Evaluation of expression systems and antiviral genes to inhibit LaCrosse virus replication in mosquito cells

Abstract

Novel control strategies are required to halt the resurgence of arthropod-borne diseases. One such control strategy involves the genetic modification of mosquitoes to reduce vector competence. Mosquito transgenesis is required to test this strategy. While stably transformed mosquitoes have been generated, transgenesis remains a difficult and laborious procedure. Until mosquito transgenesis is a routine procedure, genetic cassettes with potential for altering pathogen transmission in mosquitoes need to be tested in cell culture. In the studies presented here, several strategies for intracellular immunization for LaCrosse (LAC) virus were evaluated. Antisense strategies are effective at interfering with LAC replication when expressed from a Sindbis (SIN) expression system. While SIN virus-based expression systems are invaluable in the evaluation of antiviral strategies, they are not practical for long-term expression in mosquito populations. Evaluation of DNA-based expression of antiviral constructs is necessary before transgenic mosquitoes can be designed with reduced vector competence. Plasmids using Drosophila metallothionein (Mtn) and heat shock protein 70 (hsp70) promoters to express LAC antisense RNA were used to generate stably transformed cell lines, which were analyzed for plasmid integration, synthesis and cellular localization of antisense transcripts, and interference potential. Although LAC-specific RNAs are effective at interfering with virus replication when expressed from a SIN virus expression system, they are not effective when expressed from DNA constructs using Drosophila Mtn or hsp70 promoters. Cell lines transformed with the Mtn and hsp70-based plasmids showed little potential for interfering with LAC virus. Transcript numbers in these cell lines were much lower than is likely needed for antisense interference. Also, expression of the antisense transcript was only seen in ~30% of cells by fluorescence in situ hybridization (FISH). LAC replication in the remaining cells could account for the virus production detected in challenged cell lines. A DNA-based SIN expression system using a baculovirus IE1 promoter to drive expression of a replicon RNA capable of expressing anti-LAC S segment RNA was also evaluated for interference potential. Higher transcript production was evident in the replicon-expressing cell lines as compared to those using the Mtn promoter. These cell lines were capable of interfering with LAC replication as evidenced by lower titers of LAC virus from transformed vs. non-transformed cells. Interference was dependent upon the dose of LAC virus used for challenge. However, all transformed cell lines tested inhibited LAC replication when challenged at a dose similar to that a mosquito would receive from an infectious blood meal in nature. Interference from these cell lines may be mediated by the dsRNA molecules produced in the SIN replication cycle, rather than by classical antisense effects. A protein-based strategy for intracellular immunization was also tested for interference with LAC virus. Human MxA protein was expressed in mosquito cell culture and evaluated for interference potential against LAC virus. MxA localization in mosquito cells was similar to its localization in mammalian cell lines. Expression of MxA in mosquito cells interfered with LAC replication, as seen by immunofluorescence (IFA) analysis. However. MxA expression did not interfere with replication of a recombinant SIN virus. MxA-expressing transgenic mosquitoes may be able to inhibit LAC virus replication, resulting in decreased transmission. An alternate DNA-based expression strategy involves the use of Aedes densonucleosis (AeDNV) transducing viruses to infect mosquito larvae. A low packaging efficiency and genetic recombination resulting in the production of wild-type infectious virus are obstacles in the current system for producing densovirus transducing particles. A double subgenomic SIN virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of AeDNV from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by northern analysis of RNA from infected C6/36 (Aedes albopictus) cells, and by indirect IFA in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells expressing p7NSl-GFP. a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced comparable titers of transducing virus to the standard two-plasmid method. Employing an RNA virus expression system to supply AeDNV structural proteins eliminated the possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks. We are exploring ways to use the TE/3'2J/VP packaging system to improve the efficiency of producing AeDNV transducing viruses. The knowledge gained from these studies will aid in the design of antiviral gene expression in mosquitoes. Antiviral gene expression can be accomplished by mosquito transgenesis or by infection with an AeDNV transducing virus. Specific antiviral genes and DNA-based expression systems will be necessary to evaluate genetic control strategies for arbovirus transmission in caged mosquito populations.