Project description:The dengue virus (DENV) cause frequent epidemics infecting ~390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control transmission of DENV by the mosquito vectors, Aedes aegypti and Ae. albopictus, using Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naïve Ae. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV2. Analysis of host transcript profiles by RNAseq revealed that the presence of wMelPop had profound effects on mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 contained low frequency mutations (~25%) within the coding region of transmembrane domain-1 (TMD1) of E protein. Mutations with >97 % frequencies were distributed within other regions of E, NS5 RNA-dependent RNA polymerase (NS5POL) domain, the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naïve C6/36 cells showed syncytia formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naïve C6/36 cells; whereas, in the mutant DENV2- infected BHK-21 or Vero cells, the virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to WT and compensatory mutations in NS3 gene appeared. Our results suggest that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells.
Project description:Use of the bacterium Wolbachia is an innovative new strategy designed to break the cycle of dengue transmission. There are two main mechanisms by which Wolbachia could achieve this: by reducing the level of dengue virus in the mosquito and/or by shortening the host mosquito's lifespan. However, although Wolbachia shortens the lifespan, it also gives a breeding advantage which results in complex population dynamics. This study focuses on the development of a mathematical model to quantify the effect on human dengue cases of introducing Wolbachia into the mosquito population. The model consists of a compartment-based system of first-order differential equations; seasonal forcing in the mosquito population is introduced through the adult mosquito death rate. The analysis focuses on a single dengue outbreak typical of a region with a strong seasonally-varying mosquito population. We found that a significant reduction in human dengue cases can be obtained provided that Wolbachia-carrying mosquitoes persist when competing with mosquitoes without Wolbachia. Furthermore, using the Wolbachia strain WMel reduces the mosquito lifespan by at most 10% and allows them to persist in competition with non-Wolbachia-carrying mosquitoes. Mosquitoes carrying the WMelPop strain, however, are not likely to persist as it reduces the mosquito lifespan by up to 50%. When all other effects of Wolbachia on the mosquito physiology are ignored, cytoplasmic incompatibility alone results in a reduction in the number of human dengue cases. A sensitivity analysis of the parameters in the model shows that the transmission probability, the biting rate and the average adult mosquito death rate are the most important parameters for the outcome of the cumulative proportion of human individuals infected with dengue.
Project description:Background: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it be responsible for transposon silencing in this mosquito. Results: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. Conclusions: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster.