WOLBACHIA INDUCES RESISTANCE TO DENGUE VIRUS IN MOSQUITO AEDES AEGYPTI By Peng Lu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics - Doctor of Philosophy 2014 ABSTRACT WOLBACHIA INDUCES RESISTANCE TO DENGUE VIRUS IN MOSQUITO AEDES AEGYPTI By Peng Lu Transmitted by the bite of mosquitoes, dengue virus (DENV) is the most important arboviral pathogen in humans. Currently no drug therapy or vaccine is available for dengue fever, leaving vector control as the major way to protect human populations. The endosymbiotic bacterium Wolbachia has been proposed as a potential agent for vector control, because it can not only spread within mosquito populations via manipulation of mosquito reproduction but also inhibit transmission of DENV in mosquitoes. In order to develop Wolbachia-based control strategies to prevent DENV infection, we have to better understand the interactions between DENV and Wolbachia in mosquitoes. Aedes albopictus naturally carries Wolbachia infection, however, Wolbachia-mediated viral resistance was not observed in Ae. albopictus. In this study, we demonstrated that the native Wolbachia induce a resistance to DENV in Wolbachia density-dependent manner in Ae. albopictus. A decrease in Wolbachia density within the host cells results in increased dengue infection. We provide evidence that a very low Wolbachia density in mosquito tissues where DENV resides and travels could contribute to the absence of Wolbachia-mediated resistance to DENV in Ae. albopictus. We also investigated the impact of Wolbachia infection on DENV life cycle. We report here that Wolbachia is able to inhibit the intracellular accumulation of DENV in Aedes. aegypti mosquito cells. We showed that Wolbachia infection inhibits DENV binding to mosquito Aag2 cells. We then compared the DENV negative strand RNA levels in Wolbachia infected and uninfected mosquito cells. Wolbachia infection also inhibits DENV replication in mosquito Aag2 cells. To further investigate Wolbachia-DENV interactions in mosquito, we carried out microarray and real-time PCR analyses to define host cell transcriptional responses that are induced by Wolbachia infection. Our data indicates that sixteen previously identified DENV host factors were up-regulated in mosquito cells infected with Wolbachia. An RNA interference (RNAi) screen revealed eleven host factors that are associated with Wolbachia-mediated viral resistance. Our data provides several potential targets for interrupting dengue virus infection in mosquitoes. ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor Dr. Zhiyong Xi for his scientific guidance, professional advice and consistent support. I also acknowledge my committee members, Drs. Thiem Suzanne, Edward Walker, Robert Abramovitch and Todd Ciche for providing valuable comments and encouragements on my dissertation work. I also acknowledge the help and support of my lab members, including Dr. Xiaoling Pan, Dr. Deepak Joshi, Liang Xiao, Mike McFadden and David Bevins. I would like to thank Drs. Robert Hausinger and Donna Koslowsky for keeping me on track to graduate. I thank Collen Curry from MSU genomics core for the assistance in Real-time PCR. Finally, I would like to give a special thank you to my wife, Cheng Zhang, for her love, support and encouragement. iv TABLE OF CONTENTS LIST OF TABLES............................................................................................................vii LIST OF FIGURES..........................................................................................................viii Chapter 1: Introduction.......................................................................................................1 Dengue virus and Dengue fever .......................................................................................1 Wolbachia ........................................................................................................................4 Current understanding of Wolbachia-mediated Dengue virus inhibition in mosquitoes..........................................................................................................................7 REFERENCES................................................................................................................13 Chapter 2: Wolbachia Induces Density-dependent Inhibition to Dengue Virus in Mosquitoes .......................................................................................................................20 Introduction ....................................................................................................................20 Methods...........................................................................................................................22 Mosquito rearing and cell culture maintenance.......................................................22 Introduction of Wolbachia into Aag2 cells..............................................................22 Density-dependent assay..........................................................................................23 DENV-2 Infections in Mosquitoes...........................................................................23 RNA Extraction, cDNA Synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)..................................................................24 Wolbachia Quantitative PCR (q-PCR).....................................................................24 Susceptibility of the host cells to DENV infection..................................................25 Immunofluorescence................................................................................................25 Results..............................................................................................................................26 The Wolbachia wAlbB induce strong resistance to DENV-2 in Ae. albopictus Aa23cells..................................................................................................................26 Generate Aa23 cell lines infected with Wolbachia at different densities................27 The Wolbachia wAlbB induces resistance to DENV in a density-dependent manner.....................................................................................................................27 Wolbachia density in somatic tissues of Ae. albopictus is too low to induce viral interference......................................................................................................28 Wolbachia induces expression of antimicrobial peptide DEFD in a densitydependent manner in Ae. albopictus cells................................................................29 Cells host both Wolbachia and DENV when Wolbachia density is low.................30 Discussion........................................................................................................................38 REFERENCES................................................................................................................43 Chapter 3: Wolbachia infection inhibits multiple stages of Dengue life cycle in mosquito Aedes aegypti cells.............................................................................................................49 Introduction .....................................................................................................................49 v Methods............................................................................................................................50 Cell culture and DENV-2 infection..........................................................................50 DENV-2 binding assay.............................................................................................51 RNA Extraction, cDNA synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)..................................................................51 DENV-2 RNA transfection......................................................................................52 Plaque assays for DENV-2 virus titration................................................................52 Results..............................................................................................................................53 Wolbachia inhibits the intracellular accumulation of DENV genome copies in Aag-2 cells...........................................................................................................53 Wolbachia infection inhibits DENV binding to Aag-2 cells....................................54 Wolbachia inhibits DENV replication in Aag-2 cells..............................................55 Wolbachia infection regulates expression of DENV binding proteins....................55 Discussion........................................................................................................................61 REFERENCES................................................................................................................65 Chapter 4: Identification of Wolbachia-mediated Host Factors Involved in Resistance to Dengue Virus Infection .....................................................................................................70 Introduction .....................................................................................................................70 Methods...........................................................................................................................72 Cell-Culture Maintenance and DENV-2 infection...................................................72 Microarray analysis..................................................................................................72 RNA Extraction, cDNA synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)..................................................................73 RNAi-mediated gene silencing................................................................................73 