DISPATCHES Vector Competence of Aedes caspius and Ae. albopictus Mosquitoes for Zika Virus, Spain Rafael Gutiérrez-López, Sean M. Bialosuknia, mosquitoes (collected as larvae in Huelva, Spain, because Alexander T. Ciota, Tomás Montalvo, we were unable to rear it under laboratory conditions) and F2 Josue Martínez-de la Puente, Laura Gangoso, generation of Ae. albopictus mosquitoes (collected as eggs Jordi Figuerola, Laura D. Kramer in Barcelona, Spain) to Zika virus through infectious blood meals. We used F8 generation of colonized populations of We assessed the vector competence of Aedes caspius and Ae. aegypti mosquitoes (collected in Poza Rica, Mexico) Aedes albopictus mosquitoes in Spain for the transmission as a control population and Zika virus strains CAM (2010 of Zika virus. Whereas Ae. albopictus mosquitoes were a Cambodia; GenBank accession no. JN860885) and PR (2015 competent vector, Ae. caspius mosquitoes were unable to transmit Zika virus. We also identified high levels of vertical Puerto Rico; GenBank accession no. KU501215), passaged transmission of Zika virus in Ae. albopictus mosquitoes. 4 times on Vero cells and 2 times on C6/36 cells. We propagated on C6/36 cells for 4 days, and freshly harvested supernatant was mixed 1:1 with sheep blood (Colorado Z ika virus is an emerging arbovirus of the family Flaviviridae primarily transmitted by Aedes aegypti mosquitoes, but other Aedes species mosquitoes could be Serum Company, http://www.thepeakofquality.com) and 2.5% sucrose (5). We offered to 4- to 7-day-old Ae. albopictus and Ae. competent vectors (1). Ae. aegypti mosquitoes are absent aegypti female mosquitoes infectious blood meals con- from most countries in Europe (2), and the invasive Ae. taining either the CAM or PR strain at a final concentra- albopictus mosquito and other native species could create tion of 7.6 log10 PFU/mL. Infection rates were determined novel epidemiologic scenarios for Zika virus. Indeed, by screening mosquitoes’ bodies, dissemination rates by Ae. albopictus mosquito populations from Europe are screening legs, and transmission rates by screening saliva, competent vectors for Zika virus (3,4). However, the at 3 different time points (7, 14 and 21 dpi) using Zika- vector competence for transmission of Zika virus of most specific quantitative reverse transcription PCR including mosquito species of Europe is currently unknown and may negative controls in each reaction (12) (Table 1; Appendix, vary across virus strains and mosquito populations (5). https://wwwnc.cdc.gov/EID/article/25/2/17-1123-App1. Although no autochthonous vectorborne Zika virus pdf). We calculated Zika titers from standard curves on the transmission has been reported in Spain, >316 imported basis of infectious particle standards created from matched cases of Zika virus have been confirmed (6). The confirmed virus stocks (5). cases, together with the presence of both the Ae. albopictus We further exposed 4- to 10-day-old Ae. caspius mosquito (7) and the native Ae. caspius mosquito (8,9) (a female mosquitoes to the PR strain as described. We potential vector of chikungunya virus [10] and Rift Valley conducted 3 independent trials using different Zika virus fever virus [11]), indicate a risk for Zika virus transmission concentrations at different time points (7, 14, or 21 dpi) for in Spain. Accurately quantifying this risk requires each trial (Table 1; Appendix). evaluating the competence of these mosquito species for To determine the ability of Ae. albopictus mosquitoes Zika virus. to vertically transmit Zika virus, 4- to 7-day-old females We determined vector competence at different days were infected with Zika PR as described, and noninfectious postinfection (dpi) by exposing F0 generation of Ae. caspius blood meals were offered weekly after the first oviposition. We collected eggs laid in the second oviposition and Author affiliations: Estación Biológica de Doñana, Seville, Spain hatched them for subsequent testing. We grouped second (R. Gutiérrez-López, J. Martínez-de la Puente, L. Gangoso, instar larvae in pools of 5 individuals and tested them for J. Figuerola); New York State Department of Health, Slingerlands, Zika virus (13). We estimated vertical transmission rate, New York, USA (S.M. Bialosuknia, A.T. Ciota, L.D. Kramer); measured as filial infection rate using the maximum- Agència de Salut Pública de Barcelona, Barcelona, Spain likelihood method (PoolInfRate version 4.0, https://www. (T. Montalvo); Centro de Investigación Biomédica en Red de cdc.gov/westnile/resourcepages/mosqSurvSoft.html) (13). Epidemiología y Salud Pública (CIBERESP), Barcelona We performed generalized linear models with (T. Montalvo, J. Martínez-de la Puente, J. Figuerola) binomial error distribution and logit link function to assess DOI: https://doi.org/10.3201/eid2502.171123 the effect of mosquito species, virus strains, and dpi on the 346 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 