Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system.
(9/2153)
Genetic recombination resulting in the production of wild-type infectious virus is an obstacle in the current system for producing densovirus transducing particles. In order to eliminate this problem, a double subgenomic Sindbis virus (TE/3'2J/VP) was engineered that expresses the structural proteins (VPs) of Aedes densonucleosis virus (AeDNV) from the second subgenomic promoter. Expression of AeDNV VPs from TE/3'2J/VP was confirmed by Northern analysis of RNA from infected C6/36 (Aedes albopictus) cells and by indirect immunofluorescence in infected C6/36 cells and BHK-21 cells. TE/3'2J/VP was used to infect C6/36 cells transfected with p7NS1-GFP, a plasmid expressing the nonstructural genes of AeDNV and green fluorescent protein (GFP) as a reporter gene. This infection resulted in the production of AeDNV-GFP transducing virus, which is infectious to C6/36 cells and Aedes aegypti larvae, as determined by GFP expression. The TE/3'2J/VP packaging system produced titers of transducing virus comparable to those produced by the standard two-plasmid method. The possibility of recombination resulting in wild-type infectious virus in transducing densovirus stocks was eliminated by employing an RNA virus expression system to supply AeDNV structural proteins. (+info)
Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus.
(10/2153)
Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses. (+info)
Analysis of the steps involved in Dengue virus entry into host cells.
(11/2153)
The initial steps of dengue viral entry have been divided into adsorption and penetration using acid glycine treatment to inactivate extracellular virus after attachment to baby hamster kidney (BHK) cells but prior to penetration. First, we showed that virus infection was accomplished within 2 h after adsorption. Second, the assay was used to examine the properties of dengue envelope E protein-specific monoclonal antibodies (MAbs), lectins, and heparin. We found that three MAbs, 17-2, 46-9, and 51-3, may neutralize dengue 2 virus (DEN-2) through inhibition of not only viral attachment but also of penetration. However, one MAb, 56-3.1, interfered specifically with attachment. Therefore, the functional domains of E protein involved in attachment and penetration may be different. Moreover, studies with lectins indicated that carbohydrates, especially alpha-mannose residues, present on the virion glycoproteins may contribute to binding and penetration of the virus into BHK and mosquito C6/36 cells. Finally, virus infectivity was inhibited by heparin through its blocking effects at both virus attachment and penetration. This suggests that cell surface heparan sulfate functions in both viral attachment and penetration of DEN-2 virus. In conclusion, our results further elucidated some aspects of the dengue virus entry process. (+info)
Vesicular ATPase-overexpressing cells determine the distribution of malaria parasite oocysts on the midguts of mosquitoes.
(12/2153)
In Plasmodium-infected mosquitoes, oocysts are preferentially located at the posterior half of the posterior midgut. Because mosquitoes rest vertically after feeding, the effect of gravity on the ingested blood has been proposed as the cause of such a biased distribution. In this paper, we examined the oocyst distribution on the midguts of mosquitoes that were continuously rotated to nullify the effect of gravity and found that the typical pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the posterior part of the posterior midgut. We examined whether the distribution of oocysts depends on the distribution of vesicular ATPase (V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised against recombinant Aedes aegypti V-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anopheles gambiae are localized at the posterior part of the posterior midgut. We propose that the typical distribution of oocysts on the mosquito midgut is attributable to the presence and the spatial distribution of the V-ATPase-overexpressing cells in the midgut epithelium. (+info)
Automated measurement of oxygen consumption by the yellow fever mosquito, Aedes aegypti.
(13/2153)
Oxygen consumption of single mosquitoes was measured using a differential pressure transducer (DPT) connected to two small chambers. A mosquito was placed in the experimental chamber (P1) and was separated from NaOH by 4 cm2 of marquisette mesh. The reference chamber (P2) contained the same amount of NaOH and the marquisette mesh but without a mosquito. When these two chambers were sealed, removed O2 from P1 resulted in a pressure decrease with respect to P2. This pressure differential was transduced into an output voltage that was directly proportional to the amount of O2 consumed by the mosquito. An array of eight DPTs was interfaced with an IBM 486 PC using an ADAC 5500MF analog to digital converter and software from ADAC (Direct View) to automate the recording procedure. We determined that our apparatus was sensitive enough to detect subtle differences in O2 consumption in mosquitoes subjected to different physiologic conditions. (+info)
Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.
(14/2153)
The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults. (+info)
Genomic and evolutionary analysis of Feilai, a diverse family of highly reiterated SINEs in the yellow fever mosquito, Aedes aegypti.
(15/2153)
Five short interspersed repetitive elements (SINEs) were found fortuitously in the introns of a steroid hormone receptor AaHR3-2 gene of the yellow fever mosquito, Aedes aegypti, constituting a novel family of tRNA-related SINEs named Feilai. In addition, nine other Feilai elements were found in currently available sequences in Ae. aegypti, six of which were also near genes. Approximately 5.9 x 10(4) copies of Feilai were present in Ae. aegypti, equivalent to 2% of the entire genome. An additional 35 Feilai elements were isolated from a genomic library. Of the total 49 Feilai elements, 20 were full-length. Sequence comparisons and phylogenetic analyses of the full-length elements strongly suggest that there are at least two subfamilies within the Feilai family. There is a high degree of conservation within the two subfamilies. However, sequence divergence between the subfamilies, along with the presence of highly degenerate Feilai elements, suggests that Feilai is likely a diverse family of SINEs that has existed in Ae. aegypti for a long time. Many Feilai elements were closely associated with other transposons, especially with fragments of non-LTR retrotransposons and miniature inverted-repeat transposable elements. The 500-bp sequences immediately flanking a Feilai element were highly A + T-rich, which is consistent with the fact that no Feilai has been found in the coding regions of genes. It is likely that the highly reiterated and interspersed Feilai elements are partially responsible for the pattern of short-period interspersion of the Ae. aegypti genome. The evolutionary relationship between Feilai and the Ae. aegypti genome is likely complex. (+info)
Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.
(16/2153)
Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297. (+info)