• 1.

    Musso D, Gubler DJ, 2016. Zika virus. Clin Microbiol Rev 29: 487524.

  • 2.

    Thompson PH, Dicke RJ, 1965. Sampling studies with Aedes vexans and some other Wisconsin Aedes (Diptera: Culicidae). Ann Entomol Soc Am 58: 927930.

    • Search Google Scholar
    • Export Citation
  • 3.

    Darsie RF, Ward RA, 2005. Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. Gainesville, FL: University Press of Florida.

    • Search Google Scholar
    • Export Citation
  • 4.

    Turell MJ, Dohm DJ, Sardelis MR, O'guinn ML, Andreadis TG, Blow JA, 2005. An update on the potential of North American mosquitoes (Diptera: Culicidae) to transmit West Nile virus. J Med Entomol 42: 5762.

    • Search Google Scholar
    • Export Citation
  • 5.

    Ndiaye EH, Fall G, Gaye A, Bob NS, Talla C, Diagne CT, Diallo D, Ba Y, Dia I, Kohl A, Sall AA, Diallo M, 2016. Vector competence of Aedes vexans (Meigen), Culex poicilipes (Theobald) and Cx. quinquefasciatus Say from Senegal for West and East African lineages of Rift Valley fever virus. Parasit Vectors 9: 94.

    • Search Google Scholar
    • Export Citation
  • 6.

    Weger-Lucarelli J, Rückert C, Chotiwan N, Nguyen C, Garcia Luna SM, Fauver JR, Foy BD, Perera R, Black WC, Kading RC, Ebel GD, 2016. Vector competence of American mosquitoes for three strains of Zika virus. PLoS Negl Trop Dis 10: e0005101.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lanciotti RS, Lambert AJ, Holodniy M, Saavedra S, Signor LDC, 2016. Phylogeny of Zika virus in Western Hemisphere, 2015. Emerg Infect Dis 22: 933935.

    • Search Google Scholar
    • Export Citation
  • 8.

    Dulbecco R, Vogt M, 1953. Some problems of animal virology as studied by the plaque technique. Cold Spring Harb Symp Quant Biol 18: 273279.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR, 2008. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 14: 12321239.

    • Search Google Scholar
    • Export Citation
  • 10.

    Waggoner JJ, Gresh L, Vargas MJ, Ballesteros G, Tellez Y, Soda KJ, Sahoo MK, Nuñez A, Balmaseda A, Harris E, Pinsky BA, 2016. Viremia and clinical presentation in Nicaraguan patients infected with Zika virus, chikungunya virus, and dengue virus. Clin Infect Dis 63: 15841590.

    • Search Google Scholar
    • Export Citation
  • 11.

    Patrican LA, DeFoliart GR, Yuill TM, 1985. Oral infection and transmission of La Crosse virus by an enzootic strain of Aedes triseriatus feeding on chipmunks with a range of viremia levels. Am J Trop Med Hyg 34: 992998.

    • Search Google Scholar
    • Export Citation
  • 12.

    Turell MJ, 1988. Reduced Rift Valley fever virus infection rates in mosquitoes associated with pledget feedings. Am J Trop Med Hyg 39: 597602.

    • Search Google Scholar
    • Export Citation
  • 13.

    Mitchell CJ, 1983. Mosquito vector competence and arboviruses. Harris KF, ed. Topics in Vector Research. New York, NY: Praeger Scientific, 6392.

    • Search Google Scholar
    • Export Citation
  • 14.

    Aliota MT, Peinado SA, Osorio JE, Bartholomay LC, 2016. Culex pipiens and Aedes triseriatus mosquito susceptibility to Zika virus. Emerg Infect Dis 22: 18571859.

    • Search Google Scholar
    • Export Citation
  • 15.

    Amraoui F, Atyame-Nten C, Vega-Rúa A, Lourenço-de-Oliveira R, Vazeille M, Failloux AB, 2016. Culex mosquitoes are experimentally unable to transmit Zika virus. Euro Surveill 21: 30333.

    • Search Google Scholar
    • Export Citation
  • 16.

    Chouin-Carneiro T, Vega-Rua A, Vazeille M, Yebakima A, Girod R, Goindin D, Dupont-Rouzeyrol M, Lourenco-de-Oliveira R, Failloux AB, 2016. Differential susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika virus. PLoS Negl Trop Dis 10: e0004543.

