Browsing by Author "Myles, Kevin M."

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    Accurate Strand-Specific Quantification of Viral RNA
    Plaskon, Nicole E.; Adelman, Zach N.; Myles, Kevin M. (PLOS, 2009-10-22)
    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 o'nyong-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® 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.
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    Aedes aegypti Heat Shock 70 Genes and their Inducible Promoters
    Gross, Tiffany Lauren (Virginia Tech, 2011-07-11)
    Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease. Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti. This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries. Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems. Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains. The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed. In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules.
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    Bed Bugs and Infectious Disease: A Case for the Arboviruses
    Adelman, Zach N.; Miller, Dini M.; Myles, Kevin M. (PLOS, 2013-08-01)
    Bed bug infestations (Cimicidae; Cimex lectularius) have been increasing worldwide over the last few decades [1,2]. Several factors have been posited to explain this resurgence, including widespread insecticide resistance, human population growth, and increased international travel [1]. Clinically, reactions to bed bug bites vary from unapparent, to small (,5 mm) maculopapular lesions, to large wheals (2–6 cm); other reactions include bullous rashes, dermatitis, and asthma [1,3]. However, in the developed world the psychological, social, and economic impacts of bed bugs may be the most troubling aspects of the resurgence [2]. While the bed bug invasion cuts across economic lines, those with sufficient resources are able to clear the infestations, while those without may have to live with their bed bugs into the foreseeable future [2,4].
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    Comparative genomics of chromosomal rearrangements in malaria mosquitoes
    Xia, Ai (Virginia Tech, 2010-01-25)
    To better understand the evolutionary dynamics of chromosomal inversions, a physical map for an Asian malaria vector, Anopheles stephensi, was created and compared with the maps of the major African malaria vectors A. gambiae and A. funestus No interchromosomal transposition was observed between A. gambiae and A. stephensi. Several cases of euchromatin and heterochromatin transitions weridentified between A. gambiae and A. stephensi. The study of paracentric inversions between lineages in Anopheles mosquitoes demonstrated that X chromosome has the fastest rate of inversion fixations and highest density of repetitive elements. Among the autosomes, 2R evolved faster than other autosomes. The slowly evolved autosomes have more M/SARs than rapidly evolving arms. Breakpoint regions are enriched with repetitive elements. The study revealed that fixed inversions are distributed nonrandomly and breakpoint clustering is common in lineages of A. gambiae and A. stephensi. The parallel association between the density of inversion fixations and polymorphisms suggests that polymorphic inversions can be fixed during evolution. To understand the direction of evolution in A. gambiae complex, the ancestral status of fixed inversions for this complex was identified. The presence of the 2La inversion in outgroups, A. stephensi and A. nili, confirmed the ancestral status of the 2La inversion. The presences of breakpoint structure of the 2Ro inversion in outgroup species, A. stephensi, indicated that the 2Ro is ancestral arrangement. The presence of SINE elements at the breakpoints of the 2R+p in A. gambiae PEST strain suggested that the 2R+p is a derived arrangement. Therefore, the carrier of 2Rop inversions, A. merus, was considered closest to the ancestral species. We have developed a new protocol for laser microdissection and whole genome amplification of polytene chromosomal fragments to obtain DNA for sequencing and assembly. The chromosomal regions spanning both breakpoints of the 2La in A. arabiensis and A. merus were laser microdissected from the polytene chromosomes. Subsequently, DNA samples were amplified using Illustra GenomePhi V2 DNA and Whole-pool amplification methods for obtaining amplicons. Successful amplification of our target DNA was confirmed by PCR with specific primers followed by Sanger sequencing.
