ABSTRACT

Objective.

To identify studies on the competence of Culex mosquitoes as vectors for the transmission of Zika virus (ZIKV) around the globe.

Methods.

We performed an integrative review to identify relevant articles on specific experiments to determine whether Culex mosquitoes are vectors for ZIKV. The sources we used for our research were the Brazilian Periódicos CAPES electronic portal (MEDLINE/PubMed, ScienceDirect Journals, Nature Publishing Group, SciELO, Springer Link, and 250 other databases) and gray literature.

Results.

We identified 344 studies, of which 36 were considered for this review. In 8 studies, infection in salivary glands of Culex quinquefasciatus, Culex restuans, Culex tarsalis, and Culex coronator was detected. Cx. quinquefasciatus was the most studied among those confirmed as potential ZIKV vectors, and only strains of Asian lineages (THA/2014/SV0127-14; SZ01 (2016)) and American lineages (BRPE243 (2015); PRVABC59 (2015)) can infect the salivary glands of Culex mosquitoes. The tested African strains (MR766 and DAK AR 41525) were unable to infect salivary glands.

Conclusions.

There is still a lack of compelling evidence that indicates Culex spp. are a competent ZIKV vector, but they should remain a target for further monitoring studies, especially regarding ZIKV transmission to other species. Furthermore, studies should not be limited to studying whether their salivary glands are infected.

Keywords
Public health; Zika virus; mosquito vectors; Culex.

RESUMEN

Objetivo.

Identificar estudios sobre la competencia de los mosquitos Culex como vectores de la transmisión del virus del Zika en todo el mundo.

Métodos.

Se realizó una revisión integradora para identificar artículos relevantes sobre experimentos específicos dirigidos a determinar si los mosquitos Culex son vectores del virus del Zika. Se emplearon fuentes obtenidas a partir del portal electrónico de revistas CAPES (MEDLINE/PubMed, ScienceDirect Journals, Nature Publishing Group, SciELO, Springer Link, y otras 250 bases de datos) y la literatura gris.

Resultados.

Se identificaron 344 estudios, 36 de los cuales fueron considerados para esta revisión. En 8 estudios se detectó infección en las glándulas salivales de Culex quinquefasciatus, Culex restuans, Culex tarsalis y Culex coronator. Cx. quinquefasciatus fue la especie más estudiada entre las confirmadas como potenciales vectores del virus del Zika, y solo las cepas de linajes asiáticos (THA/2014/SV0127-14; SZ01 [2016]) y americanos (BRPE243 [2015]; PRVABC59 [2015]) pueden infectar las glándulas salivales de los mosquitos Culex. Las cepas africanas analizadas (MR766 y DAK AR 41525) no fueron capaces de infectar las glándulas salivales.

Conclusiones.

Aunque faltan pruebas convincentes que indiquen que las especies de Culex spp. son un vector competente del virus del Zika, estas deben seguir monitoreándose mediante estudios adicionales, especialmente respecto de su capacidad para transmitir el virus del Zika a otras especies. Esta vigilancia no debería limitarse solamente a determinar la infección en las glándulas salivales.

Palabras clave
Salud pública; virus Zika; mosquitos vectores; Culex.

RESUMO

Objetivo.

Identificar estudos sobre a competência dos mosquitos Culex como vetores da transmissão do vírus Zika em todo o mundo.

Métodos.

Uma revisão integrativa foi realizada para identificar artigos relevantes sobre experimentos específicos para determinar se os mosquitos Culex são vetores do vírus Zika. As fontes utilizadas na pesquisa foram o portal eletrônico CAPES (MEDLINE/PubMed, ScienceDirect Journals, Nature Publishing Group, Sci-ELO, Springer Link, e outras 250 bases de dados) e a literatura cinza.

Resultados.

Foram identificados 344 artigos, dos quais 36 foram considerados para esta revisão. Oito artigos relataram infecção nas glândulas salivares de Culex quinquefasciatus, Culex restuans, Culex tarsalis e Culex coronator. Culex quinquefasciatus foi a espécie mais estudada entre as confirmadas como vetores potenciais do vírus Zika. Apenas as linhagens asiáticas (THA / 2014 / SV0127-14; SZ01 [2016]) e americanas (BRPE243 [2015]; PRVABC59 [2015]) podem infectar as glândulas salivares dos mosquitos Culex. As cepas africanas analisadas (MR766 e DAK AR 41525) não foram capazes de infectar as glândulas salivares.

Conclusões.

Ainda não há evidências convincentes para indicar que os mosquitos Culex são um vetor competente do vírus Zika. Contudo, estudos adicionais de monitoramento devem ser realizados, especialmente no que diz respeito à transmissão do vírus Zika para outras espécies de mosquitos. Além disso, os estudos não devem se limitar a estudar a infecção nas glândulas salivares.

Palavras-chave
Saúde pública; Zika virus; mosquitos vetores; Culex.

Zika virus (ZIKV) is known to be transmitted among humans mainly through bites of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) mosquitoes (¹1. Waggoner JJ and Pinsky A. Zika Virus: diagnostics for an emerging pandemic threat. J Clin Microbiol. 2016;54: 860-7.). The virus was initially isolated in a rhesus monkey in 1947. There was a second isolation from Aedes africanus (Theobald) in 1948 in an attempt to isolate yellow fever virus from mosquitoes in the Zika Forest of Uganda (²2. Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;46:509-20.). Aedes mosquitoes are considered the only competent vectors for ZIKV transmission (³3. Benelli G, Romano D. Mosquito vectors of Zika virus. Entomol Gen. 2017;36(4):309-18., ⁴4. Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux A-B. An overview of mosquito vectors of Zika virus. Microbes Infect [Internet]. 2018;20(11–12):646-60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29481868
http://www.ncbi.nlm.nih.gov/pubmed/29481... ). Transmission can occur sexually (⁵5. Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau V-M. Potential sexual transmission of Zika virus. Emerg Infect Dis [Internet]. 2015;21(2):359–61. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4313657 Accessed on November 8 2019.
http://www.pubmedcentral.nih.gov/article... ), through blood transfusion and saliva, and from mother to child during pregnancy, birth, and breast-feeding (⁶6. Abushouk AI, Negida A, Ahmed H. An updated review of Zika virus. J Clin Virol [Internet]. 2016;84:53-8. Available from: http://dx.doi.org/10.1016/j.jcv.2016.09.012 Accessed on November 8 2019.
http://dx.doi.org/10.1016/j.jcv.2016.09.... ).

