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Zika virus detection, isolation and genome sequencing through Culicidae sampling during the epidemic in Vitória, Espírito Santo, Brazil



Zika virus (ZIKV) has been isolated from many mosquito species in nature, but it is believed that the main vectors in urban environments are species of the genus Aedes. Here, we detected and isolated ZIKV in samples from Aedes aegypti, Aedes taeniorhynchus and Culex quinquefasciatus, collected during the Zika epidemic in Vitória, southeast Brazil. Using quantitative real-time polymerase chain reaction, ZIKV detection was performed in mosquito samples collected from February to April 2016.


Overall, six pools of mosquitoes were positive for ZIKV: four of Cx. quinquefasciatus, one of Ae. aegypti and one of Ae. taeniorhynchus. Their genomes were sequenced.


These results support and strengthen the hypothesis that other mosquito species can also be involved in ZIKV transmission.


In 2015–2016, the world faced the spread of a global epidemic caused by Zika virus (ZIKV). This arbovirus (arthropod-borne virus) was first identified in Uganda in 1947 from sentinel monkeys but remained unnoticed in humans until 60 years later [1]. In 2007, a Zika outbreak was reported in Micronesia, affecting 73% of the inhabitants, followed by a new outbreak in French Polynesia in 2013, which affected 70% of the population [2, 3]. Despite these reports, no severe consequences of ZIKV infections in humans had been described until its arrival in South America. In this continent, ZIKV infections were associated with birth defects, due to mother-to-child transmission of the virus, such as microcephaly and other neurological disorders, which are now collectively referred to as “congenital Zika syndrome” [4, 5]. In addition, ZIKV is also capable of triggering neurological abnormalities, such as Guillain–Barré syndrome [6]. From a mild disease before 2015 to an epidemic that has a diverse impact on population health, in 2016 the World Health Organization (WHO) declared Zika a public health emergency of international concern [7].

The ZIKV is a positive-sense, single-stranded RNA virus, with a genome length of approximately 11 kb, which codes for a single polyprotein [3] composed of three structural proteins: capsid (C), pre-membrane (prM) and envelope (E); as well as seven nonstructural proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 [8]. The structural proteins are responsible for the formation of the virus particle, while non-structural proteins are responsible for important functions in genome replication, polyprotein processing and host response [9]. ZIKV transmission primarily occurs through bites from infected mosquitoes, but unlike other arboviral diseases, other non-vector-borne transmission pathways have been reported, such as blood transfusion, sexual transmission, transplacental infection and breastfeeding [10,11,12,13]. In addition, studies have demonstrated the presence and persistence of infective ZIKV in saliva, urine and semen [8, 14].

Aedes aegypti is considered the main vector of ZIKV along with Aedes albopictus [15,16,17,18]. However, Culex quinquefasciatus, the most common mosquito in urban areas of the southern hemisphere, also known as the southern house mosquito, was implicated in ZIKV transmission during recent epidemics [19]. This mosquito’s vector competence has been recently demonstrated under laboratory conditions by three different groups [20,21,22], as well as in field samples, when ZIKV was isolated from two pools of Cx. quinquefasciatus mosquitoes from Recife (northeast region), and from several pools collected in Guadalajara, Mexico [23].

Here, we report the results of a study conducted in early 2016 to detect ZIKV in field-caught mosquitoes collected in the city of Vitória, Espírito Santo, Brazil.


Aspirations resulted in the highest number of collected mosquitoes among the three collection methods: Cx. quinquefasciatus (n = 223), Ae. aegypti (n = 12), Aedes taeniorhynchus (n = 6) and Aedes scapularis (n = 1). Culex quinquefasciatus was also the most abundant mosquito collected in CDC traps (n = 47), followed by Ae. taeniorhynchus (n = 19). No other Aedes species were collected in CDC traps. New Jersey traps collected more Ae. taeniorhynchus (n = 20) than Cx. quinquefasciatus (n = 16) and Ae. aegypti (n = 2). Figure 1 shows the distribution of sites where mosquitoes were captured and positive pools were detected.

