Exposure to Zika and chikungunya viruses impacts aspects of the vectorial capacity of Aedes aegypti and Culex quinquefasciatus.

Zika (ZIKV) and chikungunya (CHIKV) are arboviruses that cause infections in humans and can cause clinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatus may be a potential ZIKV vector. This study aimed to evaluate fecundity, fertility, survival, longevity, and blood feeding activity in Ae. aegypti after exposure to ZIKV and CHIKV and, in Cx. quinquefasciatus exposed to ZIKV. Three colonies were evaluated: AeCamp (Ae. aegypti-field), RecL (Ae. aegypti-laboratory) and CqSLab (Cx. quinquefasciatus-laboratory). Seven to 10 days-old females from these colonies were exposed to artificial blood feeding with CHIKV or ZIKV. CHIKV caused reduction in fecundity and fertility in AeCamp and reduction in survival and fertility in RecL. ZIKV impacted survival in RecL, fertility in AeCamp and, fecundity and fertility in CqSLab. Both viruses had no effect on blood feeding activity. These results show that CHIKV produces a higher biological cost in Ae. aegypti, compared to ZIKV, and ZIKV differently alters the biological performance in colonies of Ae. aegypti and Cx. quinquefasciatus. These results provide a better understanding over the processes of virus-vector interaction and can shed light on the complexity of arbovirus transmission.

Dynamic of Mayaro Virus Transmission in Aedes aegypti, Culex quinquefasciatus Mosquitoes, and a Mice Model.

Mayaro virus (MAYV) is transmitted by Haemagogus spp. mosquitoes and has been circulating in Amazon areas in the North and Central West regions of Brazil since the 1980s, with an increase in human case notifications in the last 10 years. MAYV introduction in urban areas is a public health concern as infections can cause severe symptoms similar to other alphaviruses. Studies with Aedes aegypti have demonstrated the potential vector competence of the species and the detection of MAYV in urban populations of mosquitoes. Considering the two most abundant urban mosquito species in Brazil, we investigated the dynamics of MAYV transmission by Ae. aegypti and Culex quinquefasciatus in a mice model. Mosquito colonies were artificially fed with blood containing MAYV and infection (IR) and dissemination rates (DR) were evaluated. On the 7th day post-infection (dpi), IFNAR BL/6 mice were made available as a blood source to both mosquito species. After the appearance of clinical signs of infection, a second blood feeding was performed with a new group of non-infected mosquitoes. RT-qPCR and plaque assays were carried out with animal and mosquito tissues to determine IR and DR. For Ae. aegypti, we found an IR of 97.5-100% and a DR reached 100% in both 7 and 14 dpi. While IR and DR for Cx. quinquefasciatus was 13.1-14.81% and 60% to 80%, respectively. A total of 18 mice were used (test = 12 and control = 6) for Ae. aegypti and 12 (test = 8 and control = 4) for Cx. quinquefasciatus to evaluate the mosquito-mice transmission rate. All mice that were bitten by infected Ae. aegypti showed clinical signs of infection while all mice exposed to infected Cx. quinquefasciatus mosquitoes remained healthy. Viremia in the mice from Ae. aegypti group ranged from 2.5 × 108 to 5 × 109 PFU/mL. Ae. aegypti from the second blood feeding showed a 50% IR. Our study showed the applicability of an efficient model to complete arbovirus transmission cycle studies and suggests that the Ae. aegypti population evaluated is a competent vector for MAYV, while highlighting the vectorial capacity of Ae. aegypti and the possible introduction into urban areas. The mice model employed here is an important tool for arthropod-vector transmission studies with laboratory and field mosquito populations, as well as with other arboviruses.

Effects of gamma radiation on the vector competence of Aedes aegypti (diptera: Culicidae) to transmit Zika virus.