ZIKV is a positively enveloped RNA virus member of the Flaviviridae family, genus Flavivirus (¹1. Waggoner JJ and Pinsky A. Zika Virus: diagnostics for an emerging pandemic threat. J Clin Microbiol. 2016;54: 860-7., ⁷7. Kuno G, Chang GJJ. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. Arch Virol. 2007;152(4):687-96.). It was discovered in 1947 in the Zika Forest, in Uganda, and remained confined to some areas of Africa and Asia. In 2007, ZIKV emerged in the Yap Islands in the Federated States of Micronesia and also in the African country of Gabon. In addition, in 2013, the virus appeared in French Polynesia. By 2014, ZIKV had spread to other Pacific islands: New Caledonia, the Cook Islands, and Easter Island. In early 2015, the virus was identified in Brazil and then, later, throughout continental South America and Latin America (¹1. Waggoner JJ and Pinsky A. Zika Virus: diagnostics for an emerging pandemic threat. J Clin Microbiol. 2016;54: 860-7., ⁷7. Kuno G, Chang GJJ. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. Arch Virol. 2007;152(4):687-96.). This fast and massive spread is worrisome because there are no available drugs or vaccines for the treatment of ZIKV infection, and a possible marked, severe outcome of ZIKV infection in pregnant women is microcephaly in newborns (⁶6. Abushouk AI, Negida A, Ahmed H. An updated review of Zika virus. J Clin Virol [Internet]. 2016;84:53-8. Available from: http://dx.doi.org/10.1016/j.jcv.2016.09.012 Accessed on November 8 2019.
http://dx.doi.org/10.1016/j.jcv.2016.09.... ).

Within the genus Aedes, other anthropophilic species have been considered to be vectors, including Aedes hensilli (Farner) and Aedes vexans (Meigen). In addition, other mosquito genera have been investigated as vectors, especially Culex. (³3. Benelli G, Romano D. Mosquito vectors of Zika virus. Entomol Gen. 2017;36(4):309-18., ⁴4. Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux A-B. An overview of mosquito vectors of Zika virus. Microbes Infect [Internet]. 2018;20(11–12):646-60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29481868
http://www.ncbi.nlm.nih.gov/pubmed/29481... , ⁸8. Kauffman EB, Kramer LD. Zika Virus Mosquito Vectors: Competence, Biology, and Vector Control. J Infect Dis. 2017;216(Suppl 10):S976-90., ⁹9. Gutiérrez-Bugallo G, Piedra LA, Rodriguez M, Bisset JA, Lourenço-de-Oliveira R, Weaver SC, et al. Vector-borne transmission and evolution of Zika virus. Nat Ecol Evol. 2019;3(4):561-9. Available at: https://doi.org/10.1038/s41559-019-0836-z Accessed on November 8 2019.
https://doi.org/10.1038/s41559-019-0836-... ). Culex spp. are already considered competent vectors to transmit such flaviviruses as human-like Japanese encephalitis virus (JEV), West Nile virus (WNV), and Saint Louis encephalitis (¹⁰10. Marcondes CB, Contigiani M, Gleiser RM, Reisen W. Emergent and reemergent arboviruses in South America and the Caribbean: Why so many and why now? J Med Entomol. 2017;54(3):509-32.). This fact makes them candidates for further investigations as a vector for other flaviviruses, such as ZIKV.

In a study performed in China, Culex quinquefasciatus (Say) was identified as a potential laboratory vector for ZIKV. In that research, mosquitoes were infected through a blood meal with a ZIKV strain (SZ01) isolated from a patient, and viral RNA was found in the salivary glands, midgut, and ovary. Importantly, Cx. quinquefasciatus mosquitoes were able to infect infant mice (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ). In a session of the First International Workshop on Zika Virus, organized by the Oswaldo Cruz Foundation (FIOCRUZ) and held in Brazil in 2016, researchers from the Ageu Magalhães Institute (Recife, Pernambuco, Brazil) presented results of experiments with mosquitoes artificially fed with blood infected with the ZIKV. The virus was detected in the body and in salivary glands of Cx. quinquefasciatus 7 and 15 days after feeding, with a confirmed infection rate of 100% and 67%, respectively (¹²12. Franca RFO, Neves MHL, Ayres CFJ, Melo-Neto OP, Filho SPB. First International Workshop on Zika Virus Held by Oswaldo Cruz Foundation FIOCRUZ in Northeast Brazil March 2016 – A Meeting Report. PLoS Negl Trop Dis. 2016;10(6):e0004760.).

Currently, there are no vaccines or drugs against ZIKV (¹1. Waggoner JJ and Pinsky A. Zika Virus: diagnostics for an emerging pandemic threat. J Clin Microbiol. 2016;54: 860-7., ⁴4. Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux A-B. An overview of mosquito vectors of Zika virus. Microbes Infect [Internet]. 2018;20(11–12):646-60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29481868
http://www.ncbi.nlm.nih.gov/pubmed/29481... , ⁵5. Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau V-M. Potential sexual transmission of Zika virus. Emerg Infect Dis [Internet]. 2015;21(2):359–61. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4313657 Accessed on November 8 2019.
http://www.pubmedcentral.nih.gov/article... ), and the only precaution is to prevent mosquito bites by integrated vector control, including surveillance, biological and chemical control, source reduction, and environmental management (⁴4. Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux A-B. An overview of mosquito vectors of Zika virus. Microbes Infect [Internet]. 2018;20(11–12):646-60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29481868
http://www.ncbi.nlm.nih.gov/pubmed/29481... , ⁵5. Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau V-M. Potential sexual transmission of Zika virus. Emerg Infect Dis [Internet]. 2015;21(2):359–61. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4313657 Accessed on November 8 2019.
http://www.pubmedcentral.nih.gov/article... ). For example, the development of sanitization and management of urban water collection and vector control with DDT was responsible for vector Aedes mosquitoes disappearing after 1950, although the species has recently reinvaded European territory (⁴4. Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux A-B. An overview of mosquito vectors of Zika virus. Microbes Infect [Internet]. 2018;20(11–12):646-60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29481868
http://www.ncbi.nlm.nih.gov/pubmed/29481... ). Furthermore, the World Health Organization (WHO) also recommends the practice of safe sex for women living in areas of high virus transmission (¹³13. World Health Organization. Information for travelers visiting Zika infected countries, 2017. Available at http://www.who.int/csr/disease/zika/information-for-travelers/en/. Accessed September 29 2019.
http://www.who.int/csr/disease/zika/info... ).

Therefore, considering that the genus Aedes is the principal target among the strategies for the prevention and control of ZIKV, we reasoned that it is essential to understand whether there are other vectors capable of or even competent in transmitting ZIKV. Thus, we aimed to identify studies on the competence of the Culex mosquitoes as vectors for ZIKV transmission through a review of the literature on this subject.

METHODS

We performed a literature review in July 2019 to identify relevant articles on the vector competence of Culex spp. mosquitoes for ZIKV. The review was based on an advanced search of the Brazilian Periódicos CAPES electronic portal (www.periodicos.capes.gov.br), which includes MEDLINE/PubMed, ScienceDirect Journals, Nature Publishing Group, SciELO, Springer Link, and 250 other databases. Additional studies were identified by searching for gray literature, using the Google Scholar search engine, and with a manual search of the bibliographical references of the relevant identified publications.

