Mem Inst Oswaldo Cruz, Rio de Janeiro, 113(1) January 2018
First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes
1Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Laboratório de Ecologia de Doenças Transmissíveis na Amazônia, Manaus, AM, Brasil
2Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Programa de Pós-Graduação em Condições de Vida e Situações de Saúde na Amazônia, Manaus, AM, Brasil
BACKGROUND Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur.
OBJECTIVES In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions.
METHODS Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR.
FINDINGS The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes.
MAIN CONCLUSION Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.
Zika is a diseasecaused by an arbovirus (Zika virus, or ZIKV) of the Flaviviridae family,Flavivirus genus. It is a worldwide public health concern. ZIKV was firstdescribed in Africa in 1947 (Mukwaya & Sempala 1977), and for a long time,it was thought to cause only a benign illness. However, after its emergencein Brazil in 2015 and its spread throughout most of Latin America and the Caribbean,ZIKV infection has been associated with thousands of cases involving severecomplications such as microcephaly, Guillain-Barré syndrome, and death.This severe and unexpected epidemic led the World Health Organization (WHO)to recognise ZIKV as a Public Health Emergency of International Concern (Azevedoet al. 2016, Santos et al. 2016 29 ). Zika symptoms are mild and characterised bythe sudden onset of fever, maculopapular rash, arthralgia, myalgia, headache,nonpurulent conjunctivitis, pruritus, joint oedema, and exanthema (Azevedo etal. 2016, Nunes et al. 2016 24 , Vasconcelos & Calisher 2016). Between 2015and 2016, a total of 707,133 cases were recorded across 48 countries (Ikejezieet al. 2017). In 2016 alone, 205,578 cases and eight deaths were recorded inBrazil. The highest incidence rates in Brazil have been recorded in the Central-WestRegion (231 cases per 100,000) and the North Region (157 cases per 100,000).As of June 2017, there have been 13,353 confirmed cases of Zika, and 322 casesof microcephaly associated with congenital Zika virus infection (MS/SVS 2017 20 ).
ZIKV has been foundinfecting the salivary glands of several species of mosquitoes from the Aedesand Culex genera, but the infection susceptibility of a particularspecies seems to be strongly associated with specific virus strains (Fernandeset al. 2016, Ferreira-de-Brito et al. 2016 8 , Guedes et al. 2017 9 ). Ae. aegyptiis considered the main ZIKV vector (WHO 2016a 33 ) and is thought to be responsiblefor the 2015-2016 outbreak in Brazil (Oliveira et al. 2016 25 ). Moreover, ZIKVcan infect and be transmitted by American populations of Ae. aegyptiand Ae. albopictus (Chouin-Carneiro et al. 2016 3 , Costa-da-Silva et al.2017).
In addition totransmission by mosquito bite, ZIKV can be sexually transmitted between humans;ZIKV has been detected in human semen (Musso et al. 2015 22 ). In monkeys, ZIKVcan generate viraemia sufficient to infect competent mosquito vectors when introducedintrarectally or intravaginally (Musso et al. 2015 22 , Haddow et al. 2017 11 , Hastings& Fikrig 2017). Zika-positive Ae. aegypti males have been collectedin the field which suggests that vertical and/or venereal transmission of ZIKVmay occur between mosquitoes (Ferreira-de-Brito et al. 2016 8 , Thangamani et al.2016). The maintenance of arboviruses in nature is greatly enhanced when venerealtransmission occurs in conjunction with other transmission mechanisms. Venerealtransmission has been demonstrated in Ae. aegypti infected with the Chikungunyavirus (Mavale et al. 2010 18 ).
This study showsthat venereal transmission of ZIKV by Ae. aegypti can occur under controlledlaboratory conditions. This is an important finding because it may partiallyexplain the high dispersion rate of infected mosquitoes during Zika epidemics,and it highlights the importance of mosquito control programs.
