S. Higgs

Genetically Engineered Resistance to Dengue-2 Virus Transmission in Mosquitoes

The control of arthropod-borne virus diseases such as dengue may ultimately require the genetic manipulation of mosquito vectors to disrupt virus transmission to human populations. To reduce the ability of mosquitoes to transmit dengue viruses, a recombinant Sindbis virus was used to transduce female Aedes aegypti with a 567-base antisense RNA targeted to the premembrane coding region of dengue type 2 (DEN-2) virus. The transduced mosquitoes were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus.

Mosquito feeding modulates Th1 and Th2 cytokines in flavivirus susceptible mice: an effect mimicked by injection of sialokinins, but not demonstrated in flavivirus resistant mice

Culex pipiens and Aedes aegypti mosquitoes were fed on C3H/HeJ mice and systemic cytokine production was quantified from stimulated lymphocytes harvested four to ten days after feeding. Mosquito feeding on C3H/HeJ mice significantly down regulated IFNγ production seven to ten days post feeding by Cx. pipiens and seven days after Ae aegypti feeding. Th2 cytokines, IL‐4 and IL‐10, were significantly up regulated 4–7 days after Cx. pipiens and Ae. aegypti feeding. The immunosuppressive effect of Cx. pipiens feeding on systemic cytokine production was not evident in congenic flavivirus resistant (C3H/RV) mice, as systemic IFNγ and IL‐2 were significantly up regulated at days 7 and 10, correlating with a significant decrease in IL‐4 10 days after feeding by Cx. pipiens mosquitoes. Inoculation of 5–1000 ng of sialokinin‐I into C3H/HeJ mice mimicked the effect of Ae. aegypti feeding by down regulating Th1 cytokines and significantly up regulating Th2 cytokines four days post inoculation. Injections of sialokinin‐II resulted in only moderate effects on IFNγ and IL‐4 production seven and ten days after injection. Thus natural feeding by two arbovirus vectors had a profound T cell modulatory effect in vivo in virus susceptible animals which was not demonstrated in the flavivirus resistant host. Moreover, sialokinin‐I and sialokinin‐II mimicked the effect of mosquito feeding by modulating the host T cell response. These results may lend new insight into specific aspects of the role of the mosquito vector in potentiating virus transmission in the mammalian host.

Potentiation of vesicular stomatitis New Jersey virus infection in mice by mosquito saliva.

Saliva of arthropod vectors can modulate vertebrate host immunological functions in many ways. To investigate if vesicular stomatitis New Jersey virus (VSNJ) infection could be potentiated by arthropod saliva, mice in three different age groups (3 days, 3 weeks, or > 8 months) were exposed to VSNJ‐infected mosquitoes or were needle injected with an equivalent dose of VSNJ (titre 1.5–3 logs). Previous studies have demonstrated that VS viruses do not replicate in mice older than 3 weeks of age. Infection was monitored by examining serum for the presence of VSNJ at 2 days postinfection (PI) or for neutralizing antibody on days 7 and 14 PI. All 3‐day‐old mice succumbed to viral infection by mosquito transmission or delivery by injection. Ninety‐four percent of the 3‐week‐old mice bitten by infected mosquitoes developed antibody, whereas antibody was detected in only 13% of inoculated mice. Adult mice developed neutralizing antibody (73%) when fed upon by infected mosquitoes, but only 11% developed antibody when virus was injected. Day 2 serum samples from 3‐week and adult age groups were negative by virus isolation. These data indicate that mosquito mediated delivery of VSNJ exacerbates virus infection in mice older than 3 weeks.

Ectopic gene expression and homeotic transformations in arthropods using recombinant Sindbis viruses

BACKGROUND The morphological diversity of arthropods makes them attractive subjects for studying the evolution of developmental mechanisms. Comparative analyses suggest that arthropod diversity has arisen largely as a result of changes in expression patterns of genes that control development. Direct analysis of how a particular gene functions in a given species during development is hindered by the lack of broadly applicable techniques for manipulating gene expression. RESULTS We report that the Arbovirus Sindbis can be used to deliver high levels of gene expression in vivo in a number of non-host arthropod species without causing cytopathic effects in infected cells or impairing development. Using recombinant Sindbis virus, we investigated the function of the homeotic gene Ultrabithorax in the development of butterfly wings and beetle embryos. Ectopic Ultrabithorax expression in butterfly forewing imaginal discs was sufficient to cause the transformation of characteristic forewing properties in the adult, including scale morphology and pigmentation, to those of the hindwing. Expression of Ultrabithorax in beetle embryos outside of its endogenous expression domain affected normal development of the body wall cuticle and appendages. CONCLUSIONS The homeotic genes have long been thought to play an important role in the diversification of arthropod appendages. Using recombinant Sindbis virus, we were able to investigate homeotic gene function in non-model arthropod species. We found that Ultrabithorax is sufficient to confer hindwing identity in butterflies and alter normal development of anterior structures in beetles. Recombinant Sindbis virus has broad potential as a tool for analyzing how the function of developmental genes has changed during the diversification of arthropods.

Development of a Sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of Aedes aegypti following per os infection.