The search strategy was drawn from the descriptors (“Culex” and “vector”) AND (zik*), marking “any field” for the search field of the thesauri, in the advanced-search area of the Periódicos CAPES electronic portal. All studies from the literature search were analyzed to eliminate duplicates from the manual search strategies, specifically by comparing authors, titles, and name of the journal, along with their volume, number, and year of publication. After the duplicates were removed, the remaining studies were examined based on their titles and abstracts. At this stage, the eligibility criteria for the articles were: original studies; mentions of ZIKV and a mosquito from the genus Culex; establishes Culex spp. as a vector of ZIKV through specific results in experiments identifying the virus at their organisms; and in Portuguese, Spanish, or English. Reviews and opinion letters were discarded. The documents selected as potentially relevant went to the next step, where they were judged carefully from the reading of the full text. Only those that met all the eligibility criteria mentioned above were included in this review. The studies from each database were placed in spreadsheets in the Microsoft Excel program (version 2010), to eliminate duplicates and to create a database for references.

RESULTS

The study designs were classified as cohort studies (10 articles), case-control studies (26 articles), and a conference abstract (1 article). One of the documents presented two different study classifications, case-control and cohort.

Study selection

We identified 333 studies in the bibliographic search in the Periódicos CAPES website, 7 studies through gray literature, and 7 additional studies from the manual search of the bibliographical references of the relevant publications, for a total of 347 studies. Three duplicate papers were discarded, and then 278 articles were excluded after reading the title. We incorporated 66 studies whose titles were not clear concerning inclusion criteria into the next stage, which included reading the abstracts to avoid missing any article that matched our criteria (all the articles were written in English, so no study was excluded based on language). In the end, after discarding 26 articles by reading the abstract and 4 articles by reading the full text, we included 36 studies in this review. Figure 1 is a flow diagram of the methodology steps. Table 1 summarizes the articles used in this review.

Source: prepared by the authors

Vector competence

Thirteen studies concluded that Culex spp. are not ZIKV competent vectors because they cannot be orally infected (¹⁴14. 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.

15. Foxi C, Puggioni G, Meloni G, Rossi R, Rocchigiani AM, Vento L, et al. Entomological surveillance of Zika virus in Sardinia, Italy, 2016. Vet Ital. 2018;54(3):243-9.

16. 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;214(9):1349-56.

17. 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 [Internet]. 2016;16(10):673–6. Available at: https://www.liebertpub.com/doi/10.1089/vbz.2016.2058 Accessed on November 8 2019.
https://www.liebertpub.com/doi/10.1089/v...

18. Dodson BL, Rasgon JL. Vector competence of Anopheles and Culex mosquitoes for Zika virus. PeerJ. 2017;5:e3096.

19. Duchemin JB, Mee PT, Lynch SE, Vedururu R, Trinidad L, Paradkar P. Zika vector transmission risk in temperate Australia: A vector competence study. Virol J. 2017;14(1):1-10.

20. Hart CE, Roundy CM, Azar SR, Huang JH, Yun R, Reynolds E, et al. Zika virus vector competency of mosquitoes, Gulf Coast, United States. Emerging Infectious Diseases. 2017;23(3):559-60. doi: 10.3201/eid2303.161636.

21. Yee DA, Dejesus-Crespo R, Hunter FF, Bai F. Assessing natural infection with Zika virus in the southern house mosquito, Culex quinquefasciatus, during 2016 in Puerto Rico. Med Vet Entomol. 2018;32(2):255-8. doi: 10.1111/mve.1228928.

22. Dodson BL, Pujhari S, Rasgon JL. Vector competence of selected North American Anopheles and Culex mosquitoes for Zika virus. PeerJ. 2018; 15;6:e4324. doi: 10.7717/peerj.4324

23. Lourenço-de-Oliveira R, Marques JT, Sreenu VB, Nten CA, Aguiar ERGR, Varjak M, et al. Culex quinquefasciatus mosquitoes do not support replication of Zika virus. J Gen Virol. 2018;99(2):258-64. DOI 10.1099/jgv.0.000949 1.

24. Hery L, Boullis A, Delannay C, Vega-Rúa A. Transmission potential of African, Asian and American Zika virus strains by Aedes aegypti and Culex quinquefasciatus from Guadeloupe (French West Indies). Emerg Microbes Infect. 2019;8(1):699-706.

25. Ho ZJM, Hapuarachchi HC, Barkham T, Chow A, Ng LC, Lee JMV, et al. Outbreak of Zika virus infection in Singapore: an epidemiological, entomological, virological, and clinical analysis. Lancet Infect Dis. 2017;17(8):813-21.-²⁶26. Tien WP, Lim G, Yeo G, Chiang SN, Chong CS, Ng LC, et al. SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes. Parasites and Vectors. 2017;10(1):1-7.). These studies used a variety of ZIKV strains, including PRVABC59 from Puerto Rico (2015), FSS13025 from Cambodia (2010), MEX1-7 from Mexico City, Mexico (2015), DAKAR 41525 from Senegal (1985), PF13/251013-18 from French Polynesia (2013), BRPE243 from Pernambuco, Brazil (2015), SPH2015 from São Paulo, Brazil (2015), NC-2014-5132 from New Caledonia (2014), and strains with GenBank number KU955592 from Senegal, KU647676 from Martinique, and KX694533 from Malaysia. Five studies (from Sardinia (Italy) (¹⁵15. Foxi C, Puggioni G, Meloni G, Rossi R, Rocchigiani AM, Vento L, et al. Entomological surveillance of Zika virus in Sardinia, Italy, 2016. Vet Ital. 2018;54(3):243-9.), Chiapas (Mexico) (¹⁶16. 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;214(9):1349-56.), San Juan (Puerto Rico) (²¹21. Yee DA, Dejesus-Crespo R, Hunter FF, Bai F. Assessing natural infection with Zika virus in the southern house mosquito, Culex quinquefasciatus, during 2016 in Puerto Rico. Med Vet Entomol. 2018;32(2):255-8. doi: 10.1111/mve.1228928.), and Singapore (²⁵25. Ho ZJM, Hapuarachchi HC, Barkham T, Chow A, Ng LC, Lee JMV, et al. Outbreak of Zika virus infection in Singapore: an epidemiological, entomological, virological, and clinical analysis. Lancet Infect Dis. 2017;17(8):813-21., ²⁶26. Tien WP, Lim G, Yeo G, Chiang SN, Chong CS, Ng LC, et al. SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes. Parasites and Vectors. 2017;10(1):1-7.)) analyzed field-caught mosquitoes, not ones from laboratory colonies. The blood meal titers used to feed the mosquitoes in colonies ranged from 1 × 10⁴ to 1 × 10⁷ focus-forming units per milliliter (FFU/ml), 1 × 10^7.3 to 1 × 10^8.4 plaque-forming units per milliliter (PFU/ml), and 1 × 10^5.6 to 1 × 10^7.52/ml 50% tissue culture infectious doses (TCID₅₀). Culex annulirostris (Skuse) was identified as not susceptible to ZIKV infection in a single study (²⁷27. Hall-Mendelin S, Pyke AT, Moore PR, Mackay IM, McMahon JL, Ritchie SA, et al. Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia. PLoS Negl Trop Dis [Internet]. 2016;10(9):1-14. Available at: http://dx.doi.org/10.1371/journal.pntd.0004959 Accessed on November 8 2019.
http://dx.doi.org/10.1371/journal.pntd.0... ), unlike Cx. quinquefasciatus, Culex pipiens (Linnaeus), and Culex tarsalis (Coquillett), each of which was identified as being infected in at least in one additional paper.