Fig. 1
figure 1

Location of the city of Vitória, capital of Espírito Santo State, indicating both mosquito collection sites (blue dots) and the places where ZIKV-positive samples were detected (red stars). The map was produced using ArcGIS v.10.6 (Esri, Redlands, CA, USA)

A total of 346 mosquitoes were analyzed in 95 pools, of which 25 (26%) contained mosquito females that had recently taken a blood meal, and 70 (74%) contained mosquito females without visible blood remains in the abdomen. Six pools from the second group (non-fed mosquitoes) were positive for ZIKV (Table 1).

Table 1 Zika virus infection of potential vectors collected in Vitória-ES

ZIKV was isolated after the first passage in Vero cells from three out of the six positive pools collected in Vitória (two were obtained from Cx. quinquefasciatus and one from Ae. aegypti). None of these pools showed signs of recent blood-feeding. These genomes were named ZIKV/C. quinquefasciatus/Brazil/ES01/2016 (Cxq_ES1), ZIKV/C. quinquefasciatus/Brazil/ES24/2016 (Cxq_ES24) and ZIKV/Ae. aegypti/Brazil/ES32/2016 (Aea_ES32). Sequencing efforts obtained partial ZIKV genomes, which were aligned against all ZIKV genomes available from Faria et al. [24]. ZIKV partial genomes are available in the figshare website, as NGS reads ( These genomes presented a 44% (4745) coverage breath for Cxq_ES1, 57% (6157) for Cxq_ES24 and 55% (5948) for Aea_ES32 (Fig. 2). The coverage depth obtained ranged from 19× for Aea_ES32 to 1029x for Cxq_ES1, with Cxq_ES24 sequenced at an intermediate depth of approximately 222×.

Fig. 2
figure 2

Circular representation of the ZIKV genome (PE243: KX197192) and its corresponding annotation (black and gray external rings, red arrow denotes the UTRs of ZIKV) along with blastN (default parameters) of partial genomes obtained from Cx. quinquefasciatus (bluish rectangles) and Ae. aegypti (greenish rectangles)

Phylogenetic analysis of all available ZIKV genomes revealed that two sequenced ZIKV genomes (Cxq_ES24 and Aea_ES32) clustered with an early clade of ZIKV detected in Haiti in 2014 and in Pernambuco state in 2015 (Fig. 3) [full maximum likelihood tree can be seen on the Interactive Tree of Life website (]. In addition, the Cxq_ES1 ZIKV genome clustered within one of the major ZIKV branches that caused the 2015 epidemic in Brazil (Fig. 3). Of note, Cxq_ES24 draft genome differed by seven single nucleotide polymorphisms (SNPs) from the reference PE243 genome [25], while Aea_ES32 differed by 12 SNPs from the PE243 ZIKV genome. Because Cxq_ES1 clustered together with strains of another clade, we compared it with the Paraíba ZIKV genome (KX280026) and observed the presence of seven SNPs. Considering all complete and draft genomes of ZIKV used in the alignments, the sequences obtained in this study showed non-exclusive SNPs, that is, such variants also occurred in most ZIKV genomes from the 2015–2016 epidemic (data not shown).

Fig. 3
figure 3

ZIKV maximum likelihood phylogenetic reconstruction of the Asian genotype: blue branches represent an early offset of ZIKV in Brazil where the CXq_ES24 and Aea_ES32 genomes clustered and red branches denote one of the clades of ZIKV which emerged during the 2015 epidemic where CXq_ES1 clustered. SH-like branch support is depicted above or in front of the corresponding nodes and only aLRT branch support above 0.80 are shown


In this study, we report the detection of ZIKV in field-caught mosquitoes from Vitória, (ES), during part of the 2016 epidemic. ZIKV was found in three different species, namely Ae. aegypti, Ae. taeniorhynchus and Cx. quinquefasciatus. This is the first report of ZIKV detection in species other than Ae. aegypti in southeast Brazil.

