First report of naturally infected Aedes aegypti with chikungunya virus genotype ECSA in the Americas

Background The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. Methodology/Principal findings During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The complete sequence of CHIKV genome was obtained from this sample and phylogenetic analysis revealed that this isolate belongs to the East-Central-South-African (ECSA) genotype. Conclusions Our study describes the first identification of a naturally CHIKV-infected Ae. aegypti in Brazil and the first report of a CHIKV from ECSA genotype identified in this species in the Americas. These findings support the notion of Ae. aegypti being a vector involved in CHIKV outbreaks in northeast of Brazil.

Introduction Chikungunya viral disease is caused by an arbovirus (Alphavirus genus) from Togaviridae family [1,2]. The most frequent symptoms during human infection are fever and joint pain, but headache, muscle pain, joint swelling and rash are also observed [3,4]. Chikungunya virus (CHIKV) is transmitted to humans by Aedes mosquitoes in sylvatic (animal-mosquito-man) [5] or urban transmission cycles (man-mosquito-man) [6]. This virus is endemic in Africa and Asia, and has spread to several European countries and more recently to America [7]. In Brazil, the first autochthonous case was described in 2014 in Amapá, a state in the North Region [8]. Since then, CHIKV has expanded its distribution in Brazil and 13,236 cases were confirmed in 2015, most of them occurring in states within the Northeast Region [9][10][11].
Three CHIKV genotypes have been identified since its discovery in 1952 [2]. The East-Central-South-African (ECSA) and West African genotypes circulate in sub-Saharan Africa, whereas Ae. aegypti is the vector of the Asian genotype in human urban transmission cycles in Southeast Asia and in the Americas. Asian and ECSA genotypes were described in Brazil concomitantly with autochthonous cases caused by both genotypes [12]. Currently, all Brazilian states are infested by both of the main CHIKV vectors, Ae. aegypti and Ae. albopictus [13,14]. The presence of both species raises concern about the establishment of sustained CHIKV outbreaks throughout the entire country [15]. Although neither of these vectors have yet been found to be naturally infected with CHIKV in Brazil, it is known that Brazilian populations of both species are highly competent vectors for CHIKV when infected under laboratory conditions [16]. Moreover, it is not known if other Culicidae species occurring in urban area in Brazil are capable to transmit CHIKV. Therefore, entomological surveillance is essential for understanding the role of vector species in CHIKV transmission, especially in new endemic countries such as Brazil. Recently, Aracaju, a Northeast Region city and the capital of Sergipe state with 571,149 inhabitants [17], has experienced an increase in CHIKV cases since the detection of the first autochthonous transmission in 2015. According to the State Department of Health from the Sergipe government, the highest number of CHIKV cases in Aracaju has been reported in 2016, being 6,810 suspected and 4,743 confirmed until epidemiological week  [18].
In this alarming scenario, we hypothesize that the city of Aracaju is a potential site for detecting vector species naturally infected by CHIKV, ZIKV and DENV. During the third week of February 2016, we collected adult mosquitoes, inside and outside homes situated in urban areas of Aracaju, where residents were complaining of symptoms consistent with CHIKV or related arboviruses (DENV and ZIKV). Our results revealed that the ECSA genotype of CHIKV is naturally infecting Ae. aegypti in the Americas and showed the first identification of a field-caught mosquito infected with CHIKV in Brazil. These findings confirm the vectorial capacity of Ae. aegypti for transmitting CHIKV and suggests that this species is an important CHIKV vector which can be involved in the recent outbreaks since this virus was introduced in this country.

Fieldwork and identification
During a multidisciplinary coordinated investigation involving the ZIKV São Paulo task force (Zika Network) and the Central Public Health Laboratories (LACEN-Aracaju, Sergipe, Brazil), medical interviews, clinical diagnosis and mosquito collections were performed during one week in Aracaju. Houses in six different neighborhoods were selected based on the locations of clinically ill patients (suspected symptoms of arboviral infection) provided by LACEN. All neighborhoods were mostly residential, composed by heterogeneous house sizes of very low-to low-income, except by São José neighborhood, with middle-income dwellings. After an authorized employee from LACEN obtained permission of the residents, mosquitoes were collected by the entomological team. Peridomiciliary and intradomiciliary areas (all the rooms) of these houses and adjacent residences were aspirated during the morning after the dawn or afternoon until the dusk period using vacuum aspirators manufactured by Horst Armadilhas. Captured mosquitoes were placed into a collection vial previously identified by date and neighborhood and for each home inspected, one vial was used.
The collected mosquitoes were ice anesthetized and sorted by species using a morphological identification key as reference when necessary [19], sex and collected region. Males were pooled in 4 to 11 individuals of the same species and both engorged and non-engorged females were individually divided into head, thorax and abdomen using a McPherson-Vannas Scissors #501234 (World Precision Instruments, Sarasota, FL) to avoid contamination from possible blood inside the mosquito midgut. Samples were stored individually into a 1.5 mL microtube and immediately frozen at -80˚C for further analyses.

