cx. pipiens, with an Asian genotype of ZIKV and showed an absence of transmission up to 21 days post infection.

Mosquito experimental infections

In May and June 2016, we performed mosquito experimental infections on two laboratory mosquito colonies used in this study: Cx. pipiens collected in Tabarka, Tunisia, in 2010 [3] and Cx. quinquefasciatus collected in San Joaquin Valley in California, United States, in 1950 [4]. The latter is a colony of reference in studies on this mosquito [5]. Testing these colonies experimentally should allow us to determine whether the two species are genetically capable of transmitting ZIKV.

About 200 female mosquitoes of each species were successfully fed, with a total of 188 Cx. pipiens mosquitoes and 170 Cx. quinquefasciatus examined for vector competence. Mosquitoes were orally infected with an Asian genotype ZIKV (strain NC-2014–5132), originally isolated from a patient in New Caledonia in April 2014. The ZIKV strain is phylogenetically closely related to those currently circulating in Brazil [6]. One week-old female mosquitoes were provided with a blood meal containing a suspension of ZIKV [2] at a titre of 107.2 plaque-forming units (PFU)/mL. Engorged females were kept in cardboard containers and maintained at 28 °C with 10% sucrose solution as food. We analysed 40–48 mosquitoes each time at 3, 7, 14 and 21 days post-infection (dpi), to estimate three parameters describing vector competence: (i) infection rate, which measures the proportion of mosquitoes with an infected body (including the midgut) among the number of analysed mosquitoes; this parameter indicates if the mosquito is able to be infected after the infectious blood meal; (ii) dissemination efficiency, which corresponds to the percentage of mosquitoes with an infected head among the number of analysed mosquitoes; it measures the ability of the virus to cross the midgut barrier, penetrate the mosquito haemocoel and infect internal organs; and (iii) transmission efficiency, which estimates the overall proportion of mosquitoes presenting virus in saliva among the number of tested mosquitoes. Head/body homogenates and saliva were titrated by PFU assay on Vero E6 cell monolayers as previously described [7].

Vector competence analysis

To confirm that the mosquitoes had ingested the virus, two engorged mosquitoes from each species were homogenised and the virus was titrated just after blood feeding: the two Cx. pipiens mosquitoes had ingested 6.4 × 104 viral particles and Cx. quinquefasciatus, 9 × 104.

Viral infection rate

Viral infection rates were similar for both Culex populations at 3, 7 and 21 dpi (Fisher’s exact test: p > 0.05); they were respectively 0/42, 1/47 and 5/40 for Cx. quinquefasciatus and 1/48, 3/47 and 6/46 for Cx. pipiens. However, at 14 dpi, 7/41 of the Cx. quinquefasciatus mosquitoes were infected, whereas none of the 47 Cx. pipiens mosquitoes were (Fisher’s exact test, p = 0.003). When estimating the number of viral particles in the mosquito body, no difference was detected between the two mosquito species at each time point (Kruskal–Wallis test, p > 0.05) with higher viral loads detected in both species at 21 dpi: mean of 44 (standard deviation (SD): 60) for Cx. quinquefasciatus and 56 (SD: 90) for Cx. pipiens. Viral loads ranged from 10 to 36 particles for other time points.

Viral dissemination efficiency

Only a few Cx. quinquefasciatus mosquitoes were able to disseminate the virus at 14 dpi (1/41 mosquitoes analysed) and at 21 dpi (3/40). Upon examination of these mosquitoes, no more than 15 viral particles were detected in mosquito heads. For Cx. pipiens, no mosquitoes were detected with virus in the heads.

Viral transmission efficiency

No mosquitoes were found with ZIKV in saliva. Therefore, the tested Cx. quinquefasciatus and Cx. pipiens were able to be infected, Cx. quinquefasciatus only was able to disseminate virus at a low level, and both species were unable to transmit ZIKV up to 21 dpi.