In another 16 studies, ZIKV infection was confirmed but without infected salivary glands or transmission (²⁷27. Hall-Mendelin S, Pyke AT, Moore PR, Mackay IM, McMahon JL, Ritchie SA, et al. Assessment of Local Mosquito Species Incriminates Aedes aegypti as the Potential Vector of Zika Virus in Australia. PLoS Negl Trop Dis [Internet]. 2016;10(9):1-14. Available at: http://dx.doi.org/10.1371/journal.pntd.0004959 Accessed on November 8 2019.
http://dx.doi.org/10.1371/journal.pntd.0...

28. 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. Eurosurveillance. 2016;21(35):1-4.

29. 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. Eurosurveillance. 2016; 2016;21(35):pii=30328. DOI: http://dx.doi.org/10.2807/1560-7917. ES.2016.21.35.30328
http://dx.doi.org/10.2807/1560-7917...

30. Diallo D, Sall AA, Diagne CT, Faye OO, Faye OO, Ba Y, et al. Zika virus emergence in mosquitoes in Southeastern Senegal, 2011. PLoS One. 2014;9(10):4-11.

31. Fernandes RS, Campos SS, Ferreira-de-Brito A, Miranda RM de, Barbosa da Silva KA, Castro MG de, 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. doi:10.1371/journal.pntd.0004993

32. Weger-Lucarelli J, Rückert C, Chotiwan N, Nguyen C, Garcia Luna SM, Fauver JR, et al. Vector Competence of American Mosquitoes for Three Strains of Zika Virus. PLoS Negl Trop Dis. 2016;10(10):1-16. e0005101. doi:10.1371/journal.pntd.0005101

33. Fu S, Song S, Liu H, Li Y, Li X, Gao X, et al. ZIKA virus isolated from mosquitoes: a field and laboratory investigation in China, 2016. Sci China Life Sci. 2017; 60(12):1364-71. doi: 10.1007/s11427-017-9196-8

34. Pompon J, Morales-Vargas R, Manuel M, Tan CH, Vial T, Tan JH, et al. A Zika virus from America is more efficiently transmitted than an Asian virus by Aedes aegypti mosquitoes from Asia. Sci Rep. 2017;7(1):1-8. doi:10.1038/s41598-017-01282-6

35. Fernandes RS, Campos SS, Ribeiro PS, Raphael LMS, Bonaldo MC, Lourenço-de-Oliveira R. Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the northeast region of Brazil are refractory to the virus. Mem Inst Oswaldo Cruz. 2017;112(8):577-9.

36. Heitmann A, Jansen S, Lühken R, Leggewie M, Badusche M, Pluskota B, et al. Experimental transmission of zika virus by mosquitoes from central Europe. Eurosurveillance. 2017;22(2):1-7. pii: 30437. doi: 10.2807/1560-7917.ES.2017.22.2.30437.

37. Liu Z, Zhou T, Lai Z, Zhang Z, Jia Z, Zhou G, et al. Competence of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes as Zika virus vectors, China. Emerg Infect Dis. 2017; 23:1085-91. doi: 10.3201/eid2307.161528

38. Kenney JL, Romo H, Duggal NK, Tzeng WP, Burkhalter KL, Brault AC, et al. Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for zika virus. Am J Trop Med Hyg. 2017;96(5):1235-40. doi:10.4269/ajtmh.16-0865

39. Richard V, Paoaafaite T, Cao-Lormeau V-MM. Acquittal of Culex quinquefasciatus in transmitting Zika virus during the French Polynesian outbreak. Acta Trop. 2017;173:200–1. doi: 10.1016/j. actatropica.2017.04.036

40. Main BJ, Nicholson J, Winokur OC, Steiner C, Riemersma KK, Stuart J, et al. Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus. PLoS Negl Trop Dis. 2018;12(6):e0006524. doi: 10.1371/journal. pntd.0006524

41. Heitmann A, Jansen S, Lühken R, Leggewie M, Schmidt-Chanasit J, Tannich E. Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes. J Vis Exp. 2018;(138):3-7. doi: 10.3791/57980.-⁴²42. Ayres CFJ, Guedes DRD, Paiva MHS, Morais-Sobral MC, Krokovsky L, Machado LC, et al. Zika virus detection, isolation and genome sequencing through Culicidae sampling during the epidemic in Vitória, Espírito Santo, Brazil. Parasites and Vectors. 2019;12(1):1–9. doi: https://doi.org/10.1186/s13071-019-3461-4
https://doi.org/10.1186/s13071-019-3461-... ). In a study conducted in Kédougou, Senegal (³⁰30. Diallo D, Sall AA, Diagne CT, Faye OO, Faye OO, Ba Y, et al. Zika virus emergence in mosquitoes in Southeastern Senegal, 2011. PLoS One. 2014;9(10):4-11.), the ZIKV was identified in a single female Culex perfuscus (Edwards) mosquito. After this identification, the authors assumed a potential transmission capacity, but they did not perform any specific identification tests on the salivary glands. The infected virus strains in Culex. spp. were: H/PF from French Polynesia (2013), BE H 815744 from Brazil (2015), ZJ03 (GenBank No. 820899.2) from China (2016), GDZJ1685-2 from China (2016), PRVABC59 from Puerto Rico (2015), KFF993678 from Thailand (2013), RIO-U1 from Rio de Janeiro (2016), RIO-S1 from Rio de Janeiro (2016), PE243/2015 from the state of Pernambuco in Brazil (2015), Haiti 1225 from Haiti (2014), SPH2015 from São Paulo (2015), and strain MR766 from Uganda (1947). The infected mosquitoes were Cx. quinquefasciatus, Cx. pipiens, and Cx. tarsalis, with minimal blood meal titers of 5.7 log₁₀ PFU/ml, 5.45 ± 0.38 log₁₀ copies/ml and 7.0 log₁₀ TCID₅₀/ml.

Finally, seven studies identified ZIKV in the salivary glands of Cx. restuans, Cx. quinquefasciatus, Cx. tarsalis, and Cx. coronator (Dyar and Knab) (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.

44. Dibernardo A, Turell MJ, Lindsay LR, Loomer C, Iranpour M. Vector Competence of Some Mosquito Species From Canada For Zika Virus. J Am Mosq Control Assoc. 2017;33(4):276-81. doi: 10.2987/17-6664.1.

45. Elizondo-Quiroga D, Medina-Sánchez A, Sánchez-González JM, Eckert KA, Villalobos-Sánchez E, Navarro-Zúñiga AR, et al. Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico. Sci Rep. 2018;8(1):809. doi:10.1038/s41598-017-18682-3

46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768

47. Phumee A, Chompoosri J, Intayot P, Boonserm R, Boonyasuppayakorn S, Buathong R, et al. Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes. Sci Rep. 2019;9(1):5257. doi: 10.1038/s41598-019-41727-81–9.