One of the limitations of the Sterile Insect Technique (SIT), conventionally performed by ionizing radiation, regards separating males from females, which is not 100% effective. Some irradiated females may be released together with males in the field at SIT. The present study aimed to evaluate the influence of ionizing radiation on the ability of Aedes aegypti mosquitoes to transmit the Zika virus after exposing female pupae to a 40 Gy of gamma radiation. The results suggest that the genetic damage induced by exposure of females to this dose level promotes their total sterility, but it does not influence their vector competence. However, our data point out that ionizing radiation may decrease the proportion of infective mosquitoes.

Potential Nosocomial Infections by the Zika and Chikungunya Viruses in Public Health Facilities in the Metropolitan Area of Recife, Brazil.

Since 2015, the Dengue, Zika, and Chikungunya viruses gained notoriety for their impact in public health in many parts of the globe, including Brazil. In Recife, the capital of Pernambuco State, the introduction of ZIKV impacted human population tremendously, owing to the increase in the number of neurological cases, such as the Guillain−Barré and congenital Zika disorders. Later, Recife was considered to be the epicenter for ZIKV epidemics in Brazil. For arboviral diseases, there are some risk factors, such as climate changes, low socioeconomic conditions, and the high densities of vectors populations, that favor the broad and rapid dispersion of these three viruses in the city. Therefore, continuous arbovirus surveillance provides an important tool for detecting these arboviruses and predicting new outbreaks. The purpose of the present study was to evaluate the circulation of DENV, ZIKV, and CHIKV by RT-qPCR in mosquitoes collected in health care units from the metropolitan area of Recife (MAR), during 2018. A total of 2321 female mosquitoes (357 pools) belonging to two species, Aedes aegypti and Culex quinquefasciatus, were collected from 18 different healthcare units, distributed in five cities from the MAR. Twenty-three pools were positive for ZIKV, out of which, seventeen were of C. quinquefasciatus and six were of A. aegypti. Positive pools were collected in 11/18 health care units screened, with Cq values ranging from 30.0 to 37.4 and viral loads varying from 1.88 × 107 to 2.14 × 109 RNA copies/mL. Nosocomial Aedes- and Culex-borne transmission of arbovirus are widely ignored by surveillance and vector control programs, even though healthcare-associated infections (HAI) are considered a serious threat to patient safety worldwide. Although the results presented here concern only the epidemiological scenario from 2018 in MAR, the potential of hospital-acquired transmission through mosquito bites is being overlooked by public health authorities. It is, therefore, of the ultimate importance to establish specific control programs for these locations.

Effects of gamma radiation on the reproductive viability of Aedes aegypti and its descendants (Diptera: Culicidae).

This work evaluated the genetic damage in descendants of male pupae of Aedes (Stegomyia) aegypti (Diptera: Culicidae) separately exposed to 20, 30, and 40 Gy of gamma radiation in the context of Sterile Insect Technique (SIT). Despite the transmission of the dominant lethal mutation, the employed dose levels did not promote a marked reduction in adult mosquito emergence and fertility. This study emphasized that semi-sterilizing doses < 50 Gy for SIT of Aedes aegypti are not recommended.

Aedes aegypti Strain Subjected to Long-Term Exposure to Bacillus thuringiensis svar. israelensis Larvicides Displays an Altered Transcriptional Response to Zika Virus Infection.

Bacillus thuringiensis svar. israelensis (Bti) larvicides are effective in controlling Aedes aegypti; however, the effects of long-term exposure need to be properly evaluated. We established an Ae. aegypti strain that has been treated with Bti for 30 generations (RecBti) and is still susceptible to Bti, but females exhibited increased susceptibility to Zika virus (ZIKV). This study compared the RecBti strain to a reference strain regarding: first, the relative transcription of selected immune genes in ZIKV-challenged females (F30) with increased susceptibility detected in a previous study; then, the whole transcriptomic profile using unchallenged females (F35). Among the genes compared by RT-qPCR in the ZIKV-infected and uninfected females from RecBti (F30) and the reference strain, hop, domeless, relish 1, defensin A, cecropin D, and gambicin showed a trend of repression in RecBti infected females. The transcriptome of RecBti (F35) unchallenged females, compared with a reference strain by RNA-seq, showed a similar profile and only 59 differentially expressed genes were found among 9202 genes analyzed. Our dataset showed that the long-term Bti exposure of the RecBti strain was associated with an alteration of the expression of genes potentially involved in the response to ZIKV infection in challenged females, which is an important feature found under this condition.