The identification of ZIKV mosquito vectors has been the subject of a heated debate. The proof-of-principle study performed by Guo et al. [21] demonstrated that transmission by Cx. quinquefasciatus is feasible, but only evidence collected in natural settings, such as during outbreaks, can demonstrate how this species contributes to ZIKV spread. Unfortunately, despite the very high incidence of Zika in human populations, there is a lack of data regarding the spatial and temporal association of human cases and local vector abundance, preventing us from concluding which mosquito species plays the dominant role in ZIKV transmission in this urban context. From the 81 countries reporting recent ZIKV outbreaks, it was only possible in three (Brazil, Mexico and USA) to detect ZIKV circulating in vector populations. In Brazil, ZIKV has been detected in Ae. aegypti from Rio de Janeiro [26] and in Ae. aegypti and Cx. quinquefasciatus from Recife [20]. In Mexico, 15 out of 58 pools of Ae. aegypti were found positive for ZIKV in Chiapas state [27]. In Guadalajara, Mexico, ZIKV has been isolated from several mosquito species, especially Cx. quinquefasciatus [23]. Before the recent ZIKV outbreaks, Culex perfuscus was found naturally infected with ZIKV in Senegal and this species displayed a MIR tenfold higher than Ae. aegypti [28].

The results obtained here give further support for the role of both Ae. aegypti and Cx. quinquefasciatus in the ZIKV transmission. Our group also reported in a previous paper that Cx. quinquefasciatus plays an important role in ZIKV spread in Brazil through laboratory and field experiments [20]. On the other hand, several research groups have conducted vector competence studies of Culex and Aedes mosquitoes and demonstrated that Culex species are not competent to transmit ZIKV [29,30,31,32,33,34]. The nature of these conflicting results should be investigated in more detail, as different explanations have been suggested [35, 36]. A study conducted in Florida, USA, to identify the pattern of host use by mosquito species, suggested that Cx. quinquefasciatus plays a secondary role in ZIKV transmission, and that Ae. aegypti and Ae. albopictus are the most likely ZIKV vectors. The authors concluded that the relative importance of each species in spreading the virus may vary according to specific location and population [37].

Considering that Cx. quinquefasciatus may play a role in ZIKV transmission, it is crucial to highlight the distinct biological traits displayed by Culex and Aedes mosquitoes. The population distribution of these mosquitoes differs in most Brazilian cities and most tropical countries; Cx. quinquefasciatus is approximately 20 times more abundant than Aedes as shown previously [38, 39]. In addition, Cx. quinquefasciatus females show different feeding behavior when compared to Ae. aegypti females. Usually, an Ae. aegypti female can feed on multiple hosts until its gonotrophic cycle is complete [40], while Cx. quinquefasciatus usually feed on only one host [41]. This behavior increases the potential for Ae. aegypti to become infected compared to Cx. quinquefasciatus.

On the other hand, Cx. quinquefasciatus mosquitoes in the southern hemisphere exhibit a highly anthropophilic feeding behaviour. In Brazil, this species is solely responsible for the active transmission of Wuchereria bancrofti, the lymphatic filariasis causative agent [42]. It has also been implicated as a potential vector in the transmission of Oropouche virus (OROV), in the state of Mato Grosso [43], which was considered the second most common arbovirus in Brazil [43, 44].

In this study, we detected a pool of Ae. taeniorhynchus positive for ZIKV, but we were unable to isolate and sequence the ZIKV from this pool. Aedes taeniorhynchus is a common species found along the Brazilian coastline, and it is implicated as the primary vector of canine heartworm disease in the northeastern and southeastern regions of the country [45]. In addition, it has been identified as the primary vector of Venezuelan equine encephalitis [46] and as a potential vector for maintaining the transmission cycle of West Nile fever (WNV) in the Galapagos [47]. Further studies are necessary to better understand if Ae. taeniorhynchus play a role in ZIKV transmission among human.