48. Epelboin Y, Talaga S, Epelboin L, Dusfour I. Zika virus: An updated review of competent or naturally infected mosquitoes. PLoS Negl Trop Dis. 2017;11(11):1-22: e0005933. Doi: 10.1371/journal. pntd.0005933-⁴⁹49. Ciota AT, Bialosuknia SM, Zink SD, Brecher M, Ehrbar DJ, Morrissette MN, et al. Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence – Emerg Infect Dis. 2017;23(7):1110-7. doi: 10.3201/eid2307.161633). Detection of the virus genetic material occurred via real-time polymerase chain reaction (RT-PCR or RT-qPCR), with minimal blood meal titers of 4.0 log₁₀ PFU/ml and 5.7 plaque-forming unit equivalents per milliliter (3.5 ± 0.1 log₁₀ ZIKV PFUe/ml titer for freshly fed). These seven studies were performed in Hainan (province), China (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ); Recife (city in the state of Pernambuco), Brazil (¹²12. Franca RFO, Neves MHL, Ayres CFJ, Melo-Neto OP, Filho SPB. First International Workshop on Zika Virus Held by Oswaldo Cruz Foundation FIOCRUZ in Northeast Brazil March 2016 – A Meeting Report. PLoS Negl Trop Dis. 2016;10(6):e0004760., ⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.); Winnipeg/Beausejour (city and nearby town in the province of Manitoba), Canada (⁴⁴44. Dibernardo A, Turell MJ, Lindsay LR, Loomer C, Iranpour M. Vector Competence of Some Mosquito Species From Canada For Zika Virus. J Am Mosq Control Assoc. 2017;33(4):276-81. doi: 10.2987/17-6664.1.); Guadalajara (city in the state of Jalisco), Mexico (⁴⁵45. Elizondo-Quiroga D, Medina-Sánchez A, Sánchez-González JM, Eckert KA, Villalobos-Sánchez E, Navarro-Zúñiga AR, et al. Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico. Sci Rep. 2018;8(1):809. doi:10.1038/s41598-017-18682-3); Florida (state), United States of America (⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768); and Thailand (⁴⁷47. Phumee A, Chompoosri J, Intayot P, Boonserm R, Boonyasuppayakorn S, Buathong R, et al. Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes. Sci Rep. 2019;9(1):5257. doi: 10.1038/s41598-019-41727-81–9.). The study in Mexico (⁴⁵45. Elizondo-Quiroga D, Medina-Sánchez A, Sánchez-González JM, Eckert KA, Villalobos-Sánchez E, Navarro-Zúñiga AR, et al. Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico. Sci Rep. 2018;8(1):809. doi:10.1038/s41598-017-18682-3) analyzed mosquitoes collected from the metropolitan area of Guadalajara but did not disclose the strains. This design was different from the studies in Brazil, Canada, China, Thailand, and the United States, which used mosquitoes from existing laboratory colonies. The Brazilian paper (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.) also analyzed field-caught mosquitoes from Recife, Pernambuco, Brazil.

DISCUSSION

In our work, we found 36 relevant studies. This was 18 more works than the already identified articles in five reviews in the literature that examined the capacity of Culex spp. to transmit ZIKV (³3. Benelli G, Romano D. Mosquito vectors of Zika virus. Entomol Gen. 2017;36(4):309-18., ⁴4. Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux A-B. An overview of mosquito vectors of Zika virus. Microbes Infect [Internet]. 2018;20(11–12):646-60. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29481868
http://www.ncbi.nlm.nih.gov/pubmed/29481... , ⁸8. Kauffman EB, Kramer LD. Zika Virus Mosquito Vectors: Competence, Biology, and Vector Control. J Infect Dis. 2017;216(Suppl 10):S976-90., ⁹9. Gutiérrez-Bugallo G, Piedra LA, Rodriguez M, Bisset JA, Lourenço-de-Oliveira R, Weaver SC, et al. Vector-borne transmission and evolution of Zika virus. Nat Ecol Evol. 2019;3(4):561-9. Available at: https://doi.org/10.1038/s41559-019-0836-z Accessed on November 8 2019.
https://doi.org/10.1038/s41559-019-0836-... , ⁴⁸48. Epelboin Y, Talaga S, Epelboin L, Dusfour I. Zika virus: An updated review of competent or naturally infected mosquitoes. PLoS Negl Trop Dis. 2017;11(11):1-22: e0005933. Doi: 10.1371/journal. pntd.0005933). Cx. quinquefasciatus was identified in 6 articles, while Cx. restuans, Cx. tarsalis, and Cx. coronator were identified as possibly competent in 1 study each.

As stated before, researchers from the Ageu Magalhães Institute have detected ZIKV in salivary glands of Cx. quinquefasciatus at 7 and 15 days after feeding, and confirmed an infection rate of 100% and 67%, respectively (¹²12. Franca RFO, Neves MHL, Ayres CFJ, Melo-Neto OP, Filho SPB. First International Workshop on Zika Virus Held by Oswaldo Cruz Foundation FIOCRUZ in Northeast Brazil March 2016 – A Meeting Report. PLoS Negl Trop Dis. 2016;10(6):e0004760.). Additionally, one Chinese paper (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ) was the first to demonstrate the presence of ZIKV (Strain SZ01) in the salivary glands of Cx. Quinquefasciatus; it was also the only study to show the transmission capacity to another species. This work reported a transmission rate of 89% to mice; these animals had viral RNA in their brain at 10 days post-engorgement, with a titer of 7.85 RNA log₁₀ copies/ml. On the other hand, this study did not provide experiments with the same methodology with Ae. aegypti as a positive control.

One study demonstrated the presence of ZIKV in the salivary glands of Cx. quinquefasciatus mosquitoes in concentrations similar (P > 0.05) to those found in Ae. aegypti, both fed with 1 × 10⁶ PFU/ml (log₁₀) blood meal titers (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.). Even with a minimum blood meal titer of 1 × 10⁴ PFU/ml (log₁₀), Cx. quinquefasciatus salivary glands were infected. These findings indicate that Cx. quinquefasciatus can produce virus in the salivary gland even when fed with low titers of viral particles. This ability better mimics what occurs in nature, where mean human viremia is lower than 2.5 log₁₀ PFU/ml (⁴⁹49. Ciota AT, Bialosuknia SM, Zink SD, Brecher M, Ehrbar DJ, Morrissette MN, et al. Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence – Emerg Infect Dis. 2017;23(7):1110-7. doi: 10.3201/eid2307.161633). Likewise, the study in the Brazilian state of Pernambuco (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.) investigated wild-caught mosquitoes from the city of Recife. They observed no traces of recent feeding and used electron microscopy to detect ZIKV in the salivary glands. That paper did not perform experiments to analyze ZIKV transmission from Cx. quinquefasciatus to another species.