Intrathoracic inoculation of mosquitoes

One batch of 100 one-week-old females of each mosquito species, Cx. quinquefasciatus and Cx. pipiens were inoculated intrathoracically with ca 2,530 PFU of the same ZIKV strain (NC-2014–5132). This dose corresponds to 10 times the maximum number of viral particles detected in mosquitoes analysed for vector competence. Viral dissemination was analysed by estimating viral load in mosquito heads at 3, 7 and 14 dpi. Viral dissemination was observed at 3 dpi (1/23) for Cx. quinquefasciatus, and at 7 dpi (3/21) and 14 dpi (1/24) for Cx. pipiens. No viral transmission (ZIKV in saliva) was detected in either species up to 14 dpi. Thus bypassing the midgut barrier by inoculating a high dose of ZIKV suspension in mosquitoes favoured neither viral dissemination nor transmission.

Background

First discovered in 1947 in Uganda, ZIKV became a major public health concern after its emergence in Yap Island, Micronesia, in 2007 [8] and French Polynesia in 2013–14 [9]. Its arrival in Latin America in 2015 led to a rapid regional spread of outbreaks of ZIKV infection associated with unusually severe effects, Guillain–Barré syndrome [10] and microcephaly in newborns [11]. Up to the first six months of 2016, more than two million people have been infected, in at least 45 countries in Latin America and the Caribbean [12].

The virus (genus Flavivirus, family Flaviviridae) circulated originally in an enzootic cycle between arboreal canopy-dwelling Aedes mosquitoes and non-human primates [13]. In addition to forested habitats, ZIKV has also been isolated in urban settings, with Ae. aegypti being the main vector [14]. Ae. aegypti mainly colonises tropical areas and can share the same regions with Ae. albopictus, which has also succeeded in invading some temperate countries [15].

The aim of our study was to assess the putative role of two mosquito species from the Culex pipiens complex, namely Cx. pipiens and Cx. quinquefasciatus, in ZIKV transmission. Because they are commonly found in temperate and tropical regions [16], respectively, they could strongly increase the risk of urban ZIKV outbreaks occurring.

Discussion

Members of the Cx. pipiens species complex are among the most widely distributed mosquitoes in the world and can act as disease vectors [17]. The species complex comprises several members including Cx. pipiens and Cx. quinquefasciatus, which are the most abundant Culicinae mosquitoes in temperate and tropical regions, respectively [16]. Cx. pipiens is the most ubiquitous mosquito species in temperate regions, occurring in rural and domestic environments [16] and can be found in nature in two biological forms, pipiens and molestus, which are morphologically indistinguishable [18]. The Tabarka strain, used in this study, is a mix of both forms [3] and has been shown to be a primary vector of West Nile virus (WNV) in the Mediterranean basin [19]. Cx. quinquefasciatus is mainly associated with human habitats and can experimentally transmit WNV, making it an ideal vector for domestic/urban transmission of WNV in tropical regions [20]. Our results show that laboratory colonies of Cx. quinquefasciatus and Cx. pipiens were unable to transmit an Asian genotype of ZIKV. Using mosquito colonies for vector competence studies can be considered as a proxy for measuring the genetic ability of one species to transmit a given pathogen [21]. In addition, the experimental ability to transmit a pathogen – vector competence – can vary according to specific combinations of virus and mosquito genotypes, which can be affected by environmental factors such as temperature [22]. The mosquito midgut barrier is the site where the initial steps such as viral attachment, penetration and replication take place before the release of newly produced virions into the mosquito haemocoel. We have shown that bypassing this midgut barrier, by inoculating viral particles into the haemocoel, did not favour viral dissemination nor transmission. Thus, our results strongly suggest that the Cx. quinquefasciatus and Cx. pipiens colonies were unable to transmit ZIKV, as has already been suggested for natural populations of Cx. quinquefasciatus collected during an outbreak of ZIKV infection in Mexico [23] and demonstrated for laboratory colonies of Culex mosquitoes [24,25].

