Jonathan O. Carlson

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.

Sindbis virus-induced silencing of dengue viruses in mosquitoes.

Aedes aegypti were injected intrathoracically with double subgenomic Sindbis (dsSIN) viruses with inserted sequences derived from the genome of one or more of the four dengue (DEN) virus serotypes. Mosquitoes were highly resistant to challenge with homologous DEN viruses from which the effector sequences were derived, and resistance to DEN viruses was independent of the orientation of the effector RNA. dsSIN viruses designed to express RNA derived from the premembrane coding region of DEN‐2 prevented the accumulation of DEN2 RNA, and C6/36 cells were highly resistant to DEN‐2 virus when challenged at 2, 5 or 8 days after the initial dsSIN virus infections, even though the dsSIN‐derived RNA had sharply declined at the later time points. Initiation of resistance occurred prior to or within the first 8 h after challenge with DEN‐2 virus. We conclude that DEN viruses are inhibited by a mechanism similar to post‐transcriptional gene silencing (PTGS) or RNA interference (RNAi) phenomena described in plants and invertebrates, respectively. The potential occurrence of PTGS or RNAi in mosquitoes and mosquito cells suggests new ways of inhibiting the replication of arthropod‐borne viruses in mosquito vectors, studying vector–virus interactions, and silencing endogenous mosquito genes.

The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain

Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma-leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.

Densoviruses for control and genetic manipulation of mosquitoes.

Mosquito densoviruses (MDV) are parvoviruses that replicate in the nuclei of mosquito cells and cause the characteristic nuclear hypertrophy (densonucleosis) that gives them their name. Several MDV that differ in pathogenicity both in vitro and in vivo have been isolated. MDV have a number of features that make them potentially attractive as biological control agents for mosquito-borne disease. They are nonenveloped and relatively stable in the environment. They are highly specific for mosquitoes and they infect and kill larvae in a dose dependent manner in the aqueous larval habitat. Infected larvae that survive to become adult mosquitoes exhibit a dose-dependent shortening of lifespan and many do not survive longer than the extrinsic incubation period for arboviruses. Thus they may have a significant impact on transmission of pathogens. Infected females can transmit the virus vertically by laying infected eggs in new oviposition sites. Studies on how MDV affect populations are relatively limited. Population cage studies suggest that they will persist and spread in populations and limited field studies have shown similar preimaginal mortality in wild populations to that seen in laboratory studies. The availability of infectious clones of MDV genomes allows the development of densovirus vectors for expressing genes of interest in mosquito cells and mosquitoes. Recently short hairpin RNA expression cassettes that induce RNA interference have been inserted into densovirus genomes. These expression cassettes should be useful for both research and disease-control applications.

Jaagsiekte Sheep Retrovirus Proviral Clone JSRV JS7 , Derived from the JS7 Lung Tumor Cell Line, Induces Ovine Pulmonary Carcinoma and Is Integrated into the Surfactant Protein A Gene

ABSTRACT Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRVJS7) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRVJS7 is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.

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.

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [ Afanasiev et al. (1994) Exp Parasitol 79: 322–339; Afanasiev et al. (1999) Virology 257: 62–72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139–159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations ( Afanasiev et al., 1999 ; Allen‐Muira et al. (1999) Virology 257: 54–61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day‐old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10–0.15 m inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA.

We have isolated a cDNA from Aedes aegypti that is transcribed in the larval midgut in response to metal exposure, and in the adult female midgut in response to iron or cadmium exposure, or a blood meal. The cDNA encodes a protein, designated Aedes aegypti intestinal mucin 1 (AEIMUC1), which has similarities with invertebrate intestinal mucins and peritrophins, and vertebrate mucins. Proline, serine and threonine comprise 30% of the amino acid composition of AEIMUC1, a characteristic of mucins. AEIMUC1 contains three cysteine‐rich domains, two of which flank a proline/serine/threonine‐rich domain, a feature shared by many mucin genes. This is the first report on the isolation of a metal‐responsive gene from an aquatic insect.

Rhythms and synchronization patterns in gene expression in the Aedes aegypti mosquito

BackgroundAedes aegypti is arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock.ResultsHerein we analyze the transcription profiles of 21,494 messenger RNAs using an Ae. aegypti Agilent® microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways.ConclusionThese results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

Analysis of Integration Sites of Jaagsiekte Sheep Retrovirus in Ovine Pulmonary Adenocarcinoma

ABSTRACT Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.