Dibernardo et al. (⁴⁴44. Dibernardo A, Turell MJ, Lindsay LR, Loomer C, Iranpour M. Vector Competence of Some Mosquito Species From Canada For Zika Virus. J Am Mosq Control Assoc. 2017;33(4):276-81. doi: 10.2987/17-6664.1.) detected ZIKV in 3 of 58 (5%) of salivary glands of Cx. restuans just after intrathoracic inoculation. Cx. tarsalis was refractory using the same methodology. The authors believe that Cx. restuans could transmit by bite but also suggested the presence of salivary and midgut barriers for Cx. restuans and Cx. tarsalis. They concluded that Cx. restuans is not a competent ZIKV vector due to its feeding behavior.

Elizondo-Quiroga et al. (⁴⁵45. Elizondo-Quiroga D, Medina-Sánchez A, Sánchez-González JM, Eckert KA, Villalobos-Sánchez E, Navarro-Zúñiga AR, et al. Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico. Sci Rep. 2018;8(1):809. doi:10.1038/s41598-017-18682-3) detected ZIKV in wild-caught Cx. tarsalis, Cx. coronator, and Cx. quinquefasciatus. However, this study was not conclusive regarding the ZIKV infection titers in the saliva of the collected mosquitoes because the mosquitoes that presented the lowest maximum titer of infection were Ae. aegypti, the primary ZIKV vector. Unfortunately, for none of those mosquitoes did the authors indicate whether the mosquitoes had any trace of recent feeding or which ZIKV strains were involved. Such deficiencies impair a more detailed assessment of the competence of these Culex spp.

RNA analysis was performed in saliva from Cx. quinquefasciatus eluted from filter paper at 14 days postinfection (DPI) in a first experiment and then at 16 DPI in a second experiment (⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768). The ZIKV titers were 5.6 ± 4.5 log₁₀ ZIKV PFUe/ml and 5.02 log₁₀ ZIKV PFUe/ml, respectively. This same research group (⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768) neither investigated transmission to another species nor applied the same methodology for Ae. aegypti (as a positive control). The lack of positive controls diminishes the reliability of the results. On the other hand, this paper intriguingly showed that viruses isolated from Culex saliva can form plaques in Vero cells. These data prove that biologically active virus can be obtained from the saliva of those mosquitoes.

A Thai study investigated vertical transmission of ZIKV to larvae in Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus, with positive results for all the species (⁴⁷47. Phumee A, Chompoosri J, Intayot P, Boonserm R, Boonyasuppayakorn S, Buathong R, et al. Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes. Sci Rep. 2019;9(1):5257. doi: 10.1038/s41598-019-41727-81–9.). The ZIKV strain virus/H. sapiens-tc/THA/2014/SV0127-14 infected the salivary glands of these mosquitoes fed with 1.7x10⁵ FFU/ml blood meal titers. These Thai investigators (⁴⁷47. Phumee A, Chompoosri J, Intayot P, Boonserm R, Boonyasuppayakorn S, Buathong R, et al. Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes. Sci Rep. 2019;9(1):5257. doi: 10.1038/s41598-019-41727-81–9.) did not demonstrate any kind of transmission to other species but applied the same methodology for Ae. aegypti as a positive control.

Six studies that examined mosquito colonies reported that only when infection rates (IRs) from bodies were over 50% was salivary gland infection noted (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... , ¹²12. Franca RFO, Neves MHL, Ayres CFJ, Melo-Neto OP, Filho SPB. First International Workshop on Zika Virus Held by Oswaldo Cruz Foundation FIOCRUZ in Northeast Brazil March 2016 – A Meeting Report. PLoS Negl Trop Dis. 2016;10(6):e0004760., ⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59., ⁴⁴44. Dibernardo A, Turell MJ, Lindsay LR, Loomer C, Iranpour M. Vector Competence of Some Mosquito Species From Canada For Zika Virus. J Am Mosq Control Assoc. 2017;33(4):276-81. doi: 10.2987/17-6664.1., ⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768, ⁴⁷47. Phumee A, Chompoosri J, Intayot P, Boonserm R, Boonyasuppayakorn S, Buathong R, et al. Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes. Sci Rep. 2019;9(1):5257. doi: 10.1038/s41598-019-41727-81–9.). In addition, we found 13 other studies with IRs under 50% without any detected transmission capacity (²⁸28. 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. Eurosurveillance. 2016;21(35):1-4.

29. 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. Eurosurveillance. 2016; 2016;21(35):pii=30328. DOI: http://dx.doi.org/10.2807/1560-7917. ES.2016.21.35.30328
http://dx.doi.org/10.2807/1560-7917...

30. Diallo D, Sall AA, Diagne CT, Faye OO, Faye OO, Ba Y, et al. Zika virus emergence in mosquitoes in Southeastern Senegal, 2011. PLoS One. 2014;9(10):4-11.

31. Fernandes RS, Campos SS, Ferreira-de-Brito A, Miranda RM de, Barbosa da Silva KA, Castro MG de, 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. doi:10.1371/journal.pntd.0004993-³²32. Weger-Lucarelli J, Rückert C, Chotiwan N, Nguyen C, Garcia Luna SM, Fauver JR, et al. Vector Competence of American Mosquitoes for Three Strains of Zika Virus. PLoS Negl Trop Dis. 2016;10(10):1-16. e0005101. doi:10.1371/journal.pntd.0005101, ³⁴34. Pompon J, Morales-Vargas R, Manuel M, Tan CH, Vial T, Tan JH, et al. A Zika virus from America is more efficiently transmitted than an Asian virus by Aedes aegypti mosquitoes from Asia. Sci Rep. 2017;7(1):1-8. doi:10.1038/s41598-017-01282-6

35. Fernandes RS, Campos SS, Ribeiro PS, Raphael LMS, Bonaldo MC, Lourenço-de-Oliveira R. Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the northeast region of Brazil are refractory to the virus. Mem Inst Oswaldo Cruz. 2017;112(8):577-9.

36. Heitmann A, Jansen S, Lühken R, Leggewie M, Badusche M, Pluskota B, et al. Experimental transmission of zika virus by mosquitoes from central Europe. Eurosurveillance. 2017;22(2):1-7. pii: 30437. doi: 10.2807/1560-7917.ES.2017.22.2.30437.

37. Liu Z, Zhou T, Lai Z, Zhang Z, Jia Z, Zhou G, et al. Competence of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes as Zika virus vectors, China. Emerg Infect Dis. 2017; 23:1085-91. doi: 10.3201/eid2307.161528

38. Kenney JL, Romo H, Duggal NK, Tzeng WP, Burkhalter KL, Brault AC, et al. Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for zika virus. Am J Trop Med Hyg. 2017;96(5):1235-40. doi:10.4269/ajtmh.16-0865

39. Richard V, Paoaafaite T, Cao-Lormeau V-MM. Acquittal of Culex quinquefasciatus in transmitting Zika virus during the French Polynesian outbreak. Acta Trop. 2017;173:200–1. doi: 10.1016/j. actatropica.2017.04.036

40. Main BJ, Nicholson J, Winokur OC, Steiner C, Riemersma KK, Stuart J, et al. Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus. PLoS Negl Trop Dis. 2018;12(6):e0006524. doi: 10.1371/journal. pntd.0006524-⁴¹41. Heitmann A, Jansen S, Lühken R, Leggewie M, Schmidt-Chanasit J, Tannich E. Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes. J Vis Exp. 2018;(138):3-7. doi: 10.3791/57980.). One article, with a range of 15% to 70% of mosquitoes with intrathoracic inoculation, demonstrated virus-positive bodies but no detectable viral RNA or infectious virus in saliva (³⁸38. Kenney JL, Romo H, Duggal NK, Tzeng WP, Burkhalter KL, Brault AC, et al. Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for zika virus. Am J Trop Med Hyg. 2017;96(5):1235-40. doi:10.4269/ajtmh.16-0865).

