Microbiology, Immunology, and Pathology, Colorado State University
Phillips, A., Mossel, E., Sanchez-Vargas, I., Foy, B., Olson, K. Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors. J. Vis. Exp. (45), e2363, doi:10.3791/2363 (2010).
Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.
The following protocol is written for production and use of an ATS based on the Sindbis virus MRE16. The ATS is designed to express GFP in the mosquito vector Aedes aegypti. cDNA Infectious clones of a number of alphaviruses (Sindbis virus, Chikungunya virus, O'nyong-nyong virus, western equine encephalitis virus) are currently available that have been designed as ATS's (Table 1). For best results plasmid DNA is amplified in bacteria such as SURE competent bacteria (Stratagene/Agilent Technologies) to avoid unwanted recombination and loss of the viral cDNA. All infectious clone plasmids have a bacteriophage T7 or SP6 promoter at the immediate 5' end of the viral cDNA for transcription of full-length genomic RNA and a unique restriction endonuclease at the 3' end for runoff transcription. For each ATS, users must use the appropriate bacteriophage DNA dependent RNA polymerase and unique restriction endonuclease for in vitro transcription of the viral RNA genome.
1. Generation of Infectious RNA from cDNA Clone.
2. Generation of Virus from Infectious RNA
3. per os Infection of Mosquitoes
4. Intrathoracic Inoculation of Mosquitoes
Intrathoracic inoculation is an alternative method for infecting the arthropod. This method is used when dissemination through the midgut is not required. (Method described below but the technique is not shown in this visual experiment).
5. Monitoring Infection of Mosquitoes
6. Transmission Assay (Forced Salivation to Demonstrate Virus Transmission)
BIOSAFETY NOTE: This protocol describes a method for generating, identifying, and monitoring infected mosquitoes. Based on your facilities (i.e. a chill table, containment facility, etc.) and IBC approval, you may be limited to examining them only after removing wings and legs to prevent escape. SINV-based ATS's can be generated and used in a BSL2 environment. The use of ATS's based on other alphaviruses such as Chikungunya virus or western equine encephalitis viruses will require BSL3 facilities.
7. Representative Results
An overview of expression of a marker gene (GFP) using the ATS 5'dsMRE16/GFP is shown in Figure 1. Expression of GFP in BHK-21 and C6/36 (Aedes albopictus) cells is shown in Figure 2 at specific times after infection of cells with 5'dsMRE16/GFP virus at 0.01 multiplicity of infection. Figure 3 is an overview of alphavirus and ATS virus infection in the mosquito when the virus is delivered through an infective blood meal. Figure 4 shows preparation of a blood meal with ~107 pfu/mL of ATS virus followed by oral infection of Aedes aegypti. Whole body view of infected mosquito and selected body parts at 10 days post infection using an epifluorescence microscope (Figure 5). Detection of GFP and dsRED in midguts of infected mosquitoes following infection with ATS 5'dsMRE16 viruses expressing each fluorescent marker gene (Figure 6). Transmission assay using mosquitoes infected with 5'dsMRE16/GFP ATS virus (Figure 7).
Table 1. ATS's currently engineered for gene expression in mosquitoes.
|Sindbis Virus||TE/3'2J and TE/5'2J||Aedes aegypti||12|
|5'dsTR339||A. aeg ypti||2|
|Chikungunya virus||3'dsCHIKV and
|A. aegypti/A. albopictus||20|
|O'nyong nyong virus||5'dsONNV||Anopheles gambiae||1,9|
|Western equine encephalitis virus||5'dsWEEV.McMillan||Culex tarsalis||Stauft et al., unpublished|
Table 2. Advantages/Disadvantages of using ATS's for gene expression in mosquitoes.
|Rapid production of high-titer virus|
|Broad host range (SINV)|
|High RNA replication rate|
|High transgene expression levels|
|Relative ease of manipulating genes|
|Stable, persistent gene expression in insects|
|Minimal pathology in insects|
|Short-term expression mode in vertebrate cells|
|Strong cytopathic effects on vertebrate cells|
|Gene size restrictions (<1Kb, ideally)|
|Some alphaviruses require BSL3 containment|
Figure 1: Overview of ATS virus production in BHK-21 cells using p5'dsMRE/GFP SINV expressing GFP. Following in vitro transcription, genomic ATS RNA is electroporated into BHK-21 cells where virus is rescued. The ATS virus can then be used to infect mosquitoes. SGP = subgenomic promoter. Three ATS RNA species are transcribed in the cells, the genomic, subgenomic 1 and subgenomic 2 RNAs. C (capsid), E2 and E1 glycoproteins are assembled with ATS genome to generate infectious virus.
Figure 2: Expression of GFP in mosquito (C6/36) cells following infection with SINV 5'dsMRE16/GFP virus.
Figure 3: Overview of an ATS virus infection in mosquitoes leading to virus transmission.
Figure 4: ATS SINV 5'dsMRE16 infection by exposing mosquitoes to an infective blood meal.
Figure 5: ATS SINV 5'dsMRE16/GFP infection at 10 days post infection. Whole body detection of GFP shows that virus has escaped the midgut and disseminated to secondary tissues. Figure also shows GFP expression in selected body parts that also is indicative of a disseminated infection.
Figure 6: ATS SINV 5'dsMRE16/GFP and 5'dsMRE16/dsRED infection of midguts at specific times post-infection. Midguts usually have distinct foci of infection early after ingesting a blood meal containing virus that spreads throughout the posterior midgut at later times post-infection.
Figure 7: Detection of ATS 5'dsMRE16/GFP in mosquito saliva as early as 8 days post infection. Assay is designed to show transmission of the ATS virus and shows that the 5'dsMRE16/GFP virus can stably express GFP throughout the extrinsic incubation period in the mosquito. The left panel shows a mosquito salivating into a capillary tube, the center panel shows C6/36 cells infected with saliva at 1 day and right panel 3 days post infection.
The methods presented here enable researchers to follow the infection of an alphavirus in mosquito cell culture and the adult female mosquito. The advantages and disadvantages of using ATS viruses are summarized in Table 2. An ATS infectious clone can be genetically manipulated to express any GOI and have been used to express single chain antibodies, suppressors of RNAi, antisense RNAs targeting endogenous genes, and pro-apoptotic proteins. If a reporter is not used, then the imaging portion of this method is not relevant. However, many of the same protocols are necessary for ATS virus rescue, propagation, and mosquito infection. There are limitations in the maximal size of the transgene when inserted into an alphavirus transducing system. The size constraints vary depending on the parental strain used to generate the ATS, but are usually about 1kb although larger reporter genes such as firefly luciferase have been used in constructing ATS for use in mosquitoes. Research labs with a modest amount of virology background should be able to use ATS's following these protocols. A number of research labs have already done so using SINV-based ATS's.
No conflicts of interest declared.
This work is supported by the National Institutes of Health (NIH R01 AI46435) and the RMRCE (NIH AI065357).
|QIAprep Spin Miniprep Kit||Qiagen||27104|
|MAXIscript Kit||Ambion||AB1312||Be sure to use a kit containing the appropriate polymerase for your construct.|
|Cap Analogue (m7G(5’)ppp(5’)G)||Ambion||AM8050|
|Nanoject II nanoliter injector||Drummond Scientific||3-000-204|
|Electro Cell Manipulator||Harvard Apparatus||ECM 630|