Mosquitoes- Larvae and pupae of Ae. aegypti mosquitoes were collected in January,2016 from water tanks and discarded containers located around households inNova Cidade and Adrianópolis neighbourhoods, around the city of Manaus,Amazonas state, Brazil. Field-collected specimens were reared through two generationsunder laboratory conditions. Individual F2 adult females were stored at -80ºCand the individual batch egg / female were separated and kept for breeding andassays. Total RNA from each F2 female was extracted with TRIzol®Reagent (Thermo Fisher, Waltham, MA) following manufacturer's instructions,and tested for the presence of ZIKV (Lanciotti et al. 2008), CHIKV (Lanciottiet al. 2007) and DENV (Gurukumar et al. 2009 10 ) by reverse transcription real-timepolymerase chain reaction (RT-qPCR). Batch eggs from F2 individual adult femalesthat tested negative for all three viruses were used to establish the virus-freemosquito colony, named AaM3V-. The mosquito colony was maintainedunder laboratory conditions at 27ºC with 70% relative humidity. Larvaewere reared in plastic containers containing tap water and were fed with fishfood. Adults were kept in plastic cages and offered a 10% sucrose solutionad libitum.
ZIKV strain- The ZIKV used in this study was isolated from a female patient presentingwith classical symptoms of arbovirus infection. Zika infection was confirmedby RT-qPCR (Lanciotti et al. 2008). This strain was obtained after the secondpassage on Ae. albopictus C6/36 cells kept at 28ºC in Leibovitz'sL-15 medium supplemented with 2% foetal bovine serum (FBS) and an antibiotic/antimycotic.
After three daysof infection on C6/36 cells, viral titre was determined by flow cytometry, basedon a previously published protocol for the Dengue virus (Medina et al. 2012 19 ).Briefly, we used the anti-flavivirus 4G2 monoclonal antibody, followed by anti-mouseIgG Alexa 488-conjugated secondary antibody, diluted at 1:2,000 and 1:3,000,respectively. After incubation and washing, cells were analysed by flow cytometry,counting at least 100,000 events.
Intrathoracicmicroinjection of mosquitoes - One group containing 50 (2-day-old) malemosquitoes was intrathoracically inoculated with a 0.2-µL suspension ofZIKV at a titre of 107 infectious units/mL (FACS IU/mL), followingRosen and Gubler (1974). Microinjection was made using the Nanoject II injector(Drummond Scientific Company). Mosquitoes were subsequently maintained at 27ºCwith 70% relative humidity, and offered a 10% sucrose solution ad libitum.Four days after injection, the male mosquitoes were transferred to a cage containing100 (6-day-old) virgin females and left to copulate for five days at a proportionof one male to two females. After the 5-day copulation period, all females wereindividually placed in 1 mL of TRIzol® Reagent and stored at-80ºC prior to viral detection by RT-qPCR (Fig.1A). Two independent biological experimental replicates were made for thisassay.
Oral infectionof mosquitoes - One group containing 50 (2-day-old) virgin females was fedusing a Parafilm® membrane. A suspension of ZIKV at a titre of107 infectious units/mL (FACS IU/mL) was used for blood feeding.Fully engorged females were transferred to plastic cages and maintained at 27ºCwith 70% relative humidity, and offered a 10% sucrose solution ad libitum.Nine days after blood feeding, the females were placed in cages with Ae.aegypti AaM3V- virgin 2-day-old males and left to copulate forfour days at a proportion of one male to two females. After the 4-day copulationperiod, 20 surviving males were placed individually in 1 mL of TRIzol®Reagent and stored at -80ºC (Fig. 1B).Two experimental biological replicates were made.
Four days postinfection(dpi) was judged to be sufficient time for ZIKV to disseminate throughout thebody and into the seminal fluid of the male mosquitoes, and 9 dpi was judgedto be sufficient time for the arbovirus to be transmitted venereally and bedetectable in the female mosquitoes.
ZIKV detectionin mosquitoes - Mosquitoes were individually homogenised in 1 mL of TRIzol®Reagent and RNA was extracted following manufacturer's instructions. Viral RNAwas detected using TaqMan® Fast Virus 1-Step Master Mix in aStepOnePlus Real Time PCR System (Applied Biosystems) using ZIKV primers andprobes described previously (Lanciotti et al. 2008). The RT-qPCR conditionswere: 50ºC for 5 min, 95ºC for 20 s, and 45 cycles at 95ºC for3 s, and 60ºC for 30 s with fluorescence acquisition. For all RT-qPCR assays,the MS2 RNA bacteriophage was introduced prior to RNA extraction in order totrack false-negative reactions due to PCR inhibition, as described previously(Naveca et al. 2017 23 ).