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    Complete Genome Sequence and Pathogenicity of Two Swine Parainfluenzaviruses Isolated from Pigs in the United States
    Qiao, Dan (Virginia Tech, 2009-05-21)
    Members of the family Paramyxoviridae are non-segmented, negative-strand RNA viruses. A large and diverse host species are infected by paramyxoviruses, including avian, porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. In the last few decades, many novel paramyxoviruses have emerged causing catastrophic illnesses in different aquatic and terrestrial species of animals and some of them also made the species jump to humans. Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated in the 1980s and 1990s from the brain of pigs that experienced respiratory and central nervous system disease from South and North Central United States. To understand their importance as swine pathogens, molecular characterization and pathogenicity studies were undertaken. The complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92 was 15480 nt in length consisting of six non-overlapping genes coding for the nucloeo- (N), phospho- (P), matrix (M, fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) proteins in the order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus replication and found to be conserved in most members of Paramyxoviridae were also found in swine viruses. These include: conserved and complementary 3â leader and 5â trailer regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal sequences. The length of each gene of these two viruses was similar except for the F gene, in which ISU92 had an additional 24 nt "U" rich 3â untranslated region (UTR). The P gene of these viruses were predicted to express the P protein from the primary transcript and edit a portion of its mRNA to encode V and D proteins and the C protein was expected to be expressed from alternate translation initiation from the P gene as in Respiroviruses. Sequence specific features related to virus replication and host specific amino acid signatures in P, F, HN and L proteins indicated that these viruses probably originated from bovine parainfluenzavirus 3. Pairwise comparisons of deduced amino acid sequences of swine viral proteins with members of Paramyxoviridae and phylogenetic analysis based on individual genes as well as predicted amino acid sequences suggested that these viruses were novel members of the genus Respirovirus of the Paramyxovirinae subfamily and genotype A of bovine parainfluenzavirus type 3. The mild clinical signs and undetectable gross and microscopic lesions observed in swine parainfluenzavirus (sPIV3)-infected pigs indicate the inapparent nature of these viruses in pigs. Limited seroprevalence studies in serum samples collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA revealed that sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can facilitate its development as a vaccine vector. The screening ELISA developed by us could be used to detect seroprevalence of sPIV3 in animal and human populations.
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    Cooler Temperatures Destabilize RNA Interference and Increase Susceptibility of Disease Vector Mosquitoes to Viral Infection
    Adelman, Zach N.; Anderson, Michelle A. E.; Wiley, Michael R.; Murreddu, Marta G.; Samuel, Glady Hazitha; Morazzani, Elaine M.; Myles, Kevin M. (PLOS, 2013-05)
    Background: 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. Methodology/Principal Findings: We utilized transgenic "sensor'' strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor'' strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18 degrees C as compared with 28 degrees C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18 degrees C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses. Conclusions/Significance: This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.
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    Cross-protection and Potential Animal Reservoir of the Hepatitis E Virus
    Sanford, Brenton Joel (Virginia Tech, 2012-05-29)
    HEV is an important public health concern due largely to water-borne outbreak. Recent research confirms individual cases of zoonotic transmission due to human exposure to contaminated animal meats. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. In addition to humans, strains of HEV have been genetically identified from pigs, chickens, rats, mongoose, deer, rabbits and fish. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In the first study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. Sera from all pigs were tested for HEV RNA and IgG anti-HEV, and fecal samples were also tested for HEV RNA each week. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia and fecal virus shedding of challenge viruses were not detected, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenge with homologous and heterologous genotypes 3 and 4 HEV, respectively. Immunogenic epitopes are located within the open reading frame 2 (ORF 2) capsid protein and recombinant ORF 2 antigens are capable of preventing HEV infection in non-human primates and chickens. In the second study we expressed and purified N-truncated ORF 2 antigens based on swine, rat, and avian HEV strains. Thirty pigs were randomly divided into groups of 6 pigs each and initially vaccinated with 200µg swine ORF 2 antigen, rat ORF 2 antigen, avian ORF 2 antigen, or PBS buffer (positive and negative control groups) and booster with the same vaccine 2 weeks later. At 4 wks, after confirming seroconversion to IgG anti-HEV antibody with ELISA, all groups except the negative control were challenged with swine genotype 3 HEV (administered intravenously). The protective and cross-protective abilities of these antigens were determined following swine genotype 3 challenge by evaluating both serum and fecal samples for HEV RNA using nested RT-PCR and IgG anti-HEV using ELISA. The results from these two studies have important implications for future development of an effective HEV vaccine. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that selected goat sera were capable of neutralizing HEV in cell culture. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in 7 out of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the fecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains.