In order to obtain detailed information about the viral strains circulating in this region we performed isolation of ZIKV from field-caught mosquitoes and sequenced their genome. Bioinformatics analysis showed that these strains were clustered into two distinct clades. The first comprised Cxq_ES24 and Aea_ES32 along with the virus Brazil PE243 2015 (from Pernambuco state), Haiti 1225 2014 and SPH2015 (from São Paulo state). This clade encompasses the first group of ZIKV strains identified in northeast Brazil. The second clade clustered Cxq_ES1 with several other strains reported in the southeast and northeast Brazil. Interestingly, the detection of strains from the first clade in other Brazilian state, besides Pernambuco and São Paulo, shows that those variants also are circulating and spreading very rapidly in comparison to the other epidemic clades [24].

Previous data suggest that ZIKV is a very fast-evolving and adaptive virus, and we should not expect it to fit into previously described simple models (i.e. one pathogen-one vector). Instead, the scientific community should keep an open mind [48] and focus on understanding the ecology of ZIKV and its interactions with different host cells (i.e. human, non-human vertebrates and mosquitoes) to elucidate its amplification cycles in urban environments and to prevent new ZIKV outbreaks and its associated neurological diseases.


Results found in this study confirm the detection of ZIKV in field-caught mosquitoes from Vitória, Espírito Santo (ES) in three different mosquito species (Ae. aegypti, Ae. taeniorhynchus and Cx. quinquefascitus). Additionally, this is the first report of ZIKV detection in species other than Ae. aegypti in the studied area. Furthermore, it was observed that ZIKV strains were clustered into two distinct clades, showing that these variants are co-circulating and spreading very rapidly. Thus, it is of utmost importance to detect and monitor the emergence and spread of ZIKV in the country to implement successful surveillance strategies.


Study site

Vitória is the capital of Espírito Santo, a state located in the southeastern region of Brazil (Fig. 3). This municipality is a river-oceanic archipelago of 63,396 km2, consisting of 35 islands and a mainland region with an estimated population of almost 360,000 inhabitants [23, 49]. Approximately 40% of its area is covered by hills. Vitória comprises 80 neighborhoods, grouped into nine administrative regions. The city is considered the second best Brazilian capital with respect to quality of life indicators. The climate in Vitória is tropical with two distinguishable periods: the wet season from October to December and the sub-dry season from January to September. The exception is August, which is the driest month of the year.

Mosquito collections

Adult female mosquitoes were collected from February to April 2016 using three different methods: New Jersey and CDC light traps and battery-powered aspirators. Thirty-seven CDC light traps, with white lights, were installed for surveillance and control of Culex spp. in 35 neighborhoods in Vitória. In addition, nine New Jersey light traps were installed in other districts that had reported high numbers of Zika cases. All traps were installed outdoors at public building properties, to facilitate access for the community health agents at the time of sample collection. Traps were turned on late in the afternoon and disconnected early the next morning after sample collection.

Battery-powered aspirators were also employed during daytime for capturing adult female mosquitoes in schools, hospitals, basic healthcare units and residences, to block Zika transmission in areas where Zika cases had been reported. Mosquitoes caught in New Jersey and CDC light traps were collected by vector control staff members, while municipal health agents involved in combating endemic diseases performed aspirations. Samples were identified from the traps and transported to the laboratory immediately after collection. After being anaesthetized at −20 °C for 10 min, mosquitoes were placed on Petri dishes on ice and sorted by species, sex, date and location. Females were pooled into 1.5 ml microtubes with a maximum of 20 females per tube. Then, 100 µl of RNAlaterTM stabilization solution was added to each sample, which were then covered with cotton balls and stored at −20 °C. In September 2016, samples were shipped to Aggeu Magalhães Institute (FIOCRUZ-PE) in Recife, in portable thermal iceboxes filled with dry ice.

