Biomedical subjects

K E Olson

Publications and source records attributed to K E Olson.

At least 37 records · Page 2Linked to original sources

PubMed1998

Development of a chimeric sindbis virus with enhanced per Os infection of Aedes aegypti.

The TE/3'2J double subgenomic Sindbis (dsSIN) viruses have been used to stably express genes in Aedes aegypti nerve and salivary gland tissues. However, because these viruses inefficiently infect Ae. aegypti when administered by the per os route, TE/3'2J viruses must be intrathoracically inoculated into the mosquitoes to infect these tissues. A Malaysian Sindbis (SIN) virus isolate (MRE16) does efficiently infect Ae. aegypti midgut tissues after ingestion, and approximately 95% of these mosquitoes also develop disseminated infections within 14 days. We have sequenced the entire 26S RNA of MRE16 virus and have developed a chimeric SIN cDNA infectious clone, designated MRE1001, which contains sequence elements of TE/3'2J and MRE16 virus. MRE1001 virus efficiently infects midgut cells, and greater than 90% of infected mosquitoes develop disseminated infections after 14 days extrinsic incubation. The chimeric MRE1001 cDNA clone should allow identification of viral determinants of midgut infection and dissemination and lead to the development of new SIN virus expression systems.

Aedes↗

PubMed1998

Effect of heavy metals on Aedes aegypti (Diptera: Culicidae) larvae.

Studies were conducted to determine the biological effects of heavy metals on the development of Aedes aegypti. Embryos immersed in 32 ppm Cu or 5 ppm Cd did not hatch. The arrest of hatching was in part reversible by removal of the heavy metals. The mortality rate of third-instar larvae exposed to heavy metals for 24 h was metal and dose dependent; the 50% lethal concentration (LC50) endpoints were 3.1, 16.5, and 33 ppm for Hg, Cd, and Cu, respectively. Interestingly, a proportion of Aedes aegypti third-instar larvae exposed to either Cu or Cd for 24 h failed to produce a dissectable peritrophic matrix. This failure to produce a dissectable peritrophic matrix also was metal and dose dependent. These results are discussed in the context of Aedes aegypti as a model system for investigating the molecular biological effects of heavy metals in aquatic insects.

Aedes↗

PubMed1998

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

The 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.

Aedes↗

PubMed1998

Molecular and genetic ecotoxicologic approaches to aquatic environmental bioreporting.

Molecular and population genetic ecotoxicologic approaches are being developed for the utilization of arthropods as bioreporters of heavy metal mixtures in the environment. The explosion of knowledge in molecular biology, molecular genetics, and biotechnology provides an unparalleled opportunity to use arthropods as bioreporter organisms. Interspecific differences in aquatic arthropod populations have been previously demonstrated in response to heavy metal insult in the Arkansas River (AR) California Gulch Superfund site (CGSS). Population genetic analyses were conducted on the mayfly Baetis tricaudatus. Genetic polymorphisms were detected in polymerase chain reaction amplified 16S mitochondrial rDNA (a selectively neutral gene) of B tricaudatus using single-strand conformation polymorphism analysis. Genetic differences may have resulted from impediments to gene flow in the population caused by mortality arising from exposure to heavy metal mixture pollution. In laboratory studies a candidate metal-responsive mucinlike gene, which is metal and dose specific, has been identified in Chironomus tentans and other potential AR-CGSS bioreporter species. Population genetic analyses using the mucinlike gene may provide insight into the role of this selectable gene in determining the breeding structure of B. tricaudatus in the AR-CGSS and may provide mechanistic insight into determinants of aquatic arthropod response to heavy metal insult. Metal-responsive (MR) genes and regulatory sequences are being isolated, characterized, and assayed for differential gene expression in response to heavy metal mixture pollution in the AR-CGSS. Identified promoter sequences can then be engineered into previously developed MR constructs to provide sensitive in vitro assays for environmental bioreporting of heavy metal mixtures. The results of the population genetic studies are being entered into an AR geographic information system that contains substantial biological, chemical, and geophysical information. Integrated spatial, structural, and temporal analyses of these parameters will provide invaluable information concerning environmental determinants that restrict or promote gene flow in bioreporter populations.

Aedes↗

PubMed1998

Engineered resistance in Aedes aegypti to a West African and a South American strain of yellow fever virus.