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    Cytokine-bearing Influenza Vaccine: Adjuvant Potential of Membrane-bound Immunomodulators
    Herbert, Andrew S. (Virginia Tech, 2009-04-27)
    Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. Our group has developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Here we report methodologies for the construction of membrane-bound cytokine fusion constructs in which our cytokine of interest (mouse GM-CSF, mouse IL-2, mouse IL-4) was fused to the membrane anchoring regions of viral Hemagglutinin (HA). Progeny virions, produced from influenza infected MDCK cells expressing membrane-bound cytokines, readily incorporated membrane-bound cytokines during budding and these cytokines on the virus particles retained bioactivity following viral inactivation. In vivo vaccination studies in mice showed enhanced antibody titers and improved protection following lethal challenge in those mice vaccinated with IL-2 and IL-4-bearing CYT-IVAC's compared to the conventional wild-type vaccine without membrane-bound cytokines. In addition, the immune response induced by IL-2 and IL-4-bearing CYT-IVACs was skewed toward Th1 (cellular) mediated immunity compared to the Th2 (humoral) dominated response induced with wild-type vaccination. Cellular mediated immunity afforded by IL-2 and IL-4 CYT-IVACs was manifested as enhanced influenza specific T cell proliferation and activation. In conclusion, we have developed a novel methodology to introduce bioactive membrane-bound cytokines directly into virus particles in order to augment the immunogenicity of inactivated, whole virus influenza vaccines.
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    The Development of New Tools to Investigate Alphavirus Replication Kinetics
    Plaskon, Nicole Elyse (Virginia Tech, 2009-08-13)
    Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. The maintenance of this transmission cycle depends on the establishment of a life-long persistent infection in the invertebrate vector host. Although alphavirus replication has been extensively studied in vertebrate models, the strand-specific replication kinetics of alphaviruses during persistent infections of the invertebrate host have not been reported. We investigated the strand-specific replication of different alphavirus genotypes in invertebrate cells. By comparing different detection strategies and chemistries, we identified an optimal ssqPCR assay design for strand-specific quantification of viral RNAs in infected cells and tissues. We found that primer sets incorporating the use of a non-target tag sequence were able to avoid real-time PCR detection of amplicons that were falsely-primed during reverse-transcription. We also determined that DNA hydrolysis probes increased the sensitivity of ssqPCR assays when compared to a double-stranded DNA-specific dye, SYBR Green. Using this information, we determined the replication kinetics of two different genotypes of o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) in infected mosquito cells. We found that (-) strand viral RNAs persisted in invertebrate cells for up to 21 days after infection. We also found that significantly less (-) strand RNA was present in cells infected with opal variants of both ONNV and CHIKV than sense variants at several time points post infection, suggesting that the opal codon has a functional role in (-) strand RNA regulation. We also report the development of an ONNV replicon expression system. In total, the tools we developed for this report will facilitate future replication studies in the mosquito that may shed light on questions regarding the regulatory role of the opal codon and the persistence of (-) strand RNAs during long-term infections. The strand-specific replication kinetics of ONNV and CHIKV genotypes reported here will serve as a foundation for such investigations.
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    Discovery, Characterization, and Functional Analysis of micro RNAs in Culicidae
    Mead, Edward (Virginia Tech, 2009-05-13)
    MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs. Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations. Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages. Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases.
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    Double Subgenomic Alphaviruses Expressing Multiple Fluorescent Proteins Using a Rhopalosiphum padi Virus Internal Ribosome Entry Site Element
    Wiley, Michael R.; Roberts, Lisa O.; Adelman, Zach N.; Myles, Kevin M. (PLOS, 2010-11-10)
    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/3′2J/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.
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    Exogenously-introduced Homing Endonucleases Catalyze Double-stranded DNA Breaks in Aedes aegypti
    Traver, Brenna E. (Virginia Tech, 2009-01-13)
    Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti.
