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Since its introduction into the United States in 1999, West Nile virus (WNV) has spread rapidly throughout North America. Although the virus is principally transmitted by mosquito vectors in the genus Culex, WNV has been identified from an exceptionally broad range of vector species when compared with most arboviruses.1,2 This virus has been detected in at least 75 different arthropod species3 including 60 North American mosquito species4–7 (http://www.cdc.gov/ncidod/dvbid/westnile/mosquitoSpecies.htm). Laboratory studies have evaluated various North American mosquitoes to become infected with and transmit WNV.8–15 In Texas, Culex pipiens quinquefasciatus is the primary vector of WNV,16 whereas other Culex spp. mosquitoes, such as Cx. pipiens and Cx. tarsalis are important in other regions.
In this study, we compared three species of field collected mosquitoes, Culex p. quinquefasciatus, Aedes aegypti, and Ae. albopictus, collected in the city of Houston, Texas, and on Galveston Island, Texas. Both Aedes species are peridomestic and anthropophilic with known susceptibility to infection with several arboviruses, and Ae. albopictus is regarded as a likely bridge vector for WNV because of feeding on avian hosts.17
To accurately determine the susceptibility of adult mosquitoes to become orally infected with WNV from the natural populations, and to minimize any potential effect of colonization, eggs and larvae were collected from the field and reared to adults. Adult female mosquitoes, either F0 or F1, were presented with a blood meal containing WNV at 5–7-days post-eclosion. The three species of mosquitoes were collected over a three-year period from 2002 to 2004 and were used to determine infection, dissemination rates, and the 50% oral infectious dose (OID50).
Culex p. quinquefasciatus were collected as egg rafts from Houston, Texas, in July 2003. The F0 mosquitoes were orally infected with WNV, in two separate experiments, by exposure to a serially diluted infectious blood meal (undiluted to 10–5), to determine the OID50. Culex p. quinquefasciatus were collected the following year as egg rafts in the Houston area in May 2004. The F0 mosquitoes were exposed to one infectious blood meal to determine infection and dissemination rates.
Aedes aegypti eggs and larvae were collected from Galveston Island in the summer of 2003. The F1 mosquitoes were exposed to two separate infectious blood meals that were serially diluted (undiluted to 10–5) to determine OID50. Infection and dissemination rates for Ae. aegypti were determined by analysis of mosquitoes orally exposed to two undiluted infectious blood meals.
Aedes albopictus were collected once in Houston and twice on Galveston Island. Mosquitoes (H = Houston) were collected as eggs in the summer and fall of 2002. To determine OID50, F1 mosquitoes were exposed to two separate blood meals containing serially diluted (undiluted to 10–5) WNV. Aedes albopictus (G = Galveston) were collected in the summer of 2003. The F1 mosquitoes were exposed to a serially diluted (undiluted to 10–4) infectious blood meal and were analyzed to determine OID50. Infection and dissemination was determined by examining two groups of mosquitoes fed either the undiluted or 10–1 dilution blood meals. Aedes albopictus (G) were collected again in May 2004. A serially diluted blood meal (undiluted to 10–4) was presented to F0 mosquitoes to determine OID50. Mosquitoes exposed to the undiluted infectious blood meal were used to determine infection and dissemination rates.
A 2002 Houston isolate of WNV designated strain 114 (GenBank accession number AY187013) was prepared as a mixed brain/liver homogenate from an infected Blue jay (Cyanocitta cristata).18 Stock virus was produced following a single passage in Vero (green monkey kidney) cell culture, the titer of the stock was determined by plaque assay to be 2 x 108 plaque-forming units/mL.18 The large number of mosquitoes examined and the period of time over which the mosquitoes were collected prohibited exposing the mosquitoes to the same blood meal preparation. Preparations of meals using standardized techniques18 were designed to minimize the introduction of artificial variation between feeds. To determine the oral dose that resulted in a 50% infection rate (OID50), serial 10-fold dilutions of the supernatant in defibrinated sheep blood were prepared. The diluted blood meal was presented at 5–7-days post-eclosion. Individual mosquitoes were triturated and titrated as serial 10-fold dilutions on Vero cells.19 Percent dissemination was determined separately by titrating 10-fold serial dilutions of heads and bodies on Vero cells in 96-well plates.