First report of the L1014F kdr mutation in wild populations of Anopheles arabiensis in Cabo Verde, West Africa.

BACKGROUND: Due to the lack of vaccines, malaria control mainly involves the control of anopheline vectors (Anopheles spp.) using chemical insecticides. However, the prolonged and indiscriminate use of these compounds has led to the emergence of resistance in Anopheles populations in Africa. Insecticide resistance surveillance programs are less frequent in Cabo Verde than in other African countries. This study aimed to investigate the circulation of the L1014F and L1014S alleles in natural populations of Anopheles arabiensis collected from two sampling sites in the city of Praia, Cabo Verde. METHODS: Anopheles larvae were collected from the two sampling sites and reared in the laboratory until the adult stage. Mosquitoes were first morphologically identified by classical taxonomy and then by molecular species identification using molecular markers. All Anopheles arabiensis were subjected to PCR analysis to screen for mutations associated to resistance in the Nav gene. RESULTS: A total of 105 mosquitoes, all belonging to the Anopheles gambiae complex, were identified by classical taxonomy as well as by molecular taxonomy. Molecular identification showed that 100% of the An. gambiae senso lato specimens analyzed corresponded to An. arabiensis. Analysis of the Nav gene revealed the presence of L1014S and L1014F alleles with frequencies of 0.10 and 0.19, respectively. CONCLUSIONS: Our data demonstrated, for the first time, the presence of the L1014F allele in the An. arabiensis population from Cabo Verde, as well as an increase in the frequency of the kdr L1014S allele reported in a previous study. The results of this study demonstrate the need to establish new approaches in vector control programs in Cabo Verde.

Aedes aegypti continuously exposed to Bacillus thuringiensis svar. israelensis does not exhibit changes in life traits but displays increased susceptibility for Zika virus.

BACKGROUND: Aedes aegypti can transmit arboviruses worldwide, and Bacillus thuringiensis svar. israelensis (Bti)-based larvicides represent an effective tool for controlling this species. The safety of Bti and lack of resistance have been widely reported; however, little is known regarding the impact of the extensive use of these larvicides on the life traits of mosquitoes. Therefore, this study investigated biological parameters, including susceptibility to arbovirus, of an Ae. aegypti strain (RecBti) subjected to 29 generations of exposure to Bti compared with the RecL reference strain. METHODS: The biological parameters of individuals reared under controlled conditions were compared. Also, the viral susceptibility of females not exposed to Bti during their larval stage was analysed by oral infection and followed until 14 or 21 days post-infection (dpi). RESULTS: RecBti individuals did not display alterations in the traits that were assessed (fecundity, fertility, pupal weight, developmental time, emergence rate, sex ratio and haematophagic capacity) compared to RecL individuals. Females from both strains were susceptible to dengue serotype 2 (DENV-2) and Zika virus (ZIKV). However, RecBti females showed significantly higher rates of ZIKV infection compared with RecL females at 7 (90% versus 68%, Chi-square: χ2 = 7.27, df = 1, P = 0.006) and 14 dpi (100% versus 87%, Chi-square: χ2 = 7.69, df = 1, P = 0.005) and for dissemination at 7 dpi (83.3% versus 36%, Fisher's exact test: P < 0.0001, OR = 0.11, 95% CI 0.03-0.32). Quantification of DENV-2 and ZIKV viral particles produced statistically similar results for females from both strains. CONCLUSIONS: Prolonged exposure of Ae. aegypti larvae to Bti did not alter most of the evaluated biological parameters, except that RecBti females exhibited a higher vector susceptibility for ZIKV. This finding is related to a background of Bti exposure for several generations but not to a previous exposure of the tested females during the larval stage. This study highlights mosquito responses that could be associated with the chronic exposure to Bti in addition to the primary larvicidal effect elicited by this control agent.

Multiple Introductions Followed by Ongoing Community Spread of SARS-CoV-2 at One of the Largest Metropolitan Areas of Northeast Brazil.