RNA extraction of samples
Total RNA was extracted from each individual female thorax and from pooled whole male mosquitoes using QIAmp Viral RNA Mini Kit (Qiagen, USA) following manufacturer's recommendations. About 5 to 10 μL of extracted thoracic RNA from each female was pooled in a species-specific manner and the remaining volume of each RNA sample was stored at -80˚C for subsequent individual confirmations and further analyses.
The one step qRT-PCR conditions, including total RNA sample volume and cycling for ZIKV and CHIKV detection reactions were previously described [2]. For DENV detection, 20μL reactions were used as following: 10 μL of QuantiTect Probe RT-PCR Master mix, 0.25 μL of QuantiTect RT mix, 1 μL of 10 μM of each primer, 0.4 μL of 10 μM probe, 2.4 μL of DNA RNA free water and 5 μL of total RNA sample. The thermocycler conditions for DENV were 50˚C for 20 min and 95˚C for 15 min followed by 45 cycles of 95˚C for 15 sec, 60˚C for 15 sec and 40˚C for 30 sec. For each qRT-PCR experiment, a positive control consisting of a specific viral RNA (DENV2, ZIKV or CHIKV) was added for cDNA synthesis and detection validation. Each sample, as well as negative (DEPC treated water as template) and positive controls, were analyzed in technical duplicates.
For positive pools, individual RNA samples were retested to confirm which mosquitoes from the positive pool were infected.

Sequencing of CHIKV
Ae. aegypti RNA from CHIKV positive female was prepared for NGS sequencing using a sequence-independent single-primer amplification (SISPA) method as described previously [23]. Libraries were sequenced with an Illumina MiSeq desktop sequencer using a version 3 kit (2 x 150 cycles). Additionally for mosquito sample that was positive for CHIKV we used the Illumina TruSeq RNA Access enrichment method to get coding-complete genomes as described previously [24]. CHIKV specific probes were designed against Brazilian isolate KP164569.

Phylogenetic analysis
For phylogenetic characterization of sequenced CHIKV detected in positive mosquitoes from Sergipe-Brazil, additional CHIKV sequences from Brazil and other countries, representing all known genotypes, were recovered from Genbank (http://www.ncbi.nlm.nih.gov) (S1 Table). All sequences used in this work are presented in the format: genotype/accession number/country/year of isolation in the phylogenetic tree. Sequences were aligned using Clustal X2 [25] and the alignment was manually edited using jalview.
Viral phylogenies based on full-length nucleotide sequences were estimated using Maximum Likelihood (ML) implemented in FastTree 2 [26] and Bayesian Inference (BI) analysis with BEAST package v.1.8.2 [27], using the general time-reversible with gamma-distributed rate variation substitution model (GTR+G), as described by Akaike's information criterion (AICc) in jModelTest 0.1 [28]. For the BI, a relaxed lognormal molecular clock model and the nonparametric Skygrid coalescent model were employed. The evolutionary analysis were computed using two independent runs for 100 million MCMC steps and the convergence of the MCMC chains was inspected using TRACER v.1.6 (http://tree.bio.ed.ac.uk). Posterior trees were summarized discarding the first 10% of the sampled trees and choosing the Maximum Clade Credibility (MCC) was summarized using TreeAnnotator v.

Collected species and frequency of occurrence
Six neighborhoods were inspected in Aracaju city during February 16 th to 20 th , 2016 (Fig 1).
A total of 248 mosquitoes were collected in 39 properties and we identified 4 species during the triage procedure and identification (Table 1).
Fifteen properties presented the co-occurrence of multiple species. The co-occurrence of Ae. aegypti-Cx. quinquefasciatus was the most registered, but we also found other combinations of concurrences as well. For instance, Cx. quinquefasciatus/Ae. aegypti/Ae. taeniorhynchus or Ae. scapularis were found in the same home. However, the co-occurrence of more than three species in the same property was not observed.

Detection of CHIKV, ZIKV and DENV1-4
We tested the mosquitoes for DENV, ZIKV and CHIKV because Aracaju is endemic for DENV and human cases of ZIKV and CHIKV infection were recently confirmed in this city in 2016. All eight male and fifteen female pools of Cx. quinquefasciatus, as well as the one Ae. scapularis female pool and the Ae. taeniorhynchus female pool, were negative for CHIKV, ZIKV and DENV serotypes 1-4. Together, these pools represented a total of 198 mosquitoes ( Table 2). Negative results were also observed for ZIKV and DENV1-4 detection in all male and female Ae. aegypti pools. However, one Ae. aegypti female pool was positive for CHIKV from a total of thirteen pools tested. All male pools presented negative for CHIKV (Table 2). Following the pool analysis, we tested the individual thoracic samples that composed the CHIKV positive pool. We were able to detect one positive female, confirming our initial analysis ( Table 2). The positive female was collected in São Conrado neighborhood (Fig 1), and the mean Ct values obtained in qRT-PCR was 21.76 for the individual thorax from the positive sample. Therefore, of thirty-eight females and twelve males of Ae. aegypti analyzed, only one female was infected with CHIKV.