    • Search Google Scholar
    • Export Citation
  • 17.

    Miller BR, Monath TP, Tabachnick WJ, Ezike VI, 1989. Epidemic yellow fever caused by an incompetent mosquito vector. Trop Med Parasitol 40: 396399.

    • Search Google Scholar
    • Export Citation
  • 18.

    Becker N, 2003. Mosquitoes and Their Control. New York, NY: Kluwer Academic/Plenum Publishers.

  • 19.

    Turell MJ, Wilson WC, Bennett KE, 2010. Potential for North American mosquitoes (Diptera: Culicidae) to transmit Rift Valley fever virus. J Med Entomol 47: 884889.

    • Search Google Scholar
    • Export Citation
  • 20.

    Turell MJ, Britch SC, Aldridge RL, Kline DL, Boohene C, Linthicum KJ, 2013. Potential for mosquitoes (Diptera: Culicidae) from Florida to transmit Rift Valley fever virus. J Med Entomol 50: 11111117.

    • Search Google Scholar
    • Export Citation
  • 21.

    Kramer LD, Ebel GD, 2003. Dynamics of flavivirus infection in mosquitoes. Adv Virus Res 60: 187232.

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American Aedes vexans Mosquitoes are Competent Vectors of Zika Virus

Alex GendernalikArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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James Weger-LucarelliArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Selene M. Garcia LunaArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Joseph R. FauverArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Claudia RückertArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Reyes A. MurrietaArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Nicholas BergrenArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Demitrios SamarasArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Chilinh NguyenArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Rebekah C. KadingArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Gregory D. EbelArthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado.

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Starting in 2013–2014, the Americas have experienced a massive outbreak of Zika virus (ZIKV) which has now reached at least 49 countries. Although most cases have occurred in South America and the Caribbean, imported and autochthonous cases have occurred in the United States. Aedes aegypti and Aedes albopictus mosquitoes are known vectors of ZIKV. Little is known about the potential for temperate Aedes mosquitoes to transmit ZIKV. Aedes vexans has a worldwide distribution, is highly abundant in particular localities, aggressively bites humans, and is a competent vector of several arboviruses. However, it is not clear whether Ae. vexans mosquitoes are competent to transmit ZIKV. To determine the vector competence of Ae. vexans for ZIKV, wild-caught mosquitoes were exposed to an infectious bloodmeal containing a ZIKV strain isolated during the current outbreak. Approximately 80% of 148 mosquitoes tested became infected by ZIKV, and approximately 5% transmitted infectious virus after 14 days of extrinsic incubation. These results establish that Ae. vexans are competent ZIKV vectors. Their relative importance as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary in ecologically distinct environments.

Introduction

Zika virus (ZIKV; Flaviviridae: Flavivirus) is a mosquito-borne virus currently spreading in the Western Hemisphere. It has now reached ≥ 49 countries and territories in the Americas including the United States and can cause febrile illness, severe neurological complications, and congenital malformations (e.g., microcephaly).1 Aedes (Stegomyia) aegypti Linnaeus and Aedes (Stegomyia) albopictus Skuse are the main mosquito vectors of ZIKV.1 Travel-associated cases have now been confirmed in all U.S. states and much of Europe (http://www.cdc.gov/zika/geo/active-countries.html). It is therefore important to define the competence of other potential ZIKV vectors that could feed on viremic individuals.

The inland floodwater mosquito, Aedes (Aedimorphus) vexans (Meigen), has a worldwide distribution and is known to aggressively bite humans.2,3 These mosquitoes are capable of transmitting West Nile virus, St. Louis encephalitis virus, Western and Eastern equine encephalitis viruses, Rift Valley fever virus (RVFV), and the dog heartworm parasite, Dirofilaria immitis.4,5 We therefore sought to determine the vector competence of field-caught Ae. vexans mosquitoes to transmit ZIKV after oral exposure.