RT-qPCR reactionswere analysed by Ct values being Ct ≤35 positive for ZIKV RNA presence. Similar studies have considered Ct values≤36 (Smartt et al. 2017 30 ) and ≤38 (Ferreira-de-Brito et al. 2016 8 , Burkhalter & Savage 2017) as positivefor ZIKV presence in mosquitoes. The Ct is the qPCR cycle where the reportersignal crosses the background fluorescence, and specific amplification is detected.Moreover, the Ct values are inversely linked to the initial quantity of targetnucleic acid and can be used to measure its relative concentration in the reaction.Although our experiments were not designed to estimate quantitative data, theobtained Ct values were used as an indirect approach to evaluate viral replication.
We did not evaluatethe sensitivity of this assay in vectors, but Lanciotti et al. (2008) 15 reporteda sensitivity of 25 copies per reaction. This assay employs a Taqman probe,together with primers specifically designed to ZIKV, which enhances specificity.Furthermore, we used a ZIKV isolate that was previously submitted to nucleotidesequencing that confirms the viral species and genotype.
Data analysis- Data were analysed using StepOne V2.3 software (Applied Biosystems). Infectionrates were calculated by dividing the number of positive mosquitoes for ZIKVRNA by the number of mosquitoes tested for ZIKV infection. A Student t-testwas used to compare the intra-experimental differences between the two biologicalreplicates.
Ethics statement- Mosquito field collections were approved by SisBio (Sistema de Autorizaçãoe Informação em Biodiversidade - Permission and Information inBiodiversity System) number 12186. This study was approved by the BrazilianNational Ethics Committee (CONEP, 3726).
Venereal transmissionof ZIKV by Ae. aegypti males infected via intrathoracic microinjection -To determine whether infected Ae. aegypti males can transmit ZIKV touninfected females, 100 adult females were made available for copulation withinfected males. Ct values ranged from 13 to 35 in females after mating withintrathoracically infected males (Fig. 2A). Themean infection rate in females was 45%, with 40% infected in the first experimentand 50% infected in the replicate (t-test, p = 0.21) (Table).
Venereal transmissionof ZIKV by orally infected Ae. aegypti females - To determine whether infectedAe. aegypti females can transmit ZIKV to uninfected males, 20 survivingmales (10 from each experimental replicate) were tested for ZIKV transmittedvenereally by orally infected females. Ct values ranged from 29 to 35 in malesmated with orally infected females (Fig. 2B).The mean infection rate in males was 35%, with 50% infected in the first experimentand 20% infected in the second experiment (t-test, p = 0.34).
To complete thelife-cycle in nature, the virus must replicate in a mosquito's tissues. Virusparticles then spread throughout the mosquito's body, and once they reach thesalivary glands, they can be transmitted to vertebrate hosts. Some studies havebeen conducted to ascertain the steps involved in the replication process ofDENV, a closely related Flavivirus (Pang et al. 2001 26 , Salazar et al.2007). In this process, DENV-2 spreads from the midgut at 2 dpi, it disseminatesto the salivary glands and other organs at 4 dpi, and it can still be detectedat up to 21 dpi (Salazar et al. 2007 28 ). Few details on the ZIKV replication processin Ae. aegypti are known. This mosquito species has been susceptibleto ZIKV (Li et al. 2012, Chouin-Carneiro et al. 2016 3 , Guedes et al. 2017 9 ). Afterthree or four days of ZIKV challenge, the infection was detected in the midgut,and by seven and 14 days, it is detected in salivary glands. However, the infectionrates may change according to the combination of the mosquito population andvirus strain. Decreased viral titres were observed by day 14 in the midgut,but ZIKV RNA was detected in the saliva indicating that virus replication occursin salivary glands during the late phase of infection, suggesting viral transmission(Guedes et al. 2017 9 ).
