Emerging Microbes & Infections 2020, VOL. 9 https://doi.org/10.1080/22221751.2020.1830718 Evidence of Zika virus horizontal and vertical transmission in Aedes albopictus from Spain but not infectious virus in saliva of the progeny Ana I. Nuñeza, Sandra Talaveraa, Lotty Birnberga, Raquel Rivasa, Núria Pujola, Marta Verdúna, Carles Arandaa,b, Miguel Berdugoc and Núria Busquetsa a IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain; b Consell Comarcal del Baix Llobregat, Servei de Control de Mosquits, Barcelona, Spain; cInstitut de Biología evolutiva de Barcelona, Universidad Pompeu Fabra-CSIC, Dr. Aigüader 88, Barcelona, 08003, Spain ABSTRACT Aedes albopictus mosquitoes have been experimentally demonstrated to be a competent vector for Zika virus (ZIKV) in different countries, but there are still some gaps related to the importance of Ae. albopictus in ZIKV transmission. Recent studies on Spanish Ae. albopictus populations showed controversial results for ZIKV transmission and no studies have been performed yet to detect infectious ZIKV in saliva of progeny of infected female mosquitoes. Herein, the horizontal transmission (HT) and vertical transmission (VT) of ZIKV in field-collected Ae. albopictus mosquitoes from Spain were evaluated for ZIKV strains (African I and Asian lineages) to better estimate the risk of ZIKV transmission by Ae. albopictus. The two field-collected Ae. albopictus populations assayed were infected by all tested ZIKV strains, however differences in terms of vector competence were detected depending on strain-population combination. Moreover, a higher susceptibility to the African I lineage strain than to the Asian lineage strain was observed in both mosquito populations. On the other hand, VT was demonstrated for both ZIKV lineages, detecting the virus in both males and females of the progeny of infected females, although importantly ZIKV dissemination and transmission were not detected in the infected females from the offspring. The results of the present study demonstrate that Spanish Ae. albopictus populations could sustain virus transmission in case of ZIKV introduction, but VT would play a poor role in the ZIKV epidemiology. Overall, our results provide helpful information to health authorities to establish efficient surveillance and vector control programmes for ZIKV. ARTICLE HISTORY Received 7 August 2020; Revised 23 September 2020; Accepted 26 September 2020 KEYWORDS Zika virus; Aedes albopictus; vector competence; vertical transmission Introduction Due to the recent outbreaks in the Americas and Zika virus (ZIKV) is an emergent arthropod-borne since it has been demonstrated to cause microcephaly virus that belongs to Flavivirus genus within Flavi- in neonates, the virus has become a threat to the viridae family [1]. It was isolated for the first time public health [1]. in 1947 from a rhesus monkey in the Zika forest Zika virus is maintained in a sylvatic zoonotic cycle (Uganda) [2], and afterwards in an Aedes africanus among primatophilic mosquitoes and non-human pri- pool from the same localization in 1948 [3]. Here- mates [10]. The epidemic cycle occurs between after, since the 1960s, ZIKV was sporadically found humans and anthropophilic mosquitoes maintaining outside the African continent and until 2007, when the virus in the urban sites [1]. The viral infection in ZIKV emerged in the Yap Island (Micronesia) [4] humans generally occurs by the bite of infected causing the first large outbreak. Then, it spread to females, but sexual [11] and perinatal [12] ZIKV trans- the Pacific islands in 2013 [5] and reached the mission have also been reported in humans. In this American continent in 2015 [6], where it finally sense, ZIKV vertical transmission (VT) has also been affected more than 45 countries [1,7]. All phyloge- shown in mosquitoes; ZIKV was detected in adult netic analyses performed by now, confirm that mosquitoes, which came from reared field-eggs col- ZIKV was originated in the African continent and lected of some mosquitoes from Aedes genus then spread to Asia, Pacific Islands and the Americas [1,13,14]. In addition, ZIKV was detected in field col- [8]. Currently, ZIKV is classified into three lineages: lected adult males of Aedes furcifer [13], Aedes aegypti Asian, African I, and African II [9]. The Asian lineage and Culex quinquefasciatus mosquitoes indicating caused the spread of ZIKV into the Americas [1]. venereal or