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    Foodborne Transmission and Molecular Mechanism of Cross-species Infection of Hepatitis E Virus (HEV)
    Feagins, Alicia R. (Virginia Tech, 2010-11-17)
    Hepatitis E virus (HEV), the causative agent of hepatitis E, is an emerging virus of global distribution. At least four distinct genotypes of HEV exist worldwide: genotype 1 and 2 HEV strains are responsible for waterborne epidemics; genotype 3 and 4 HEV strains are responsible for sporadic occurrences of acute hepatitis E. Genotype 3 and 4 HEVs are zoonotic and have a more expanded host range than genotypes 1 and 2 which are restricted to humans. Genotype 3 and 4 HEV isolates obtained from animal tissues are genetically very similar, or identical in some cases, to human HEV recovered from hepatitis E patients. The objectives of this dissertation research were to assess the zoonotic foodborne transmission of HEV and elucidate the viral determinants of HEV host range. To determine the risk of HEV foodborne transmission, 127 packages of commercial pig liver were tested for HEV RNA. Eleven percent of them were positive for HEV RNA and the contaminating virus remained infectious. We also demonstrated that medium-to-rare cooking condition (56°C) does not completely inactivate HEV, although frying and boiling of the contaminated livers inactivated the virus. To reduce the risk of foodborne HEV transmission, commercial pig livers must be thoroughly cooked for consumption. To determine the host range of genotype 4 HEVs, pigs were inoculated with a genotype 4 human HEV. All pigs developed an active HEV infection indicating that genotype 4 human HEVs can cross species barriers and infect pigs. To identify viral determinant(s) of species tropism, ORF2 alone or in combination with its adjacent 5′ junction region (JR) and 3′ non-coding region (NCR), were swapped between genotypes 1 and 4, 3 and 4, and 1 and 3 to produce 5 chimeric viruses. Chimeric viruses containing ORF2 or JR+ORF2+3' NCR from genotype 4 human HEV in the backbone of genotype 3 swine HEV were viable in vitro and infectious in vivo. Chimeric viruses containing the JR+ORF2+3'NCR of genotypes 3 or 4 HEV in the backbone of genotype 1 human HEV were viable in vitro but non-infectious in pigs, suggesting that ORF1 may also be important for host range.
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    Gene editing in Aedes aegypti
    Aryan, Azadeh (Virginia Tech, 2013-10-08)
    Aedes aegypti (Ae. aegypti) is one of the most important vectors of dengue, chikungunya and yellow fever viruses. The use of chemical control strategies such as insecticides is associated with problems including the development of insecticide resistance, side effects on animal and human health, and environmental concerns. Because current methods have not proven sufficient to control these diseases, developing novel, genetics-based, control strategies to limit the transmission of disease is urgently needed. Increased knowledge about mosquito-pathogen relationships and the molecular biology of mosquitoes now makes it possible to generate transgenic mosquito strains that are unable to transmit various parasites or viruses. Ae. aegypti genetic experiments are enabled, and limited by, the catalog of promoter elements available to drive transgene expression. To find a promoter able to drive robust expression of firefly (FF) luciferase in Ae. aegypti embryos, an experiment was designed to compare Ae. aegypti endogenous and exogenous promoters. The PUb promoter was found to be extremely robust in expression of FF luciferase in different stages of embryonic development from 2-72 hours after injection. In subsequent experiments, transformation frequency was calculated using four different promoters (IE1, UbL40, hsp82 and PUb) to express the Mos1 transposase open reading frame in Mos1-mediated transgenesis. Germline transformation efficiency and size of transgenic cluster were not significantly different when using endogenous Ae. aegypti PUb or the commonly used exogenous Drosophila hsp82 promoter to express Mos1 transposase. This study also describes the development of new tools for gene editing in the Ae. aegypti mosquito genome and the use of these tools to design an efficient gene drive system in this mosquito. Homing endonucleases (HEs) are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. The activities of four HEs (Y2-I-AniI, I-CreI, I-PpoI, and I-SceI) were investigated for their ability to catalyze the excision of genomic segments from the Ae. aegypti genome. All four enzymes were found to be active in Ae. aegypti; however, the activity of Y2-I-AniI was higher compared to the other three enzymes. Single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways were identified as mechanisms to repair HE-induced dsDNA breaks. TALE nucleases (TALENs) are a group of artificial enzymes capable of generating site-specific DNA lesions. To examine the ability of TALENs for gene editing in Ae. aegypti, a pair of TALENs targeted to the kmo gene were expressed from a plasmid following embryonic injection. Twenty to forty percent of fertile G0 produced white-eyed progeny which resulted from disruption of the kmo gene. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. A small deletion of one to seven bp occurred at the TALEN recognition site. These results show that TALEN and HEs are highly active in the Ae. aegypti germline and can be used for gene editing and gene drive strategies in Ae. aegypti.