RNA extraction and RT-qPCR

After registering feeding status, mosquitoes were re-pooled into groups of a maximum of 10 specimens. RNA extractions and ZIKV molecular detection (RT-qPCR description and protocol) are described in Guedes et al. [20] and in Lanciotti et al. [3]. All samples were tested in duplicate. A reference sample (ZIKV BRPE243/2015 RNA) was used as a positive control, and a negative control consisted of RNA from a known ZIKV-negative mosquito pool. Both positive and negative controls were used in all RT-qPCR assays.

Minimum infection rate (MIR)

To estimate viral infection rates in mosquito samples, we calculated the minimum infection rate (MIR), which is the number of positive pools divided by the total number of specimens tested and multiplied by 1000 as previously described [50].

Spatial analysis

Collection sites were georeferenced based on an address geocoding method that transforms residential addresses registered in a tabular database into geographic coordinates, by first using a database of streets stored in a digital cartographic base and then converting them to geographical coordinates (latitude and longitude) using QGIS, a free geographical information system (GIS) software. Therefore, georeferenced information was derived by registering each address displaying an associated geographical coordinate for each location. ArcGIS v.10.6 was used to create a map.

Isolation and sequencing of ZIKV from field-collected samples

ZIKV-positive pools from non-fed mosquitoes were used to recover ZIKV strains according to the protocol described in Guedes et al. [20]. To obtain ZIKV genomic sequences from positive samples, we performed the conditions described in the ZIBRA project protocol V3 [51]. PCR products were quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, USA). MiSeq (Illumina, San Diego, CA, USA) sequencing libraries were prepared with a Nextera XT Library Prep Kit (Illumina) using 2 ng of input cDNA derived from the ZIKV multiplex PCR, following manufacturer’s instructions. MiSeq Reagent Kit V3 (Illumina; 150 cycles) was used in a paired-end strategy, resulting in 75 bp reads separated from each other by ca. 350 bp. Sequencing was performed in a MiSeq instrument (Illumina) from the Technological Platform Core at Aggeu Magalhães Institute.

ZIKV genome analysis

Low quality raw reads, adaptors and ZIKV primers were trimmed with Trimmomatic v 0.36 [52], using the ILLUMINACLIP parameter. Bowtie2 [53] was used to map reads against the reference genome PE243 (KX197192.1) and consensus sequences were generated with Integrated Genome Viewer (IGV) [54, 55], considering only regions with coverage depth higher than 5x.

Phylogenetic analysis

Phylogenetic reconstruction was performed with PhyML 3.0 [56] using the GTR + I + G nucleotide substitution models suggested by the SMART model selection [57]. The tree topology search was performed with NNIs and SPRs [58]. Branch support was evaluated with the aLRT [59]. A total of 255 draft and complete available ZIKV genomes, were collected from NCBI ( up to December 2017, aligned with Mafft online service [60] and manually edited to keep only the ZIKV coding region. Figure generation was performed with FigTree (



Zika virus


quantitative real-time polymerase chain reaction


World Health Organization


Centers for Disease Control and Prevention


threshold cycle


minimum infection rate


single nucleotide polymorphisms


Oropouche virus


West Nile virus


  1. Basu R, Tumban E. Zika virus on a spreading spree: what we now know that was unknown in the 1950’s. Virol J. 2016;13:165.

    Article  Google Scholar 

  2. 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;360:2536–43.

    Article  CAS  Google Scholar 

  3. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008;14:1232–9.

    Article  CAS  Google Scholar 

  4. Del Campo M, Feitosa IM, Ribeiro EM, Horovitz DD, Pessoa AL, Franca GV, et al. The phenotypic spectrum of congenital Zika syndrome. Am J Med Genet A. 2017;173:841–57.

    Article  Google Scholar 

  5. Miranda-Filho DB, Martelli CM, Ximenes RA, Araujo TV, Rocha MA, Ramos RC, et al. Initial description of the presumed congenital Zika syndrome. Am J Public Health. 2016;106:598–600.