Materials and Methods

Aedes vexans mosquitoes were collected from August to early October 2016 using Centers for Disease Control and Prevention light traps baited with dry ice. Twenty traps were placed at 105°3 20.10 W, 40°34 44.27 N in Fort Collins, CO. Traps were placed on the nights of August 17, 23, and September 8, 2016. Traps were set at approximately 1,600 hours and were collected by 1,000 hours the following morning, at which point Ae. vexans females were identified morphologically.3 Mosquitoes were housed at 27°C, 80% relative humidity, and 16:8 (light/dark) photoperiod for no more than 7 days before further manipulation.

Vector competence experiments were performed as previously described.6 In brief, mosquitoes were given an infectious bloodmeal consisting of a 1:1 solution of cell culture supernatant containing freshly grown ZIKV and defibrinated calf blood. ZIKV strain PRVABC59 (accession no. KU501215) was freshly grown for each biological replicate by infecting semiconfluent monolayers of Vero cells at a multiplicity of infection (MOI) of 0.01 and harvesting virus 5 days postinfection.7 ZIKV titers in the resulting bloodmeals were determined by plaque assay (Table 1) according to standard methods.8 Three biological replicates were performed, hereafter referred to as BR1, BR2, and BR3 (Table 1). Fourteen days after feeding of the infectious bloodmeal, mosquitoes were cold-anesthetized, and legs, midguts, and salivary secretions were collected as described previously.6 Samples were preserved at −80°C until screening by plaque assay on Vero cells. Midguts shown to be virus positive indicated infection, virus-positive legs indicated dissemination, and saliva shown to be virus positive indicated transmission. Infection, dissemination, and transmission rates are defined as the proportion of mosquitoes with infectious virus in their midguts, legs, and saliva, respectively, out of the total number of blood-fed mosquitoes. All saliva samples shown positive for virus on plaque assays were confirmed to be ZIKV positive by quantitative reverse transcription polymerase chain reaction9 (data not shown).

Table 1

Competence of Aedes vexans mosquitoes as vectors of Zika virus, number of virus positive/number bloodfed (%; 95% CI)

Experiment Infected Disseminated Transmitted Bloodmeal titer
Genomes/mL PFU/mL
BR1 38/58 (66) 2/58 (3) 1/58 (2) 4.15E + 10 7.00E + 06
BR2 53/58 (91) 14/58 (24) 4/58 (7) 3.05E + 10 1.30E + 07
BR3 27/32 (84) 8/32 (25) 2/32 (6) 2.76E + 10 1.70E + 07
Total 118/148 (80; 48.3 to 112.37) 24/148 (16; −13.53 to 48.19) 7/148 (5; −1.57 to 11.57)    

CI = confidence interval; PFU = plaque-forming units.

Results and Discussion

Infection rates ranged from 66% (38/58) in BR1 to 91% (53/58) in BR2, dissemination rates ranged from 3% (2/58) in BR1 to 25% in BR3 (8/32), and transmission rates ranged from 2% (1/58) in BR1 to 7% (4/58) in BR2 (Table 1). High variability between replicates is likely related to genetic variability between generations of wild Ae. vexans mosquitoes and/or environmental fluctuations (shortening of photoperiod, temperature changes, and precipitation) that were naturally occurring over the summer months.

To our knowledge, our study is the first to assess the vector competence of wild Ae. vexans mosquitoes for transmission of ZIKV. We demonstrated that wild-caught Ae. vexans from northern Colorado were highly susceptible to infection by ZIKV. However, dissemination and transmission rates were relatively low, indicating the existence of a moderately strong midgut escape and salivary gland barriers. More notably, however, was the low transmission rate compared with infection and dissemination rates. Since we did not collect salivary glands as part of this study, we cannot determine whether low transmission was due to infection of or egress from this tissue. Moreover, the data presented here indicate that Ae. vexans are susceptible to ZIKV infection, but virus transmission potential appears to be low. Additionally, it is important to note that mosquitoes were fed a relatively high bloodmeal concentration of ZIKV. Viremias greater than 7 log10 genomes/mL have been reported,10 which is considerably lower than what was used here. However, several previous reports have determined that mosquitoes are less susceptible to artificial-membrane infections as opposed to infection from viremic animals.1113

