Detailed Analyses of Zika Virus Tropism in Culex quinquefasciatus Reveal Systemic Refractoriness

Abstract

The role of Culex quinquefasciatus in Zika virus transmission has been debated since the epidemic of Zika occurred in the Americas in 2015 to 2016. The majority of studies have found no evidence that C. quinquefasciatus or other Culex species are competent vectors of Zika virus, and the few studies that have proposed Zika vector status for C. quinquefasciatus have relied predominantly on quantitative real-time PCR (qRT-PCR) for viral detection. We assessed the infectious range of pre- and post-epidemic Zika virus isolates in order to classify mosquito samples based on titer infectiousness and demonstrated that two strains of C. quinquefasciatus, including one previously found to be competent, are highly resistant to infection with these Zika isolates compared to Aedes aegypti and are not competent for virus transmission. Further dissection of the dynamics of Zika exposure in both A. aegypti and C. quinquefasciatus revealed that while virus transmission by C. quinquefasciatus is blocked at the levels of the midgut and salivary glands, viral RNA persists in these tissues for prolonged periods post-exposure. We assessed Zika entry dynamics in both Aedes and Culex cells, and our results suggest that Zika virus infection in Culex cells may be blocked downstream of cell entry. These findings strongly suggest that C. quinquefasciatus is not a vector of Zika virus and additionally inform the use of qRT-PCR in vector competence assays as well as our understanding of barriers to arbovirus infection in non-susceptible mosquito species. IMPORTANCE Understanding which mosquito species transmit an emerging arbovirus is critical to effective vector control. During the Zika virus epidemic in 2015 to 2016, Aedes mosquitoes were confirmed as vectors. However, studies addressing the vector status of Culex quinquefasciatus mosquitoes presented conflicting evidence and remain an outstanding source of confusion in the field. Here, we established a robust cell-based assay to identify infectious titers of Zika virus and assessed the virus titers in C. quinquefasciatus by quantitative real-time PCR (qRT-PCR). We found that while low levels of virus were detected in C. quinquefasciatus, these titers did not correspond to infectious virus, and these mosquitoes did not transmit virus in the saliva. We also present evidence that the virus may enter Culex cells before infection is disrupted. Our findings are important for future studies incriminating vector species using qRT-PCR for virus detection and offer new information on how virus transmission is blocked by mosquitoes.

Molecular survey of Zika virus in the animal-human interface in traditional farming

“…The rapid spread and severity of the ZIKV epidemic that occurred in the Americas in 2015-2016 has raised suspicions that Cx. quinquefasciatus is an additional vector of this virus (38,39). Some studies suggest that Cx.…”

“…It is important to emphasize that the previously mentioned experimental studies were conducted under different conditions that may affect or bias the result, such as the origin of the mosquito colonies or animals used for the study, the virus strain and concentration to which they were exposed, and the mode of inoculation (39,68,69). This last aspect is important when it comes to mosquito-borne viruses because it has been observed that vertebrates experimentally exposed via mosquitoes or in combination with mosquito saliva have a higher infection rate compared with needle inoculations (70,71)…”

Which Plagues are Coming Next?

“…Although Aedes mosquitos are the confirmed vector for ZIKV, Culex genera mosquitoes, mostly Culex quinquefasciatus, are theorized to be capable to transmit Zika, further expanding its geographic reach [34]. However, recent studies did not support the ability for Culex mosquitoes to transmit ZIKV [35,36]. In conjunction with its vectors wide global reach, a high proportion of Zika cases are asymptomatic and those who are symptomatic mirror symptoms to dengue and the flu (e.g., fever, rash, muscle and joint pain) compounding the barriers to diagnosis, treatment, and epidemic mitigation efforts.…”

The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses

“…Several flaviviruses that are transmitted by various species of Culex mosquitoes and can cause human illness are associated with birds, including West Nile virus (WNV), Usutu virus, and St. Louis encephalitis virus [ 12 , 13 , 14 ]. To date, no Culex species has been unequivocally confirmed as a competent vector for ZIKV transmission [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. The two major vectors of ZIKV for humans, Aedes aegypti and Aedes albopictus , have a wide and expanding geographical range across all continents except Antarctica [ 22 , 23 , 24 ].…”