Plaque assay.............................................................................................................74 Results..............................................................................................................................74 Differentially expressed profiles in Wolbachia infected and uninfected Aedes aegypti cells..............................................................................................................74 Select candidate Wolbachia-mediated host factors involved in resistance to DENV infection........................................................................................................75 Identify Wolbachia-associated DENV host factors by RNAi screen ......................76 Discussion........................................................................................................................85 REFERENCES................................................................................................................88 Chapter 5: Conclusions and future directions ...................................................................93 Conclusions .....................................................................................................................93 Future directions .............................................................................................................95 REFERENCES................................................................................................................99 vi LIST OF TABLES Table 2.1: The native Wolbachia does not inhibit dissemination of DENV-2 to mosquito heads in Ae. albopictus......................................................................................................31 Table 3.1: Differentially expressed DENV binding proteins in W-Aag2 and R-Aag2 cells....................................................................................................................................57 Table 4.1: Candidate Wolbachia-mediated host factors involved in resistance to DENV infection ............................................................................................................................78 vii LIST OF FIGURES Figure 1.1: Overview of dengue virus life cycle..................................................................3 Figure 1.2: Examples of cytoplasmic incompatibility caused by Wolbachia......................8 Figure 2.1: wAlbB induces a strong resistance to DENV-2 in both Ae. albopictus Aa23 (A) and Ae. aegypti Aag2 cell lines (B) ............................................................................32 Figure 2.2: Generation of Aa23 cells with different Wolbachia density...........................33 Figure 2.3: Wolbachia induces density-dependent inhibition to DENV-2 in Aa23 cell lines....................................................................................................................................34 Figure 2.4: Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to DENV............................................................................................................35 Figure 2.5: The expression of DEFD was induced by Wolbachia in density-dependent manner in Aa23 cell lines..................................................................................................36 Figure 2.6: Localization of Wolbachia and DENV-2 in Aa23 cells..................................37 Figure 3.1: DENV infection of W-Aag2 and R-Aag2 cells...............................................58 Figure 3.2: Wolbachia infection inhibits DENV binding to mosquito cells......................59 Figure 3.3: Wolbachia infection inhibits DENV replication in mosquito cells.................60 Figure 4.1: Functional classification of differentially expressed genes in the Aag2 cell line in response to Wolbachia infection.............................................................................81 Figure 4.2: Characterizing Wolbachia-regulated DENV host factors by RNA interference (RNAi) screen....................................................................................................................82 Figure 5.1: A current model of Wolbachia-mediated viral interference in Ae. aegypti.....94 viii Chapter 1: Introduction Dengue virus and Dengue fever In recent decades, dengue fever, caused by infection with dengue virus (DENV), has become one of the most important emerging infectious diseases. The World Health Organization (WHO) estimates that there are 50-100 million dengue fever cases worldwide every year, and over 2.5 billion people are at risk of dengue infection (WHO, 2012). DENV infection is usually characterized by a high fever, severe joint pain, skin rash and flu-like symptoms, but a small proportion of infections develop into more serious conditions such as dengue hemorrhagic fever, or dengue shock syndrome. At present, no licensed vaccine or antiviral drug is available for dengue fever. Vector control is the primary method to reduce or prevent DENV transmission. DENV is an RNA virus in the family Flaviviridae, genus Flavivirus. The DENV genome is a single strand positive sense RNA of approximately 11 kb in length. This RNA contains short untranslated regions (UTR) at both the 3’ and 5’ end, and a long open reading frame (ORF) encoding 10 genes. The whole viral genome is translated as one polypeptide chain that is then co- and post-translationally cleaved by viral and cellular proteases into three structural and seven nonstructural (NS) proteins (Murray et al., 2008). The structural proteins include a capsid (C) protein that binds and stabilizes the genomic RNA, a premembrane/membrane (PrM/M) protein that blocks immature virus fusion and may stabilize the envelope (E) protein conformation , and an E protein that facilitates virus binding, membrane fusion and viral assembly (Mukhopadhyay et al., 1 2005). The NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) are involved in viral transcription, replication, assembly and attenuation of host immune responses (Murray et al., 2008, Bollati et al., 2010). Dengue virions enter susceptible cells through clathrin-mediated endocytosis (van der Schaar et al., 2008, Acosta et al., 2008). After internalization, dengue virions are delivered to endosomes, where the low pH triggers the fusion of viral and endosomal membranes. Then the viral nucleocapsid, which consists of the viral genomic RNA and C protein, is released into the cytoplasm. In the cytoplasm, the viral RNA translation occurs at the rough endoplasmic reticulum (ER), giving rise to a single long polyprotein that is cleaved into 3 structural proteins and 7 NS proteins by host and viral proteases (Whitehorn and Simmons, 2011). The non-structural proteins form a replication complex that synthesizes new viral RNA. Viral replication starts with synthesis of the negativestrand RNA, which then serves as a template for the transcription of positive-strand RNA. The newly synthesized positive-sense RNAs are used for RNA replication, translation, or assembly of new Dengue virions, which most likely form on the ER membrane (Mackenzie and Westaway, 2001). New virions are further processed in the trans-Golgi network and are released from the cell through the cellular secretory pathway (Figure 1.1). DENV is transmitted to humans by the bite of Aedes genus mosquitoes, primarily the urban-dwelling mosquito Aedes aegypti and, to a lesser extent, Aedes albopictus. After a mosquito bites a dengue-infected-person, DENV first infects the mosquito midgut epithelial tissue, then disseminates from the midgut to hemolymph and finally infects the salivary gland at approximately 7-14 days post-feeding. (Salazar et al., 2007). Once 2 Figure 1.1: Overview of dengue virus life cycle (1) Virus particles attach to cell. (2) Dengue virions enter the cell by receptor-mediated endocytosis. (3) After fusion of the viral membrane with lysosomes, nucleocapsid enters into cytoplasm. (4) Viral genome is translated into single open reading frame. (5) Polyprotein is processed by host and viral proteases to produce individual proteins. (6) By using the positive-sense RNA as template, the viral RNA replication proteins synthesize negative-strand RNA. (7) The negative-strand RNA serves as template for synthesis of new positive-strand RNA. (8) Structural proteins and viral RNA assemble into immature viral particles on the ER membrane. (9) Newly assembled viral particles are further processed in trans-Golgi network, where they mature and change into infectious form. (10) Mature dengue virions are released into the extracellular environment. Modified from Mukhopadhyay et al., 2005 3 DENV infects the salivary gland, it can be transmitted by the infected mosquito to a human host during blood feeding. Wolbachia Wolbachia is a maternally transmitted endosymbiotic bacterium that infects millions of arthropod and nematode species worldwide. Estimated frequencies of Wolbachia infected insect species is up to 65 percent (Hilgenboecker et al., 2008). Wolbachia belong to Rickettsiales, a group of obligate bacteria having mutualistic, commensalistic and parasitic interactions with various hosts. While the related genera Anaplasma, Ehrlichia, and Rickettsia, generally have life cycles including an invertebrate vector and mammalian host, Wolbachia does not infect vertebrates (Werren et al., 2008). Surrounding by a vacuole in cytoplasm, Wolbachia is bacilliform or coccoid in morphology with size ranging from 0.8 μm to 1.5 μm in length (Stouthamer et al., 1999). Wolbachia was first described in the mosquito Culex pipiens in 1924 (Saridaki and Bourtzis, 2010), and has since been detected in many species of arthropods and filarial nematodes. Similar to most Rickettsiales, Wolbachia genome sizes are relatively small (1.08–1.7 Mb) (Werren et al., 2008), but they contain high numbers of mobile and repetitive elements (Saridaki and Bourtzis, 2010). Based on phylogenetic analysis of several genes including 16S ribosomal DNA, wsp, ftsZ, and others, Wolbachia has so far been taxonomically subdivided into 13 supergroups (namely A- N) (Doudoumis et al., 2013). The most common Wolbachia supergroups found in arthropods are A and B, while in filarial nematodes, supergroups C and D are dominant. 