Both mosquito species can tolerate environments highly charged with organic matter and high levels of chemical pollutants including insecticides [26]. Repeatedly confronted with insecticidal molecules, mosquito populations have developed resistance to insecticides, making vector control more difficult [27]. As Aedes and Culex mosquitoes do not share the same breeding sites, control measures targeting each of them are basically different. On the basis of our results, we consider that vector control should continue to focus on larval and adult habitats specific to Aedes mosquitoes, in order to efficiently control ZIKV vectors. While a vaccine is pending, surveillance and vector control should be reinforced against Ae. aegypti and Ae. albopictus, species that are able to transmit dengue virus, chikungunya virus and ZIKV.


Acknowledgements

The authors thank Myrielle Dupont-Rouzeyrol for providing the ZIKV strain and Mylène Weill for the Cx. quinquefasciatus SLab strain. We warmly thank Richard Paul for correcting the manuscript. The study is funded by a FP7 Health 2011 grant ‘Dengue research Framework for resisting epidemics in Europe’ (DENFREE) and the PTR grant no. 528 ‘Risk of reemergence of urban yellow fever in Brazil: role of the invasive mosquito Aedes albopictus. FA was supported by the Institut Pasteur and CAN by the AXA Research Fund.

Conflict of interest

None declared.

Authors’ contributions

FA and CAN designed and performed the research. AVR and RLO participated in producing reagents for mosquito experiments. MV produced viral stocks and performed mosquito inoculations. ABF designed the research, analysed the data and wrote the paper. All authors reviewed the paper.


References

  1. Fauci AS , Morens DM. 2016. Zika Virus in the Americas--Yet Another Arbovirus Threat. N Engl J Med. 2016 Feb 18;374(7):601-4.

  2. 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. DOI: 10.1371/journal.pntd.0004543 PMID: 26938868

  3. Krida G, Rhim A, Daaboub J, Failloux AB, Bouattour A. New evidence for the potential role of Culex pipiens mosquitoes in the transmission cycle of West Nile virus in Tunisia.Med Vet Entomol. 2015;29(2):124-8. DOI: 10.1111/mve.12107 PMID: 25586151

  4. Georghiou GP. Research on mosquito resistance to insecticides at the University of California, Riverside.Proc Pap Annu Conf Calif Mosq Control Assoc. 1966;34:71-3.PMID: 5964233

  5. Pocquet N, Milesi P, Makoundou P, Unal S, Zumbo B, Atyame C,  et al.  Multiple insecticide resistances in the disease vector Culex p. quinquefasciatus from Western Indian Ocean. PLoS One. 2013;8(10):e77855. DOI: 10.1371/journal.pone.0077855 PMID: 24204997

  6. Zanluca C, Melo VC, Mosimann AL, Santos GI, Santos CN, Luz K. First report of autochthonous transmission of Zika virus in Brazil.Mem Inst Oswaldo Cruz. 2015;110(4):569-72. DOI: 10.1590/0074-02760150192 PMID: 26061233

  7. Jupille H, Seixas G, Mousson L, Sousa CA, Failloux AB. Zika Virus, a New Threat for Europe?PLoS Negl Trop Dis. 2016;10(8):e0004901. DOI: 10.1371/journal.pntd.0004901 PMID: 27505002

  8. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS,  et al.  Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009;360(24):2536-43. DOI: 10.1056/NEJMoa0805715 PMID: 19516034

  9. Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP,  et al.  Zika virus, French polynesia, South pacific, 2013. Emerg Infect Dis. 2014;20(6):1085-6. DOI: 10.3201/eid2006.140138 PMID: 24856001

  10. Brasil P, Sequeira PC, Freitas AD, Zogbi HE, Calvet GA, de Souza RV,  et al.  Guillain-Barré syndrome associated with Zika virus infection. Lancet. 2016;387(10026):1482. DOI: 10.1016/S0140-6736(16)30058-7 PMID: 27115821

  11. Duarte Dos Santos CN, Goldenberg S. Zika Virus and Microcephaly: Challenges for a Long-Term Agenda.Trends Parasitol. 2016;32(7):508-11. DOI: 10.1016/j.pt.2016.03.008 PMID: 27105931

  12. United States Centers for Disease Control and Prevention (CDC). All countries & territories with active Zika virus transmission. Atlanta, GA: CDC. [Accessed 16 Aug 2016]. Available from: http://www.cdc.gov/zika/geo/active-countries.html