A double subgenomic Sindbis (dsSIN) virus, MRE/3′2 J/GFP, was constructed to efficiently express green fluorescent protein (GFP) in the midgut of Aedes aegypti following per os infection. The MRE/3′2 J/GFP RNA genome contained the nonstructural genes and cis‐acting sequences of the dsSIN virus, TE/3′2 J/GFP, but had the structural genes of MRE16 SIN virus. MRE/3′2 J/GFP virus, unlike TE/3′2 J/GFP virus, efficiently infected mosquitoes orally. At 1–2 days postinfection, GFP was observed as multiple foci of expression on the lumenal side of the midgut. At 10–12 days postinfection, thirteen of fifteen mosquitoes infected with MRE/3′2 J/GFP virus had high levels of GFP expression in the mosquito midgut. The MRE3′2 J dsSIN expression system should be an important tool for efficient gene expression in Ae. aegypti midguts.

Viral expression systems and viral infections in insects

The development of techniques to manipulate reliably the arthropod genome in a predictable manner remains an elusive goal. To date, all successful transformations of mosquitoes have been due to illegitimate recombination, and transformation efficiencies are too low for routine use. The routine generation of transgenic mosquitoes will undoubtedly be achieved, but when transformation technology is finally perfected, it would be beneficial to have a battery of sequences of known utility (e.g. disruption of pathogen transmission) for ready production of mosquitoes with the desired characteristic. In the absence of heterologous gene expression from within the mosquito genome, virus expression systems can be exploited to introduce and express sequences in mosquito cells for functional analysis. The Sindbis (SIN) alphavirus expression systems have proven to be exceptionally useful for expression of heterologous genes and for gene knock out. Additionally, viral vectors such as Aedes aegypti densonucleosis parvovirus and pseudotyped retroviral expression vectors (Carlson et al., 1995; Chapter 37) may facilitate mosquito transformation.

Alphavirus expression systems: Applications to mosquito vector studies

In this review, Steve Higgs, Ann Powers and Ken Olson describe how alphavirus expression systems, based on infectious cDNA clones of Sindbis virus, constitute a novel RNA virus delivery system suitable for the efficient expression of heterologous gene products or RNA sequences in mosquito cells or adult mosquitoes. The technique permits ready assessment of molecular genetic-based methods of intracellular interference to infection and provides a powerful new tool for molecular biological studies in arthropods.

Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus

Abstract Sindbis virus expression vectors have been used successfully to express and silence genes of interest in vivo in several mosquito species, including Aedes aegypti, Ae. albopictus, Ae. triseriatus,Culex pipiens, Armigeres subalbatus and Anopheles gambiae. Here we describe the expression of an endogenous gene, defensin, in Ae. aegypti using the orally infectious Sindbis virus, MRE/3′2J expression vector. We optimized conditions to infect mosquito larvae per os using C6/36 Ae. albopictus cells infected with the recombinant virus to maximize virus infection and expression of defensin. Infection with the parental Sindbis virus (MRE/3′2J) did not induce defensin expression. Mosquito larvae infected by ingestion of recombinant Sindbis virus-infected C6/36 cells expressed defensin when they emerged as adults. Defensin expression was observed by western analysis or indirect fluorescent assay in all developmental stages of mosquitoes infected with MRE/3′2J virus that contained the defensin insert. The multiplicity of infection of C6/36 cells and the quantity of infected cells consumed by larvae played an important role in defensin expression. Parental viruses, missing the defensin insert, and/or other defective interfering virus may have contributed to these observations.

Sindbis Virus Expression Systems for the Manipulation of Insect Vectors

In this chapter we will introduce and describe the use of RNA virus expression systems based on the Alphavirus, Sindbis (SIN; Togaviridae). We will describe the unique biological properties of the mosquito-borne SIN virus that have been exploited to efficiently transduce mosquito cells and mosquitoes and obtain stable, cytoplasmic expression of a number of heterologous genes in the arthropod. Additionally, we will discuss the use of SIN virus expression systems as powerful tools for studying basic virus-vector interactions and identifying molecular strategies for interfering with the transmission of vector-borne diseases (VBD), such as dengue fever and LaCrosse encephalitis.

Use of the Sindbis replicon system for expression of LaCrosse virus envelope proteins in mosquito cells

SummaryThe Sindbis replicon expression system was used to express La Crosse (LAC) virus envelope glycoprotein genes in both mammalian and mosquito cell culture. Replicon expressed LAC proteins had correct molecular mass (Mr) and were antigenically similar to wild type LAC envelope proteins. In addition, LAC G1 and G2 proteins colocalized when expressed from separate constructs in both mammalian and mosquito cells suggesting that they were trafficked through the cell similarly to wild type LAC proteins. A truncated form of the G1 protein was secreted from mosquito cells when expressed alone. The truncated G1 protein was also secreted from mosquito cells when expressed with the G2 protein, but to a lesser extent than when expressed alone, suggesting that the G2 protein sequestered G1 protein intracellularly. The Sindbis replicon system is a powerful tool for the study of LAC virus protein maturation within mosquito cells and mosquitoes.