4 Wolbachia is obligate intracellular bacterium that reside within host-derived vacuoles. Typically, Wolbachia is transmitted vertically through the cytoplasm of eggs. The horizontal transmission of Wolbachia, which is fairly rare, allows those bacteria to move across species boundaries, resulting in the wide distribution of Wolbachia (Werren, 1997). Wolbachia establishes diverse symbiotic associations with their hosts, ranging from obligate mutualism in filarial nematodes, to commensal, or parasitic associations in arthropod hosts. To increase transmission frequencies, Wolbachia can induce host reproductive disorders including cytoplasmic incompatibility (CI), parthenogenesis, feminization and male-killing in arthropods. CI, the most common phenotype in mosquitoes, occurs between infected males and uninfected females or infected females harboring different Wolbachia strains, and results in zygotic death (O'Neill and Karr, 1990, Zabalou et al., 2004). The CI mechanism is not yet fully understood, but it can be interpreted under a 'modification/rescue' model (Lassy and Karr, 1996, Callaini et al., 1997, Werren, 1997). According to this model, Wolbachia modifies sperm in infected males during early spermatogenesis, such that karyogamy failure occurs, leading to early embryo death (Xi et al., 2005). However, the modification can be rescued if the female carries a compatible Wolbachia strain, resulting in normal progeny development. There are two basic types of CI, unidirectional and bidirectional CI (Werren et al., 2008). Unidirectional CI occurs in mating between infected males and aposymbiotic females or single infected females and superinfected (infection with multiple Wolbachia strains) males (Figure 1.2 A, C). Bidirectional CI occurs in both directions when the individuals are infected by different Wolbachia strains (Figure 1.2 B). 5 Besides reproductive manipulation of arthropod hosts, Wolbachia is known to confer resistance against pathogens in insect hosts (Bian et al., 2010, Moreira et al., 2009). The phenotype was observed in Drosophila, where Wolbachia can induce resistance to DENV, Drosophila C virus (DCV), Flock house virus and Cricket Paralysis virus (Hedges et al., 2008, Teixeira et al., 2008). As observed in Drosophila, Wolbachia (wAlbB, wMelPop-CLA, and wMel strains) was able to induce a strong inhibition to replication and dissemination of DENV in the transinfected Ae. aegypti (Bian et al., 2010, Walker et al., 2011). Plasmodium infection intensity was also significantly decreased in Anopheles mosquitoes transinfected with Wolbachia (wAlbB and wMelPop strain) (Bian et al., 2013, Hughes et al., 2011). Recently, Wolbachia has attracted growing scientific and public attention as a control tool for arboviral diseases. The manipulation of hosts’ reproductive systems, as well as vector pathogen-refractory phenotypes place Wolbachia among the most promising tools for mosquito-borne disease control. Several strategies for Wolbachiabased mosquito control have been proposed (Bourtzis, 2008). One such strategy is population replacement, in which wild mosquito populations would be replaced with Wolbachia-infected populations that are not competent to transmit pathogens. The first field release experiment utilizing Wolbachia (wMel strain) transinfected Ae. aegypti was conducted in Australia. In the study, wMel Wolbachia infection successfully invaded two natural Ae. aegypti populations within a few months following releases of infected adult mosquitoes (Hoffmann et al., 2011). One year post-release, field collected wMel mosquitoes showed significantly reduced DENV replication and dissemination compared to Wolbachia-uninfected mosquitoes (Frentiu et al., 2014). Indeed, new field trials will be 6 carried out in other dengue-endemic countries, including China, Brazil, Indonesia, and Vietnam In addition to a population replacement control strategy, another potential strategy is population suppression. Similar to Sterile Insect Technique (SIT), this strategy is also known as the incompatible insect technique (IIT) that uses Wolbachia-infected males to induce sterility within a mosquito population. Releases of incompatible males into a wild population successfully eliminated Culex pipiens fatigans in a village in Burma (Laven, 1967). A recent study also showed the potential of Wolbachia to eradicate Aedes polynesiensis, the primary vector of Lymphatic filariasis, in the South Pacific with population suppression (Brelsfoard et al., 2008). Current understanding of Wolbachia-mediated Dengue virus inhibition in mosquitoes The Wolbachia-mediated pathogen-refractory phenotype varies depending on the pathogen type, Wolbachia strain, infection type (natural, laboratory introduced ) and host factors (Rainey et al., 2014). For example, Wolbachia (wAlbB strain) infected Ae. albopictus cell line Aa23 is more resistant to DENV compared to Wolbachia-cured controls. In contrast, Wolbachia-induced DENV resistance was not observed in Ae. albopictus mosquitoes, which naturally carry two Wolbachia strains (wAlbA and wAlbB) (Bian et al., 2010). Wolbachia (wPip strain) native infected Culex quinquefasciatus 7 Figure 1.2: Examples of cytoplasmic incompatibility caused by Wolbachia (A) Unidirectional CI occurs in mating between infected males and aposymbiotic females. (B) Crosses of individuals infected with different, non-compatible Wolbachia strains result in bidirectional CI. (C) Unidirectional CI results from incompatible crosses between single infected and superinfected individuals. Modified from Dobson, 2003, Sinkins and Gould, 2 8 mosquitoes are less susceptible to West Nile virus (WNV) infection compared to aposymbiotic controls (Glaser and Meola, 2010). The underlying mechanism involved in Wolbachia-mediated DENV resistance in mosquitoes is still not clearly known; however, increasing numbers of studies suggest that the resistance is related to enhanced mosquito innate immunity. First, transcriptome analyses showed that Wolbachia infection activated a number of immune genes, for example cecropins, defensins, and several Toll and Imd pathway genes, in transinfected mosquitoes. Moreover, we found a positive relationship between Wolbachia density and the amount of defensin transcript, supporting the hypothesis that host immune level could contribute to Wolbachia induced viral resistance(Lu et al., 2012). Second, knockdown of the immune gene TEP1 by injection of specific dsRNA into wMelPop transiently infected An. gambiae has been shown to partially rescue the Wolbachia-mediated Plasmodium inhibition, which indicates up-regulation of innate immune responses contribute to this pathogen resistance phenotype(Kambris et al., 2010). In accordance with this observation, we found knockdown of antimicrobial peptides Defensin D and Cecropin C compromise resistance to DENV in Wolbachia transinfected Ae. Aegypti(Pan et al., 2012). Recently, we investigated the molecular mechanism of Wolbachia-mediated Toll pathway activation (Pan et al., 2012). In this study, we found Wolbachia wAlbB strain infection elevates the level of reactive oxygen species (ROS), and the increased level of ROS resulted in activation of the Toll pathway in Ae. aegypti. In addition, Pinto et al. reported that recombinant Wolbachia surface protein (WSP) stimulates immune gene expression in two different mosquito cell lines in a dosage dependent manner (Pinto et al., 2012). 9 In insects, RNA interference (RNAi) is an important innate immune response to virus infection. Mosquitoes have three major RNAi pathways: small interfering RNA (siRNA) pathway, microRNA (miRNA) pathway and Piwi RNA (piRNA) pathway (Ding and Voinnet, 2007). It is likely that siRNA pathway is not required for Wolbachiainduced antiviral protection. First, DENV replication was inhibited by Wolbachia in the C6/36 cell line that lacks a functional antiviral siRNA response (Frentiu et al., 2010, Brackney et al., 2010). Second, Wolbachia infection rendered siRNA mutant D. melanogaster more resistant to West Nile Virus infection (Glaser and Meola, 2010). Furthermore, Hedges et al. reported Wolbachia-mediated viral interference was present in all siRNA Dropshilia mutants (dcr-z, r2de/CyO and AGO2), indicating Wolbachiamediated antiviral protection is independent of siRNA pathway (Hedges et al., 2012). Although the siRNA pathway is not critical for Wolbachia-induced antiviral protection, the miRNA pathway may be involved in the interaction between Wolbachia and DENV (Zhang et al., 2013). Recently, Hussain et al. reported differential expression of miRNAs in Ae. aegypti mosquitoes transinfected with Wolbachia (wMelPop-CLA strain), and found a host miRNA (aae-miR-2940) was expressed only in Wolbachiainfected mosquitoes (Hussain et al., 2011, Zhang et al., 2013). Two target genes (metalloprotease m41 ftsh and AaDnmt2) of aae-miR-2940 were identified in Ae. aegypti. Metalloprotease m41ftsh gene was found to be up-regulated in the Wolbachia-infected host. Knockdown of this gene or inhibition of miR-2940 led to reduced Wolbachia density in mosquitoes and cells. Wolbachia-induced aae-miR-2940 negatively regulated the expression of the gene AaDnmt2, which in turn inhibited DENV replication. Conversely, overexpression of AaDnmt2 in mosquito cells led to an increase in DENV 10 replication, but a decrease in Wolbachia density (Zhang et al., 2013). These finding indicate the miRNA pathway is important for maintenance