Sequencing and phylogeny of CHIKV
The total RNA from CHIKV positive sample was selected for sequencing using Illumina sequencing platform. The whole sequence of the CHIKV genome was obtained and deposited in GenBank with accession number: KY055011.
Identity analysis of the whole CHIKV genome generated in this study with representative sequences of all known CHIKV genotypes obtained in GenBank (S1 Table) had percentages identities ranging from 84.24% to 99.99%, while between CHIKV sequence previously isolated in 2014-2015 in Bahia state showed percentages identities ranging from 99.91% to 99.99% (S2 Table). The mutations previously associated with CHIKV fitness increase in Ae. aegypti and Ae. albopictus [29,30] were not detected in envelope protein 1 (E1) from sequenced CHIKV.
Maximum likelihood analysis and Bayesian Inference comparing the CHIKV sequence isolated in Sergipe with sequences previously characterized (S1 Table) produced trees with similar topologies (Fig 2A and 2B, respectively).  The CHIKV-mosquito sequence was closely-related to other sequences from Bahia-Brazil isolated in 2014-2015 and with sequences from Africa, characterized as ECSA genotype (Fig  2). The consistency of the results is supported by the high bootstrap values and posterior probability observed in the trees (Fig 2A and 2B, respectively).

Discussion
The identification of the vectors in endemic areas is essential to better design surveillance programs and defines efficient vector control strategies. In Brazil, it is well known that Ae. aegypti is the main DENV vector and although Ae. albopictus is also a potential vector in this country, ecological characteristics and adaptations favor the first species to sustain DENV outbreaks in urban areas [31]. Nevertheless, the recent introductions of CHIKV and ZIKV in Brazil and severe epidemics reported, have raised fundamental questions regarding the establishment of viral circulation in relation to the mosquito species occurring in endemic places [32].
A recent study showed that American populations of Ae. aegypti and Ae. albopictus, including Brazilian ones, are highly competent to transmit different CHIKV genotypes when orally infected in the laboratory [16]. Based on their results, the authors highlighted the imminent risk for an expansion of CHIKV outbreaks in tropical and subtropical areas of this continent [16]. In fact, Ae. aegypti transmitting CHIKV Asian genotype in the Americas was first described in Mexico, during an outbreak in Ciudad Hidalgo, Chiapas State [33].
CHIKV was first isolated in Brazil from infected patients in 2014 and the circulation of two genotypes was observed: Asian genotype in Oiapoque (Amapá state), North Region of Brazil and ECSA genotype in Feira de Santana (Bahia state), Northeast Region of Brazil [12]. It is important to highlight that Aracaju is in the Northeast Region and is approximately 300 km away from Feira de Santana. Coherently, we report here the first detection of a natural infection of Ae. aegypti mosquito by CHIKV genotype ECSA in Brazil and the Americas.
In the present study, Ae. albopictus was not found in the inspected houses although this species is endemic in Aracaju city [14]. However, we found two unusual species intra domiciliary, Ae. scapularis and Ae. taeniorhynchus, but the occurrence was much less frequent than Ae. aegypti and Cx. quinquefasciatus. The collected females from Ae. scapularis (n = 2) and Ae. taeniorhynchus (n = 2) were negative for ZIKV, CHIKV or DENV 1-4 serotypes. These females were engorged suggesting they are biting humans inside their homes. Moreover, it is known that Ae. scapularis presents vectorial competence for Rocio encephalitis, Ilheus and Melao viruses [34][35][36] while Ae. taeniorhynchus for Venezuelan equine encephalitis and West Nile viruses [37,38]. Nevertheless, it is unknown if these mosquito species are competent vectors in Brazil for the three arboviruses assayed in this study and if they can act as secondary vectors during outbreaks.
Recently, Ae. aegypti was shown to be a ZIKV vector in Rio de Janeiro city. In this study ZIKV-positive Cx. quinquefasciatus or Ae. albopictus were not identified [39]. We found Ae. aegypti as a potential vector of CHIKV in Aracaju city, as seen in outbreaks already reported elsewhere [5,[40][41][42]. None of the other species examined were positive for CHIKV, ZIKV or DENV serotypes 1-4, mainly Cx. quinquefasciatus that was abundant in our sampling effort.
The surveillance of the Aedes mosquitoes should be expanded in order to prevent new CHIKV outbreaks in Brazil, since this country presents adequate conditions for the establishment of an endemic situation, which can also exposes other countries at risk. The mutations that enhance the fitness of the CHIKV genotype ECSA in Aedes mosquitoes were previously described [29,30] and they were not found in the genotype characterized in our study. However, new mutations that improve vector competence can be acquired since the ECSA genotype is being detected in other regions in Brazil [43], which suggests that different populations of Aedes mosquitoes are interacting with this recently introduced genotype, mainly Aedes aegypti in urban areas.
Our findings constitute the first description of Ae. aegypti-CHIKV genotype ECSA interaction in Brazil. These results reinforce the role of this species as an important vector of CHIKV in urban areas of northeast regions in Brazil.
Supporting information S1 Table. Available