vertical transmission in the field [10,15]. CONTACT Núria Busquets nuria.busquets@irta.cat CReSA, Campus Universitat Autònoma de Barcelona, Cerdanyola del Vallés, Barcelona, 08193, Spain Supplemental data for this article can be accessed at https://doi.org/10.1080/22221751.2020.1830718 © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrest- ricted use, distribution, and reproduction in any medium, provided the original work is properly cited. EMERGING MICROBES AND INFECTIONS 2237 Moreover, ZIKV VT has also been experimentally ZIKV epidemiology. These studies provide relevant demonstrated in Ae. aegypti [16–18] and Ae. albopic- and helpful information to health authorities to tus mosquitoes [17]; and venereal transmission, in design the strategies of the ZIKV surveillance Ae. aegypti mosquitoes [19]. Zika virus VT has been programmes. suggested as an important mechanism to maintain the virus in the environment during hostile conditions [16]. However, the epidemiological importance of Material and methods ZIKV VT in mosquitoes in nature and the trans- Mosquito rearing mission by the progeny of infected females is still unknown since no studies have been performed yet Eggs from two populations of Ae. albopictus from El to detect infectious ZIKV in saliva of progeny of Prat de Llobregat and Rubí (Catalonia, Spain [Fig infected females. S1]), were collected to evaluate the VC of ZIKV. In Recently, numerous Aedes mosquitoes from all addition, F0-F1 of field-collected Ae. albopictus continents have been shown to be experimentally sus- females from Rubí municipality were also used for ceptible to transmit the virus, such as Ae. vittatus, the VT assays. Larvae were reared in trays containing Aedes vexans [1], Aedes polynesiensis [20] and Ae. dechlorinated water supplemented with yeast (Gaye- Albopictus [21–25]. The Asian tiger mosquito species, lord- Hauser, Saint-Genis-Laval, France) until the Ae. albopictus, which was originated in the forests of adult stage. Emerging adults were maintained in the Southeastern Asia, is widely distributed in tropical laboratory under controlled environmental con- and subtropical regions. Nowadays, due to its global ditions: 28°C, 80% of relative humidity and a light/ expansion, it has been catalogued as an invasive mos- dark cycle of 12:12 h. Adult mosquitoes were provided quito species [26]. This mosquito species, which is an with 10% sucrose solution ad libitum. anthropophilic and day-time feeder [1], is known to be a competent vector of several arboviruses, such as chi- Zika virus strains kungunya virus (CHIKV), West Nile virus (WNV) or dengue virus (DENV) [27]. Its current expansion and Two ZIKV strains (Dakar and Martinique) were used establishment in Eastern Spain since 2004 [28] along in the present study to evaluate the VC of local mos- the Mediterranean coast and Balearic Islands, and its quito populations. The Dakar strain (UVE/ZIKV/ anthropophilic feeding behaviour, threat seriously 1984/SN/Dakar ArD 411662, African I lineage, pas- the public health of the country. It has been exper- sage 4, Genbank reference: KU955592) was isolated imentally demonstrated that Mediterranean Ae. albo- from an Aedes taylori mosquito species in Senegal in pictus mosquitoes are competent vectors for ZIKV 1984 and the Martinique strain (MRS_OPY_Martini- [22,25,29,30]. However, differences in vector compe- que_ PaRi_2015, Asian lineage, passage 3, Genbank tence (VC) among Ae. albopictus populations have reference: KU647676) was isolated from human been observed for ZIKV [31]. Moreover, recent studies serum in 2015. Both ZIKV strains were passaged one with Spanish Ae. albopictus populations have shown more time in our laboratory and titrated in Vero controversial results in terms of ZIKV susceptibility cells (ATCC, ref. CCL-81) to obtain the 50% tissue and transmission [30,32,33]. Additionally, three culture infective dose per mL (TCID50/ml). cases of ZIKV autochthonous transmission have been reported in France [34], emphasizing the Oral infection of mosquitoes for vector importance to determine if Spanish field-collected competence assays Ae. albopictus mosquito populations can transmit the virus in order to develop an efficient vector sur- The VC experiment was performed using the same veillance and control programmes by the competent protocol recently reported by Vazeille et al. [25]. authorities. The results of ZIKV transmission studies Briefly, a