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Culex quinquefasciatus mosquitoes do not support replication of Zika virus.

ABSTRACT: The rapid spread of Zika virus (ZIKV) in the Americas raised many questions about the role of Culex quinquefasciatus mosquitoes in transmission, in addition to the key role played by the vector Aedes aegypti. Here we analysed the competence of Cx. quinquefasciatus (with or without Wolbachia endosymbionts) for a ZIKV isolate. We also examined the induction of RNA interference pathways after viral challenge and the production of small virus-derived RNAs. We did not observe any infection nor such small virus-derived RNAs, regardless of the presence or absence of Wolbachia. Thus, Cx. quinquefasciatus does not support ZIKV replication and Wolbachia is not involved in producing this phenotype. In short, these mosquitoes are very unlikely to play a role in transmission of ZIKV.

SUBMITTER: Lourenco-de-Oliveira R

PROVIDER: S-EPMC5882084 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Several mosquito species have been described as vectors for the Zika virus (ZIKV), such as those in the Aedes, Anopheles, Mansonia and Culex genera. Our previous survey studies were found the ZIKV RNA positive in both male, female and larvae of Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes collected from active ZIKV infected patients' homes in Thailand. Therefore, the aims of this study were to investigate whether ZIKV could be vertically transmitted in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. Laboratory and field colonies of these mosquito species were maintained and artificially fed with ZIKV in human blood. Fully engorged mosquitoes (F0) were selected and reared for the vertical transmission study. The subsequent mosquito generations were fed with human blood without the virus. ZIKV in the mosquitoes was detected by hemi-nested RT-PCR and sequencing. C6/36 cells were used to isolate ZIKV from samples that tested positive by hemi-nested RT-PCR. Moreover, ZIKV was identified by immunocytochemical staining 7 days after infection in several organs of infected F0 females, including the salivary glands, midguts, yoke granules and facet cells of the eye. The localization of the ZIKV antigen was identified by the presence of the specific antibody in the salivary glands, midguts, yoke granules and facet cells. ZIKV was detected in female and male Cx. quinquefasciatus until the F6 and F2 generations, respectively. The isolated virus showed cytopathic effects in C6/36 cells by 5 days postinfection. The results suggested that the vertical transmission of ZIKV occurs in Cx. quinquefasciatus in the laboratory. However, we were able to detect the presence of ZIKV in Ae. aegypti in only the F1 generation in both male and female mosquitoes, and Ae. albopictus mosquitoes were not able to vertically transmit the virus at all. Data obtained from this study could be valuable for developing a better understanding of the role of Cx. quinquefasciatus as a potential vector for ZIKV transmission in Thailand and may be useful in creating more effective mosquito vector control strategies in the future.

Project description:Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher's exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Project description: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.

Project description:Newly emerging or re-emerging arthropod-borne viruses (arboviruses) are important causes of human morbidity and mortality worldwide. Arboviruses such as Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) have undergone extensive geographic expansion in the tropical and sub-tropical regions of the world. In the Americas the main vectors of DENV, ZIKV, and CHIKV are mosquito species adapted to urban environments, namely Aedes aegypti and Aedes albopictus, whereas the main vector of WNV is Culex quinquefasciatus. Given the widespread distribution in the Americas and high permissiveness to arbovirus infection, these mosquito species may play a key role in the epidemiology of other arboviruses normally associated with sylvatic vectors. Here, we test this hypothesis by determining the vector competence of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus to Mayaro (MAYV) virus, a sylvatic arbovirus transmitted mainly by Haemagogus janthinomys that has been causing an increasing number of outbreaks in South America, namely in Brazil. Using field mosquitoes from Brazil, female mosquitoes were experimentally infected, and their competence for infection and transmission rates of MAYV was evaluated. We found consistent infection rate for MAYV in Ae. aegypti (57.5%) and Ae. albopictus (61.6%), whereas very low rates were obtained for Cx. quinquefasciatus (2.5%). Concordantly, we observed high potential transmission ability in Ae. aegypti and Ae. albopictus (69.5% and 71.1% respectively), in contrast to Cx. quinquefasciatus, which could not transmit the MAYV. Notably, we found that very low quantities of virus present in the saliva (undetectable by RT-qPCR) were sufficiently virulent to guarantee transmission. Although Ae. aegypti and Ae. albopictus mosquitoes are not the main vectors for MAYV, our studies suggest that these mosquitoes could play a significant role in the transmission of this arbovirus, since both species showed significant vector competence for MAYV (Genotype D), under laboratory conditions.

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Culex quinquefasciatus mosquitoes do not support replication of Zika virus.

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