80 research outputs found

    The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti

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    <p>Abstract</p> <p>Background</p> <p>The RNA interference (RNAi) pathway acts as an innate antiviral immune response in <it>Aedes aegypti</it>, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: <it>Togaviridae</it>, genus: <it>Alphavirus</it>) is an arbovirus that infects <it>Ae. aegypti </it>in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of <it>Ae. aegypti</it>. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.</p> <p>Results</p> <p>As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the <it>Aa</it>-<it>dcr2 </it>gene. In midgut tissue of the transgenic Carb/dcr16 line, <it>Aa</it>-<it>dcr2 </it>expression was reduced ~50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, <it>Aa</it>-<it>dcr2 </it>expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes.</p> <p>Conclusions</p> <p>We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by <it>Ae. aegypti</it>, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of <it>Ae. aegypti </it>modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.</p

    A positively selected mutation in the WNV 2K peptide confers resistance to superinfection exclusion in vivo

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    AbstractMolecular epidemiologic studies of North American (NA) West Nile virus (WNV; Flaviviridae, Flavivirus) have documented the displacement of the introduced NY99 genotype with WN02. In addition, these studies have shown that particular substitutions are under positive selection. One occurs in the C-terminus of the NS4A coding sequence and results in a valine to methionine substitution at position nine of the 2K peptide. 2K-V9M confers the ability to overcome superinfection exclusion in vitro. We hypothesized that WNV strains bearing 2K-V9M have higher fitness than wildtype in Culex quinquefasciatus mosquitoes. Although infection rates and viral titers were not significantly different, virus dissemination rates were significantly higher with WNV 2K-V9M. As a super-infecting virus, WNV 2K-V9M was more successful than wildtype, however, in a mixed infection, 2K-V9M was not. These data support observations that 2K-V9M confers a context-specific selective advantage in mosquitoes and provides an in vivo mechanism for its positive selection

    An insight into the sialotranscriptome of the West Nile mosquito vector, <it>Culex tarsalis</it>

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    <p>Abstract</p> <p>Background</p> <p>Saliva of adult female mosquitoes help sugar and blood feeding by providing enzymes and polypeptides that help sugar digestion, control microbial growth and counteract their vertebrate host hemostasis and inflammation. Mosquito saliva also potentiates the transmission of vector borne pathogens, including arboviruses. <it>Culex tarsalis </it>is a bird feeding mosquito vector of West Nile Virus closely related to <it>C. quinquefasciatus</it>, a mosquito relatively recently adapted to feed on humans, and the only mosquito of the genus <it>Culex </it>to have its sialotranscriptome so far described.</p> <p>Results</p> <p>A total of 1,753 clones randomly selected from an adult female <it>C. tarsalis </it>salivary glands (SG) cDNA library were sequenced and used to assemble a database that yielded 809 clusters of related sequences, 675 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 283 protein sequences, 80 of which code for putative secreted proteins.</p> <p>Conclusion</p> <p>Comparison of the <it>C. tarsalis </it>sialotranscriptome with that of <it>C. quinquefasciatus </it>reveals accelerated evolution of salivary proteins as compared to housekeeping proteins. The average amino acid identity among salivary proteins is 70.1%, while that for housekeeping proteins is 91.2% (P < 0.05), and the codon volatility of secreted proteins is significantly higher than those of housekeeping proteins. Several protein families previously found exclusive of mosquitoes, including only in the <it>Aedes </it>genus have been identified in <it>C. tarsalis</it>. Interestingly, a protein family so far unique to <it>C. quinquefasciatus</it>, with 30 genes, is also found in <it>C. tarsalis</it>, indicating it was not a specific <it>C. quinquefasciatus </it>acquisition in its evolution to optimize mammal blood feeding.</p

    Role of genetic and environmental factors in the increased blood pressures of Bolivian blacks