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    Genetic factors affecting the RNA interference pathway of Aedes aegypti mosquitoes
    Haac, Mary Etna Richter (Virginia Tech, 2013-12-30)
    Aedes aegypti mosquitoes are the vectors of many significant arboviruses that cause tremendous social and economic impact. RNA interference (RNAi) plays a crucial role in the vector competence of mosquitoes and is often targeted in studies involving mosquito innate immunity, genetics-based vector control strategies, and the development of viral-resistant transgenic mosquitoes. In general, RNA interference is induced by double stranded RNA (dsRNA) and results in the inhibition of cognate gene expression. There are several different RNA interference pathways, with distinct functions and mechanisms. The micro RNA pathway is important for endogenous gene regulation and development. The endogenous small interfering RNA (endo-siRNA) pathway functions in gene regulation and protection of the genome from the deleterious effects of transposable elements. The exogenous siRNA (exosiRNA) pathway is a major contributor to mosquito innate immunity and vector competence by limiting viral replication during infection. Lastly, the piwi RNA (piRNA) pathway primarily functions in protecting the genome from the deleterious effects of transposable elements. While the structure and function of many genes involved in Drosophila RNAi have been characterized, the corresponding mosquito orthologs have only been peripherally described or remain unknown. Thus, the overall purpose of this study is to improve the understanding of mosquito RNAi mechanisms by identifying and analyzing genetic factors involved in the various pathways. This research especially focuses on characterizing and analyzing putative doubleiii stranded RNA binding proteins (dsRBPs) important to the function of the RNAi initiator and effector complexes. Two genes, r2d2 and r3d1 are orthologs of Drosophila genes known to have important roles in the RNAi initiator complex. A third member of the same family, which we refer to as extra loquacious (exloqs), appears to have no known orthologs outside of the Aedes genus. Structural characterization of these genes included identification of single nucleotide polymorphisms (SNPs), novel exons and alternative splice variants. RT-PCR assays were utilized to examine differential expression of all three genes in specific tissues and developmental stages. Sub-cellular fractionation assays enabled intracellular localization of the RNAi proteins within Ae. aegypti cells. Co-immunoprecipitation of tagged dsRBPs revealed protein-protein interactions between specific dsRBPs and known RNAi factors. In addition, an exo-siRNA sensor was designed and tested in-vivo and in-vitro with the purpose of facilitating the identification of novel genetic factors involved in this anti-viral pathway. Lastly, TALENbased gene disruption was successfully employed to knockout the exloqs gene in Ae. aegypti mosquitoes, enabling further analysis into the function of this gene. The research described in this document provides further insight into mosquito innate immunity and gene regulation, which is important to the advancement of genetics-based vector control strategies.
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    Germline excision of transgenes in Aedes aegypti by homing endonucleases
    Aryan, Azadeh; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N. (Nature Publishing Group, 2013-04)
    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.
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    The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes
    Haac, Mary Etna; Anderson, Michelle A. E.; Eggleston, Heather; Myles, Kevin M.; Adelman, Zach N. (2015-04-20)
    Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.
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    Identification of Candidate Iron Transporters From the ZIP/ZnT Gene Families in the Mosquito Aedes aegypti
    Tsujimoto, Hitoshi; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N. (Frontiers, 2018-04-12)
    Mosquito-transmitted viral pathogens, such as dengue and Zika, afflict tens of thousands of people every year. These viruses are transmitted during the blood-feeding process that is required for mosquito reproduction, the most important vector being Aedes aegypti. While vertebrate blood is rich in protein, its high iron content is potentially toxic to mosquitoes. Although iron transport and sequestration are essential in the reproduction of vector mosquitoes, we discovered that culicine mosquitoes lack homologs of the common iron transporter NRAMP. Using a novel cell-based screen, we identified two ZIP and one ZnT genes as candidate iron transporters in the mosquito A. aegypti, the vector of dengue, Zika, and chikungunya. We determined the organ-specific expression pattern of these genes at critical time points in early reproduction. The result indicates modulation of these genes upon blood feeding, especially a ZIP13 homolog that is highly up-regulated after blood feeding, suggesting its importance in iron mobilization during blood digestion and reproduction. Gene silencing resulted in differential iron accumulation in the midgut and ovaries. This study sets a foundation for further investigation of iron transport and control strategies of this viral vector.