of Wolbachia density as well as the resistance to DENV in Wolbachia transinfected mosquitoes. Evidence from recent transinfected hosts supports the hypothesis that Wolbachia interfere with viruses by elevating host basal immunity, while results from its natural host Drosophila suggest the up-regulation of immune genes is not the only mechanism responsible for Wolbachia-mediated viral interference (Rances et al., 2012). In D. melanogaster, Wolbachia (wMelPop and wMel strains) could not induce transcription of antimicrobial peptides (AMPs), Toll pathway and melanization genes, but DENV inhibition occurred in the host (Rances et al., 2012). D. simulans, which naturally carries Wolbachia, showed no upregulation of immune effector genes compared to uninfected counterparts (Bourtzis et al., 2000, Wong et al., 2011). It has been hypothesized that the immune activation occurring in Wolbachia transinfected mosquitoes may be the consequence of the recent association with Wolbachia, and insects carrying long-term Wolbachia infections have evolved a certain degree of immune tolerance to Wolbachia (Blagrove et al., 2012). Competition over common host nutrients such as cholesterol could also contribute to Wolbachia-mediated DENV resistance. Cholesterol is vital to a host, Wolbachia, and DENV. In insects, cholesterol plays a vital role in cellular signaling and membrane structure (Eaton, 2008). Wolbachia resides within cholesterol-rich Golgi-related vesicles, and requires cholesterol for its replication and propagation (Lin and Rikihisa, 2003, Cho et al., 2011). Both insect and Wolbachia are unable to synthesize cholesterol de novo, 11 thus both depend on and compete for dietary sterols as key nutrients (Clayton, 1964, Caragata et al., 2013). DENV is also dependent on host cholesterol for cellular entry and replication (Lee et al., 2008). Therefore, it is possible that changes in host cholesterol metabolism inflicted by Wolbachia influence DENV propagation. In Ae. aegypti, Wolbachia infection was found to reduce total cholesterol levels by 15-25% (Caragata et al., 2014). A recent study investigated whether competition for cholesterol is involved in Wolbachia-mediated pathogen resistance (Caragata et al., 2013). A significant increase in DCV titer was observed in both wMelCS- and wMelPop- infected D. melanogaster flies reared on cholesterol-enriched diets at five days post-infection, indicating the competition for cholesterol contributes to Wolbachia-mediated viral interference (Caragata et al., 2013). 12 REFERENCES 13 REFERENCES ACOSTA, E. G., CASTILLA, V. & DAMONTE, E. B. (2008) Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrinmediated endocytosis. J Gen Virol, 89, 474-84. 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Insect Biochem Mol Biol, 35, 903-10. ZABALOU, S., RIEGLER, M., THEODORAKOPOULOU, M., STAUFFER, C., SAVAKIS, C. & BOURTZIS, K. (2004) Wolbachia-induced cytoplasmic incompatibility as a means for insect pest population control. Proc Natl Acad Sci U S A, 101, 15042-5. ZHANG, G., HUSSAIN, M., O'NEILL, S. L. & ASGARI, S. (2013) Wolbachia uses a host microRNA to regulate transcripts of a methyltransferase, contributing to dengue virus inhibition in Aedes aegypti. Proc Natl Acad Sci U S A, 110, 1027681. 19 Chapter 2: Wolbachia Induces Density-dependent Inhibition to Dengue Virus in Mosquitoes Introduction Dengue fever and dengue hemorrhagic fever are emerging globally as a major public health problem in more than 100 countries. 2.5 to 3.0 billion people, or two fifths of the world’s populations, are currently living in dengue-endemic areas. Each year there are an estimated 100 million new dengue cases and 22,000 deaths (Gubler, 1998). The number of cases reported to WHO has increased nearly nine times in the past 4 decades, and it continues to expand into temperate climates. Its fatal form, dengue hemorrhagic fever, has expended from Southeast Asia to 28 countries in the Western Hemisphere. Dengue virus (DENV) is transmitted to humans by the mosquitoes Aedes aegypti and Aedes albopictus. Currently no drug therapy or vaccines are available for dengue fever. Vector control is the primary intervention tool, but this barrier is weakened by increased pesticide resistance. This situation justifies seeking alternative and innovative approaches for the development of new prevention options. Wolbachia-based dengue control strategy is showing great potential because it could provide a solution that will be more sustainable, economical and environmental friendly than the other methods (Iturbe-Ormaetxe et al., 2011). Wolbachia spp. are intracellular alpha-proteobacteria closely related to Rickettsia. Maternally inherited infections with Wolbachia occur in more than 65% of all the insect species and approximately 28% of the surveyed mosquito species (Werren et al., 2008, Kittayapong et al., 2000). Through the cytoplasmic incompatibility (CI) mechanism, Wolbachia can induce early embryo death when a Wolbachia-infected male mates with an uninfected female (Serbus et al., 20 2008). Since infected females can successfully mate with uninfected males, Wolbachia can spread quickly in a population. This has been observed to occur naturally in Drosophila simulans(Turelli and Hoffmann, 1991), and demonstrated in Ae. aegypti through both laboratory cage studies and a recent field trial (Hoffmann et al., 2011, Xi et al., 2005). Another important feature of Wolbachia is its ability to induce resistance to a number of human pathogens, including DENV, in its insect hosts (Walker et al., 2011, Hughes et al., 2011, Kambris et al., 2010, Hedges et al., 2008, Teixeira et al., 2008, Bian et al., 2010). In the transinfected Ae. aegypti, all the three different types of Wolbachia, wAlbB, wMelPop-CLA, and wMel, show a significant inhibition to replication and dissemination of DENV, resulting in either complete or partial block of viral transmission (Walker et al., 2011, Bian et al., 2010, Moreira et al., 2009). Recent studies further show that Wolbachia induces production of reactive-oxygen species (ROS) which then activate Toll-pathway to induce expression of antiviral effectors (Pan et al., 2012). In the Drosophila host, native Wolbachia can also confer resistance to DENV and the other pathogens (Hedges et al., 2008). This resistance appears to be induced by the nonimmune related mechanisms because the tested immune genes do not show differential expression in response to Wolbachia(Bourtzis et al., 2000, Rances et al., 2012). Inhibition of dengue virus replication was also observed in cell lines, and the extent of inhibition is related to bacterial density (Frentiu et al., 2010). Ae. albopictus is generally considered as the secondary vector of dengue fever. This mosquito species naturally carries two types of Wolbachia, wAlbA and wAlbB, which distribute throughout both germ line and somatic tissues in mosquitoes (Dobson et al., 1999). However, Wolbachia-mediated viral interference was not observed in Ae. albopictus (Bian et al., 2010), consistent with the fact that it is a competent vector for at least 22 arboviruses (Gratz, 2004). The 21 recent study shows a transinfected Ae. albopictus line that carries wMel is resistant to DENV (Blagrove et al., 2012), which excludes the possibility that Ae. albopictus lacks the genetic background for Wolbachia to induce viral interference. Thus, although both wAlbB and Ae. albopictus own the machinery to induce viral interference, the latter does not occur for unknown reasons. In this work, we used an Aa23 cell line, initially established from eggs of Ae. albopictus (O'Neill et al., 1997), to study the mechanism underlying the lack of Wolbachia-mediated resistance to DENV. We confirmed that wAlbB induces a strong resistance to DENV in Aa23 cell line. Moreover, the levels of resistance strongly correlate with density of Wolbachia in Aa23 cells. Further comparison of genome copy of wAlbB in both somatic and germ line tissues between dengue resistant transinfected Ae. aegypti and susceptible Ae. albopictus suggests that Wolbachia density is too low to induce resistance to DENV in Ae. albopictus. Methods Mosquito rearing and cell culture maintenance Houston (HOU) and HT1 strain of Ae. albopictus and WB1 strain of Ae. aegypti were maintained at 27°C and RH 85% with a 12-hr light/dark cycle. Ae. albopictus Aa23, Aa23T cell lines (derived from Aa23 cells through tetracycline treatment) and Ae. aegypti Aag2 cell line were cultured as previously described (Dobson et al., 2002, Sim and Dimopoulos, 2010). Those cells were maintained in Schneider's Drosophila Medium (Invitrogen) supplemented with 10 % (v/v) heat-inactivated fetal bovine serum (FBS). Introduction of Wolbachia into Aag2 cells 22 W-Aag2 cell line was generated by introducing Wolbachia from Aa23 cells into Aag2 cell line using shell vial technique as previously described (Dobson et al., 2002)with a slight modification. In brief, two 75-cm2 flasks of confluent Aa23 cells were shaken, centrifuged at 1,000 x g for 10min, re-suspended in 1.5 ml Schneider's Drosophila Medium in a 50ml conical tube, and lysed by vortexing with 3-mm-diameter glass beads. The lysate was centrifuged at 2,500x g for 10min, and the supernatant was filtered through a 5µm syringe filter (Millipore). 500µl of the filtrate was overlaid on Aag2 cells grown in 12-well plate to 80% confluence. The plate was centrifuged at 2,000 x g at 15°C for 1h and then incubated at 26 °C overnight. After that, the cells were transferred to a 25 ml cell flask with fresh medium. Wolbachia was stably maintained in W-Aag2 for more than 17 passages before used for dengue infection. Density-dependent assay To generate Aa23 cells with different Wolbachia densities, the cell culture was treated with rifampicin to make a final concentration at 5µg/ml, 0.5 µg/ml and 0.05µg/ml in 24-well plates. For each dose, cells were treated for four time periods: 5h, 10h, 40h and 70 hr. After treatment, the old medium was removed and cells were washed with fresh medium for one time. After adding new medium, cells were grown for another three days. Then, the cells were passaged to another 24 well-plate with new medium, and grown for one week. Two days after the 2nd passage, cells were infected with DENV-2 at multiplicity of infection (MOI) of 0.1. At Day 5 post infection, cells were collected to measure the genome copy of Wolbachia and DENV2. To study Wolbachia-density dependent expression of Defensin D (DEFD), cells were grown for 7 days in fresh medium after rifampicin treatment. Then, cells were collected to measure both the genome copy of Wolbachia and the expression of DEFD (Gao et al., 1999). DENV-2 Infections in Mosquitoes 23 The New Guinea C strain of DENV serotype 2 (DENV-2) was propagated in C6/36 cells as previously described (Troyer et al., 2001). Virus was harvested 7 days post infection by collection of supernatants and centrifugation at 3,000 x g. The virus suspension was mixed in 1:1 with commercial ox defibrinated blood (Colorado Serum Company). The blood meal was warmed at 37°C in water bath for 30 min and then used to feed 7-day-old mosquitoes as described previously(Das et al., 2007). RNA Extraction, cDNA Synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) The total RNA or viral genomic RNA were extracted from either cell lines or mosquitoes tissues by RNeasy Mini Kit (QIAGEN Sciences, Germantown, MD, USA) and then the cDNA were transcript using QuantiTect reverse Transcription Kit (QIAGEN Sciences, Germantown, MD, USA). Real-time PCR was performed using the QuantiTect SYBR Green PCR Kit (QIAGEN Sciences, Germantown, MD, USA) and ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). DENV-2 genomic RNA was measured by qRT-PCR using primers directed to NS5 gene (Molina-Cruz et al., 2005). The dengue copy was normalized with host actin gene (Tortosa et al., 2008). A standard curve was generated for each of NS5 and actin by analyzing 101 to 108 copies/reaction of two plasmids containing each individual fragment (Tortosa et al., 2008). DENV-2 in mosquito head was diagnosed by RT-PCR with the same NS5 primers. DEFD was amplified with the primers: For (5′GTCTGTTGCCAACTCTCTTT -3′) and Rev (5′- CACAAGCACTGTCACCAAC -3′). Wolbachia Quantitative PCR (q-PCR) qPCR was performed to measure the Wolbachia density in the cell lines and mosquitoes (Tortosa et al., 2008). Genomic DNA was extracted from either Aa23 cells (two to six biological 24 replicates) or mosquito tissues (ten biological replicates). The primers specifically directed to Wolbachia surface protein (wsp) of wAlbA and wAlbB were used in PCR to measure the Wolbachia genome copy, which was normalized with the host actin for Aa23 cells (Tortosa et al., 2008) or ribosomal protein S6 (RPS6) gene for both Ae. albopictus and Ae. aegypti mosquitoes. RPS6 was amplified with the primers: For (5’-GAAGTTGAACGTATCGTTTC-3’) and Rev (5’GAGATGGTCAGCGGTGATTT-3’). A standard curve was generated for each of wAlbA, wAlbB and actin by analyzing 101 to 108 copies/reaction of the of the two plasmids containing each individual fragment (Tortosa et al., 2008). Another plasmid containing the RPS6 fragment was cloned to generate another standard curve for RPS6. Susceptibility of the host cells to DENV infection The susceptibility of Aa23, Aa23T, W-Aag2 and Aag2 cell lines for DENV-2 was measured by plaque assay with a slight modification (Das et al., 2007). DENV-2 virus stock (2x 107 PFU/ml; determined previously in C6/36 cells) was serially diluted in ten-fold increments (from 101 to 106) using cell medium, and then inoculated into each of the four mosquito cells in 24-well plates. After incubation at 26°C for 7 days, the plates were assayed for plaque formation by peroxidase immunostaining, using mouse hyperimmune ascitic fluid and a goat anti-mouse HRP conjugate as the primary and secondary antibody. Immunofluorescence The viral antigen and Wolbachia wsp protein in Aa23 cells were detected simultaneously using an indirect immunofluorescence assay (IFA). Confluent rifampicin -treated Aa23 cells were grown on chamber slides (Thermo), and then were infected with DENV-2 at 0.1 MOI. After incubation for 2 days, cells were fixed for 30 min at 4°C in 4% (w/v) paraformaldehyde in PBS, containing 0.5% (v/v) Triton X-100. Then, samples were incubated in PBS and 10% (v/v) 25 goat serum at room temperature for 1 h. Subsequently, the slides were incubated with a mouse anti-dengue complex monoclonal antibody at a 1:300 dilution and a rabbit anti-wsp polyclonal antibody (GenScript) at a 1:500 dilution in PBS at room temperature for 1 h. After wash, the slides were incubated with Alexa-488 anti-mouse and Alexa-594 anti-rabbit antibodies (Molecular Probes, Invitrogen) at a 1:1000 dilution in PBS at room temperature for 1 h. To stain cell nucleus, the samples were incubated with DAPI for 10 min. Immunostaining was examined under with an Olymupus Fluoview 1000 confocal microscope. Results The Wolbachia wAlbB induce strong resistance to DENV-2 in Ae. albopictus Aa23 cells We previously reported that the Wolbachia wAlbB does not inhibit dissemination of DENV-2 to the mosquito head in the naturally infected Ae. albopictus (Bian et al., 2010). This could indicate that wAlbB does not induce resistance to DENV in the host genetic background of Ae. albopictus. To test that possibility, we compared susceptibility for DENV-2 between wAlbBinfected Aa23 cell line and its aposymbiotic Aa23T. Seven days after viral infection, the viral titer in Aa23T reached to 1.1 x 107 PFU/ml while no virus particle was detected in Aa23 cell line (Fig. 2.1A). A parallel assay was conducted to measure the Wolbachia-mediated viral interference in Ae. aegypti cell line Aag2. Like Ae. aegypti mosquito, Aag2 cell line is uninfected by Wolbachia. We introduced wAlbB from Aa23 into Aag2 through a shell-vial technique, generating a Wolbachia-infected cell line W-Aag2. As expected, viral titer in the Aag2 cell line (1.3 x 106 PFU/ml) is significantly higher than in the W-Aag2 cell line (12.5 PFU/ml; P<0.01, Student’s t-Test; Fig. 2.1B). These results confirm that wAlbB can induce resistance to DENV-2 even in Ae. albopictus host background. 26 Generate Aa23 cell lines infected with Wolbachia at different densities To study how the density of wAlbB can influence its induced resistance to DENV in Aa23 cell line, we used sub-lethal doses of rifampicin to partially remove Wolbachia and generated cell cultures with Wolbachia at different densities. Three dosages (0.05 µg/ml, 0.5 µg/ml and 5 µg/ml) with four treatment times (4 h, 10 h, 40 h and 70 h), resulting in a total of 12 treatments, were designed to make Wolbachia density cover a broad range. A mock treatment (control) was included to treat cells with only methanol solvent for the above four time periods. As a result (Fig. 2.2), we generated a serial of cell cultures with different Wolbachia densities, with a highest at 953.2 wsp/actin (from 0.05 µg/ml and 4 h) and a lowest at 18.0 wsp/actin (from 5 µg/ml and 70 h). Wolbachia density significantly decreased with an increase in treatment dose. Within each dosage, no significant difference in Wolbachia density is observed among four treatment times. This indicates a treatment for 4 h can effectively remove Wolbachia and a further increase in treatment time will not significantly enhance the inhibitory effect of rifampicin on Wolbachia. For example, a treatment at dosage of 5 µg/ml for 4 h can dramatically reduce Wolbachia to a very low level (75.2 wsp/actin), compared to the mock treatment (1,888.3 wsp/actin). Further increasing treatment time at this dose will not significantly change the Wolbachia density, resulting in 74.1 wsp/actin (10 h), 52.4 wsp/actin (40 h), and 18.0 wsp/actin (70 h, Fig. 2.2). The Wolbachia wAlbB induces resistance to DENV in a density-dependent manner To study the relationship between Wolbachia density and dengue infection level, cell cultures derived from the above 12 treatments were inoculated with DENV-2. Five days after infection, we measured genome copies of DENV-2 in the cells. Wolbachia density in the cells and its corresponding viral infection level were recorded in parallel to examine their interactions. 27 We observed that genome copy of DENV-2 in cells increased with a decrease in Wolbachia density. Moreover, there is a negative correlation between the genome copy of Wolbachia and DENV-2 (r = −0.82; P<0.001). The level of DENV-2 (y) can be predicted by the genome copy of Wolbachia (x) via a model: y = −0.004x+3.847 (Fig. 2.3). These results confirm that Wolbachia induces a density-dependent viral inhibition in Aa23 cells. Wolbachia density in somatic tissues of Ae. albopictus is too low to induce viral interference Within mosquito vectors, DENV needs replicate and pass through midguts and salivary glands before transmission occurs. Wolbachia-mediated density-dependent viral inhibition in mosquito cells leads to a hypothesis that the Wolbachia density in the somatic tissues of Ae. albopictusis too low to induce viral interference in this mosquito species. Thus, we measured Wolbachia density in both somatic tissues (midgut, salivary gland, fatbody) and germ line tissue (ovary) of the HOU strain of Ae. albopictus (carrying wAlbA and wAlbB). As shown in Fig. 2.4A, wAlbB is significantly higher than wAlbA in all the four tissues. While none of wAlbA could be detected in midgut, salivary gland and fatbody, only 5.4 wsp/actin of wAlbA presents in ovary. There are 0.3, 5.3, and 12.3 wsp/actin of wAlbB in midgut, salivary gland and fatbody, respectively, compared to 57.9 wsp/actin of wAlbB in ovary. To better compare Wolbachia density between the HOU strain of Ae. albopictus and the