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    The tendency toward hypertension or higher blood pressure is more common in blacks than whites. The factors that account for these differences are attributed to both environmental and genetic factors. To clarify this issue, an anthropological study of black and nonblack populations in the lowland village of Chicaloma, northeastern Bolivia at a midaltitude of 1,800 m was conducted. The study included 159 subjects, of which 79 were black and 80 were nonblack, 17–78 years. The study suggests the following: (1) the socioeconomic status of blacks as measured by an ownership index is greater than that of nonblacks, (2) blacks had higher average systolic and diastolic blood pressures than nonblacks and showed an age-associated increase in blood pressures, (3) the prevalence of hypertension was higher for blacks (7–6%) than nonblacks (1.3%), but three times lower than among blacks in the United States, (4) skin reflectance is inversely related to blood pressures so that contrary to what has been suggested the darker the skin color, the higher the blood pressures even at comparable levels of affluence. These findings together suggest that genetic factors predispose black individuals to increased blood pressures, but the expression of clinical hypertension is influenced by adverse unaccounted environmental factors. Am. J. Hum. Biol. 11:489–498, 1999. © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35096/1/8_ftp.pd

    Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

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    A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti

    Development of a Panel of Genome-Wide Ancestry Informative Markers to Study Admixture Throughout the Americas

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    Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions (R2>0.9 for ancestral components with significant between-subject variance). Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region

    The Genetic Basis for Salivary Gland Barriers to Arboviral Transmission

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    Arthropod-borne viruses (arboviruses) infect mosquito salivary glands and then escape to saliva prior to virus transmission. Arbovirus transmission from mosquitoes can be modulated by salivary gland infection barriers (SGIBs) and salivary gland escape barriers (SGEBs). We determined the influence of SGIBs and SGEBs by estimating the quantitative genetic contributions of Aedes aegypti half-sib families (Mapastepec, Mexico) infected with three dengue 2 (DENV2), two chikungunya (CHIKV), and two Zika (ZIKV) genotypes. We determined virus titer per salivary gland and saliva at seven days post-infection and virus prevalence in the half-sib population. CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB. However, virus titer and prevalence due to additive genetic factors in the half-sib family displayed a significant narrow-sense heritability (h2) for SGIB in two of the three DENV2 genotypes and one CHIKV and one ZIKV genotype. SGEBs were detected in all seven virus strains: 60–88% of DENV2 and 48–62% of CHIKV or ZIKV genotype infections. SGEB h2 was significant for all CHIKV or ZIKV genotypes but not for any of the DENV2 genotypes. SGIBs and SGEBs exhibited classical gene-by-gene interaction dynamics and are influenced by genetic factors in the mosquito and the virus

    Analysis of Salivary Glands and Saliva from <i>Aedes albopictus</i> and <i>Aedes aegypti</i> Infected with Chikungunya Viruses

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    Chikungunya virus (CHIKV) is a medically important mosquito-borne virus transmitted to humans by infected Aedes (Stegomyia) species. In 2013&#8315;2014, Ae. aegypti transmitted CHIKV to humans in the Caribbean and in 2005&#8315;2006, Ae. albopictus transmitted CHIKV on La R&#233;union Island (Indian Ocean basin). CHIKV LR2006 OPY1 from the La R&#233;union epidemic was associated with a mutation (E1:A226V) in the viral E1 glycoprotein that enhanced CHIKV transmission by Ae. albopictus. CHIKV R99659 from the Caribbean outbreak did not have the E1:A226V mutation. Here, we analyzed the salivary glands and saliva of Ae. albopictus strains from New Jersey, Florida, Louisiana and La R&#233;union after infection with each virus to determine their transmission potential. We infected the Ae. albopictus strains with blood meals containing 3&#8315;7 &#215; 107 PFU/mL of each virus and analyzed the mosquitoes nine days later to maximize infection of their salivary glands. All four Ae. albopictus strains were highly susceptible to LR2006 OPY1 and R99659 viruses and their CHIKV disseminated infection rates (DIR) were statistically similar (p = 0.3916). The transmission efficiency rate (TER) was significantly lower for R99659 virus compared to LR2006 OPY1 virus in all Ae. albopictus strains and Ae. aegypti (Poza Rica) (p = 0.012) suggesting a salivary gland exit barrier to R99659 virus not seen with LR2006 OPY1 infections. If introduced, LR2006 OPY1 virus poses an increased risk of transmission by both Aedes species in the western hemisphere
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