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Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

ABSTRACT: Aedes mosquitoes are vectors for many pathogenic viruses. Cell culture systems facilitate the investigation of virus growth in the mosquito vector. We found Zika virus (ZIKV) growth to be consistent in A. albopictus cells but hypervariable in A. aegypti cell lines. As a potential explanation of this variability, we tested the hypothesis that our cells harbored opportunistic viruses. We screened Aedes cell lines for the presence of insect specific viruses (ISVs), Cell-fusing agent virus (CFAV) and Phasi charoen-like virus (PCLV). PCLV was present in the ZIKV-growth-variable A. aegypti cell lines but absent in A. albopictus lines, suggesting that these ISVs may interfere with ZIKV growth. In support of this hypothesis, PCLV infection of CFAV-positive A. albopictus cells inhibited the growth of ZIKV, dengue virus and La Crosse virus. These data suggest ISV infection of cell lines can impact arbovirus growth leading to significant changes in cell permissivity to arbovirus infection.

SUBMITTER: Schultz MJ

PROVIDER: S-EPMC8005325 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

Schultz Michaela J MJ Frydman Horacio M HM Connor John H JH

Virology 20180403

Aedes mosquitoes are vectors for many pathogenic viruses. Cell culture systems facilitate the investigation of virus growth in the mosquito vector. We found Zika virus (ZIKV) growth to be consistent in A. albopictus cells but hypervariable in A. aegypti cell lines. As a potential explanation of this variability, we tested the hypothesis that our cells harbored opportunistic viruses. We screened Aedes cell lines for the presence of insect specific viruses (ISVs), Cell-fusing agent virus (CFAV) an ...[more]

PMID: 29625404

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Project description:BackgroundAedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections, which arboviruses encounter more commonly in nature, has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures.Methodology/principle findingsWe generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies.Conclusions/significanceOur findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.

Project description:BackgroundMany arboviral outbreaks have occurred in various locations in Kenya. Entomological surveys are suitable methods for revealing information about circulating arboviruses before human outbreaks are recognized. Therefore, mosquitoes were collected in Kenya to determine the distribution of arboviruses.MethodsVarious species of mosquitoes were sampled from January to July 2012 using several collection methods. Mosquito homogenates were directly tested by reverse transcription-polymerase chain reaction (RT-PCR) using various arbovirus-targeted primer pairs.ResultsWe collected 12,569 mosquitoes. Although no human-related arboviruses were detected, Culex flavivirus (CxFV), an insect-specific arbovirus, was detected in 54 pools of 324 Culex quinquefasciatus individuals collected during the rainy season. Of these 54 positive pools, 96.3% (52/54) of the mosquitoes were collected in Busia, on the border of western Kenya and Uganda. The remaining two CxFV-positive pools were collected in Mombasa and Kakamega, far from Busia. Phylogenetic analysis revealed minimal genetic diversity among the CxFVs collected in Mombasa, Kakamega, and Busia, even though these cities are in geographically different regions. Additionally, CxFV was detected in one mosquito pool collected in Mombasa during the dry season. In addition to Culex mosquitoes, Aedes (Stegomyia) and Anopheles mosquitoes were also positive for the Flavivirus genus. Cell fusing agent virus was detected in one pool of Aedes aegypti. Mosquito flavivirus was detected in three pools of Anopheles gambiae s.l. collected in the dry and rainy seasons.ConclusionsAlthough no mosquitoes were positive for human-related arbovirus, insect-specific viruses were detected in various species of mosquitoes. The heterogeneity observed in the number of CxFVs in Culex mosquitoes in different locations in Kenya suggests that the abundance of human-related viruses might differ depending on the abundance of insect-specific viruses. We may have underestimated the circulation of any human-related arbovirus in Kenya, and the collection of larger samples may allow for determination of the presence of human-related arboviruses.

Project description:Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo)viruses including dengue and chikungunya virus (CHIKV) but in contrast to human hosts, arbovirus-infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge of the molecular interactions of arboviruses with their mosquito hosts is incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well-known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against Sindbis virus. Amongst these, the three DEAD-box RNA helicases AAEL004419/Dhx15, AAEL008728, and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explored the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected and CHIKV-infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most enriched gene ontology (GO) term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production indicated that Dhx15 silencing indeed functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolytic genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mosquito cells.

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Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

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Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

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