Abstract
Recent reports of Zika virus (ZIKV) isolates from Culex species mosquitoes have resulted in concern regarding a lack of knowledge on the number of competent vector species for ZIKV transmission in the new world. Although observations in the field have demonstrated that ZIKV isolation can be made from Culex species mosquitoes, the detection of ZIKV in these mosquitoes is not proof of their involvement in a ZIKV transmission cycle. Detection may be due to recent feeding on a viremic vertebrate, and is not indicative of replication in the mosquito. In this study, susceptibility of recently colonized Culex species mosquitoes was investigated. The results showed a high degree of refractoriness among members of Culex pipiens complex to ZIKV even when exposed to high-titer bloodmeals. Our finding suggests that the likelihood of Culex species mosquitoes serving as secondary vectors for ZIKV is very low, therefore vector control strategies for ZIKV should remain focused on Aedes species mosquitoes. Our demonstration that Culex quinquefasciatus from Vero Beach, FL, is refractory to infection with ZIKV is especially important and timely. Based on our data, we would conclude that the autochthonous cases of Zika in Florida are not due to transmission by C. quinquefasciatus, and so control efforts should focus on other species, logically Aedes aegypti and Aedes albopictus.
Introduction
T
The isolation of ZIKV from Culex species mosquitoes (Phillips 2016) is cause for concern, and the role of mosquitoes in the Culex pipiens complex has been extensively discussed because of their medical importance and close association with human civilization as “common house mosquitoes” (Moraes 2016). Detection of ZIKV in these mosquitoes may be due to recent feeding on a viremic human, especially if sampling was in proximity to the homes of infected people. Historically, successful field isolations of ZIKV have been mainly made from Aedes species mosquitoes despite the fact Culex species mosquitoes in the same locations have also been tested in the same studies (Dick et al. 1952, Haddow et al. 1964, Marchette et al. 1969).
Sylvatic cycles involve species such as Aedes africanus, from which the virus was first isolated in 1948 (Dick et al. 1952), most urban transmission probably involves A. aegypti, although a 2007 outbreak in Gabon involved Aedes albopictus (Grard et al. 2014). Although ZIKV has been detected in other species, including C. perfuscus (Diallo 2014), laboratory experiments have focused on determining vector competence of various Aedes species mosquitoes for the African lineage of the virus (Boorman and Porterfield 1956, Wong et al. 2013, Diagne et al. 2015).
Recently, Chouin-Carneiro et al. (2016) evaluated the susceptibility of A. aegypti and A. albopictus collected in the Americas to Asian lineage ZIKV. The introduction of an arbovirus into a new location can result in an increase of competent vector species that promote viral transmission and maintenance. For example, the introduction of West Nile virus (WNV) into North America resulted in virus isolations from more than 60 different species of mosquitoes (Huang et al. 2014). Knowledge regarding the relative importance of competent vector species is critical not only for our understanding of transmission cycles of arboviruses but also for the development of vector control strategies for arboviruses. A particularly good example was the role of A. albopictus in transmitting WNV in Louisiana, where the Culex quinquefasciatus population was targeted by vector control and successfully suppressed (Palmisano et al. 2005, Vanlandingham et al. 2016).
In this report, three populations of Culex species mosquitoes collected from three locations in the United States and colonized for less than 2 years were tested for their susceptibility to ZIKV through per os infection. Detection of infectious viruses and viral nucleic acids was performed to determine whether infection of ZIKV was established. The results provide the important evidence to exclude C. pipiens and C. quinquefasciatus mosquitoes as important vector species for ZIKV transmission.
Materials and Methods
Cells and virus
African green monkey kidney epithelial Vero76 cells maintained in L-15 media were used in this study for propagation of virus stocks and titration of homogenized tissues as previously described (Huang et al. 2014). ZIKV PRVABC59 strain of the Asian lineage currently circulating in the Americas was isolated from a human case in Puerto Rico in 2015. This isolate was passaged in Vero cells three times and was kindly provided to us by Centers for Disease Control and Prevention and used as seeds for the production of viral stocks. A stock at 7.22 logTCID50/mL was used throughout the study. This is comparable to viremic titers typically detected in humans (Lanteri et al. 2016) and so represents a legitimate challenge to evaluate vector competence.
Mosquitoes and per os infection
Three recently colonized Culex species mosquitoes, F15 of C. pipiens from Anderson, CA, F7 of C. pipiens from Ewing township, Mercer County, NJ, and F7 of C. quinquefasciatus from Vero Beach, FL, were tested in this study. C. pipiens was collected in Anderson, CA, in July 2014. Colonization of C. pipiens from Ewing, NJ, and C. quinquefasciatus from Vero Beach, FL, were both initiated in August 2015. Maintenance of colonies was achieved by nectar solution and bloodmeals delivered through cotton pledgets under a 16 h:8 h light:dark photo regimen at 28°C.
Eight- to 10-day-old female mosquitoes were deprived of sugar and water 48 and 24 h before per os infection, respectively. Viremic bloodmeals were made by mixing equal volumes of ZIKV stock and defibrinated sheep blood (Colorado Serum Company, CO) and administered to mosquitoes in gallon-size cartons through cotton pledgets at room temperature for 1 h (Huang et al. 2015). Control groups received bloodmeals prepared by mixing mock tissue culture media and defibrinated sheep blood in the same manner. Engorged mosquitoes were collected under cold anesthetization and returned to the cartons. Three engorged mosquitoes in each group were immediately frozen after receiving bloodmeals to confirm the ingestion of ZIKV.
