Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti.
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Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the vitellogenin (Vg) gene of Aedes aegypti for its ability to express potential antipathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg-promoter fragment driving the expression of the Defensin A (DefA) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the Vg-DefA transgene insertion was stable. The Vg-DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20-22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission. (+info)
Pathogen-specific resistance to Rift Valley fever virus infection is induced in mosquito cells by expression of the recombinant nucleoprotein but not NSs non-structural protein sequences.
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Rift Valley fever virus (RVFV) is an arbovirus of the BUNYAVIRIDAE: family, causing recurrent disease outbreaks in Africa. Natural vertebrate hosts include cattle and humans. Several mosquito species belonging to the AEDES: and CULEX: genera act as vectors of this phlebovirus. To test whether pathogen-derived resistance against RVFV could be induced by expressing genomic sequences in mosquito cells, as has been shown for La Crosse and dengue 2 viruses, we generated various recombinant Semliki Forest viruses expressing the S segment (or its genes) in the genomic or antigenomic sense. Expression of the N but not the NSs gene interfered with the production of RVFV in mosquito cells and this phenomenon was RNA- but not protein-dependent. These results raise questions on the molecular mechanisms involved in virus resistance. (+info)
Molecular and evolutionary analysis of two divergent subfamilies of a novel miniature inverted repeat transposable element in the yellow fever mosquito, Aedes aegypti.
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A novel family of miniature inverted repeat transposable elements (MITEs) named Pony was discovered in the yellow fever mosquito, Aedes aegypti. It has all the characteristics of MITEs, including terminal inverted repeats, no coding potential, A+T richness, small size, and the potential to form stable secondary structures. Past mobility of PONY: was indicated by the identification of two Pony insertions which resulted in the duplication of the TA dinucleotide targets. Two highly divergent subfamilies, A and B, were identified in A. aegypti based on sequence comparison and phylogenetic analysis of 38 elements. These subfamilies showed less than 62% sequence similarity. However, within each subfamily, most elements were highly conserved, and multiple subgroups could be identified, indicating recent amplifications from different source genes. Different scenarios are presented to explain the evolutionary history of these subfamilies. Both subfamilies share conserved terminal inverted repeats similar to those of the Tc2 DNA transposons in Caenorhabditis elegans, indicating that Pony may have been borrowing the transposition machinery from a Tc2-like transposon in mosquitoes. In addition to the terminal inverted repeats, full-length and partial subterminal repeats of a sequence motif TTGATTCAWATTCCGRACA represent the majority of the conservation between the two subfamilies, indicating that they may be important structural and/or functional components of the Pony elements. In contrast to known autonomous DNA transposons, both subfamilies of PONY: are highly reiterated in the A. aegypti genome (8,400 and 9, 900 copies, respectively). Together, they constitute approximately 1. 1% of the entire genome. Pony elements were frequently found near other transposable elements or in the noncoding regions of genes. The relative abundance of MITEs varies in eukaryotic genomes, which may have in part contributed to the different organizations of the genomes and reflect different types of interactions between the hosts and these widespread transposable elements. (+info)
Comparison of rapid centrifugation assay with conventional tissue culture method for isolation of dengue 2 virus in C6/36-HT cells.
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A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results suggest that the rapid centrifugation assay may be useful for detection of dengue virus in clinical specimens. (+info)
Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay.
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We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases. (+info)
Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei.
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We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito. These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division. The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins. Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced. Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21. (+info)
Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes.
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Genetic modification of the vectorial capacity of mosquito vectors of human disease requires promoters capable of driving gene expression with appropriate tissue and stage specificity. We report on the characterization in transgenic Aedes aegypti of two mosquito gut-specific promoters. A 1.4-kb DNA fragment adjacent to the 5' end of the coding region of the Ae. aegypti carboxypeptidase (AeCP) gene and a corresponding 3.4-kb DNA fragment at the 5' end of the Anopheles gambiae carboxypeptidase (AgCP) gene were linked to a firefly luciferase reporter gene and introduced into the Ae. aegypti germ line by using Hermes and mariner (Mos1) transposons. Six independent transgenic lines were obtained with the AeCP construct and one with the AgCP construct. Luciferase mRNA and protein were abundantly expressed in the guts of transgenic mosquitoes in four of the six AeCP lines and in the AgCP line. Expression of the reporter gene was gut-specific and reached peak levels at about 24 h post-blood ingestion. The AeCP and AgCP promoters can be used to drive the expression of genes that hinder parasite development in the mosquito gut. (+info)
Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti.
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Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F(1) intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F(2) offspring were analyzed for midgut infection and escape barriers. In P(1) and F(1) parents and in 207 F(2) individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I-III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)(n) microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F(2) females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for approximately 30% of the phenotypic variance (sigma(2)(p)) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (sigma(2)(g)). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III. (+info)