Our observations, in addition to the wide distribution and aggressive biting nature of Ae. vexans, suggest this mosquito as an unlikely, but potential contributor to the spread of ZIKV in areas within and outside the geographical range of Ae. aegypti and Ae. albopictus. Aedes vexans was more competent at transmitting ZIKV than other North American species tested to date: Culex pipiens quinquefasciatus Say, Culex pipiens pipiens L., and Culex tarsalis Coquillett, and Aedes (Protomacleaya) triseriatus (Say) mosquitoes were shown to be highly refractory to infection by ZIKV.6,14,15 By comparison, infection, dissemination, and transmission rates in Ae. aegypti have been observed to be as high as 76.7% (23/30), 46.7% (14/30), and 10% (3/30) and 50% (15/30), 6.7% (2/30), and 3.3% (1/30) in Ae. albopictus16 (data modified from Chouin-Carneiro and others). Under certain situations, the moderate competence of Ae. vexans for transmission of ZIKV demonstrated in this study may be enough to contribute to local transmission. High population densities of poorly competent vectors have been observed to maintain virus transmission17 and such population densities have been shown to occur with Ae. vexans.2,18 Furthermore, the competence of Ae. vexans for transmission of RVFV was also demonstrated to vary substantially from incompetent (Colorado, California) to highly competent (Florida) areas, highlighting the importance of future studies to evaluate populations of Ae. vexans from multiple geographic areas.19,20 Finally, vector competence contributes relatively weakly to vectorial capacity, particularly when compared with the host biting rate and probability of daily survival.21 Moreover, the relative importance of Ae. vexans as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary locally and that powerfully impact the epidemiological significance of Ae. vexans mosquitoes as ZIKV vectors.

  • 1.

    Musso D, Gubler DJ, 2016. Zika virus. Clin Microbiol Rev 29: 487524.

  • 2.

    Thompson PH, Dicke RJ, 1965. Sampling studies with Aedes vexans and some other Wisconsin Aedes (Diptera: Culicidae). Ann Entomol Soc Am 58: 927930.

    • Search Google Scholar
    • Export Citation
  • 3.

    Darsie RF, Ward RA, 2005. Identification and Geographical Distribution of the Mosquitoes of North America, North of Mexico. Gainesville, FL: University Press of Florida.

    • Search Google Scholar
    • Export Citation
  • 4.

    Turell MJ, Dohm DJ, Sardelis MR, O'guinn ML, Andreadis TG, Blow JA, 2005. An update on the potential of North American mosquitoes (Diptera: Culicidae) to transmit West Nile virus. J Med Entomol 42: 5762.

    • Search Google Scholar
    • Export Citation
  • 5.

    Ndiaye EH, Fall G, Gaye A, Bob NS, Talla C, Diagne CT, Diallo D, Ba Y, Dia I, Kohl A, Sall AA, Diallo M, 2016. Vector competence of Aedes vexans (Meigen), Culex poicilipes (Theobald) and Cx. quinquefasciatus Say from Senegal for West and East African lineages of Rift Valley fever virus. Parasit Vectors 9: 94.

    • Search Google Scholar
    • Export Citation
  • 6.

    Weger-Lucarelli J, Rückert C, Chotiwan N, Nguyen C, Garcia Luna SM, Fauver JR, Foy BD, Perera R, Black WC, Kading RC, Ebel GD, 2016. Vector competence of American mosquitoes for three strains of Zika virus. PLoS Negl Trop Dis 10: e0005101.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lanciotti RS, Lambert AJ, Holodniy M, Saavedra S, Signor LDC, 2016. Phylogeny of Zika virus in Western Hemisphere, 2015. Emerg Infect Dis 22: 933935.

    • Search Google Scholar
    • Export Citation
  • 8.

    Dulbecco R, Vogt M, 1953. Some problems of animal virology as studied by the plaque technique. Cold Spring Harb Symp Quant Biol 18: 273279.

    • Search Google Scholar
    • Export Citation
  • 9.

    Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR, 2008. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 14: 12321239.

    • Search Google Scholar
    • Export Citation
  • 10.

    Waggoner JJ, Gresh L, Vargas MJ, Ballesteros G, Tellez Y, Soda KJ, Sahoo MK, Nuñez A, Balmaseda A, Harris E, Pinsky BA, 2016. Viremia and clinical presentation in Nicaraguan patients infected with Zika virus, chikungunya virus, and dengue virus. Clin Infect Dis 63: 15841590.