    Article  Google Scholar 

  6. Oehler E, Watrin L, Larre P, Leparc-Goffart I, Lastere S, Valour F, et al. Zika virus infection complicated by Guillain–Barre syndrome—case report, French Polynesia, December 2013. Euro Surveill. 2014;19:20720.

    Article  Google Scholar 

  7. Lowe R, Barcellos C, Brasil P, Cruz OG, Honorio NA, Kuper H, et al. The Zika virus epidemic in Brazil: from discovery to future implications. Int J Environ Res Public Health. 2018;15:E96.

    Article  Google Scholar 

  8. Lindenbach BD, Rice CM. Molecular biology of flaviviruses. Adv Virus Res. 2003;59:23–61.

    Article  CAS  Google Scholar 

  9. Shi W, Zhang Z, Ling C, Carr MJ, Tong Y, Gao GF. Increasing genetic diversity of Zika virus in the Latin American outbreak. Emerg Microbes Infect. 2016;5:e68.

    Article  Google Scholar 

  10. Aubry M, Finke J, Teissier A, Roche C, Broult J, Paulous S, et al. Seroprevalence of arboviruses among blood donors in French Polynesia, 2011–2013. Int J Infect Dis. 2015;41:11–2.

    Article  Google Scholar 

  11. Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill. 2014;19:20751.

    Article  Google Scholar 

  12. Dupont-Rouzeyrol M, Biron A, O’Connor O, Huguon E, Descloux E. Infectious Zika viral particles in breastmilk. Lancet. 2016;387:1051.

    Article  Google Scholar 

  13. Foy BD, Kobylinski KC, Chilson Foy JL, Blitvich BJ, Travassos da Rosa TA, Haddow AD, et al. Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis. 2011;17:880–2.

    Article  Google Scholar 

  14. Bonaldo MC, Ribeiro IP, Lima NS, Dos Santos AA, Menezes LS, da Cruz SO, et al. Isolation of infective Zika virus from urine and saliva of patients in Brazil. PLoS Negl Trop Dis. 2016;10:e0004816.

    Article  Google Scholar 

  15. Araujo AP, Araujo Diniz DF, Helvecio E, de Barros RA, de Oliveira CM, Ayres CF, et al. The susceptibility of Aedes aegypti populations displaying temephos resistance to Bacillus thuringiensis israelensis: a basis for management. Parasit Vectors. 2013;6:297.

    Article  Google Scholar 

  16. 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:e0004543.

    Article  Google Scholar 

  17. Grard G, Caron M, Mombo IM, Nkoghe D, Mboui Ondo S, Jiolle D, et al. Zika virus in Gabon (Central Africa)—2007: a new threat from Aedes albopictus? PLoS Negl Trop Dis. 2014;8:e2681.

    Article  Google Scholar 

  18. Smartt CT, Stenn TMS, Chen TY, Teixeira MG, Queiroz EP, Souza Dos Santos L, et al. Evidence of Zika virus RNA fragments in Aedes albopictus (Diptera: Culicidae) field-collected eggs from Camacari, Bahia, Brazil. J Med Entomol. 2017;54:1085–7.

    Article  Google Scholar 

  19. Ayres CF. Identification of Zika virus vectors and implications for control. Lancet Infect Dis. 2016;16:278–9.

    Article  Google Scholar 

  20. Guedes DR, Paiva MH, Donato MM, Barbosa PP, Krokovsky L, Rocha S, et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017;6:e69.

    Article  Google Scholar 

  21. 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:e102.

    Article  Google Scholar 

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

    Article  Google Scholar 

  23. Elizondo-Quiroga D, Medina-Sanchez A, Sanchez-Gonzalez JM, Eckert KA, Villalobos-Sanchez E, Navarro-Zuniga AR, et al. Zika virus in salivary glands of five different species of wild-caught mosquitoes from Mexico. Sci Rep. 2018;8:809.

    Article  Google Scholar 

  24. Faria NR, Quick J, Claro IM, Theze J, de Jesus JG, Giovanetti M, et al. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Nature. 2017;546:406–10.