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    Identifying Novel Contributors to RNA Interference in Aedes aegypti
    Saadat, Angela P. (Virginia Tech, 2015-09-02)
    Aedes aegypti is an important vector of human pathogens including the viruses yellow fever, dengue and chikungunya. The small interfering RNA (siRNA) pathway is a critical immune response for controlling viral replication in Aedes aegypti. The goal of this research is to identify components of the Aedes aegypti genome that influence this pathway. A transgenic mosquito strain that reports the status of the siRNA pathway via enhanced green fluorescent protein (EGFP) intensity was employed to differentiate silencing abilities among individuals. Extreme EGFP expression phenotypes, representing efficient and poor silencing abilities, were enriched over five generations. Transcriptome sequencing and analyses were performed from pools of individuals from each enriched phenotype, revealing potential RNAi contributors. 1,120 transcripts were significantly different (FDR<0.0001) among the extreme phenotypes. Four genes were chosen, amplified, sequenced for SNP analysis. These analyses were performed on samples obtained by crossing enriched, extreme phenotype F0 individuals, intercrossing their progeny, then selecting individuals representing the extreme phenotypes from the F2 population. Though further verification is needed, findings from these analyses imply the regions of Aedes aegypti, Liverpool strain (AAEL) gene identifiers AAEL005026, AAEL013438 and AAEL011704 amplified do not contribute to the two extreme, opposite RNAi silencing in the sensor strain used here. SNP analyses of AAEL000817 indicate this gene either influences extreme RNAi phenotypes or is closely linked to a gene(s) that contributes to RNAi in Aedes aegypti. The 1,120 genes identified can be validated or eliminated as potential targets in the quest to mitigate the impact of Aedes aegypti.
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    Modulation of Alphaviruses by Small RNAs
    Morazzani, Elaine M. (Virginia Tech, 2011-08-31)
    Mosquito-borne diseases remain a significant burden on global public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although infection of the vertebrate host is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. It is well known that invertebrates rely on small RNA pathways as an adaptive antiviral defense. The canonical antiviral response in these organisms involves dicer enzymes that cleave viral double-stranded RNA replicative intermediates (RIs) into small interfering RNAs (siRNAs; ~21-24 nucleotides). One strand of the siRNA duplex guides the targeting and destruction of complementary viral RNAs when loaded and retained in a multi-protein complex called the RNA-induced silencing complex. Here, we show that mosquito vectors mount a redundant double defense against virus infection mediated by two different small RNA pathways. Specifically, we demonstrate that in addition to a canonical antiviral response mediated by siRNAs, virus infection of the mosquito soma also triggers an antiviral immune pathway directed by ping-pong-dependent PIWI-interacting RNAs (piRNAs; ~24-30 nucleotides). The complexity of mosquito antiviral immunity has important implications for understanding how viruses both induce and modulate RNA-silencing responses in mosquito vectors. In mammals, viral RIs induce a range of relatively nonspecific antiviral responses. However, it remains unclear if viral RIs also trigger RNA silencing in mammals. Mosquito-borne viruses represent an ideal model for addressing this question as their transmission cycles involve alternating replication in mammalian and invertebrate hosts. Although we report identifying a subset of virus-derived small RNAs that appear to be products of RNA silencing in two mammalian cell lines infected with the mosquito-borne chikungunya virus (CHIKV), our studies suggest these small RNAs have little biological relevance in combating virus infections. Thus, while the accumulation of virus-derived siRNAs is essential to the survival of mosquitoes infected with CHIKV, they appear to have little functional significance in mammalian antiviral immunity.