The natural maintenance cycles of many mosquito-borne viruses require establishment of persistent non-lethal infections in the invertebrate host. While the mechanisms by which this occurs are not well understood, antiviral responses directed by small RNAs are important in modulating the pathogenesis of viral infections in disease vector mosquitoes. In yet another example of an evolutionary arms race between host and pathogen, some plant and insect viruses have evolved to encode suppressors of RNA silencing (VSRs). Whether or not mosquito-borne viral pathogens encode VSRs has been the subject of debate. While at first there would seem to be little evolutionary benefit to mosquito-borne viruses encoding proteins or sequences that strongly interfere with RNA silencing, we present here a model explaining how the expression of VSRs by these viruses in the vector might be compatible with the establishment of persistence. We also discuss the challenges associated with interrogating these viruses for the presence of suppressor proteins or sequences, as well as the candidates that have been identified in the genomes of mosquito-borne pathogens thus far.
Nearly all RNA viruses produce double-stranded RNA (dsRNA) during their replication cycles--an important pathogen-associated molecular pattern recognized by the RNA interference (RNAi) pathway in invertebrates and plants. Nodamura virus (NoV) encodes a suppressor of RNA silencing termed B2, which binds to dsRNA and prevents the initiation of RNAi as well as the loading of silencing complexes. Using the published crystal structure of NoV-B2, we performed a series of molecular dynamics (MD) simulations to determine the relative electrostatic and van der Waals contributions of various residues in binding dsRNA, identifying four novel potential interactors: R56, E48, P68 and R69. Additionally, steered MD was used to simulate the binding affinity of NoV-B2 sequences bearing substitutions at positions F49, R56 or R59 to dsRNA, with F49S and R56L/R59L substitutions found to have a significant negative impact on the ability of NoV-B2 to bind dsRNA. NoV RNA1 variants were tested for self-directed replication in both vertebrate (RNAi?) and invertebrate (RNAi?) cultured cells. Consistent with a role in dsRNA binding, NoV replication in F49C and F49S variant constructs was affected negatively only in RNAi? cells. Thus, we used a combination of MD simulations and experimental mutagenesis to further characterize residues important for NoV-dsRNA interactions.
The Culicine mosquito, Aedes aegypti, is both a major vector of arthropod-borne viruses (arboviruses) and a genetic model organism for arbovirus transmission. TALE nucleases (TALENs), a group of artificial enzymes capable of generating site-specific DNA lesions, consist of a non-specific FokI endonuclease cleavage domain fused to an engineered DNA binding domain specific to a target site. While TALENs have become an important tool for targeted gene disruption in a variety of organisms, application to the mosquito genome is a new approach. We recently described the use of TALENs to perform heritable genetic disruptions in A. aegypti. Here, we provide detailed methods that will allow other research laboratories to capitalize on the potential of this technology for understanding mosquito gene function. We describe target site selection, transient embryo-based assays to rapidly assess TALEN activity, embryonic microinjection and downstream screening steps to identify target site mutations.
The preservation of a homomorphic sex-determining chromosome in some organisms without transformation into a heteromorphic sex chromosome is a long-standing enigma in evolutionary biology. A dominant sex-determining locus (or M-locus) in an undifferentiated homomorphic chromosome confers the male phenotype in the yellow fever mosquito Aedes aegypti. Genetic evidence suggests that the M-locus is in a nonrecombining region. However, the molecular nature of the M-locus has not been characterized. Using a recently developed approach based on Illumina sequencing of male and female genomic DNA, we identified a novel gene, myo-sex, that is present almost exclusively in the male genome but can sporadically be found in the female genome due to recombination. For simplicity, we define sequences that are primarily found in the male genome as male-biased. Fluorescence in situ hybridization (FISH) on A. aegypti chromosomes demonstrated that the myo-sex probe localized to region 1q21, the established location of the M-locus. Myo-sex is a duplicated myosin heavy chain gene that is highly expressed in the pupa and adult male. Myo-sex shares 83% nucleotide identity and 97% amino acid identity with its closest autosomal paralog, consistent with ancient duplication followed by strong purifying selection. Compared with males, myo-sex is expressed at very low levels in the females that acquired it, indicating that myo-sex may be sexually antagonistic. This study establishes a framework to discover male-biased sequences within a homomorphic sex-determining chromosome and offers new insights into the evolutionary forces that have impeded the expansion of the nonrecombining M-locus in A. aegypti.
The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.
Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.
In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20-40% of fertile survivors produced kmo alleles that failed to complement an existing kh(w) mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1-7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.
Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue haemorrhagic fever and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an antipathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti heat shock protein 70 (hsp70) genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ~25-50-fold in whole adults by 4 h after heat-shock, with significant activity (~20-fold) remaining at 24 h. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ~2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of antipathogen molecules.
A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD(50) bioassays, we determined that resistance ratios for Richmond strain bed bugs were ?5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.
Dengue viruses (DENV) are endemic in over 100 countries worldwide, and annually 50 to 100 million people are infected by one of the four DENV serotypes, whereas over 2.5 billion people are at risk for infection. West African countries lack the surveillance to determine the true incidence of dengue; hence, this disease is likely significantly underestimated. In Mali, ?14 million people are potentially at risk of acquiring a dengue infection.
Double subgenomic Sindbis virus (dsSINV) vectors are widely used for the expression of proteins, peptides, and RNA sequences. These recombinant RNA viruses permit high level expression of a heterologous sequence in a wide range of animals, tissues, and cells. However, the alphavirus genome structure and replication strategy is not readily amenable to the expression of more than one heterologous sequence. The Rhopalosiphum padi virus (RhPV) genome contains two internal ribosome entry site (IRES) elements that mediate cap-independent translation of the virus nonstructural and structural proteins. Most IRES elements that have been characterized function only in mammalian cells but previous work has shown that the IRES element present in the 5 untranslated region (UTR) of the RhPV genome functions efficiently in mammalian, insect, and plant systems. To determine if the 5 RhPV IRES element could be used to express more than one heterologous sequence from a dsSINV vector, RhPV 5 IRES sequences were placed between genes for two different fluorescent marker proteins in the dsSINV, TE/32J/mcs. While mammalian and insect cells infected with recombinant viruses containing the RhPV sequences expressed both fluorescent marker proteins, only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence. Thus, we report development of a versatile tool for the expression of multiple sequences in diverse cell types.
The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus onyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (-) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (-) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR(R) Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.
Heat shock genes are highly evolutionarily conserved and are expressed to varying degrees in all organisms in response to stress. Heat shock 70 (hsp70) genes have been well characterized in a number of organisms, most notably Drosophila melanogaster, but not as yet for any of the major arthropod-borne viral mosquito vectors. To identify hsp70 genes in the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae), basic local alignment searches of the Ae. aegypti genome were performed using D. melanogaster Hsp70 protein sequences as query. Two clusters of six previously unannotated AaHsp70 genes were identified and found to be organized into three pairs of nearly identical open reading frames, which mapped to two genomic scaffolds. Consistent with a designation as heat shock genes, no detectable level of expression of AaHsp70 genes was observed under normal rearing conditions (28 degrees C), with robust expression observed with a heat shock of 37-39 degrees C. Northern analysis showed heat-inducible expression of putative AaHsp70 genes at all life stages and in all tissues tested in a time- and temperature-dependent manner. Monitoring of AaHsp70 gene expression levels in field-caught Ae. aegypti may serve as a general marker for stress. In addition, promoter sequences from AaHsp70 genes may be used to control the expression of transgenes in an inducible manner.
The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.
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