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S Higgs

Publications and source records attributed to S Higgs.

At least 37 records · Page 2Linked to original sources

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.

Aedes↗

Effects of scopolamine on a novel choice serial reaction time task.

Rats were trained on a novel attentional task adapted from the five-choice serial reaction time test first developed by Carli et al. [1983; Behav. Brain. Res., 9, 361-80]. The novel task required rats to detect the occurrence of brief light flashes in one of two spatial locations following trial initiation by a lever press. Blank trials were interleaved with stimulus trials and the rat had to make a different response to indicate the absence of a light. Occasional light-alone trials were also presented in which the visual stimulus appeared without forewarning. Pre-exposure to food for 60 min prior to test increased response omissions for all trials and slowed correct response latency, but failed to significantly alter accuracy. A decrease in light-stimulus duration (1-0.4 s) decreased accuracy, increased the reporting of blank trials and the number of light-alone trial omissions. Scopolamine (0.03-0.1 mg/kg) and scopolamine methylbromide (0.1 mg/kg) failed to affect accuracy, but increased light-alone trial omissions and lengthened correct response latency. The results confirm that the novel task is able to distinguish between motivational and attentional manipulations, and imply that scopolamine affected performance via nonattentional mechanisms.

Animals↗

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.

Animals↗

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Aedes↗

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 IFN gamma 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 IFN gamma 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 IFN gamma 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.

Aedes↗

Selection of refractory and permissive strains of Aedes triseriatus (Diptera: Culicidae) for transovarial transmission of La Crosse virus.

The genetic basis of transovarial transmission of La Crosse virus in Aedes triseriatus (Say) was investigated through selection experiments on 2 mosquito strains. One strain was subject to selection for transovarial transmission refractoriness, the other for permissiveness to transovarial transmission. Response to selection for a low filial infection rate was rapid, decreasing from 18 to 3% in 3 generations. However, no response to selection for permissiveness was observed in the other strain; the average filial infection rates through 4 generations fluctuated between 25 and 40%. By contrast, the transovarial transmission rate in both strains showed a consistent response to selection in both directions. These patterns are consistent with a model in which transovarial transmission is controlled by a single genetic locus and permissiveness is conditioned by dominant alleles; whereas the filial infection rate is nongenetic and influenced by stochastic factors in the mosquito and virus.

Aedes↗

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↗

Evidence for early opioid modulation of licking responses to sucrose and intralipid: a microstructural analysis in the rat.

The behavioural mechanisms underlying the effects of the opioid antagonist naloxone (0.3-3 mg/kg i.p.), and the opioid agonists morphine (0.3-3 mg/kg s.c.), and U-50, 488H (0.3-3 mg/kg s.c.) on ingestive behaviour were investigated using a microstructural analysis of licking patterns for sucrose solutions and Intralipid (fat emulsions) in a brief contact test. Naloxone dose-dependently decreased the total number of licks and the number of bouts for sucrose and Intralipid, but did not affect mean bout duration. Morphine dose-dependently increased the total number of licks and the number of bouts for both test fluids. For Intralipid but not for sucrose drinking, morphine actually decreased mean bout duration. U-50, 488H significantly affected total licks, although the dose-effect relationship showed an inverted U-shaped function. There was a dose-dependent increase in mean bout duration following administration of U-50, 488H and an increase in bout number, although only the lowest dose differed significantly from the control condition. The results show that microstructural analysis can distinguish between the effects of naloxone, morphine and U-50, 488H on licking behaviour and indicate that selective opioid receptor subtypes may be differentially involved in ingestive processes.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

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↗

Antineophobic effect of the neuroactive steroid 3alpha-hydroxy-5beta-pregnan-20-one in male rats.

The neuroactive steroid 3alpha-hydroxy-5beta-pregnan-20-one (pregnanolone) and benzodiazepine receptor (BZR) agonists share sedative, anxiolytic, and anticonvulsant properties. Recent evidence suggests that like BZR agonists, pregnanolone may also modulate feeding responses. The present experiments examined the behavioral mechanisms responsible for any hyperphagic effect of pregnanolone. The effect of pregnanolone (1-10 mg/kg i.p.) on the intake and microstructure of licking for two sucrose solutions (1 and 3%) in well familiarized nondeprived male rats under either light or dark conditions was examined. Pregnanolone had no effect on either intake or the duration or number of bouts of licking in these experiments, although in all cases the intrabout lick rate was significantly reduced at the highest dose. Pregnanolone (1-10 mg/kg) also failed to increase intake of a sweet wet mash in familiarized nondeprived male rats. However, in a food choice test where both novel and familiar food items were available, pregnanolone (1-3 mg/kg) significantly increased the time spent eating the novel food. These results suggest that unlike BZR agonists, which enhance feeding responses directly, pregnanolone may facilitate feeding secondarily via an attenuation of anxiety.

Animals↗

Effects of benzodiazepine receptor ligands on the ingestion of sucrose, intralipid, and maltodextrin: an investigation using a microstructural analysis of licking behavior in a brief contact test.