    Article  CAS  Google Scholar 

  25. Donald CL, Brennan B, Cumberworth SL, Rezelj VV, Clark JJ, Cordeiro MT, et al. Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil. PLoS Negl Trop Dis. 2016;10:e0005048.

    Article  Google Scholar 

  26. Ferreira-de-Brito A, Ribeiro IP, Miranda RM, Fernandes RS, Campos SS, Silva KA, et al. First detection of natural infection of Aedes aegypti with Zika virus in Brazil and throughout South America. Mem Inst Oswaldo Cruz. 2016;111:655–8.

    Article  Google Scholar 

  27. 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;214:1349–56.

    Article  CAS  Google Scholar 

  28. 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:e109442.

    Article  Google Scholar 

  29. Amraoui F, Atyame-Nten C, Vega-Rua A, Lourenco-de-Oliveira R, Vazeille M, Failloux AB. Culex mosquitoes are experimentally unable to transmit Zika virus. Euro Surveill. 2016;21:30333.

    Article  Google Scholar 

  30. 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;21:30328.

    Article  Google Scholar 

  31. Fernandes RS, Campos SS, Ferreira-de-Brito A, Miranda RM, Barbosa da Silva KA, 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:e0004993.

    Article  Google Scholar 

  32. 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. 2016;10:e0004959.

    Article  Google Scholar 

  33. Huang YJ, Ayers VB, Lyons AC, Unlu I, Alto BW, Cohnstaedt LW, et al. Culex species mosquitoes and Zika virus. Vector Borne Zoonotic Dis. 2016;16:673–6.

    Article  Google Scholar 

  34. Weger-Lucarelli J, Ruckert 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:e0005101.

    Article  Google Scholar 

  35. Jupatanakul N, Sim S, Dimopoulos G. The insect microbiome modulates vector competence for arboviruses. Viruses. 2014;6:4294–313.

    Article  CAS  Google Scholar 

  36. Lambrechts L, Chevillon C, Albright RG, Thaisomboonsuk B, Richardson JH, Jarman RG, et al. Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors. BMC Evol Biol. 2009;9:160.

    Article  Google Scholar 

  37. Stenn T, Peck KJ, Rocha Pereira G, Burkett-Cadena ND. Vertebrate hosts of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus (Diptera: Culicidae) as potential vectors of Zika virus in Florida. J Med Entomol. 2019;56:10–7.

    Article  Google Scholar 

  38. Azmi SA, Das S, Chatterjee S. Seasonal prevalence and blood meal analysis of filarial vector Culex quinquefasciatus in coastal areas of Digha, West Bengal, India. J Vector Borne Dis. 2015;52:252–6.

    PubMed  Google Scholar 

  39. Nunes VdN. Avaliação da metodologia de aspiração de mosquitos adultos para monitoramento da infestação por Aedes aegypti em área endêmica de dengue em Recife/PE. Recife: Aggeu Magalhães Institute; 2013.

    Google Scholar 

  40. Farjana T, Tuno N. Multiple blood feeding and host-seeking behavior in Aedes aegypti and Aedes albopictus (Diptera: Culicidae). J Med Entomol. 2013;50:838–46.

    Article  Google Scholar 

  41. Barata EA, Costa AI, Chiaravalloti Neto F, Glasser CM, Barata JM, Natal D. Aedes aegypti (L.) population in an endemic area of dengue in Southeast Brazil. Rev Saude Publica. 2001;35:237–42 (In Portuguese).

    Article  CAS  Google Scholar 

  42. Regis L, Silva-Filha MH, de Oliveira CM, Rios EM, da Silva SB, Furtado AF. Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife. Mem Inst Oswaldo Cruz. 1995;90:115–9.