total of 879 Ae. albopictus adult females of allow to decide which mosquito species are relevant 7–9 days old, respectively, were starved for 24 h and in the ZIKV epidemiology so that they must be con- artificially fed with washed rabbit erythrocytes mixed sidered a target for ZIKV surveillance and reduced to with adenosine 5’-triphosphate (ATP) (5 × 10−3 M) avoid the spread of the virus and mitigate its locally (Sigma-Aldrich, St. Louis, MO) and containing circulation. For all the above-mentioned reasons, in ZIKV from a frozen viral stock (final concentration: the present study, we evaluated: (i) the VC of two 7 log10 TCDI 50/mL of Dakar and Martinique strains) field-collected Spanish Ae. albopictus mosquito popu- (Table 1). The infectious blood was provided to mos- lations for the African I and Asian ZIKV lineages, quitoes by an artificial Hemotek feeding system set at and (ii) VT and viral transmission of the offspring 37.5°C ± 0.5 (Discovery Workshop, Accrington, UK). of intrathoracically inoculated Ae. albopictus females Females were fed during 30 min. Then, blood- with the African I and Asian ZIKV lineages to better engorged females were anaesthetized on ice, selected, understand ZIKV VT and its importance in the and maintained in groups of 10 in cardboard cages 2238 A. I. NUÑEZ ET AL. Table 1. Summary of vector competence assays. Number of mosquitoes tested per time point Mosquito species tested Population ZIKV strains tested Titer of ZIKV (TCID 50/ml) 7 dpea 14 dpea 21 dpea Total Ae. albopictus El Prat de Llobregat Martinique 7 log10 TCID50/ml 20 20 17 57 El Prat de Llobregat Dak84 7 log10 TCID50/ml 11 13 12 36 Ae. albopictus Rubí Martinique 7 log10 TCID50/ml 28 27 22 77 Rubí Dak84 7 log10 TCID50/ml 28 27 25 80 a Dpe: days post-exposure. (Watkins & Doncaster, Leominster, UK) under the inoculated females were sacrificed at 7 days old and above-mentioned rearing conditions. Throughout whole bodies were kept at −80°C. Meanwhile, adult the experiment, mosquitoes were maintained with females from the same generation were anaesthetized 10% sucrose solution ad libitum. After a period of 7, on ice and dissected at 14 days old to allow the virus 14, and 21 days post exposure (dpe) to infectious reach salivary glands. Males were sacrificed earlier blood females were anaesthetized on ice. The legs than females since they die before females and only and wings were dissected and the saliva was extracted infection of the bodies was evaluated. Females were using a tip with 5 μl of FCS (fetal calf serum), where dissected, legs and wings were detached from the the proboscis was introduced during 30 min, and col- body to allow saliva extraction and heads were also lected in 45 μl of Dulbecco’s modified Eagle’s medium collected. All female mosquito samples (head, body (DMEM) (Gibco, MA, USA). The head, body and sal- and saliva samples) were stored at −80 °C until viral iva samples were stored in 300 μl of DMEM sup- isolation following the same protocol described plemented with 2% FCS and 1 X anti–anti (Gibco above. Finally, the filial infection rate (FIR) was used Life Technologies 100 ml 15240-062 100X) at −80°C to estimate the proportion of infected progeny until virus isolation. To evaluate the VC four rates among all eggs produced by the inoculated females were assessed: (i) the infection rate (IR), which rep- and the IR, DIR and TR of the progeny of the females resents the proportion of females with infected body inoculated with ZIKV were determined. among tested females; (ii) the disseminated infection (DIR), that refers to the proportion of females with infected head among females with infected body; (iii) the transmission rate (TR), which is the ratio ZIKV detection between females with infectious saliva among females The head and body samples obtained from the exper- with disseminated infection; and lastly (iv), the trans- imental infections were thawed and homogenized in mission efficiency (TE) that is the percentage of 0.5 ml of DMEM at 30 Hz for 1 min using TissueLyser females with infectious saliva among the total number II (Qiagen GmbH, Stockach, Germany). Virus detec- of mosquito females tested [31]. The experimental tion in head and body samples was performed by infections were carried out at Institut de Recerca i Tec- inoculating on 96 well plates with Vero CCL-81 nologia Agroalimentàries- Centre de Recerca en Sanitat cells, as described by Valleize et al. [25]. To