Abstract
Efficient gene delivery technologies play an essential role in the gene functional analyses that are necessary for basic and applied researches. Mosquitoes are ubiquitous insects, responsible for transmitting many deadly arboviruses causing millions of human deaths every year. The lack of efficient and flexible gene delivery strategies in mosquitoes are among the major hurdles for the study of mosquito biology and mosquito-pathogen interactions. We found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type baculovirus species, can efficiently transduce mosquito cells without viral propagation, allowing high level gene expression upon inducement by suitable promoters without obvious negative effects on cell propagation and viability. AcMNPV transduces into several mosquito cell types, efficiently than in commonly used mammalian cell lines and classical plasmid DNA transfection approaches. We demonstrated the application of this system by expressing influenza virus neuraminidase (NA) into mosquito hosts. Moreover, AcMNPV can transduce both larvae and adults of essentially all blood-sucking mosquito genera, resulting in bright fluorescence in insect bodies with little or no tissue barriers. Our experiments establish baculovirus as a convenient and powerful gene delivery vector in vitro and in vivo that will greatly benefit research into mosquito gene regulation, development and the study of mosquito-borne viruses.
Highlights
Mosquitoes are primary vectors for the transmission of many human diseases such as chikungunya (CHIKV), dengue (DENV), filarial, malaria, yellow fever, and the recent Zika virus (ZIKV) outbreaks, all of which continue to pose public health risks and contribute to significant global economic losses[1,2]
To examine if Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can enter into mosquito host C6/36 cells and deliver an effective gene expression cassette, we utilized recombinant baculovirus containing mCherry under the control of Heliothis zea nudivirus 1 (HzNV-1) virus early gene pag[1] promoter[23] and sv[40] promoter[24] that was originally designed for mammalian and insect cell systems respectively (Fig. 1a)
We found that baculovirus treated with GP64-neutralizing antibody did not show mCherry fluorescence at 48 h post-transduction compared to a negative control (Fig. 1e), indicating that baculovirus enters mosquito hosts through the GP64 surface protein
Summary
Mosquitoes are primary vectors for the transmission of many human diseases such as chikungunya (CHIKV), dengue (DENV), filarial, malaria, yellow fever, and the recent Zika virus (ZIKV) outbreaks, all of which continue to pose public health risks and contribute to significant global economic losses[1,2]. In 1995, AcMNPV was found possible to transfer genes into mammalian cells and efficiently expressed by a promoter functional in the target cells[6,14] It has since been successfully exploited in gene transfer applications for many mammalian cell lines, primary cells, progenitor cells, induced pluripotent (iPS) and stem cells, and is well-known as BacMam system[15,16]. We first examined and compared the strength of various baculovirus-incorporated promoters in C6/36 cells, and further showed strong baculovirus-mediated foreign gene expression in larvae and in almost all adult mosquito tissues without an obvious tissue barrier This BacMos system combines simplicity and flexibility, and serves as an alternative to existing inefficient or tedious approaches. We believe BacMos has the potential to become a powerful tool for gene regulation analysis, which may significantly assist in advancing studies of mosquito biology and pathogen interactions
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