transinfected Ae. aegypti WB1 strain (carrying wAlbB), we designed one set of primers that can amplify a conserved region of Ribosomal protein S6 (RPS6) in both mosquito species, which was used to normalize the Wolbachia copy in qRT-PCR. We observed that wAlbB density is significantly lower in the midgut than in the ovary and salivary gland for both mosquito species (P<0.05, Student's t-Test). However, wAlbB density in fatbody is similar to that in salivary gland. Importantly, a significant lower density of wAlbB presents in midgut, 28 fatbody and salivary gland (P<0.001, Student's t-Test) of HOU mosquitoes as compared with that of WB1 mosquitoes. Specifically, the Wolbachia genome copies in midgut, fatbody and salivary gland of HOU are 80-, 18-, and 24-fold less than that of WB1, respectively (Fig. 2.4B). In ovary, wAlbB density is also 5.6-fold lower in HOU than in WB1 (P<0.001, MannWhiteney U test). To confirm a lack of Wolbachia-mediated viral interference in Ae. albopictus, we compared mosquito susceptibility for DENV-2 at a serial diluted viral titers between the HOU and its aposymbiotic strain HT1 (initially derived from Houston strain through tetracycline treatment). We have previously observed that Wolbachia suppresses viral dissemination in the HOU mosquitoes after mosquitoes took infectious blood meal at a viral titer of 107 PFU/ml (Bian et al., 2010). However, it is possible that Wolbachia induces only a weak resistance to DENV in Ae. albopictus which could be overcome when a very high viral titer was used in the infection assay. As shown in Table 2.1, Wolbachia does not inhibit viral dissemination even mosquitoes took blood with a low viral titers. The head infection rate of both HOU and HT1 strain was dropped dramatically when mosquitoes fed with dengueinfected blood at diluted viral titers. No significant difference in head infection rate was observed between HOU and HT1 in all the three diluted viral titers (P>0.05, Fisher's exact test). These results confirm that Wolbachia does not suppress viral dissemination in the naturally infected Ae. albopictus. Wolbachia induces expression of antimicrobial peptide DEFD in a density-dependent manner in Ae. albopictus cells We previously show that the boosted host immunity boosted plays important roles in Wolbachia-mediated viral inference and antimicrobial peptide defensin is involved in this anti- 29 dengue resistance (Pan et al., 2012).To test whether the above Wolbachia-density dependent viral inhibition in Aa23 cells relates to host immunity, we measured both Wolbachia density and the expression of DEFD in the antibiotic treated Aa23 cells. As shown in Fig. 2.5, there is a positive correlation between Wolbachia density and the amount of DEFD transcript (r = 0.92; P = 0.0001). This supports that host immune level could contribute to Wolbachia-density dependent viral inhibition. Cells host both Wolbachia and DENV when Wolbachia density is low To study whether the above antibiotic treatment leads to a uniform change in both Wolbachia and DENV at the cellular level, cells at two days post-DENV infection were assayed by double immunofluorescent staining to visualize the distribution of Wolbachia and DENV-2 using antibodies against the wsp of Wolbachia and envelop protein of DENV-2. As expected, we observed Wolbachia was largely removed by rifampicin treatment, resulting in more cells infected by DENV-2 (Fig. 2.6A–C). However, such a reduction in Wolbachia density appears not to be even, resulting in that certain cells host much more Wolbachia than the others (Fig. 2.6A–C). Importantly, we observed that colocalization of DENV-2 and Wolbachia can occur in the same cells when Wolbachia density is low in those cells (Fig. 2.6D–E). For those cells that were heavily infected by DENV-2, however, they typically do not contain Wolbachia (Fig. 2.6G–I). 30 Table 2.1: The native Wolbachia does not inhibit dissemination of DENV-2 to mosquito heads in Ae. albopictus. The HOU strain of Ae. albopictus and its aposymbiotic strain HT1 were infected with the blood containing DENV-2 at titers of 106, 105, and 104 PFU/ml. At day 14 post infection, heads of ten mosquitoes were collected and used for diagnosis of DENV-2 by RT-PCR. Data from two experiments were pooled together. 31 Figure 2.1: wAlbB induces a strong resistance to DENV-2 in both Ae. albopictus Aa23 (A) and Ae. aegypti Aag2 cell lines (B). wAlbB is a native infection in Aa23 cells while Aa23T cells were initially generated by tetracycline treatment of Aa23 cell to remove Wolbachia infection. There is no Wolbachia in Aag2 cells. W-Aag2 was generated from Aag2 cells by introducing wAlbB from Aa23 using a shell-vial technique. Five days after inoculated with DENV-2, the cells were tested for dengue infection by plaque assay. Error bars are standard errors. **, P<0.01; ***, P<0.001in Student’s t-Test. 32 Figure 2.2: Generation of Aa23 cells with different Wolbachia density. Cells were treated using sub-lethal doses of rifampicin for a different time periods. Three dosages (0.05 µg/ml, 0.5 µg/ml and 5 µg/ml) and four time periods (4 h, 10 h, 40 h and 70 h) were used. The genome copies of wsp were measure by q-PCR, normalized by host gene actin. Error bars are standard errors of the mean of at least three biological replicates. Statistical significance is represented by letters above each column, with different letters signifying distinct statistical groups. Student's t test: a vs. b, P<0.001; b vs. c, P<0.001; d vs. a, P<0.05. 33 Figure 2.3: Wolbachia induces density-dependent inhibition to DENV-2 in Aa23 cell lines. Cell cultures derived from twelve different treatments in Fig. 2.2 were used in the assay. Five days after these cells were infected with DENV-2, viral genome copies were measured by qRTPCR. Actin was used as a host gene to normalize the data. There is a negative linear correlation between Wolbachia density and DENV copy. Each point is the mean of at least three biological replicates. 34 Figure 2.4: Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to DENV. (A) The density of wAlbA is significantly lower than wAlbB in midgut, fatbody, salivary gland and ovary of Ae. albopictus. The copy number of the Wolbachia wsp was normalized by Ae. albopictus actin; (B) Wolbachia density in somatic tissues is significantly lower in the Ae. albopictus HOU strain than in the transinfected Ae. aegyptiWB1 strain. The copy number of the Wolbachia wsp was normalized by one conserved RPS6 in both Ae. albopictus and Ae. aegypti. In all the assays, midguts, salivary glands, fatbodies and ovaries of 7day-old non blood fed females were dissected and used for extraction of total genomic DNA. ***, P<0.001; **, P<0.01 in Student's t-Test. Error bars are standard errors of the mean of ten biological replicates. 35 Figure 2.5: The expression of DEFD was induced by Wolbachia in density-dependent manner in Aa23 cell lines. Seven days after rifampicin treatment, cells were collected to measure Wolbachia densities and DEFD expression. Because treatment with 5 µg/ml of rifampicin for 70 h resulted in both Wolbachia infection and DEFD expression at lowest level, it serves as a reference for all the other treatments to calculate a fold increase in both Wolbachia density and DEFD expression. Each point is the mean of at least three biological replicate. 36 Figure 2.6: Localization of Wolbachia and DENV-2 in Aa23 cells. Double immunofluorescence staining of cells showing the localization of dengue virus (green) and Wolbachia (red). Cells were probed simultaneously with polyclonal anti-wsp antibody (Wolbachia) and monoclonal anti-DENV-2 antibody, followed by Alexa 594 (red) and Alexa 488 (green) conjugated antibodies, respectively. DNA (blue) is stained with DAPI. In panels (A, B, and C), the red, green and blue channels are merged. A and B show Aa23 cells with mock treatment and treatment with 5 µg/ml of rifampicin for 5 hr, respectively, followed by dengue infection. C is Aa23T cells without dengue infection (negative control). D to F or G to I is the same sample with different channel merged: D and G show only red and blue channel merged, E and H show only green and blue channel merged, F and I show all the red, green and blue merged. Aa23 cells treated with 5 µg/ml of rifampicin for 10 hr (D to F) and 40 hr (G to I), followed by dengue infection, are shown. 37 Discussion Wolbachia can induce a resistance to DENV and other arboviruses in Ae. aegypti (Bian et al., 2010, Moreira et al., 2009). This raises a puzzle as to why mosquitoes that naturally carry Wolbachia, such as Ae. albopictus, can still serve as vectors for the arboviruses. Here, we demonstrate that wAlbB is able to induce resistance to DENV in Ae. albopictus Aa23 cell line, and such viral interference occurs in Wolbachia density-dependent manner. We also show that Wolbachia density in somatic tissues of Ae. albopictus is significantly lower than the transinfected Ae. aegypti WB1. Such a low density of Wolbachia is predicted not to induce viral interference in Ae. albopictus. Consistently, Wolbachia does not suppress viral dissemination in Ae. albopictus even when mosquitoes take an infectious blood meal at a very low viral titer. Moreover, we observed a positive correlation between Wolbachia density and the expression of the antimicrobial peptide DEFD. When Wolbachia density is low, DENV can colocalize with Wolbachia in the same cell hosts. Consistent with previous studies, Wolbachia distributes in both somatic and germ line tissues in Ae. albopictus (Dobson et al., 1999). We further show that only wAlbB presents in midgut, salivary gland and fatbody while wAlbA was detected in only ovary. In this work, we focus on the above three somatic tissues because midgut and salivary gland are the two major sites for DENV to replicate and migrate through in order for mosquitoes to be