    Article  CAS  Google Scholar 

  43. Cardoso BF, Serra OP, Heinen LB, Zuchi N, Souza VC, Naveca FG, et al. Detection of Oropouche virus segment S in patients and in Culex quinquefasciatus in the state of Mato Grosso, Brazil. Mem Inst Oswaldo Cruz. 2015;110:745–54.

    Article  CAS  Google Scholar 

  44. Vasconcelos PF, Travassos Da Rosa JF, Guerreiro SC, Degallier N, Travassos Da Rosa ES, Travassos Da Rosa AP. 1st register of an epidemic caused by Oropouche virus in the states of Maranhao and Goias, Brazil. Rev Inst Med Trop Sao Paulo. 1989;31:271–8 (In Portuguese).

    Article  CAS  Google Scholar 

  45. Ahid SMM, Lourenco-de-Oliveira R. Mosquitos vetores potenciais de dirofilariose canina na Região Nordeste do Brasil. Rev Saúde Pública. 1999;33:560–5 (In Portuguese).

    Article  CAS  Google Scholar 

  46. Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC. Venezuelan equine encephalitis. Annu Rev Entomol. 2004;49:141–74.

    Article  CAS  Google Scholar 

  47. Eastwood G, Goodman SJ, Cunningham AA, Kramer LD. Aedes taeniorhynchus vectorial capacity informs a pre-emptive assessment of West Nile virus establishment in Galapagos. Sci Rep. 2013;3:1519.

    Article  CAS  Google Scholar 

  48. Hunter FF. Linking Only Aedes aegypti with Zika virus has world-wide public health implications. Front Microbiol. 2017;8:1248.

    Article  Google Scholar 

  49. IBGE. 2017. Accessed 4 Aug 2017.

  50. Chow VT, Chan YC, Yong R, Lee KM, Lim LK, Chung YK, et al. Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing. Am J Trop Med Hyg. 1998;58:578–86.

    Article  CAS  Google Scholar 

  51. Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc. 2017;12:1261–76.

    Article  CAS  Google Scholar 

  52. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.

    Article  CAS  Google Scholar 

  53. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.

    Article  CAS  Google Scholar 

  54. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–6.

    Article  CAS  Google Scholar 

  55. Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14:178–92.

    Article  CAS  Google Scholar 

  56. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59:307–21.

    Article  CAS  Google Scholar 

  57. Lefort V, Longueville JE, Gascuel O. SMS: Smart Model Selection in PhyML. Mol Biol Evol. 2017;34:2422–4.

    Article  CAS  Google Scholar 

  58. Hordijk W, Gascuel O. Improving the efficiency of SPR moves in phylogenetic tree search methods based on maximum likelihood. Bioinformatics. 2005;21:4338–47.

    Article  CAS  Google Scholar 

  59. Anisimova M, Gascuel O. Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst Biol. 2006;55:539–52.

    Article  Google Scholar 

  60. Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2017.

    Article  PubMed  Google Scholar 

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We thank the Programme for Technological Development in Tools for Health (PDTIS-FIOCRUZ) for assistance with sequencing and real-time PCR.


This study was supported by the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE APQ-0078-2.02/16 and APQ-0085-2.13/16), the Brazilian National Council for Research and Development (CNPq; grant 441100/2016-3) and the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (R21AI128931).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Authors’ contributions

CFJA, DRDG, MHSP, MAVMS, CMFO, WSL and GLW designed the initial project. RSR, OAC, ALBM and RCL collected mosquito samples in Vitória, ES. MCMS, LK, LCM, MC conducted laboratory experiments. CFL and ALSO performed the spatial analysis. CFJA, DRDG, MHSP, MAVMS, CMFO, WSL and GLW analyzed the results, interpreted data, and drafted the manuscript. All authors read and approved the final manuscript.

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Correspondence to Constância Flavia Junqueira Ayres.

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Ayres, C.F.J., Guedes, D.R.D., Paiva, M.H.S. et al. Zika virus detection, isolation and genome sequencing through Culicidae sampling during the epidemic in Vitória, Espírito Santo, Brazil. Parasites Vectors 12, 220 (2019).

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