summar- Animal (IRTA-CReSA) BLS3 facilities. ize, Vero cells inoculated with the samples were main- tained using DMEM, which was supplemented with 2% FCS and 1 X anti–anti, during seven days at 37 ° Intrathoracical ZIKV inoculation of mosquitoes C and 5% CO2. After this period, to observe the cyto- for vertical transmission assays pathic effect (CPE), cells were stained with a 0.2% Aedes albopictus (F0-F1) females from Rubí munici- crystal violet solution (C.I. 42555, Merck KGaA, pality were used for the VT assays. A total of 69, 29, Darmstadt, Germany) and 2% of formaldehyde (Elec- and 38 females of 5–7 days old were intrathoraci- tron Microscopy Sciences, Hatfield, PA, USA) during cally-inoculated with the Dakar (African I lineage) 30 min. Saliva samples were titrated on six well plates and Martinique (Asian lineage) strains at 7.5 log10- with Vero CCL-81 cells using a method described pre- TCID50/ml and with the Martinique strain at 8.2 log10- viously by Arias-Goeta et al. [35]. After seven days of TCID50/ml, respectively. All intrathoracically incubation under agarose overlay, the agar was inoculated females were maintained at the rearing removed, and the cells were stained with crystal violet conditions mentioned above. All females were starved solution for 30 min. Viral titers from saliva samples for 24 h and fed with fresh rabbit blood at 7- and 14- were expressed as plaque forming units per volume days post-inoculation (dpi) to obtain the eggs of the (PFUs/ml). The saliva samples from the VT exper- first (E1) and second (E2) ovipositions. The eggs iment were titrated in 96-well plates in a Vero cell were dried during 7 days at rearing conditions and monolayer and incubated for seven days at same con- then hatched for subsequent mosquito rearing. ditions above-mentioned until cytopathic effect Finally, adult males from the offspring of the evaluation. EMERGING MICROBES AND INFECTIONS 2239 Statistical analyses the infection, but the viral dissemination varied depending on the virus strain. The midgut escape The statistical differences between mosquito popu- barrier was stronger for the Martinique strain (Asian lations, virus strains and dpe were analysed using a lineage) than for the Dakar strain (African I lineage) generalized linear model with a binomial family distri- in both populations due to its low dissemination at bution (logistic regression). In this model, we analysed all tested time-points. both nominal effect and all factor interactions. Time With regards to the El Prat de Llobregat popu- post exposure was treated as a factor and not as a con- lation, the TE (positive saliva samples among all tinuous variable. Post-hoc analyses to compare blood feed mosquitoes) for Dakar strain (African I between mosquito populations among ZIKV lineages lineage) showed 23% and 58.3% at 14 and 21 dpe, at each time point were performed with a χ 2 test respectively, while Martinique strain (Asian lineage) using Monte Carlo permutations to determine the p- was not detected in any saliva sample. Moreover, in value. The statistical analyses were conducted using the Rubí population, Dakar strain (African I lineage) R 3.6 [36]. The p-value < 0.05 was considered statisti- presented 18.5% and 20% of positive saliva among cally significant. The TR and TE were not analysed by the assayed mosquito samples at 14 and 21 dpe, the logistic regression model due to the lack of var- while Martinique strain (Asian lineage) presented iance in the response variable driven by an excess of 0. 3.7% and 4.5% at 14 and 21 dpe, respectively. Further- more, Dakar strain (African I lineage) TE for both Results populations was higher at 21 dpe than at 14 dpe. Regarding to viral titers in saliva of those female Evidence that vector competence of field- mosquitoes that were able to expectorate ZIKV within collected Ae. albopictus populations for Zika the saliva, both Ae. albopictus populations which were virus depends on strain-population tested, showed higher viral titers in saliva samples for combination the Dakar strain (mean = 1712.4 PFU/ml) than for the The feeding rates were 58.5% (277/473) and 27.3% Martinique strain (mean = 186.8 PFU/ml), in which (111/406) for Ae. albopictus from Rubí and El Prat the size of the positive samples was low (2). The de Llobregat populations, respectively. Infected bodies viral loads in the saliva of positive mosquitoes are with ZIKV were detected from both