Differences in infection and dissemination rates among species were tested for significance using Fisher’s exact test in SPSS version 11.5 (SPSS, Inc., Chicago, IL). The OID50 values were calculated based on the percent of mosquitoes infected after ingesting an estimated 5-µL blood meal containing various titers of virus (Figure 1
). Regression lines were produced using PriProbit (version 1.63) by Masayuki Sakuma (Kyoto University, Kyoto, Japan).
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). Although the blood meal titer presented to Cx. p. quinquefasciatus was lower (5.2 log10 50% tissue culture infectious doses [TCID]50/µL) than the viral titer presented to Ae. aegypti(6.4 log10 TCID50/µL) or Ae. albopictus (5.7 or 6.6 log10 TCID50/µL), 100% of the Cx. p. quinquefasciatus produced a disseminated WNV infection (Table 1). West Nile virus infected 73% of Ae. aegypti and produced a disseminated infection in 86% of the infected mosquitoes (Table 1). Variable susceptibility to WNV infection was observed between the F0 and F1 generations of Ae. albopictus collected in Galveston. Only 13% of the mosquitoes from the F0 generation presented with a blood meal containing 6.65 log10 TCID50/5 µL were infected (Table 1). Aedes albopictus (F1) mosquitoes were exposed to a lower titer blood meal (5.72 ± 0.71 log10 TCID50/5 µL) compared with the F0 generation; however, 69% of the F1 mosquitoes tested were positive for WNV (Table 1). The infection rate of Cx. p. quinquefasciatus was significantly different from those of Ae. aegypti and Ae. albopictus F0. However, the infection rate of Ae. albopictus F1 was not significantly different from that of Cx. p. quinquefasciatus or from that of Ae. aegypti. There was no significant difference among the dissemination rates for any of the species tested.
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). Culex p. quinquefasciatus (F0) collected in Houston were found to be the most susceptible to WNV infection with the lowest OID50 value (F0 = 5.33 ± 0.16 log10 TCID50/5 µL). Aedes aegypti (F1) collected in Galveston were the least susceptible to WNV infection with an OID50 value of 6.37 ± 0.84 log10 TCID50/5 µL. The OID50 for Ae. albopictus (F1) collected in the city of Houston was intermediate between the other two species, with a titer of 6.17 ± 0.11 log10 TCID50/5 µL (Figure 1).
Aedes albopictus collected on Galveston Island were found to have varying susceptibility to WNV infection on the basis of the generation examined. The F0 mosquitoes were least susceptible to oral infection with an OID50 of 6.96 log10 TCID50/5 µL, whereas the F1 generation was found to be more susceptible to WNV infection with an OID50 of 4.68 log10 TCID50/5 µL (Figure 1).
The relative susceptibility to WNV infection of three mosquito species from the Houston area was evaluated. Mosquito surveillance and control efforts focus on Culex p. quinquefasciatus; however because of the close association of Ae. albopictus and Ae. aegypti with humans, their anthropophilic feeding preference, and abundance around Houston they may represent a threat to humans. Both Aedes species are naturally infected in the United States with WNV. West Nile virus infected Ae. albopictus have been found since 2000 and WNV-infected Ae. aegypti have been found since 2002 (http://www.cdc.gov/ncidod/dvbid/westnile/mosquitoSpecies.htm).