In two studies (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59., ⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768), the infection of salivary glands declined over time, findings that are different from the Chinese study mentioned earlier (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ). Thus, there are inconsistent results with Cx. quinquefasciatus, with a marked decrease from day 8 to day 12 postexposure, but an apparent increase again at days 16 and 18 postexposure. Another aspect in these three articles is that they all used different strains: the Chinese study (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ) used SZ01, from an infected patient who had returned from Samoa to China; the Brazilian paper (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.) used BRPE243/2015, from Pernambuco; and the United States paper (⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768) used PRCABC59, from Puerto Rico (2015). Another study about vector competence of Aedes mosquitoes argued that in Ae. albopictus and Ae. aegypti, ZIKV transmission can be relatively dependent on the virus strain (⁴⁹49. Ciota AT, Bialosuknia SM, Zink SD, Brecher M, Ehrbar DJ, Morrissette MN, et al. Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence – Emerg Infect Dis. 2017;23(7):1110-7. doi: 10.3201/eid2307.161633). Thus, it is important to note that diverse strains, especially strains isolated from patients, can present distinct behaviors. This phenomenon may represent one factor responsible for the varied results found in the literature.

With regards to blood meal titers, only one work (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.), which used 4.0 log₁₀ ZIKV PFU/ml, reached an IR of 36% at 7 DPI and 10.53% at 15 DPI. The majority of studies that reached some IR used blood meal titers greater than 5.0 log₁₀ ZIKV PFU/ml, 5.0 log₁₀ ZIKV RNA copies/ml, or 1 × 10^6.7 TCID₅₀/ml. The results were refractory at lower blood meal concentrations. As described above, 13 studies did not detect the infection capacity, dissemination, or transmission of any Culex spp., even with higher blood meal titers. Some authors (²⁸28. 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. Eurosurveillance. 2016;21(35):1-4., ³⁸38. Kenney JL, Romo H, Duggal NK, Tzeng WP, Burkhalter KL, Brault AC, et al. Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for zika virus. Am J Trop Med Hyg. 2017;96(5):1235-40. doi:10.4269/ajtmh.16-0865, ⁴⁴44. Dibernardo A, Turell MJ, Lindsay LR, Loomer C, Iranpour M. Vector Competence of Some Mosquito Species From Canada For Zika Virus. J Am Mosq Control Assoc. 2017;33(4):276-81. doi: 10.2987/17-6664.1.) suggested that the random inability to transmit in Culex mosquitoes may be linked to a gut barrier of some Culex spp., a place where viral particles attack and initiate penetration and replication. However, Amraoui et al. (²⁸28. 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. Eurosurveillance. 2016;21(35):1-4.) did not demonstrate that inoculation of viral particles into the hemocoel tissue of Cx. quinquefasciatus favored viral ZIKV dissemination or transmission. A study from the United States (⁴⁶46. Smartt CT, Shin D, Kang S, Tabachnick WJ. Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. Front Microbiol. 2018; 9:768. doi: 10.3389/fmicb.2018.00768) warned about the existence of specific populations with regard to variability in transmission competence. The authors concluded that some Cx. quinquefasciatus populations may be capable of salivating ZIKV under environmental and other unknown conditions. This statement is noteworthy. It cannot be ignored that some mosquito populations may be more prone to ZIKV infection and dissemination. All these aspects definitely merit further studies that could reveal new intervention approaches.

Another particular condition revealed by Ciota et al. (⁴⁹49. Ciota AT, Bialosuknia SM, Zink SD, Brecher M, Ehrbar DJ, Morrissette MN, et al. Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence – Emerg Infect Dis. 2017;23(7):1110-7. doi: 10.3201/eid2307.161633) in a study on the vector competence of Aedes mosquitoes was that there were significant differences in the proportion of infected mosquitoes with equivalent ZIKV titers but two different types of meals. Fresh blood meals resulted in a significantly higher IR than did stocked meals frozen and stored at -80°C and then thawed before preparation (P < 0.0001). Thus, considering the documents of Table 1 that indicated the transmission capacity, the Brazilian work in Pernambuco (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.) stored the viral stocks at -80°C and subsequently thawed them to prepare the blood meal. However, another paper (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ) was unclear about storage conditions and used a stock of virus that had been passaged twice in C6/36 cells prior to the infectious feed. This method suggests the use of a recently prepared blood meal.

Roundy et al. (⁵⁰50. Roundy CM, Azar SR, Brault AC, Ebel GD, Failloux A-B, Fernandez-Salas I, et al. Lack of evidence for Zika virus transmission by Culex mosquitoes. Emerg Microbes Infect. 2017;6(10):e90. doi: 10.1038/emi.2017.85) noted that the criterion (iii) proposed by Barnett (⁵¹51. Barnett HC. The incrimination of arthropods as vectors of disease. In: Strouhal H, Beier M, eds. Proceedings of the 11th International Congress of Entomology. 1962:341–5.) for incrimination of an arthropod vector (repeated demonstration of natural infection of the vector) has only been fulfilled for Ae. aegypti and Ae. albopictus. Furthermore, as can be seen in our review, only one study (¹¹11. 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 [Internet]. 2016;5(9):e102-5. Available from: http://dx.doi.org/10.1038/emi.2016.102 Accessed on November 8 2019.
http://dx.doi.org/10.1038/emi.2016.102... ) demonstrated ZIKV transmission to other species (criteria (iv) for incrimination of an arthropod (⁵¹51. Barnett HC. The incrimination of arthropods as vectors of disease. In: Strouhal H, Beier M, eds. Proceedings of the 11th International Congress of Entomology. 1962:341–5.)). However that study used a ZIKV strain not tested in any other work found for this review, and it did not include Ae. aegypti positive control tests. Thus, the defining evidence for Culex spp. as a ZIKV vector is still lacking.