infectious, and fatbody is the main immune organ to defend against foreign invaders (Beerntsen et al., 2000). Our results indicate that the Wolbachia infection in midgut and fatbody of Ae. albopictus may be too low to suppress viral replication in midgut and the subsequent dissemination into the other parts of mosquito body. This is supported by the lack of difference in the head infection rate between the HOU and HT1 strain. Comparison of wAlB infection between the dengue resistant 38 WB1 and the susceptible HOU strain shows that there is a general reduction in Wolbachia density in HOU strain and the magnitude of reduction in midgut is more than the other tissues. We also observed a significant higher Wolbachia density in salivary gland than in midgut in Ae. albopictus. This suggests that the future study should examine whether there is Wolbachiamediated resistance to DENV in salivary gland or saliva. We observe a linear negative correlation between Wolbachia density and genome copy of DENV-2 in Aa23 cell line. Based on the model (y = −0.004x+3.847), Wolbachia density at 961.8 wsp/actin is required to completely clean DENV in Aa23 cells. With the observed Wolbachia copy in midgut (0.3 wsp/actin) and salivary gland (5.3 wsp/actin) of Ae. albopictus HOU strain, Wolbachia would have no inhibition to DENV-2 in midgut and reduced DENV-2 by 0.6% in salivary gland in this mosquito species. However, it is possible that our model will be more appropriate to be used in the in vitro system. Wolbachia strains, host cells or tissues and environment may influence the viral inhibition, resulting in a modified linear correlation. Even that, a general pattern of Wolbachia density dependent viral inhibition should present in different systems. When wMelPop-CLA was introduced into two Ae. albopictus cell lines, RML12 and C6/36, the cell line with a high Wolbachia density (C6/36) shows a strong inhibition to DENV-2 (Frentiu et al., 2010). In D. simulans, Wolbachia strains that grow to high density provide the highest protection from virus infection (Osborne et al., 2009). All these indicate that Wolbachia density-dependent viral inhibition occurs for both native and non-native Wolbachia and cross different host cell types. The linear negative correlation between Wolbachia density and dengue copy could lead to a simple hypothesis, in which Wolbachia secretes antimicrobial effector molecules into a host cell to directly inhibit pathogens. Such effectors should have a broad-spectrum activity against a 39 variety of pathogens, including virus, plasmodium and worm. Consistently, a bacterium was recently reported to produce ROS and induce resistance to malaria parasites in Anopheles gambiae mosquitoes (Cirimotich et al., 2011). The Type IV Secretion System, which presents within Wolbachia (Masui et al., 2000, Wu et al., 2004), was reported to secret different factors into host to mediate interactions between the other intracellular bacteria and their hosts (Stein et al., 2000). Our preliminary studies show that induction of DEFD expression occurs in Wolbachia-density dependent manner, providing us support to further test this hypothesis. Currently there are two major modes to explain the mechanisms of Wolbachia-mediated viral interference (Teixeira et al., 2008, Rances et al., 2012). The first is Wolbachia boosts mosquito basal immunity against viral infection, while the second is that Wolbachia and virus compete for the same host resource. It appears that Wolbachia density dependent viral inhibition could be explained by both modes. In the first mode, a high Wolbachia density could cause high oxidative stress in host, leading to production of more ROS for a local antiviral immune response. The low Wolbachia density in somatic tissues of Ae. albopictus is consistent with the previous observation that Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides in Ae. albopictus(Blagrove et al., 2012). In the second mode, more Wolbachia could lead to less host resource for virus. While it appears to be straightforward for the second mode to fit the density-dependent data, it will be challenging to identify the common host components needed by both Wolbachia and a variety of pathogens. Our results indicate cell lines could be used as a simple in vitro system to study the mechanism of Wolbachia-mediated viral interference. DENV was strongly inhibited in Wolbachia-infected Aa23 and Aag-2 cells. Recent studies identified hundreds of host proteins that are required for DENV-2 propagation in cell lines (Sessions et al., 2009). Evidence indicates 40 that these host proteins form dengue-associated protein interaction networks, consisting of highly interconnected proteins with closely related functions in each of replication/transcription/translation, immunity, transport and metabolism (Guo et al., 2010). Wolbachia could perturb the above network to influence dengue infection. The cell culture provides us an ideal tool to study the interaction of Wolbachia with these dengue host proteins/networks. In the field, a difference in Wolbachia density in mosquito vectors may directly influence the disease transmission. Wolbachia density was reported to differ largely among Ae. albopictus collected from the field in different locations in Thailand (Ahantarig et al., 2008). It is still unknown to what extent this will affect the vector status and vector competence of Ae. albopictus. However, conflicting results were reported on the relative susceptibility of Ae. albopictus versus Ae. aegypti to oral DENV infection (Lambrechts et al., 2010, Gratz, 2004). Some studies show that Ae. albopictus is more susceptible to DENV than Ae. aegypti, while the others show opposite results (Lambrechts et al., 2010, Rosen et al., 1985, Chen et al., 1993, Jumali et al., 1979, Vazeille et al., 2003). It was also reported a significant increase of susceptibility to DENV in Ae. albopictus with increasing generations in the laboratory (Lambrechts et al., 2010, Vazeille et al., 2003). Although generally considered a secondary vector of dengue, Ae. albopictus was the primary cause of dengue epidemics in a number of areas (Gratz, 2004, Hotta, 1998, Kobayashi et al., 2002, Metselaar et al., 1980, Effler et al., 2005). In China, Ae. albopictus has long been considered the primary dengue vector. Dengue epidemics frequently occurred without Ae. aegypti, with 22,122 new infections, including the fatal dengue hemorrhagic fever, recorded in single year (Gratz, 2004, Jin and Li, 2008, Kan, 1997). All the above suggest vector 41 competence and vector status of Ae. albopictus can vary in different locations. Future work should study the impacts of Wolbachia on vector status and vector competence of Ae. albopictus. Our results could provide important implications for the future vector-borne disease control. First, novel control strategies can be developed to manually increase Wolbachia density in mosquitoes naturally carrying this bacterium. This could lead to block pathogen transmission to human but without a need to eradicate these mosquito species. Second, it is still unknown whether Wolbachia can persistently maintain infection at a level that effectively induces complete resistance to DENV in the transinfected Ae. aegypti. We could not exclude the possibility that adaption of a recent Wolbachia to a new host will finally lead to a low Wolbachia density, resulting in that what we see today in Ae. albopictus will be what happens tomorrow in those transinfected Ae. aegypti. 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(2004) Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol, 2, E69. XI, Z., KHOO, C. C. & DOBSON, S. L. (2005) Wolbachia establishment and invasion in an Aedes aegypti laboratory population. Science, 310, 326-8. 48 Chapter 3: Wolbachia infection inhibits multiple stages of Dengue life cycle in mosquito Aedes aegypti cells Introduction Dengue viruses (DENV), a member of the family Flaviviridae, is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. DENV infection is a major public health problem in the world, approximately 2.5 billion people are now at risk of dengue(WHO, 2012). DENV is transmitted to humans by Aedes mosquitoes. Currently, effective vaccines and antiviral therapies to prevent or treat DENV infections are unavailable. Vector control is the primary intervention method, but this tool is failing to prevent the global spread of dengue (Guzman et al., 2010). Innovative approaches are needed to control vector populations. DENV is an enveloped positive-strand RNA virus with genome of approximately 11 kilobases. The DENV genome has a single open reading frame which encodes three structural proteins ( capsid (C), membrane(M) and envelope (E) protein), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A and NS5). The infection process begins with the binding of the virions to the cellular receptors on the surface of susceptible cells. After receptor binding, DENV enters cells by receptor-mediated endocytosis. The acidification of endosome lumen promotes fusion of the viral membrane with the endosome membrane, thus delivering nucleocapsid into the cell cytoplasm. Following uncoating of the nucleocapsid in the cell cytoplasm, the viral RNA replication and translation start. Immature virions assembly occurs on the surface of the endoplasmic reticulum (ER) when the newly formed nucleocapsids bud into the lumen of ER. Subsequently, the immature virions are transported through the trans-Golgi network (TGN), 49 where they mature and from infectious particles. Finally, the mature viruses are released from the cell by exocytosis. Wolbachia is a maternally transmitted intracellular symbiotic bacterium belonging to the α-proteobacteria. Wolbachia is believed to the most pervasive symbiont; it has been detected in most orders of insects and infects more than 60% of