Previous studies have reported varying infection and transmission rates for Ae. albopictus and Cx. pipiens.14,20,21 In these studies, Cx. pipiens was more susceptible than Ae. albopictus to WNV when fed on chicks with viremias < 107.5, but nearly as susceptible if exposed to viremias 
107.5. In a subsequent study,22 after feeding on meals 107.0, the cumulative mean dissemination rates for Cx. pipiens and Ae. albopictus were 74.8 ± 2.6 and 88.6 ± 21.%, respectively, and cumulative mean transmission rates were 46.8 ± 4.5 and 72.4 ± 5.5, respectively. The investigators suggested that Ae. albopictus is a more efficient vector than Cx. pipiens when fed on blood meal titers > 107.0 CID50.
Geographic variation in susceptibility of mosquitoes to viral infection is well documented, including variation of Cx. pipiens to WNV.23 Although our conclusions may not apply to populations collected in other areas, they do provide insight into the relative importance of these species in the Houston area where WNV transmission occurs throughout the year.
On the basis of OID50 values for the three Texan mosquito species tested, we conclude that Cx. p. quinquefasciatus is more susceptible to infection with WNV than either Ae. aegypti or Ae. albopictus, and this, together with infection and dissemination rates, would suggest that of the three species, Cx. p. quinquefasciatus is the more competent vector. In the context of WNV epidemiology, in a recent study,24 blood meal analyses showed that Cx. p. quinquefasciatus collected in the Houston area fed on birds (52%), mammals (52%) and mixed avian/mammals host (8.3%). Species-specific blood analysis showed that avian meals were derived from Columbiformes (52%) and Passeriformes (44%), including several species known to be important reservoir and amplifying hosts for WNV. Dogs were the most frequent mammalian blood source (72%) with human blood accounting for 0.7% of mammalian meals (0.4% of all meals). On the basis of host preference, the investigators concluded that Cx. p. quinquefasciatus is the principal vector of WNV in the Houston area. Our infection and transmission data further support this conclusion. The investigators comment that further research is needed to determine the role of other mosquitoes in transmission of the virus. Our data demonstrate that both Ae. aegypti and Ae. albopictus are readily infected and displayed disseminated infection that is consistent with the capacity for viral transmission. Although Ae. aegypti and Ae. albopictus are reluctant to feed on avians, which limits their potential to become infected because of their anthrophilic feeding preference, these species represent potential vectors of WNV to humans.
Received June 5, 2007. Accepted for publication July 22, 2007.
Acknowledgments: We thank Jing Haung for her expert technical assistance with this project.
Financial support: This study was supported in part by the Centers for Disease Control and Prevention (U50/CCU620539). Dana L. Vanlandingham was supported in part by National Institutes of Health grant T32 A107536. Charles E. McGee was supported by a Centers for Disease Control and Prevention Fellowship Training Program in Vector-Borne Infectious Diseases (TOI/CCT622892).
* Address correspondence to Stephen Higgs, Department of Pathology, University of Texas Medical Branch, Keiller 2.104, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: sthiggs{at}utmb.edu 
Authors’ addresses: Dana L. Vanlandingham, Charles E. McGee, Kimberly A. Klinger, and Stephen Higgs, Department of Pathology, University of Texas Medical Branch, Keiller 2.104 L 20762, 301 University Boulevard, Galveston, TX 77555-0609, Telephone: 409-747-2426, Fax: 409-772-2511, E-mail: sthiggs{at}utmb.edu. Nathan Vessey and Chris Fredegill, Harris County Mosquito Control, Harris County Public Health and Environmental Services, 3330 Old Spanish Trail, Building C, Houston, TX 77021, Telephone: 713-440-3025.
This article has been cited by other articles:
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  D. L. Vanlandingham, C. E. McGee, K. A. Klingler, S. E. Galbraith, A. D. T. Barrett, and S. Higgs Comparison of Oral Infectious Dose of West Nile Virus Isolates Representing Three Distinct Genotypes in Culex quinquefasciatus Am J Trop Med Hyg, December 1, 2008; 79(6): 951 - 954. [Abstract] [Full Text] [PDF]
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