Some authors (³²32. Weger-Lucarelli J, Rückert C, Chotiwan N, Nguyen C, Garcia Luna SM, Fauver JR, et al. Vector Competence of American Mosquitoes for Three Strains of Zika Virus. PLoS Negl Trop Dis. 2016;10(10):1-16. e0005101. doi:10.1371/journal.pntd.0005101, ³⁸38. Kenney JL, Romo H, Duggal NK, Tzeng WP, Burkhalter KL, Brault AC, et al. Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for zika virus. Am J Trop Med Hyg. 2017;96(5):1235-40. doi:10.4269/ajtmh.16-0865, ⁴⁰40. Main BJ, Nicholson J, Winokur OC, Steiner C, Riemersma KK, Stuart J, et al. Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus. PLoS Negl Trop Dis. 2018;12(6):e0006524. doi: 10.1371/journal. pntd.0006524) agree that the focus on prevention of ZIKV disease should remain on population control of the genus Aedes. Indeed, the probability of a Culex mosquito biting two humans in a sequence and transmitting the Zika virus is small, according to its preference for feeding on avian hosts (⁵²52. Reisen WK, Fang Y, Martinez VM. Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J Med Entomol. 2005;42(3):367–75.). Nevertheless, Culex spp. are widespread in urban centers and also feed on human blood. Considering the results of studies with collected mosquitoes, when the Culex spp. was infected, the Aedes species was infected (³⁰30. Diallo D, Sall AA, Diagne CT, Faye OO, Faye OO, Ba Y, et al. Zika virus emergence in mosquitoes in Southeastern Senegal, 2011. PLoS One. 2014;9(10):4-11., ⁴²42. Ayres CFJ, Guedes DRD, Paiva MHS, Morais-Sobral MC, Krokovsky L, Machado LC, et al. Zika virus detection, isolation and genome sequencing through Culicidae sampling during the epidemic in Vitória, Espírito Santo, Brazil. Parasites and Vectors. 2019;12(1):1–9. doi: https://doi.org/10.1186/s13071-019-3461-4
https://doi.org/10.1186/s13071-019-3461-... , ⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59., ⁴⁵45. Elizondo-Quiroga D, Medina-Sánchez A, Sánchez-González JM, Eckert KA, Villalobos-Sánchez E, Navarro-Zúñiga AR, et al. Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico. Sci Rep. 2018;8(1):809. doi:10.1038/s41598-017-18682-3). Only one study (¹⁵15. Foxi C, Puggioni G, Meloni G, Rossi R, Rocchigiani AM, Vento L, et al. Entomological surveillance of Zika virus in Sardinia, Italy, 2016. Vet Ital. 2018;54(3):243-9.) presented data that showed neither Culex spp. nor Aedes mosquitoes infected with ZIKV. Conversely, in four papers (¹⁶16. 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;214(9):1349-56., ²⁴24. Hery L, Boullis A, Delannay C, Vega-Rúa A. Transmission potential of African, Asian and American Zika virus strains by Aedes aegypti and Culex quinquefasciatus from Guadeloupe (French West Indies). Emerg Microbes Infect. 2019;8(1):699-706., ²⁵25. Ho ZJM, Hapuarachchi HC, Barkham T, Chow A, Ng LC, Lee JMV, et al. Outbreak of Zika virus infection in Singapore: an epidemiological, entomological, virological, and clinical analysis. Lancet Infect Dis. 2017;17(8):813-21., ²⁶26. Tien WP, Lim G, Yeo G, Chiang SN, Chong CS, Ng LC, et al. SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes. Parasites and Vectors. 2017;10(1):1-7.), there was no Culex mosquito infected while the official vector Ae. aegypti was infected. Two articles (²⁵25. Ho ZJM, Hapuarachchi HC, Barkham T, Chow A, Ng LC, Lee JMV, et al. Outbreak of Zika virus infection in Singapore: an epidemiological, entomological, virological, and clinical analysis. Lancet Infect Dis. 2017;17(8):813-21., ²⁶26. Tien WP, Lim G, Yeo G, Chiang SN, Chong CS, Ng LC, et al. SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes. Parasites and Vectors. 2017;10(1):1-7.) used the same collection of mosquitoes from the field with different methodologies to investigate viral ZIKV RNA. All these results with field-caught mosquitoes showed that Culex spp. and Ae. aegypti may use identical hosts (but not always). In fact, in a study with mosquitoes from field collections in Thailand (⁵³53. Khaklang S, Kittayapong P. Species composition and blood meal analysis of mosquitoes collected from a tourist island, Koh Chang, Thailand. J Vector Ecol. 2014;39(2):448-52. doi: 10.1111/jvec.12122), in Cx. quinquefasciatus there were mixed blood meals, with 7.84% from humans or monkeys, 47.06% from dogs, and 33.33% from others hosts. Comparatively, in Ae. aegypti, there were also several blood meals: 70.0% from humans plus monkeys or 13.33% only from monkeys and 10.0% from other kinds of hosts. These data demonstrate that variations in favorite hosts from place to place influence the infection rates of Culex spp. and Aedes mosquitoes. Additionally, as argued by Kauffman et al. (⁸8. Kauffman EB, Kramer LD. Zika Virus Mosquito Vectors: Competence, Biology, and Vector Control. J Infect Dis. 2017;216(Suppl 10):S976-90.), especially regarding the work from Pernambuco, Brazil (⁴³43. Guedes DRD, Paiva MHS, Donato MMA, Barbosa PP, Krokovsky L, Rocha SWS, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6(8):e69. doi:10.1038/emi.2017.59.), Cx. quinquefasciatus may serve as a secondary vector in places with abundant ZIKV infection in humans.

Additional experimental studies that use identical strains, experimental conditions, methodologies, with positive controls in Ae. aegypti and/or Ae. albopictus, and that utilize tests to prove the possibility of ZIKV transmission to other species from Culex spp., could be very decisive for discarding or confirming the contribution of these mosquitoes as a competent or incompetent ZIKV vector. According to the studies we have investigated, we feel that, besides well-implemented sanitization, the main strategies for the prevention and control of ZIKV should remain on the genus Aedes.

Conclusions

This work demonstrated the accumulation of evidence to prove the capacity of the ZIKV to infect Culex spp. However, only 7 studies out of the 36 identified for this review demonstrated the infection of Cx. restuans, Cx. quinquefasciatus, Cx. tarsalis, and Cx. coronator salivary glands. Furthermore, only 1 study showed the capacity of transmission to mice. Considering the records found here, Cx. quinquefasciatus remains the most widely studied species with confirmed salivary glands infected by ZIKV.

Additionally, only Asian or American ZIKV strains were able to infect the salivary glands of Culex mosquitoes: THA/2014/SV0127-14, SZ01, BRPE243, and PRVABC59. The MR766 and DAK AR 41525 African strains were unable to infect Culex spp. Further experimental studies that utilize the same strains, experimental conditions, use a positive Aedes control, and test ZIKV transmission to other species via Culex spp. are still needed to confirm the contribution of the Culex mosquitoes in ZIKV transmission. We believe that strategies for ZIKV control should stay focused on the genus Aedes, but responsible authorities should continue to monitor Culex spp. mosquitoes, especially regarding their ability to transmit ZIKV to other species. This surveillance should not be limited to determining whether their salivary glands are infected.

Author contributions.

All the authors (SGVR, EGR, and WCS) conceived the original idea, collected the data, analyzed the data and interpreted the results, and wrote the paper. All the authors reviewed and approved the final version.

Disclaimer.

Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the RPSP/PAJPH and/or PAHO.

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