field-collected Ae. shown in the Figure 2. albopictus populations exposed orally to infectious blood with both ZIKV strains tested at all-time points Vertical transmission was detected in Ae. (7, 14, and 21 dpe) (Figure 1, Table S1). At 7 dpe, an albopictus but the infected females of the early dissemination and transmission of the African progeny were not able to disseminate and I lineage (Dakar strain) could only be observed in transmit ZIKV the Rubi population. However, the proportion of females with infectious saliva among the total tested Infection and dissemination of the inoculated females females (TE) was higher at 21 dpe in El Prat de Llobre- were confirmed at 7 dpi. Zika virus was detected in gat population than in Rubí population (p = 0.02) for bodies of both males and females from the progeny Dakar strain. of the E1 of inoculated females for both tested ZIKV Regarding Martinique strain, Rubí population was lineages (Table 3). In addition, infected bodies were able to transmit it at 14 and 21 dpe; being the TE detected in males from the E2 of inoculated females slightly higher at 21 dpe (TE = 4.5%) than at 14 dpe for the Asian lineage. All heads and saliva samples (TE = 3.7%) (Figure 1). No viral particles of the Mar- from infected females of the progeny resulted negative tinique strain were isolated in mosquito saliva samples for both ZIKV lineages (Table S2 and Figure 3). from El Prat de Llobregat population probably may be due to the small sample size. Discussion Due to the ZIKV outbreak in 2015 in Brazil, the Spanish Ae. albopictus populations were more amount of scientific research about this virus has competent to the African I lineage than to the recently increased because of its medical importance. Asian ZIKV lineage Consequently, the number of VC studies for ZIKV When viral strains were compared we observed that has also augmented in the last years since virus trans- viral dissemination was higher for Dakar strain (Afri- mission via mosquito bite plays a crucial role to deter- can I lineage) than for Martinique strain (Asian line- mine the risk of introduction of a pathogenic agent in age) in both assayed mosquito populations along the a non-endemic country. European Ae. albopictus mos- infection (p = 1.18E-06) (Figure 1, Table 2, and S1). quitoes have been demonstrated to be competent vec- These results indicated that the midgut infection tors for ZIKV although showing heterogeneous results barrier was similar for both ZIKV strains throughout [22,25,29,30,37]. Our present study also shows the 2240 A. I. NUÑEZ ET AL. Figure 1. Infection, dissemination and transmission rates and transmission efficiency of two field-collected Ae. albopictus popu- lations exposed to Dakar and Martinique ZIKV strains, which are represented with red and green colours, respectively. The * in the figures shows the statically significant results between populations. IR: infection rate; DIR: dissemination rate; TR: transmission rate; TE: transmission efficiency. infection susceptibility of two field-collected Ae. albo- comparable since they performed the experiment pictus populations from Spain for two ZIKV lineages using the same protocol (viral dose, viral strains, (African I and Asian). Our results are particularly in environmental conditions, procedure) established in agreement with Vazeille et al. [25], which is easily the frame of the European ZIKAlliance project. Vazeille et al. [25] demonstrated that Ae. albopictus Table 2. Comparison of disseminated infection rates between populations from France (Montpellier and Corsica) mosquito population, dpe and viral strains using a generalized were more susceptible to Dakar strain (African I line- linear model with a binomial family distribution (logistic age) than to Martinique and Cambodia strains (Asian regression). lineage), which agrees with our findings. Both tested DIR populations in the present study showed significant d.f. χ2 p value differences in transmission at 21 dpe for the African Dpe 2 28.551 6.31E-07*** I lineage, as also previously presented by Vazeille Strain 1 23.6072 1.18E-06*** Population 1 0.0524 0.8189 et al. [25]. Moreover, our results showed an early Dpe*Strain 2 2.482 0.2891 transmission of the African I lineage at 7 dpe in Ae. Dpe*Population 2 1.7889 0.4088 Strain*Population 1 1.4643 0.2262 albopictus population from Rubí as also previously Dpe*Strain*Population 2 4.2258 0.1209 demonstrated by Vazeille et al. [25] for the African I ***p-value <0.0001; Dpe: days post-exposure; d.f.: degrees of freedom. lineage in an Ae. albopictus population from France EMERGING MICROBES AND INFECTIONS 2241 Figure 2. ZIKV loads in saliva (Plaque forming units [PFU/mL]) of infected female mosquitoes from two field-collected Ae. albo- pictus mosquito populations (El Prat del Llobregat and Rubí) exposed to Dakar and Martinique ZIKV strains. *Dpe: days post- exposure to the virus. Table 3. Number of eggs obtained from ZIKV inoculated females during the first and second gonotrophic cycle and filial infection rates (including males and females). Specie Generation Strains Viral doses FEF females Egg E1 Filial infection rate Egg E2 Filial infection rate Ae. albopictus F0-F1 Dak84 7.5 log10 TCID50/ml 34 559 1: 19.9 322 <1: 322 (0%) Ae. albopictus F0-F1 Martinique 7.5 log10 TCID50/ml 19 272 1: 38.8 94 1: 23.5 Ae. albopictus F1 Martinique 8.2 log10 TCID50/ml 12 140 1: 28 133 N.D. Note: FEF: full-engorged inoculated females; E1: first gonotrophic cycle; E2: second gonotrophic cycle; N.D.: No data. and Gutiérrez-López et al. [10] for the Asian lineage in introduction into the Mediterranean countries since a Spanish Ae. albopictus population. This early trans- the infected mosquito females could transmit the mission, which means a relatively short extrinsic incu- virus at least from 7 dpe. Besides, our results showed bation period, reported for Ae. albopictus mosquitoes differences in terms of VC for ZIKV between the could increase their vectorial capacity (commonly two tested mosquito populations. These differences defined as the number of infectious bites a host could also be observed when we compared our results receives daily [38]). A high vectorial capacity may with those obtained for French Ae. albopictus mosqui- increase the risk of infection in case of ZIKV toes. They seem to be better vectors for Dakar strain Figure 3. Infection and dissemination rates and transmission efficiency of the progeny of infected females from of one field-col- lected Ae. albopictus population (Rubí) for Dakar and Martinique ZIKV strains. IR: infection rate; DIR: dissemination rate; TE: trans- mission efficiency. 2242 A. I. NUÑEZ ET AL. (African I lineage) than Spanish populations at 14 dpe. interactions [42] as previously demonstrated in Ae. The TR obtained was around 80% in both assayed aegypti and Ae. albopictus mosquitoes for DENV French mosquito populations, and our transmission and ZIKV [31,39]. Lastly, besides mosquito-virus results were 60% and 35% in El Prat de Llobregat interaction, the number of generations of laboratory and in Rubí population respectively; although the colony influences VC [25,43]. It is a possible expla- transmission was similar at 21 dpe for all French nation of why the laboratory colony (from 2009) (around 80%) and Spanish mosquito populations used in the VC experiment by Hernández-Triana (100% and 71%, in El Prat de Llobregat and Rubí, et al. (2019) has not been able to transmit ZIKV. It respectively). In addition, Spanish Ae. albopictus is important to take into account that the laboratory were less competent for the Asian lineage than French colonies would not be the best option to perform Ae. albopictus, where the TR was around 60% for VC experiments since the long-term colonization Cambodia strain and 40% for Martinique strain. How- may cause genotypic and phenotypic changes in mos- ever, in our study, transmission was only detected in quitoes [43]. Rubí population for Martinique strain at 14 and To know how ZIKV could be maintained in nature 21 dpe in a low number of tested mosquitoes (TR during periods without active vectors and available ver- was 50% and 20%, respectively). Regarding the viral tebrate hosts is essential to understand the ZIKV ecol- titer in saliva samples, Rubí mosquito population ogy. In this regard, the VT has been suggested as an showed higher viral titer for the African I lineage alternative mechanism to preserve the virus in the than for the Asian lineage as previously showed environment [16]. Arboviral VT takes place through Vazeille et al. for French mosquito populations, two mechanisms: transovarial transmission and trans- besides the viral titers obtained in both studies were ovum transmission [44–46]. It is thought that the VT similar [25]. European results show VC differences of flaviviruses (e.g. DENV) generally occurs by trans- between Ae. albopictus