The focus of ourwork is to demonstrate the venereal transmission of ZIKV in Ae. aegypti mosquitoes.Dissemination or transmission rates were not measured. However, as discussedby Li et al. (2012) 17 , the proliferation and dissemination of ZIKV in Ae. aegyptiis systemic, being possible to find the virus in the midgut, salivary glands,and other tissues such as haemocytes, ganglion, and fat bodies at three-fivedays after challenge. In addition, Brazilian Ae. aegypti is extremelysusceptible to ZIKV, with high infection rates found in the midgut and salivaryglands (Ferreira-de-Brito et al. 216, Costa-da-Silva et al. 2017 5 , Guedes etal. 2017). Further, in our work, the short time of four-five days was enoughto spread the virus in the body of the mosquito, and four-five days of matingwere enough for venereal transmission to occur. Higher mating time was not necessaryto guarantee venereal transmission because females are inseminated only oncein their life (Lea 1968 16 ).
Until now, therehas been no strong evidence that ZIKV can be transmitted sexually among mosquitoes.However, it is known that female mosquitoes can become infected with arbovirusesduring haematophagy, and that females can then vertically transmit viruses totheir eggs. Vertical transmission of ZIKV has been observed in experimentallyinfected Ae. aegypti specimens (Thangamani et al. 2016 31 , Ciota et al.2017). The detection of ZIKV in Ae. furcifer males (Diallo et al. 2014)and Ae. aegypti males (Ferreira-de-Brito et al. 2016 8 ) suggests, but doesnot prove, that vertical and/or venereal transmission of Zika can occur in thesetwo species.
Our findings showedthe presence of ZIKV RNA in previously uninfected mosquitoes of both sexes followingcopulation with ZIKV-infected mates. These data strongly support the possibilitythat ZIKV is transmitted in the sexual fluids of mating mosquitoes. This posesa concern to public health because the venereal transmission of Zika among Ae.aegypti mosquitoes could potentially increase mosquito infection rates andthereby increase the spread of the virus. Furthermore, if venereal infectionoccurs in natural mosquito populations, this mode of transmission may be animportant mechanism of ZIKV maintenance in nature.
In this study,male mosquitoes were infected with ZIKV via intrathoracic microinjection andtransmitted to females during copulation. Additionally, female mosquitoes wereorally infected with ZIKV and transmitted to males during copulation. This resultindicates that ZIKV systemically disseminates throughout the mosquito body,as it does in other arboviruses from the Flaviviridae family. This studyshows that ZIKV infection of Ae. aegypti mosquitoes occurs not only duringblood feeding on infected vertebrates, but also during copulation. To the bestof our knowledge, this is the first strong evidence that ZIKV can be transmittedvenereally among Ae. aegypti mosquitoes.
Several authorsspeculate that vector to vector transmission is capable of maintaining an arboviruscirculation between interepidemic periods, when epidemiological factors, suchas the drop of viral density and the reduction of vertebrate-susceptible hosts,decreases a specific arboviral circulation. Thus, we believe that venereal transmissionas well as natural transovarian transmission may play an important role aidingthe establishment of endemic arboviral cycles.
Experts at theWHO have recently declared that "ZIKV and its associated consequences remaina significant and enduring public health challenge that require intense action,but ZIKV no longer represents a Public Health Emergency of International Concern"(WHO 2016b 34 ). In the global context, the dissemination of Aedes vectorsis of such scope that future outbreaks of ZIKV and other flaviviruses will bedifficult to foresee. Moreover, in the absence of a vaccine, our ability toblock the spread of ZIKV relies solely on vector control measures. Therefore,studies that increase our understanding of viral-host biological interactionsare of great importance and should be encouraged.
To Gervilane Ribeirofor help with mosquito colonisation.
CMRV, JWPS, FGN,FACP and VAN - Experiment conception and design, data analysis and writing;CMRV, JWPS, FGN, FACP, VAN, HCMB and JFA - conducted experiments. All authorsread and approved the final version of the manuscript.