    • Search Google Scholar
    • Export Citation
  • 11.

    Patrican LA, DeFoliart GR, Yuill TM, 1985. Oral infection and transmission of La Crosse virus by an enzootic strain of Aedes triseriatus feeding on chipmunks with a range of viremia levels. Am J Trop Med Hyg 34: 992998.

    • Search Google Scholar
    • Export Citation
  • 12.

    Turell MJ, 1988. Reduced Rift Valley fever virus infection rates in mosquitoes associated with pledget feedings. Am J Trop Med Hyg 39: 597602.

    • Search Google Scholar
    • Export Citation
  • 13.

    Mitchell CJ, 1983. Mosquito vector competence and arboviruses. Harris KF, ed. Topics in Vector Research. New York, NY: Praeger Scientific, 6392.

    • Search Google Scholar
    • Export Citation
  • 14.

    Aliota MT, Peinado SA, Osorio JE, Bartholomay LC, 2016. Culex pipiens and Aedes triseriatus mosquito susceptibility to Zika virus. Emerg Infect Dis 22: 18571859.

    • Search Google Scholar
    • Export Citation
  • 15.

    Amraoui F, Atyame-Nten C, Vega-Rúa A, Lourenço-de-Oliveira R, Vazeille M, Failloux AB, 2016. Culex mosquitoes are experimentally unable to transmit Zika virus. Euro Surveill 21: 30333.

    • Search Google Scholar
    • Export Citation
  • 16.

    Chouin-Carneiro T, Vega-Rua A, Vazeille M, Yebakima A, Girod R, Goindin D, Dupont-Rouzeyrol M, Lourenco-de-Oliveira R, Failloux AB, 2016. Differential susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika virus. PLoS Negl Trop Dis 10: e0004543.

    • Search Google Scholar
    • Export Citation
  • 17.

    Miller BR, Monath TP, Tabachnick WJ, Ezike VI, 1989. Epidemic yellow fever caused by an incompetent mosquito vector. Trop Med Parasitol 40: 396399.

    • Search Google Scholar
    • Export Citation
  • 18.

    Becker N, 2003. Mosquitoes and Their Control. New York, NY: Kluwer Academic/Plenum Publishers.

  • 19.

    Turell MJ, Wilson WC, Bennett KE, 2010. Potential for North American mosquitoes (Diptera: Culicidae) to transmit Rift Valley fever virus. J Med Entomol 47: 884889.

    • Search Google Scholar
    • Export Citation
  • 20.

    Turell MJ, Britch SC, Aldridge RL, Kline DL, Boohene C, Linthicum KJ, 2013. Potential for mosquitoes (Diptera: Culicidae) from Florida to transmit Rift Valley fever virus. J Med Entomol 50: 11111117.

    • Search Google Scholar
    • Export Citation
  • 21.

    Kramer LD, Ebel GD, 2003. Dynamics of flavivirus infection in mosquitoes. Adv Virus Res 60: 187232.

Author Notes

* Address correspondence to Gregory D. Ebel, Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, 1690 Campus Delivery, Fort Collins, CO 80523. E-mail: gregory.ebel@colostate.edu

Financial support: This work was funded by grants from the National Institute of Allergy and Infectious Diseases, National Institutes of Health under grant numbers AI067380 and AI125996. S. G. L. was supported by a grant from the Fogarty International Center under grant number 2D43TW001130-0681 and R. A. M. was supported by a grant from the National Science Foundation under grant number DGE-1450032.

Authors' addresses: Alex Gendernalik, James Weger-Lucarelli, Selene M. Garcia Luna, Joseph R. Fauver, Claudia Rückert, Reyes A. Murrieta, Nicholas Bergren, Demitrios Samaras, Chilinh Nguyen, Rebekah C. Kading, and Gregory D. Ebel, Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, E-mails: alex.gendernalik@colostate.edu, james.weger@colostate.edu, luna_marysol@hotmail.com, joseph.fauver@colostate.edu, claudia.rueckert@colostate.edu, reyes.murrieta@colostate.edu, nicholas.bergren@colostate.edu, dsamaras@rams.colostate.edu, chilinhnguyenn@gmail.com, rebekah.kading@colostate.edu, and gregory.ebel@colostate.edu.

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