Multiple epicenters of the SARS-CoV-2 pandemic have emerged since the first pneumonia cases in Wuhan, China, such as Italy, USA, and Brazil. Brazil is the third-most affected country worldwide, but genomic sequences of SARS-CoV-2 strains are mostly restricted to states from the Southeast region. Pernambuco state, located in the Northeast region, is the sixth most affected Brazilian state, but very few genomic sequences from the strains circulating in this region are available. We sequenced 101 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms that reside in Pernambuco. Phylogenetic reconstructions revealed that all genomes belong to the B lineage and most of the samples (88%) were classified as lineage B.1.1. We detected multiple viral introductions from abroad (likely from Europe) as well as six local B.1.1 clades composed by Pernambuco only strains. Local clades comprise sequences from the capital city (Recife) and other country-side cities, corroborating the community spread between different municipalities of the state. These findings demonstrate that different from Southeastern Brazilian states where the epidemics were majorly driven by one dominant lineage (B.1.1.28 or B.1.1.33), the early epidemic phase at the Pernambuco state was driven by multiple B.1.1 lineages seeded through both national and international traveling.

Sequencing of ZIKV genomes directly from Ae. aegypti and Cx. quinquefasciatus mosquitoes collected during the 2015-16 epidemics in Recife.

Zika virus (ZIKV) is a negative sense RNA virus from the Flaviviridae family, which was relatively unknown until the first human epidemic in Micronesia, in 2007. Since then, it spread to French Polynesia and the Americas. Recife, the capital of Pernambuco state and epicenter of the Zika epidemic in Brazil, experienced a large number of microcephaly cases and other congenital abnormalities associated to the ZIKV infection from, 2015 to 16. Evidences suggest that both Aedes aegypti and Culex quinquefasciatus mosquitoes from Recife are capable of replicating and transmitting the virus. Here, we conducted high throughput sequencing of ZIKV genomes directly from Ae. aegypti and Cx. quinquefasciatus mosquitoes collected during the ZIKV epidemics in Recife, in order to investigate the variability and evolution of the virus. We obtained 11 draft ZIKV genomes derived from 5 pools from each Ae. aegypti and Cx. quinquefasciatus species. Genome coverage breadth ranged from 16 to 100% and average depth from 45 to 46,584×. Two of these genomes were obtained from pools of Cx. quinquefasciatus females with no sign of blood in the abdomen. Amino acid substitutions found here were not species-specific. In addition, molecular clock dating estimated that ZIKV draft genomes obtained here were co-circulating in other regions of the country during the epidemics. Overall results highlight that viral mutations and even minor variants can be detected in genomes directly sequenced from mosquito samples and insights about natural viral genomic variability and viral evolution can be useful when designing tools for mosquito control programs.

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

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

Detection of alleles associated with resistance to chemical insecticide in the malaria vector Anopheles arabiensis in Santiago, Cabo Verde.

BACKGROUND: Mosquitoes of the Anopheles gambiae complex are the main malaria vectors worldwide. Due to the lack of a vaccine to prevent malaria, the principal way to reduce the impact of this disease relies on the use of chemical insecticides to control its vectors. However, the intensive use of such compounds has led to the emergence of insecticide resistance in several Anopheles populations in Africa. This study aimed to investigate the presence of resistance alleles in an Anopheles arabiensis population from the City of Praia, capital of the Archipelago Cabo Verde, one of the countries on the World Health Organization list of countries that are on a path to eliminate local transmission of malaria. METHODS: Larvae from the Anopheles genus were collected using a one-pint dipper in three areas of City of Praia. Larvae were fed and maintained until the emergence of adult mosquitoes, and these were morphologically identified. In addition, molecular identification was performed using IGS markers and all An. arabiensis samples were subjected to PCR to screen for mutations associated to resistance in the Ace-1, Nav and GSTE2 genes. RESULTS: From a total of 440 mosquitoes collected, 52.3% were morphologically identified as An. gambiae sensu lato (s.l.) and 46.7% as Anopheles pretoriensis. The molecular identification showed that 100% of the An. gambiae s.l. were An. arabiensis. The mutations G119S in the Ace-1 gene and L119F in the GSTE2 gene were screened but not found in any sample. However, sequencing analysis for GSTE2 revealed the presence of 37 haplotypes, 16 polymorphic sites and a high genetic diversity (π = 2.67). The L1014S mutation in the Nav (voltage-gated sodium channel gene) was detected at a frequency of 7.3%. CONCLUSION: This is the first study to investigate the circulation of insecticide resistance alleles in An. arabiensis from Cabo Verde. The circulation of the L1014S allele in the population of An. arabiensis in the city of Praia suggests that pyrethroid resistance may arise, be quickly selected, and may affect the process of malaria elimination in Cabo Verde. Molecular monitoring of resistance should continue in order to guide the development of strategies to be used in vector control in the study region.

Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.

Development of an urban molecular xenomonitoring system for lymphatic filariasis in the Recife Metropolitan Region, Brazil.

INTRODUCTION: Molecular xenomonitoring (MX)-pathogen detection in the mosquito rather than human-is a promising tool for lymphatic filariasis (LF) surveillance. In the Recife Metropolitan Region (RMR), the last LF focus in Brazil, Culex quinquefasciatus mosquitoes have been implicated in transmitting Wuchereria bancrofti parasites. This paper presents findings on the ideal mosquito collection method, mosquito dispersion, W. bancrofti infection in mosquitoes and W. bancrofti antigen in humans to aid MX development. METHODS: Experiments occurred within two densely populated urban areas of Olinda, RMR, in July and August 2015. U.S. Centers for Disease Control and Prevention (CDC) light traps were compared to battery-powered aspirators as collection methods, and mosquito dispersion was measured by mosquito mark release recapture (MMRR). Female Cx. quinquefasciatus were tested by PCR for W. bancrofti infection, and study area residents were screened by rapid tests for W. bancrofti antigen. RESULTS: Aspirators caught 2.6 times more total Cx. quinquefasciatus, including 38 times more blood-fed and 5 times more gravid stages, than CDC light traps. They also collected 123 times more Aedes aegypti. Of the 9,644 marked mosquitoes released, only ten (0.01%) were recaptured, nine of which were < 50m (34.8m median, 85.4m maximum) from the release point. Of 9,169 unmarked mosquitoes captured in the MMR, 38.3% were unfed, 48.8% blood-fed, 5.5% semi-gravid, and 7.3% gravid. PCR on 182 pools (1,556 mosquitoes) found no evidence of W. bancrofti infection in Cx. quinquefasciatus. Rapid tests on 110 of 111 eligible residents were all negative for W. bancrofti antigen. CONCLUSIONS: Aspirators were more effective than CDC light traps at capturing Ae. aegypti and all but unfed stages of Cx. quinquefasciatus. Female Cx. quinquefasciatus traveled short (< 86m) distances in this urban area. Lack of evidence for W. bancrofti infection in mosquitoes and antigen in humans in these fine-scale studies does not indicate that LF transmission has ceased in the RMR. A MX surveillance system should consider vector-specific collection methods, mosquito dispersion, and spatial scale but also local context, environmental factors such as sanitation, and host factors such as infection prevalence and treatment history.

Circulation of DENV2 and DENV4 in Aedes aegypti (Diptera: Culicidae) mosquitoes from Praia, Santiago Island, Cabo Verde.