  13. Diallo D, Sall AA, Diagne CT, Faye O, Faye O, Ba Y,  et al.  Zika virus emergence in mosquitoes in southeastern Senegal, 2011. PLoS One. 2014;9(10):e109442. DOI: 10.1371/journal.pone.0109442 PMID: 25310102

  14. Marchette NJ, Garcia R, Rudnick A. Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia.Am J Trop Med Hyg. 1969;18(3):411-5.PMID: 4976739

  15. 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

  16. Farajollahi A, Fonseca DM, Kramer LD, Marm Kilpatrick A. "Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology.Infect Genet Evol. 2011;11(7):1577-85. DOI: 10.1016/j.meegid.2011.08.013 PMID: 21875691

  17. Ciota AT, Kramer LD. Vector-virus interactions and transmission dynamics of West Nile virus.Viruses. 2013;5(12):3021-47. DOI: 10.3390/v5123021 PMID: 24351794

  18. Tempelis CH. Host-feeding patterns of mosquitoes, with a review of advances in analysis of blood meals by serology.J Med Entomol. 1975;11(6):635-53. DOI: 10.1093/jmedent/11.6.635 PMID: 235647

  19. Amraoui F, Krida G, Bouattour A, Rhim A, Daaboub J, Harrat Z,  et al.  Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region. PLoS One. 2012;7(5):e36757. DOI: 10.1371/journal.pone.0036757 PMID: 22693557

  20. Fall G, Diallo M, Loucoubar C, Faye O, Sall AA. Vector competence of Culex neavei and Culex quinquefasciatus (Diptera: Culicidae) from Senegal for lineages 1, 2, Koutango and a putative new lineage of West Nile virus.Am J Trop Med Hyg. 2014;90(4):747-54. DOI: 10.4269/ajtmh.13-0405 PMID: 24567319

  21. Vazeille-Falcoz M, Mousson L, Rodhain F, Chungue E, Failloux AB. Variation in oral susceptibility to dengue type 2 virus of populations of Aedes aegypti from the islands of Tahiti and Moorea, French Polynesia.Am J Trop Med Hyg. 1999;60(2):292-9. PMID: 10072154

  22. Zouache K, et al. Three-way interactions between mosquito population, viral strain and temperature underlying chikungunya virus transmission potential. Proc Biol Sci. 1792;2014:281.

  23. Guerbois M, Fernandez-Salas I, Azar SR, Danis-Lozano R, Alpuche-Aranda CM, Leal G,  et al.  Outbreak of Zika virus infection, Chiapas State, Mexico, 2015, and first confirmed transmission by Aedes aegypti mosquitoes in the Americas. J Infect Dis. 2016; pii:jiw302. DOI: 10.1093/infdis/jiw302 PMID: 27436433

  24. Aliota MT, Peinado SA, Osorio JE, Bartholomay LC. Culex pipiens and Aedes triseriatus Mosquito Susceptibility to Zika Virus.Emerg Infect Dis. 2016;22(10). DOI: 10.3201/eid2210.161082 PMID: 27434194

  25. Huang YS, Ayers VB, Lyons AC, Unlu I, Alto BW, Cohnstaedt LW, et al. Culex Species Mosquitoes and Zika Virus. Vector Borne Zoonotic Dis. 2016 Aug 24.

  26. Krida G, Rhaiem A, Bouattour A. Effet de la qualité des eaux sur l’expression du potentiel biotique du moustique Culex pipiens L. dans la région de Ben Arous (Sud de Tunis). [Effect of water quality on the expression of biotic potential of the mosquito Culex pipiens L. in the region of Ben Arous (South of Tunis)].Bull Soc Entomol Fr. 1997;102:143-50.French.

  27. Raymond M, Callaghan A, Fort P, Pasteur N. Worldwide migration of amplified insecticide resistance genes in mosquitoes.Nature. 1991;350(6314):151-3. DOI: 10.1038/350151a0 PMID: 2005964



Back to Table of Contents
Download (pdf)
Next

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.