Double subgenomic Sindbis (dsSIN) viruses were engineered to transduce mosquito cells with antisense RNA derived either from the premembrane (prM) or polymerase (NS5) coding regions of the 17D vaccine strain of yellow fever virus (YFV). Aedes albopictus C6/36 cells were infected at high multiplicities of infection (MOI) with each dsSIN virus. Forty-eight hours later, the transduced cells were challenged with an MOI of 0.1 of the Asibi strain of YFV. At 72-hr postchallenge, the cells were assayed by immunofluorescence for the presence of YFV antigen. Cells transduced with prM or NS5 antisense RNAs derived from the YFV genome displayed no YFV-specific antigens. In contrast, cells infected with control dsSIN viruses that expressed no antisense RNA or dengue virus-derived antisense RNAs were permissive for the challenge virus. To analyze resistance in the mosquito, five log10 50% tissue culture infective doses (TCID50) of each dsSIN virus and three log10TCID50 of either a West African (BA-55) or South American (1899/81) strain of wild-type YFV were coinoculated into Ae. aegypti. Mosquitoes transduced with effector RNAs targeting the prM or NS5 gene regions did not transmit West African YFV and poorly transmitted the South American strain of YFV.

Aedes↗

PubMed1997

Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.

Mosquito 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 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).

Animals↗

PubMed1997

Rapid characterization of genetic diversity among twelve dengue-2 virus isolates by single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) analysis was used to characterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucleotide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the reverse transcriptase-polymerase chain reaction. The viral E, premembrane (prM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 bp, and 201 bp, respectively, were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenaturing polyacrylamide gels, and SSCP patterns were observed by silver staining. The SSCP analysis revealed polymorphisms among a number of dengue-2 isolates from the same topotype, and these were markedly different between isolates of different topotype (distinct genetic group). Comparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP patterns contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Positive predictive value and negative predictive value as measures of certainty for predicting identical and different sequences were 26% and 100%, respectively. The SSCP patterns of the 12 dengue-2 isolates suggested greater genetic variation in the prM gene region than in either the E or NS5 gene regions. The SSCP analyses should allow easy, sensitive, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, SSCP screening of dengue-2 virus for genetic variability may reveal the introduction of new viral genotypes in a given geographic area. These genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.

DNA, Complementary↗

PubMed1996

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.

Aedes↗

PubMed1996

Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes.

Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.

Aedes↗

PubMed1996

Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.

Aedes↗

PubMed1995

The expression of chloramphenicol acetyltransferase in mosquitoes and mosquito cells using a packaged Sindbis replicon system.

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 x 10(6) and 6.0 x 10(6) 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 x 10(4) IFU rep5/CAT/EB or 1 x 10(5) IFU rep5/CAT/26S virus. Virus titers remained at approximately 10(5) IFU/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 x 10(-4) units CAT/10 micrograms protein; rep5/CAT/26S, 2.03 x 10(-5) units CAT/10 micrograms 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.

Aedes↗

PubMed1995

Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector.

The scorpion, Androctonus australis Hector, produces an insect-specific toxin (AaHIT) encoded by the Scotox gene. To assess the toxicity of AaHIT for mosquitoes, we have taken a novel approach to express the Scotox gene in vivo. We have engineered a double subgenomic Sindbis (dsSIN) virus that contains the Scotox gene in the viral genome and intrathoracically inoculated the virus (TE/3'2J/Scotox) into mosquitoes (Aedes aegypti, Ae. triseriatus and Culex pipiens), houseflies (Musca domestica) and ticks (Dermacentor andersoni). Mosquitoes, which normally show no pathologic effects from Sindbis (SIN) virus infections, died 1-5 days after infection with TE/3'2J/Scotox virus. Neither flies nor ticks were killed. The mosquitocidal action of AaHIT in mosquitoes makes AaHIT a potential candidate for inclusion in molecular-based methods of mosquito control. The expression of an arthropod gene in vivo demonstrates the utility of dsSIN expression vectors for future use to examine and potentially disrupt endogenous gene functions in mosquitoes.

Aedes↗

PubMed1995

Replication and expression of a recombinant Sindbis virus in mosquitoes.

