Baculovirus as an efficient vector for gene delivery into mosquitoes

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.

Figure 1

Baculovirus transduction into mosquito C6/36 cells. (a) Schematic representation of a composite expression vector for generating recombinant baculovirus. The composite transfer vector contains the sv40 and pag1 promoters, which are designed to express mCherry in mammalian and insect cells, respectively. (b) Dose-dependent entry of baculovirus. C6/36 cells were transduced with different concentrations (MOI = 1, 10, and 100) of recombinant vABspmC baculovirus. (c) Gating of mCherry-positive cells by flow cytometry, shown the efficiency of baculovirus transduction using different MOIs. (d) Quantification of mCherry-positive cells to indicate transduction efficiency. (e) GP64-mediated entry of baculovirus. C6/36 cells were treated with a baculovirus-antibody mixture (neutralizing or non-neutralizing antibodies against GP64 protein). The mCherry fluorescence images were taken at 48 h post-transduction in both panel’s (b and e). Data represent the average ± SD (standard deviation) of three biological replicates (n = 3).

Figure 2

Effect of baculovirus transduction on cellular proliferation and toxicity. (a) Schematic diagram showing the experimental design. (b) Flow cytometry analysis of cells labeled with CPD eFluor450 dye. Representative dot plots of mock, mock + CPD eFluor450 and vABspmC + CPD eFluor450 (MOI = 1) for 24 h were shown as an example for gating of populations. (c) Representative histograms of CPD eFluor450 labeling intensity in baculovirus transduced cells (Q2 population only) and MOCK cell (Q3 population only) at the indicated time points. (d) Analysis of mean CPD eFluor450 labeling intensity in mock and baculovirus transduced cells with different MOIs at various time points. (e) Cell viability. Toxicity of baculovirus transduction into C6/36 cells at varying dosages, measured every 4 days by AlamarBlue cell viability assay. (f) Persistent baculovirus-mediated gene expression. C6/36 cells were transduced with baculovirus at MOI = 1 and expression of fluorescent protein was analyzed over several days. The mCherry fluorescence images at various time-points were taken by fluorescence microscopy. Data shown represent the averages of three biological replicates, with error bars showing standard deviations.

Figure 3

Replication analysis of baculovirus in C6/36 cells. (a) mCherry fluorescence images of baculovirus transduction. C6/36 cells were transduced with vABspmC at MOI = 10 and mCherry fluorescence images were captured by fluorescence microscopy at various time-points as shown. (b) Baculovirus entry efficiency. Baculovirus was transduced into C6/36 cells or infected with MOI = 10 and MOI = 50 for 2 h into Sf21 cells before relative entry efficiencies were quantified by qPCR. (c) Replication efficiency of baculovirus. C6/36 or Sf21 cells were transduced or infected with MOI = 10, harvested at various time-points, and intracellular viral DNA was quantified by qPCR. Amounts of viral DNA at all time-points were normalized against an internal gene control—GAPDH for Sf21 or Actin for C6/36—and further normalized against viral DNA amount after 2 h post-infection or post-transduction. Data represent the average ± SD of three biological replicates (n = 3).

Figure 4

Efficiency of baculovirus-incorporated promoters in C6/36 cells. (a) Schematic representation of transfer vectors used to generate the recombinant baculoviruses expressing EGFP driven by several baculovirus, mammalian viral, and mosquito host promoters from the following genes: pag1, a HzNV-1 viral early expressing gene; p10, baculovirus late gene; cmv, cytomegalovirus; sv40, simian virus 40; cir, chimeric internal ribosome entry site (IRES) of RhPV virus and EV71 virus^,; b1, cecropin b1 gene; pub, polyubiquitin gene and a4, defensin a4 gene. (b) Fluorescence images. C6/36 cells were transduced with recombinant viruses expressing EGFP at an MOI = 1 or transfected with 500 ng of the recombinant plasmid DNA constructs used to generate recombinant baculoviruses. The images were taken fluorescence microscopy at 48 h post-transduction. (c) Flow cytometry analysis of EGFP fluorescence driven by the promoters of interest. Recombinant baculovirus-transduced or plasmid DNA-transfected C6/36 cells were collected after 48 h and mean EGFP fluorescence intensities were measured by flow cytometry to determine promoter efficiency. Data represent the average ± SD of three biological replicates (n = 3).

Figure 5

Enzymatic analysis of influenza virus NA expressed in mosquito C6/36 cells by recombinant baculovirus. (a) Schematic representation of a pABb1p10-NA3 transfer vector for generating recombinant baculovirus. The transfer vector contains the EGFP reporter gene driven by the pag1 promoter; NA (H5N3) gene under the control of dual host b1 and p10 promoters for mosquito C6/36 and insect Sf21 cells; FLAG and HIS tags were inserted at c-terminal; AcMNPV lateral ends flanking the expression cassette for homologous recombination. (b) C6/36 cells were transduced with vABb1p10-empty (virus backbone without NA3) and vABb1p10-NA3 baculovirus and the EGFP florescent pictures were taken at 48 h. (c) Western blot analysis. The cell lysates of MOCK, vABb1p10-empty and vABb1p10-NA3 were harvested at 48 h, analyzed by SDS-PAGE, followed by Western detection of NA3 protein in C6/36 cells. (d) Neuraminidase activity and Neuraminidase inhibition assays: The cell lysates of MOCK, vABb1p10-empty and vABb1p10-NA3 were mixed with or without (10 µM) NA inhibitors (Oseltamivir or Zanamivir) in the presence of 4-MUNANA substrate. Purified NA9 protein (H5N9 strain) was used as a positive control in the experiment. The experiment was performed three times and the data shown is the representative of one independent experiment done in triplicate. The error bars indicate the standard deviations.

Figure 6

Analysis of in vivo baculovirus transduction of mosquitoes. (a) Transduction of baculovirus into mosquito larvae. Four different mosquito species (A. aegypti, A. albopictus, C. tritaeniorhynchus, and A. sinensis) were microinjected with 1 × 10⁵ PFU of vBacb1EG-irEG baculovirus. EGFP expression was visualized by fluorescence microscopy at 2 days post-transduction. (b) Dose-dependent expression of a baculovirus-mediated transgene in adult mosquitoes. Four different mosquito species were microinjected intra-thoracically with several doses (1 × 10³, 10⁴ or 10⁵ PFU) of vBacb1EG-irEG baculovirus. EGFP expression was visualized by fluorescence microscopy at 6 days post-transduction. (c) Transduction of baculovirus into adult mosquitoes. Four different mosquito species were microinjected intra-thoracically with 1 × 10⁵ PFU of vBacb1EG-irEG baculovirus. EGFP expression was visualized by fluorescence microscopy as a function of time over several days as shown.

Figure 7

Tissue tropism of baculovirus in adult A. aegypti. (a) Dissection of baculovirus transduced adult mosquito. A. aegypti adult mosquitoes were microinjected intra-thoracically with 1 × 10⁵ PFU of vBacb1EG-irEG baculovirus and observed at 15 days post-transduction. EGFP fluorescence was observed in head, antennae, proboscis, leg, wing, haltere, midgut Malpighian tubules, ovary, crop, and fat body tissues of adult mosquitoes. EGFP: green fluorescence, and BF: bright field images. (b) Schematic representation of adult mosquito tissues. For simplicity, cartoon of adult mosquito showing various tissues representing respective images of (a) is shown.