mosquito populations as it ovum transmission [17,47]. The ZIKV VT has been was previously pointed out in American mosquito previously reported for Ae. albopictus [17,48] and populations [31] and as other studies have previously other species in the field [13,49], but the viral trans- described for DENV [39]. mission in the progeny of infected females had not The vector competence of Spanish Ae. albopictus been tested until now. Our results confirmed VT, populations remained controversial considering that since infected males and females of the E1 progeny of two recent studies [32,33] showed that the virus was intrathoracically inoculated Ae. albopictus for both not detected in the expectorated saliva in contrast to African I and Asian lineages were detected. These another recent study [30], which tested an Ae. albopic- results agree with a recent study performed in Ae. albo- tus population from the same region of Spain (Barce- pictus mosquitoes [48], in which they detected the lona province) and showed ZIKV transmission. Our Asian lineage of ZIKV in males and females from the results, which agree with the latest study, suggest first oviposition progeny. A recent study conducted that these contradictory results could be explained with orally infected Ae. aegypti mosquitoes with Asian by at least four main reasons. Firstly, the rearing temp- ZIKV lineage has shown that VT moderately decreased erature, which is an extrinsic factor that affects direct with gonotrophic cycles [18], which would agree with and indirectly the Ae. albopictus susceptibility to arbo- our results in Ae. albopictus, since we found higher virus [23,40,41]. The environmental factors in which prevalence in E1 progeny than in the E2 progeny. How- mosquitoes were reared, may have played a crucial ever, our results contrast with previous results obtained role for ZIKV transmission of Spanish Ae. albopictus by Thangamani et al. [16] and Phumee et al., where VT populations. The VC study performed by Hernán- in Ae. albopictus was not demonstrated for the Asian dez-Triana et al. (2019), where the rearing tempera- lineage. These contrasting results could be explained ture was low (20°C/25°C [night/day]), did not detect because of the viral load within the infected mosquitoes, transmission for ZIKV, in contrast with the results since they used a lower viral dose and the specimens of other VC studies that showed ZIKV transmission, were orally infected [50], and as it was discussed where the experiments were performed under temp- above, viral dose may affect ZIKV infection in Ae. albo- erature conditions that ranged from 26°C to 28°C. Sec- pictus mosquitoes. To our knowledge, this is the first ondly, the viral concentration used to artificially feed time that the viral transmission in the infected progeny the females could have also influenced the VC of Span- has been evaluated for ZIKV; the obtained results indi- ish Ae. albopictus mosquitoes for ZIKV. The low titer cated that all infected females of the progeny were not (1.8 × 106 PFU/ml; cf. the above-mentioned European able to disseminate the virus through hemolymph and mosquito studies and even our assay) used in the study transmit the virus, suggesting that ZIKV VT in Ae. of González et al. (2019) could explain their negative albopictus might be not very relevant for ZIKV results in terms of ZIKV transmission. Thirdly, the epidemiology. VC of Ae. albopictus females for ZIKV depends on Arboviruses (e.g. La Crosse virus and dengue mosquito population and virus lineage specific virus) could be amplified in a mosquito population EMERGING MICROBES AND INFECTIONS 2243 by a venereal transmission mechanism [51,52]. In Framework Programme [grant number ZIKAlliance, no. this case, male mosquitoes would obtain arboviruses 734548]; Agència de Gestió d’Ajuts Universitaris i de by VT from an infected female and thereby, could Recerca. transmit the virus horizontally to a non-infected female during mating as demonstrated in exper- imental studies for La Crosse virus in Aedes triseria- References tus [51]. After that, the females may develop infected [1] Boyer S, Calvez E, Chouin-Carneiro T, et al. An over- oocytes, generating infected progeny [16,51,53]. In view of mosquito vectors of Zika virus. Microbes this regard, the ZIKV detection in Ae. albopictus Infect. 2018;20:646–660. [2] Dick GWA, Kitchen SF, Haddow AJ. 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