A recombinant Sindbis virus, TE/3'2J/ANTI-S, containing LaCrosse virus small segment cDNA in antisense orientation, was inoculated into Aedes triseriatus mosquitoes. Virus replication and LAC-ANTI-S RNA expression were analysed temporally and spatially. TE/3'2J/ANTI-S virus titre peaked at 5.0 log10 TCID50 in heads 6-9 days post infection (p.i.) and decreased to 3.4 log10 TCID50 by 37 days p.i. Salivary glands contained 4.4 log10 TCID50 of virus 6 days p.i.; titres were lower in other organs. LAC-ANTI-S RNA levels paralleled virus titre. SIN E1 antigen was detected in many mosquito organ systems, but in specific cells and tissues of some organs. TE/3'2J/ANTI-S virus exhibited different cellular tropisms in salivary glands of Aedes and Culex mosquitoes.

Aedes↗

PubMed1994

Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector.

A cDNA of the small RNA genome segment of La Crosse (LAC) virus was inserted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ANTI-S virus yielded at least 4 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.

Aedes↗

PubMed1994

The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant Sindbis virus.

Genomic RNA was transcribed in vitro from the double subgenomic recombinant Sindbis (SIN) virus expression vector, pTE/3'2J/CAT, and transfected into BHK-21 cells to generate recombinant virus stocks. TE/3'2J/CAT virus was used to infect C6/36 (Aedes albopictus) cells and adult female Aedes triseriatus. When C6/36 cells were infected with TE/3'2J/CAT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 x 10(5). Approximately 4.0 log10TCID50 of the TE/3'2J/CAT virus was intrathoracically inoculated into adult female mosquitoes. Titers greater than 6.0 log10TCID50/ml were detected within 4 days pi and declined to less than 4.0 log10TCID50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 x 10(-5) units of CAT/mosquito or 1.4 x 10(10) CAT polypeptides), peaked at day 6 (4 x 10(-3) units of CAT/mosquito or 7.2 x 10(11) CAT polypeptides), and remained at peak levels to day 20. Immunofluorescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neural, midgut, ovarian, and salivary gland tissues. Alphavirus-based expression vectors should be useful for expressing heterologous genes in mosquito cells as well as adult mosquitoes.

Aedes↗

PubMed1993

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.

Journal Article↗

PubMed1992

Expression of the chloramphenicol acetyltransferase gene in Aedes albopictus (C6/36) cells using a non-infectious Sindbis virus expression vector.

Genomic RNA was transcribed in vitro from the non-infectious Sindbis (SIN) virus expression vector (pTRCAT) and introduced into C6/36 (Aedes albopictus) cells by liposome-mediated transfections. The chloramphenicol acetyltransferase (CAT) polypeptide expressed within cells was detected by an indirect immunofluorescent assay directly into 24-well polystyrene tissue culture plates. Approximately 1 in 1000 C6/36 cells showed fluorescence when the transfection was optimized. CAT enzyme activity was also assayed; in C6/36 cells CAT expression was detected as early as 8 h after transfection, peaked at 24 h, and could still be detected at 7 days. At 24 h posttransfection each transfected C6/36 cell was calculated to express 1.3 x 10(7) CAT polypeptides.

Aedes↗

PubMed1990

Detection of dengue viral RNA in mosquito vectors by mixed phase and solution hybridization.

A mixed phase hybridization technique was developed to detect dengue virus type 2 (DEN-2) RNA in pools of infected Aedes albopictus mosquitoes using radiolabelled RNA probes. This technique used a guanidine thiocyanate extraction procedure to simplify analyte preparation. The probes contained sequences complementary to portions of the NS-1 or NS-5 genes of the DEN-2 viral genome. One infected mosquito in a pool of 25 could be detected in approximately 48 h. RNAs from DEN serotypes 1-4 were extracted from cultured mosquito (C6/36) cells. The NS-1 RNA probe was highly specific for DEN-2 RNA. The NS-5 RNA probe detected both DEN-2 and DEN-4 RNA. DEN-2 RNA was also detected by molecular hybridization in concentrated solutions of guanidine thiocyanate using the NS-1 probe. Solution hybridization was 10-fold more sensitive when detecting RNA from purified DEN-2 virus than the mixed phase assay and could detect one infected mosquito in a pool of 25 within 6-8 h. Solution hybridizations were performed in 2-3 h vs 16-20 h for mixed phase hybridizations, and solution hybridizations required 5-10 times less mosquito RNA than mixed phase hybridizations to attain comparable sensitivities. However, solution hybridizations did result in a broader probe specificity than mixed phase hybridizations.

Aedes↗