Regular ArticleThe Expression of Chloramphenicol Acetyltransferase in Mosquitoes and Mosquito Cells Using a Packaged Sindbis Replicon System
Abstract
Sindbis (SIN) replicon virus was used to express chloramphenicol acetyltransferase (CAT) in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes. RNA transcribed in vitro from a SIN replicon plasmid (pSINrep5/CAT) and from SIN virus helper constructs (pDH-EB or pDH(26S)5′SIN) was coelectroporated into BHK-21 cells to generate replicon viruses, designated rep5/CAT/EB and rep5/CAT/26S. C6/36 cells infected with rep5/CAT/EB or rep5/CAT/26S virus at a multiplicity of infection of 3, expressed 3.8 × 106 and 6.0 × 106 CAT trimers per cell, respectively, at 2 days postinfection (pi). Both viruses attained peak titers by Day 2 pi. Adult female A. triseriatus mosquitoes were intrathoracically inoculated with 7 × 104 IFU rep5/CAT/EB or 1 × 105 IFU rep5/CAT/26S virus. Virus titers remained at approximately 105 IEU/ml through Day 2 pi and decreased roughly 1 log by Day 10 pi. CAT enzyme activity was detected 2 days pi (rep5/CAT/EB, 1.49 × 10−4 units CAT/10 μg protein; rep5/CAT/26S, 2.03 × 10−5 units CAT/10 μg protein) and remained near these levels through Day 10 pi. CAT was detected in the head, salivary glands, midgut, and ovaries of inoculated mosquitoes by indirect immunofluorescence or CAT activity assays. These results suggest that packaged replicon viruses can be useful for expression of heterologous genes in mosquito cells and whole mosquitoes.
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Ectopic gene expression and homeotic transformations in arthropods using recombinant Sindbis viruses
1999, Current BiologyBackground: 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.
Seoul virus (SEOV) is a member of the Hantavirus genus (family Bunyaviridae) and an etiological agent of hemorrhagic fever with renal syndrome. The medium (M) and small (S) gene segments of SEOV encode the viral envelope glycoproteins and nucleocapsid protein, respectively. We compared the immunogenicity and protective efficacy of naked DNA (pWRG7077), DNA-based Sindbis replicon (pSIN2.5), and packaged Sindbis replicon vectors (pSINrep5), containing either the M or S gene segment of SEOV in Syrian hamsters. All of the vectors elicited an anti-SEOV immune response to the expressed SEOV gene products. Vaccinated hamsters were challenged with SEOV and monitored for evidence of infection. Protection from infection was strongly associated with M-gene vaccination. A small number of S-gene-vaccinated animals also were protected. Hamsters vaccinated with the pWRG7077 vector expressing the M gene demonstrated the most consistent protection from SEOV infection and also were protected from heterologous hantavirus (Hantaan virus) infection.
DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection
1999, VirologySeoul virus (SEOV) is one of four known hantaviruses causing hemorrhagic fever with renal syndrome (HFRS). Candidate naked DNA vaccines for HFRS were constructed by subcloning cDNA representing the medium (M; encoding the G1 and G2 glycoproteins) or small (S; encoding the nucleocapsid protein) genome segment of SEOV into the DNA expression vector pWRG7077. We vaccinated BALB/c mice with three doses of the M or S DNA vaccine at 4-week intervals by either gene gun inoculation of the epidermis or needle inoculation into the gastrocnemius muscle. Both routes of vaccination resulted in antibody responses as measured by ELISA; however, gene gun inoculation elicited a higher frequency of seroconversion and higher levels of antibodies in individual mice. We vaccinated Syrian hamsters with the M or S construct using the gene gun and found hantavirus-specific antibodies in five of five and four of five hamsters, respectively. Animals vaccinated with the M construct developed a neutralizing antibody response that was greatly enhanced in the presence of guinea pig complement. Immunized hamsters were challenged with SEOV and, after 28 days, were monitored for evidence of infection. Hamsters vaccinated with M were protected from infection, but hamsters vaccinated with S were not protected.
Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes
1999, VirologyNoncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), β-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3′-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications.
Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes
1997, Insect Biochemistry and Molecular BiologyMosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3′2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3′2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3′2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3′2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 × 10−5 units of CAT enzyme (3.0 × 106 CAT trimers).
Expression analysis and knockdown of two antennal odorant-binding protein genes in Aedes aegypti
2010, Journal of Insect Science