Microstructural analysis of licking behavior in the rat was conducted (a) to describe in detail the characteristics of benzodiazepine-induced changes in ingestion and (b) to determine if the changes are consistent with an alteration in palatability. The effects of the benzodiazepine receptor (BZR) agonist midazolam (0.3-3 mg/kg), and the partial inverse agonist Ro 15-4513 (0.3-3 mg/kg), on licking for several concentrations of sucrose, Intralipid, and maltodextrin in a brief contact test were investigated. Midazolam increased the total number of licks for all 3 fluids; conversely, Ro 15-4513 decreased the total number of licks. Midazolam increased mean bout duration for sucrose and maltodextrin drinking and there was a trend toward a similar effect with Intralipid drinking. Ro 15-4513 reduced mean bout duration for all 3 test fluids. These data are discussed in terms of bidirectional changes in fluid palatability by drug actions at BZRs.

Analysis of Variance↗

Mosquito feeding-induced enhancement of Cache Valley Virus (Bunyaviridae) infection in mice.

Cache-Valley (CV) virus, an arthropod-borne bunyavirus, recently has emerged as a significant veterinary pathogen causing infertility and congenital malformations in North American ruminants. To investigate the role of vector feeding on CV infection, adult mice were injected subcutaneously with CV, CV and vector thorax extract (a source of vector saliva), or CV into the site of intense, noninfected-mosquito feeding. Mice did not become infected after injection of CV or CV and vector saliva, nor did they produce antibodies to CV. However, injection of CV into sites of mosquito feeding resulted in viremia and production of anti-CV antibody by 2 wk after infection. This enhancement of CV infection resulted after feeding by Aedes triseriatus (Say), Ae. aegypti (L.), or Culex pipiens (L.). Enhancement occurred when injection was delayed up to 4 h after mosquito feeding, but it was not observed when virus injection was performed at a site distant from mosquito feeding. These results indicate that arbovirus infection may be enhanced by mosquito-vertebrate host interactions and that replication of arboviruses in arthropod vectors may not be responsible for increased virulence of infections mediated by infected arthropods. Enhanced CV infection in pregnant mice did not result in infertility or malformed pups, indicating that the mouse is not a suitable model to study CV-induced malformations.

Aedes↗

Pantropic retroviral vectors mediate somatic cell transformation and expression of foreign genes in dipteran insects.

The control of insects that transmit disease and damage crops has become increasingly difficult. The ability to genetically engineer insects would facilitate strategies to protect crops and block arthropod vector-borne disease transmission. Transformation vectors based on insect transposable elements have been developed, but most have limited host ranges. A promising alternative is the pantropic retroviral vector, which is packaged with the envelope glycoprotein from vesicular stomatitis virus and is replication-defective. We show here that pantropic murine retroviral vectors can mediate high-level expression of foreign genes in somatically transformed insect larvae and adults of three dipteran genera. This success demonstrates the potential for germline transformation mediated by pantropic retroviral vectors.

Aedes↗

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↗

Midazolam-induced rapid changes in licking behaviour: evidence for involvement of endogenous opioid peptides.

The role of endogenous opioid peptides in the effects of midazolam on ingestive behaviour was investigated using a detailed analysis of licking behaviour in the rat. Midazolam (1.8 mg/kg i.p.) was administered in combination with either flumazenil (10 and 20 mg/kg i.p.) or naloxone (0.1 and 0.3 mg/kg i.p.). The effect on licking patterns during 60-s exposures to a range of concentrations of a fat emulsion (Intralipid) was then recorded. Midazolam significantly increased the total number of licks for Intralipid by increasing the mean bout duration. This effect is consistent with the proposal that benzodiazepines enhance palatability. Flumazenil and naloxone were ineffective when administered alone, but both drugs blocked the effect of midazolam on total number of licks by selectively attenuating mean bout duration. Midazolam also produced a significant decrease in the intrabout lick rate, probably due to the muscle relaxant effects of this drug. This decrease in the intrabout lick rate was reversed by pretreatment with flumazenil but not by naloxone. The results suggest that endogenous opioids may be important for the palatability effects of midazolam, but may not be involved in the muscle relaxant effects of this drug.

Animals↗

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↗

Hyperphagia induced by direct administration of midazolam into the parabrachial nucleus of the rat.

Benzodiazepine receptor agonists increase food intake in many different species, yet there has been little investigation of the central site of actions of these drugs on ingestive behaviour. In the present experiments, direct administration of the benzodiazepine receptor agonist midazolam (3-30 micrograms/microliter) into the parabrachial nucleus of the pons significantly increased the consumption of a wet mash diet and a 3% sucrose solution in adult non-deprived rats. The hyperphagic response was blocked by pre-treatment with the selective benzodiazepine receptor antagonist flumazenil. Injection of midazolam into the parabrachial nucleus had no effect on locomotor activity, despite the fact that in the same animals an increase in mash intake was observed following intra-parabrachial midazolam. These data suggest that benzodiazepine receptors located in the parabrachial nucleus may be an important site of action for the effects of benzodiazepines specifically on ingestive behaviour.

Analysis of Variance↗