2, February 2019 Competence of Mosquitoes for Zika Virus, Spain Table 1. Infection, dissemination, and transmission rates of mosquitoes experimentally infected with 2 Zika virus strains, Spain Days Zika virus Blood meal titers, % Infected (total % Infected % Infected postinfection Mosquito species strain* log10 PFU/mL no.) disseminating transmitting 7 Aedes aegypti CAM 7.6 24.2 (33) 75 12.5 PR 7.6 61.8 (34) 38.1 0 Ae. albopictus CAM 7.6 90.5 (21) 42 10.5 PR 7.6 97.0 (33) 31.3 0 Ae. caspius PR 7.7 21.4 (14) 0 0 14 Ae. aegypti CAM 7.6 22.6 (31) 71.4 14.3 PR 7.6 45 (40) 77.8 16.7 Ae. albopictus CAM 7.6 81.5 (27) 81.8 9.1 PR 7.6 93.3 (30) 67.9 0 Ae. caspius PR 8.7 40 (25) 0 0 21 Ae. aegypti CAM 7.6 35.7 (28) 100 40 PR 7.6 56.3 (32) 88.9 38.9 Ae. albopictus CAM 7.6 94.4 (18) 82.4 23.6 PR 7.6 96.2 (26) 96 36 Ae. caspius PR 7.6 18.5 (27) 0 0 *CAM, Zika virus 2010 Cambodia strain; PR, Zika virus 2015 Puerto Rico strain. infection, dissemination, and transmission rates. We also Ae. albopictus mosquitoes (29.4%; N = 17) were positive considered the interactions between virus strain and dpi and for Zika virus, with a filial infection rate of 72.2 (95% CI between virus strain and mosquito species. We determined 27.6–156.1) and mean viral load of 2.5 log10 PFU/mL. This differences in mean viral titers between mosquito species, value equates to a ratio of 1:14. virus strains, and dpi in mosquito body, legs, and saliva Our results suggest Ae. albopictus mosquitoes in using Kruskal-Wallis tests. Analyses were run in JMP Spain are competent vectors of Zika virus at levels similar version 9 (SAS Institute, http://www.jmp.com). to Ae. aegypti mosquitoes. We detected Zika CAM in sa- Infection rate was higher in Ae. albopictus than in Ae. liva earlier than Zika PR, which suggests that genetically aegypti mosquitoes, and Zika PR had a higher infection variable strains may have different transmission potential rate than Zika CAM. Dissemination rate increased with (5). Although a similar transmission rate was found in Ae. time (dpi) but was similar between mosquito species and albopictus mosquitoes from Spain and Italy (3), lower rates Zika strains. Transmission rate also increased with time, were measured in populations in France (4). In addition, and mosquitoes infected with Zika CAM showed a higher Ae. albopictus mosquitoes from Spain could transmit Zika transmission rate than those infected with Zika PR. Trans- virus at 7 dpi, 4 days earlier than mosquitoes in Italy (4). mission rate did not differ between Ae. albopictus and Ae. These discrepancies may be explained by variation in vec- aegypti mosquitoes (Table 1, 2). Mean viral titers in bodies tor competence between mosquito populations and virus differed between mosquito species and Zika strains, with strains (5). Although Zika virus can infect Ae. caspius mos- higher titers in Ae. albopictus compared with Ae. aegypti quitoes, it is unable to escape the midgut and be effectively mosquitoes (χ2 = 5.09, df = 1; p<0.02) and higher titers transmitted (14). for Zika PR compared with Zika CAM (χ2 = 6.92, df = 1; Zika virus is vertically transmitted by the population of p<0.009). Mean viral titers in legs were similar for both Ae. albopictus mosquitoes in Spain at substantially higher Zika strains (χ2 = 0.95, df = 1; p = 0.33), but were higher in rates than found in Ae. albopictus mosquitoes from New Ae. aegypti relative to Ae. albopictus mosquitoes (χ2 = 9.53, York and Italy (4,13) and for other flaviviruses (15). These df = 1; p<0.002). Mean viral titers did not differ in saliva results suggest that the ability of Zika virus to be trans- secretions between mosquito species (χ2 = 1.7, df = 1; p mitted vertically is highly population dependent and could = 0.19) or Zika strains (χ2 = 1.02, df = 1; p = 0.31). We contribute to maintenance of the virus in Ae. albopictus detected Zika virus infection in Ae. caspius mosquitoes at mosquitoes in Spain. 7, 14, and 21 dpi, but detected no virus dissemination or Our results confirm that populations of Ae. albopictus transmission at any point (Table 1). Five larval pools of mosquitoes increase the risk for Zika virus transmission in Table 2. Results of generalized linear models analyzing the variation in infection, dissemination, and transmission rates of Zika virus* Infection rate Dissemination rate Transmission rate Variable χ2 df p value χ2 df p value χ2 df p value Mosquito species 110.95 1 <0.001 2.08 1 0.15 2.37 1 0.12 Zika virus strain 10.43 1 0.001 1.28 1 0.26 4.91 1 0.03 dpi 0.15 1 0.70 39.61 1 <0.001 26.77 1 <0.001 Zika virus strain • dpi 1.17 1 0.28 1.34 1 0.25 6.70 1 0.01 Mosquito species • Zika virus strain 0.01 1 0.90 0.76 1 0.39 0.01 1 0.94 *Bold indicates significant effect; • indicates interaction between variables; dpi, days postinfection. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 2, February 2019 347 DISPATCHES Spain. 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