Mosquitoes were maintained for 14 days after infection and sampled at 7 and 14 days postinfection (dpi). Mosquitoes collected at 7 and 14 dpi in each experiment were divided into two groups to characterize viral infection, dissemination, and replication. Mosquitoes from the first group were subjected to dissection by placing the abdominal body section into the first tube and the head, wings, and legs into the second tube to represent the midgut and its surrounding tissues and all other secondary tissues as sites for the initial establishment of infection and the subsequent viral dissemination, respectively. Mosquitoes of the second group were collected without dissection to determine viral replication in individual mosquitoes. Titers of viremic bloodmeals, titers of engorged mosquitoes, and number of mosquitoes sampled at 7 and 14 dpi are summarized in Table 1.
dpi, days postinfection.
Processing of mosquito samples and detection of ZIKV
Samples stored at −80°C were thawed at 37°C before homogenization at 26 Hz for 4 min and titration by 50% tissue culture infectious dose (TCID50) method with Vero76 cells (Higgs et al. 2006). To confirm the presence of ZIKV genomic RNA, homogenates were further subjected to viral RNA extraction by QIAmp Viral RNA mini kit (Qiagen, Valencia, CA) followed by reverse transcription (RT) with Superscript II® reverse transcriptase (Life Technologies, Carlsbad, CA) and screening of nested PCR with Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA). ZIKV-specific primers (Integrated DNA technologies, Coralville, IA) were used for the nested RT-PCR assay as previously described (Faye et al. 2008). Amplicons were separated by electrophoresis on 1% agarose gels (Bioexpress, Kaysville, UT) at 125 V for 20 min. Infection rates were calculated by both the presence of cytopathic effect as observed by microscopic examination of cell monolayers and the detection of ZIKV viral RNA in any dissected mosquito tissues from the body tube. Dissemination was defined by the presence of cytopathic effect and the detection of ZIKV viral RNA in dissected secondary tissues, the head, wings, and legs.
Results
Infection and dissemination of ZIKV
Although ZIKV was readily detected in all three populations of Culex species mosquitoes immediately after engorgement of high-titer viremic bloodmeals at 0 dpi, detection of infectious viruses by the TCID50 method followed by seminested RT-PCR did not yield any positive results in dissected tissues or whole mosquitoes from all three populations at either 7 or 14 dpi (Table 2). Infection rates of C. pipiens from Anderson, CA, at 7 and 14 dpi were 0.0% (0/27) and 0.0% (0/34), respectively. C. pipiens from Ewing, NJ, had infection rates at 0.0% (0/20) and 0.0% (0/20) at 7 and 14 dpi, respectively. Similarly, no infection was observed in C. quinquefasciatus from Vero Beach, FL, at 7 (0.0%, 0/30) and 14 dpi (0.0%, 0/33). The lack of detection of established infection agree with the finding that none of the dissected tissues from either of the three populations at 7 and 14 dpi showed positive detection of infectious particles and viral genomic materials.
Conclusions and Discussion
Although ZIKV isolates from Culex species mosquitoes have been reported, resulting in the concern that Culex species mosquitoes can potentially serve as secondary vectors in the transmission and viral maintenance of ZIKV, our results based on recently colonized members of Culex pipiens complex showed a high degree of refractoriness at relatively higher titers of per os infection. Our findings are consistent with an earlier report demonstrating the lack of susceptibility to ZIKV among colonized C. pipiens mosquitoes (Aliota et al. 2016). The evidence has demonstrated that neither C. pipiens nor C. quinquefasciatus, which are widely distributed and referred to as northern and southern house mosquitoes, respectively, can serve as competent vector species for transmission of ZIKV. The role of other Culex species in ZIKV transmission was not determined in our studies, and we cannot exclude the possibility that Culex from Brazil may differ in their susceptibility to the ZIKV circulating in Brazil. However, based on phylogenetic analysis, ZIKV is positioned with the Aedes-transmitted flaviviruses and not with Culex-transmitted ones (Moureau et al. 2015). Vector control strategies targeting ZIKV should focus on suppressing the populations of Aedes species mosquitoes, especially A. aegypti and A. albopictus.
Footnotes
Acknowledgments
The authors would like to thank Christopher Clarkston, Ryan Dajczak, Susan Hettenbach, Brittany Hilfiker, Nick Indelicato, Sara Ortiz, Sydney Rathjen, and Kathryn Ryan for their technical assistance. The research is supported by the startup fund of the Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University (PI: D.L.V.). The funding body has no role in the design of the study, collection, analysis, and interpretation of data or writing of the article.
Author's Contributions
Y.-J.S.H., V.B.A., A.C.L., I.U., and B.W.A. performed the acquisition of mosquitoes and experimental infection. Y.-J.S.H., V.B.A., and A.C.L. analyzed the data. Y.-J.S.H., S.H., and D.L.V. designed the experiment and wrote the article.
Author Disclosure Statement
Coauthor S.H. is editor-in-chief of Vector-Borne and Zoonotic Diseases. All other authors declare that no competing financial interests exist.