Biomedical subjects

S Higgs

Publications and source records attributed to S Higgs.

At least 55 records · Page 3Linked to original sources

PubMed1996

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

Animals↗

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

Increased food intake following injection of the benzodiazepine receptor agonist midazolam into the IVth ventricle.

Despite a prolonged period of research with benzodiazepines, the central site(s) of action for the hyperphagic effects of these compounds remains to be determined. The aim of the present studies was to examine the effect of direct administration of the benzodiazepine receptor agonist midazolam into the IVth ventricle on ingestive behavior in nondeprived rats. In Experiment 1, microinjection of midazolam (3 and 30 micrograms/microliter) into the IVth ventricle was sufficient to increase consumption of a palatable mash. In Experiment 2, the hyperphagic effect was blocked by systemic administration of the selective benzodiazepine receptor antagonist flumazenil (20 mg/kg). The results indicate that a brainstem site of action may be important for the effects of benzodiazepine receptor agonists on ingestive behavior.

Animals↗

PubMed1996

Effects of the benzodiazepine receptor inverse agonist Ro 15-4513 on the ingestion of sucrose and sodium saccharin solutions: a microstructural analysis of licking behavior.

The effects of the benzodiazepine receptor partial inverse agonist Ro 15-4513 on consumption of either a 1% sucrose solution or a 0.1% sodium saccharin solution in nondeprived male rats was examined. A video-recording approach was adopted in which licks were counted in a frame-by-frame analysis. Ro 15-4513 (1-10 mg/kg) caused a significant decrease in the intake of both sucrose and saccharin solutions that was associated with a reduction in the initial rate of licking. There was a decrease in the total duration of drinking, total licks, and number of bouts for both sucrose and saccharin. For sucrose, mean bout duration was significantly reduced, although this was not so for saccharin. Intrabout lick rate, the latency to engage in drinking, and the postdrinking time were not affected for either sucrose or saccharin. These data are consistent with previous evidence that strongly suggests that benzodiazepines influence palatability.

Animals↗

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↗

PubMed1996

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.

Animals↗

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↗

PubMed1995

Molecular genetic manipulation of mosquito vectors.

Despite their central role in disease transmission, relatively little is known of the molecular biology of arthropod vectors. Modern molecular approaches will undoubtedly provide considerable information about gene regulation and expression in vectors and consequently a much better understanding of the biology and molecular biology of vectors. Such knowledge is essential for developing effective control strategies for vector-borne diseases. In this review, we focus upon techniques and approaches used at the Arthropod-Borne and Infectious Diseases Laboratory (AIDL) at Colorado State University to bioengineer mosquitoes with reduced vector competence. We have developed technologies and procedures that allow genetic manipulation of mosquitoes, including RNA and DNA virus gene-delivery vehicles and efficacious antiviral constructs, which will facilitate the development of pathogen-resistant, transformed mosquitoes. Many of the approaches, constructs, and technologies developed at AIDL will be applicable to molecular manipulation of other arthropod genomes.

Animals↗

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↗

PubMed1993

Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence.

Seven mutant viruses were derived from a Scottish strain of louping ill virus using a virus envelope-specific neutralizing monoclonal antibody. None of the mutants was neutralized and immunofluorescence microscopy confirmed that they did not bind to this antibody. Four mutants showed reduced mouse neurovirulence compared with parent virus and two mutants failed to induce protective immune responses in mice challenged with virulent tick-borne encephalitis virus. The mutants with the lowest virulence showed poor or undetectable haemagglutinating activity. The nucleotide sequence of the envelope glycoprotein gene of each of the seven mutants was determined and the deduced amino acid sequence was compared with parent virus. For each mutant, only a single amino acid codon change was detected and all the amino acid substitutions occurred within amino acid positions 308 to 311. A change from the amino acid aspartate to asparagine at amino acid position 308, which represented a potential glycosylation site, was the most effective substitution in reducing mouse neurovirulence. The results demonstrate the importance of critical sites within the envelope glycoprotein as determinants of virus virulence.

Animals↗

PubMed1992

Saliva activated transmission (SAT) of Thogoto virus: relationship with vector potential of different haematophagous arthropods.

Tick saliva (or salivary gland extract) potentiates the transmission of Thogoto (THO) virus to uninfected ticks feeding on a non-viraemic guinea-pig. This phenomenon has been named saliva activated transmission (SAT). To investigate the potential of different haematophagous arthropods to mediate SAT, guinea-pigs were infested with uninfected R.appendiculatus Neumann nymphs and inoculated with THO virus and salivary gland extract (SGE) derived from a range of ixodid (metastriate and prostriate) or argasid ticks, or mosquitoes; control guinea-pigs were inoculated with virus alone. Enhancement of THO virus transmission was observed only when SGE was derived from metastriate ticks. Comparison with the vector potential of these various arthropod species revealed that enhancement of THO virus transmission was specific for ticks which were competent vectors of the virus. The data indicate a correlation between vector competence and the ability of haematophagous arthropods to mediate SAT of THO virus.

Aedes↗

PubMed1991

Differences in fusogenicity and mouse neurovirulence of Japanese encephalitis viruses.

The fusogenic capacity in AP-61 cell monolayers of 10 strains of Japanese encephalitis (JE) virus from different geographic locations was compared. One strain, isolated from Beijing (JE-Bei), did not fuse AP-61 cells after replication (fusion from within; FFWI), whereas all other strains fused these cells by 72 h post-infection. JE-Bei also readily established a non-cytolytic persistent infection in AP-61 cells. Differences in the envelope proteins of fusogenic and non-fusogenic virus were detected by haemagglutination-inhibition tests and by antigenic analysis using monoclonal antibodies. Yields of infectious virus in either AP-61 or Vero cell cultures were similar if JE-Bei was compared with the fusogenic strain (JE-Sar) but yields of haemagglutinin were 50-100 fold higher with the non-fusogenic virus, implying excessive generation of non-infectious particles. When added directly to AP-61 cell monolayers at pH6, only JE-Bei produced significant fusion from without (FFWO) presumably reflecting the larger quantity of antigen. Cell monolayers persistently infected with JE-Bei or monolayers treated with UV-inactivated JE-Bei, were resistant to superinfection with JE, West Nile and dengue 2 viruses but were susceptible to infection with the alphavirus Sindbis. When administered intracerebrally (I/C) to newborn and weanling mice, the viruses were equally neurovirulent. However, fusogenic JE-Sar was significantly more neurovirulent than JE-Bei for weanling mice after intraperitoneal (I/P) or subcutaneous (S/C) inoculation. Mice given non-fusogenic JE-Bei, resisted the peritoneal challenge with fusogenic JE-Sar, and West Nile but not Semliki Forest virus when given 6 h after the first virus. The potential significance of cell fusion by JE virus and interference through over production of non-infectious virus, is discussed in the context of JE virus virulence.

Aedes↗

PubMed1991

17D yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein.

We have constructed a recombinant baculovirus containing cloned DNA encoding the membrane and envelope (E) proteins of 17D yellow fever vaccine virus. Spodoptera frugiperda cells infected with this recombinant baculovirus produced a 66K protein which corresponded to the estimated size of the protein encoded by the cloned inserted DNA, and a 54K protein with the same molecular size as that of the authentic 17D yellow fever virus E protein. This recombinant 54K protein was labile, producing E protein-specific breakdown products (45K to 36K). Indirect immunofluorescence, using a panel of E protein-specific monoclonal antibodies, showed that the recombinant protein was presented both inside as well as on the surface of cells and was antigenically indistinguishable from the E protein of 17D yellow fever vaccine virus.

Animals↗