Arthropod-borne viruses, such as Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV), pose a challenge to public health, due to their worldwide distribution and large-scale outbreaks. Dengue fever is currently one of the most important diseases and it is caused by four serotypes of DENV and is mainly transmitted by the mosquito Aedes aegypti. It is estimated that 50-100 million cases are reported every year worldwide. More recently, CHIKV and ZIKV, which are also transmitted by Ae. aegypti, have caused epidemics in countries in the Caribbean region, the Pacific region, and Americas. Cabo Verde faced its first dengue outbreak in 2009, with more than 21,000 reported cases and four registered deaths. The epidemic was caused by DENV-3 transmitted by Ae. aegypti mosquitoes. In addition, the country faced a Zika outbreak with more than 7,500 notified cases from October 2015 to May 2016. In the present study, we conducted a survey in mosquito samples to detect arboviruses circulating in the local vector population. Collections were performed from November 2014 to January 2015, in the City of Praia, the capital of Cabo Verde, using aspirators and BG-sentinel traps. Samples were examined by multiplex Reverse Transcription-polymerase chain reaction. A total of 161 Ae. aegypti adult females were analyzed (34 pools) and from these samples, eight pools were found positive for DENV-2 and DENV-4. Our results revealed a very high natural infection rate in the vector population and showed two different serotypes co-circulating in the island that differ from the one detected in the 2009 outbreak in Cabo Verde.

Sensitivity of RT-PCR method in samples shown to be positive for Zika virus by RT-qPCR in vector competence studies

Abstract Tissue samples from mosquitoes artificially infected with Zika virus and shown to be positive by RT-qPCR were reexamined by RT-PCR. Using these samples we compared the two methods employed in virus RNA detection for vector competence studies. Results demonstrated that, albeit useful, RT-PCR gave false negatives with low viral loads (< 106 RNA copies/ml).

Zika virus replication in the mosquito Culex quinquefasciatus in Brazil.

Zika virus (ZIKV) is a flavivirus that has recently been associated with an increased incidence of neonatal microcephaly and other neurological disorders. The virus is primarily transmitted by mosquito bite, although other routes of infection have been implicated in some cases. The Aedes aegypti mosquito is considered to be the main vector to humans worldwide; however, there is evidence that other mosquito species, including Culex quinquefasciatus, transmit the virus. To test the potential of Cx. quinquefasciatus to transmit ZIKV, we experimentally compared the vector competence of laboratory-reared Ae. aegypti and Cx. quinquefasciatus. Interestingly, we were able to detect the presence of ZIKV in the midgut, salivary glands and saliva of artificially fed Cx. quinquefasciatus. In addition, we collected ZIKV-infected Cx. quinquefasciatus from urban areas with high microcephaly incidence in Recife, Brazil. Corroborating our experimental data from artificially fed mosquitoes, ZIKV was isolated from field-caught Cx. quinquefasciatus, and its genome was partially sequenced. Collectively, these findings indicate that there may be a wider range of ZIKV vectors than anticipated.

Sensitivity of RT-PCR method in samples shown to be positive for Zika virus by RT-qPCR in vector competence studies.

Tissue samples from mosquitoes artificially infected with Zika virus and shown to be positive by RT-qPCR were reexamined by RT-PCR. Using these samples we compared the two methods employed in virus RNA detection for vector competence studies. Results demonstrated that, albeit useful, RT-PCR gave false negatives with low viral loads (< 106 RNA copies/ml).

Does Zika virus infection affect mosquito response to repellents?

The World Health Organization (WHO) recommends that people travelling to or living in areas with Zika virus (ZIKV) outbreaks or epidemics adopt prophylactic measures to reduce or eliminate mosquito bites, including the use of insect repellents. It is, however, unknown whether repellents are effective against ZIKV-infected mosquitoes, in part because of the ethical concerns related to exposing a human subject's arm to infected mosquitoes in the standard arm-in-cage assay. We used a previously developed, human subject-free behavioural assay, which mimics a human subject to evaluate the top two recommended insect repellents. Our measurements showed that DEET provided significantly higher protection than picaridin provided against noninfected, host-seeking females of the southern house mosquito, Culex quinquefasciatus, and the yellow fever mosquito, Aedes aegypti. When tested at lower doses, we observed a significant reduction in DEET-elicited protection against ZIKV-infected yellow fever mosquitoes from old and recent laboratory colonies. The reduction in protection is more likely associated with aging than the virus infection and could be compensated by applying a 5x higher dose of DEET. A substantial protection against ZIKV-infected and old noninfected mosquitoes was achieved with 5% DEET, which corresponds approximately to a 30% dose in the conventional arm-in-cage assays.