all insect species worldwide (Hilgenboecker et al., 2008). Wolbachia has been placed at the frontline of novel approaches to control Dengue infection. Through the cytoplasmic incompatibility (CI) mechanism(Werren, 1997), Wolbachia can spread quickly in a mosquito population. Another advantage of using Wolbachia for disease control is its ability to induce resistance to DENV in mosquitoes(Walker et al., 2011, Bian et al., 2010). However, the mechanism that underlying resistance remains poorly understood. In addition, the impact of Wolbachia infection on DENV life cycle has not been demostrated. Our previous studies have shown that Wolbachia induce strong resistance to DENV serotype 2 (DENV-2) in Wolbachia-infected Ae. aegypti cell line W-Aag2. Here, we explored the impact of Wolbachia infection on DENV-2 life cycle in Wolbachia infected and uninfected mosquito cell lines, W-Aag2 and R-Aag2. Materials and Methods Cell culture and DENV-2 infection Ae. aegypti W-Aag2 cell line was generated by introducing Wolbachia into Aag2 cells using centrifugation enhanced (shell vial) technique as previously described (Lu et al., 2012). WAag2 and R-Aag2 cell line (derived from W-Aag2 cells through rifampicin treatment) were 50 maintained at 25 °C in Schneider's Drosophila Medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies), and were passaged at 1:5 dilution every 6-7 days. New Guinea C strain (NGC) of DENV-2 was used in this study. DENV-2 was grown in W-Aag2 and R-Aag2 cells as previously described (Sim and Dimopoulos, 2010). Briefly, cells were seeded in 48-well plates to a confluency of 80%. W-Aag2 and R-Aag2 monolayers were infected with DENV-2 at a desired multiplicity of infection (MOI) of DENV-2 in culture medium. Plates were incubated at 25°C for the duration of the experiment. DENV-2 binding assay DENV binding assay was carried out to characterize the attachment of DENV to W-Aag2 and R-Aag2 cells. Prior to the binding experiment, the culture medium was removed, and cells were washed with cold Schneider's Drosophila Medium. Subsequently, DENV-2 was incubated with the cells at MOI of 1 or 10 for 1 hour at 4 °C. The incubation medium was collected for measuring the titers of unbound viruses. Then, the cells were washed three times with cold PBS to remove the unbound viruses. 350µl buffer RLT (Qiagen) was added to each well for RNA extraction. The gene copies of bound DENV-2 were quantified by real-time PCR. RNA Extraction, cDNA synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) The total RNA was extracted from cell lines by RNeasy Mini Kit (Qiagen), and then the cDNA transcript was produced using QuantiTect reverse Transcription Kit (Qiagen). Real-time PCR was conducted using the QuantiTect SYBR Green PCR Kit (Qiagen) and ABI Prism 51 7900HT Sequence Detection System (Applied Biosystems). DENV-2 genomic RNA was measured by qRT-PCR using primers directed to NS5 gene (Molina-Cruz et al., 2005). Negative strand viral RNA was quantified using Tagged RT-PCR, a technique shown to be able to amplify specifically negative RNA by preventing false priming (Peyrefitte et al., 2003). The primer tagF (5′-CGGTCATGGTGGCGAATAAACAAGTAGAACAACCTGGTCCAT-3') was designed to contain the DENV-targeting sequence in its 3' end and a 19-mer-long nonDENV sequence in its 5' end. After RNA extraction, the RNAs were denatured at 65°C for 3min in the presence of 20pmol of tagF primer for the negative strand-specific reverse transcription. cDNA was synthesized without addition of the RT primer mix. Real-time PCR was performed with a forward primer Tag (5'-CGGTCATGGTGGCGAATAA-3') and a DENV-targeting reverse primer, as previously described (Molina-Cruz et al., 2005). The host ribosomal protein S6 (RPS6) gene was used to normalize cDNA template. DENV-2 RNA transfection W-Aag2 and R-Aag2 cells were plated in 48-well plates 24 hours prior to transfection and were approximately 70-80% confluent at the time of transfection (1×105 cells/well). DENV2 RNA was extracted from virus infected cell culture supernatant using RNeasy Mini Kit (Qiagen). The DNV infectious RNA was transfected using TransIT-mRNA Transfection Kit (Mirus) according to the manufacturer's instructions. Briefly, DNEV-2 RNA (0.5 μg) was incubated with 1 µl mRNA Boost Reagent and 1µl TranIT-mRNA Reagent in 26 µl Schneider's Drosophila Medium for 5 minutes and then was transferred to 70-80% confluent W-Aag2 and RAag2 cells in a 48 well plate (1×105 cells/well) containing 260 µl complete medium. Plaque assays for DENV-2 virus titration 52 The DENV-2 titers were measured by plaque assays, as previously reported(Das et al., 2007, Bian et al., 2010). Briefly, C6/36 cells were seeded in 48-well plates at a density of 4-8 × 104 cells/well and maintained for 2-3 days at 32°C in 5% CO2. The virus-containing culture medium was serially diluted and inoculated into C6/36 cell. After incubation for 5 d, plaque forming uinits (PFUs) were measured by the plates by peroxidase immunostaining, using mouse hyperimmune ascitic fluid (specific for DENV-2, CDC) as the primary antibody and a goat antimouse horseradish peroxidase(HRP) conjugate as the secondary antibody. Results Wolbachia inhibits the intracellular accumulation of DENV genome copies in Aag-2 cells We previously reported that Wolbachia wAlbB induces density-dependent inhibition to DENV in mosquito cells. To further investigate the profile of DENV production in Wolbachia transinfected W-Aag2 cell line and its aposymiotic R-Aag2 (derived from W-Aag2 cells through rifampicin treatment), cells were infected with DENV-2 at an MOI of 1 and the genome copies of DENV-2 were measured by qRT-PCR at 8 time points over the course of 9 days post-infection (dpi). Results (Figure 3.1) showed that DENV RNA level declined in both R-Aag2 and W-Aag2 cells by 1 dpi, then it increased daily in R-Aag2 cells, while DENV genomic RNA level did not significantly change after 1 dpi in W-Aag2 cells. At 2 hours post infection, the mean DENV genomic RNA copies in R-Aag2 cells ( 0.0072 DENV/RPS6) was three times higher than in WAag2 cells ( 0.0023 DENV/RPS6) (P<0.01, Student's t-Test), indicating Wolbachia infection may interfere with early events DENV-2 life cycle. On 1 dpi, the mean of DENV genomic RNA copies in R-Aag2 cells ( 0.096 DENV/RPS6) was 55 fold higher than in W-Aag2 cells ( 0.0017 53 DENV/RPS6). On 9 dpi, the number of viral copies in R-Aag2 cells ( 4.3 DENV/RPS6) was 1500 times higher than in W-Aag2 cells (0.0028 DENV/RPS6). Those results indicate that Wolbachia infection inhibits intracellular accumulation of DENV genome copies in Aag-2 cells. Wolbachia infection inhibits DENV binding to Aag-2 cells To better understand of the mechanism behind Wolbachia-mediated viral interference, we analyzed the DENV binding to mosquito cells. R-Aag2 and W-Aag2 cells were incubated with DENV at multiplicity of infection (MOI) 1 and MOI 10 for 60 min at 4 °C in order to avoid virus penetration. Subsequently, cells were washed three time with ice-cold medium. RNA copies of DENV that bound to cells were determined by qRT-PCR. The amount of DENV binding to WAag2 cells (0.0033 DENV/RPS6) is significantly lower than the amount bound to R-Aag2 cells (0.013 DENV/RPS6) at a MOI of 10 ( P<0.0001, Student's t-test) (Figure 3.2.A). Similar results were obtained when mosquito cells were exposed to a low level of input virus(MOI=1). The viral genomic copy number was only 0.0006 DENV/RPS6 in W-Aag2 cell line, as compared to 0.0019 DENV/RPS6 in R-Aag2 cells ( P<0.0001, Student's t-test) (Figure 3.2.A). We also carried out plaque assays to measure titers of unbound DENV in incubation medium. When mosquito cells were incubated with DENV at 10 MOI, unbound DENV titer in the incubation medium of W-Aag2 cells (3.4×106 PFU/ml) was significantly higher than it in the incubation medium of R-Aag2 cells (1.6 ×106 PFU/ml, P<0.05, Student's t-test) (Figure 3.2.B). Similar results were observed when cells were infected with DENV at a MOI of 1. We next performed the same assay at 25 °C. The viral genomic RNA level of W-Aag2 cells was 0.0036 DENV/RPS6 at 10 MOI, as compared to 0.022 DENV/RPS6 for the R-Aag2 cells ( P<0.0001, Student's t-test). A similar pattern was also observed at a MOI of 1, where the viral genomic RNA copy number in W-Aag2 cells was 0.00058 DENV/RPS6, as compared to 0.0038 54 DENV/RPS6 ( P<0.0001, Student's t-test). Those results indicate that Wolbachia infection inhibits DENV binding to mosquito cells at both high and low MOI. Wolbachia inhibits DENV replication in Aag-2 cells DENV negative sense antigenomic RNA is a hallmark of active DENV replication (Tuiskunen et al., 2010). To decipher whether Wolbachia infection interfere with DENV RNA replication, we first delivered infectious DENV RNA into cells by transfection, which could bypass the early events of DENV infection ( binding, entry, nuclocapsid relase, and uncoating). Then we conducted tagged RT-PCR to achieve negative strand-specific amplification (Peyrefitte et al., 2003). DENV-2 RNA (0.5 µg) was transfected into W-Aag2 and R-Aag2 cells (1 ×105 cells/well) in 48 well plates. Cells were sampled at 4 hour, 1 day, 3 day and 7 day post RNA transfection, followed by RNA extraction, reverse transcription and tag-PCR amplification. No significant differences were observed in the relative amount of DENV-2 total RNA between WAag2 and R-Aa2 cells at 4 hours post-transfection (P=0.13, Student's t test) (Figure 3.3.A), indicating comparable amounts of DENV-2 RNA were transfected into two cell lines. As shown in Figure 3.3. B, negative strand RNA levels in R-Aag2 cell line were significantly higher than in W-Aag2 cell line at day1, day3 and day7 post transfection (P<0.05, Student's t test). DENV titer of supernatant was measured by plaque assay at 5 days post transfection. Viral titer in the supernatant of R-Aag2 cells (6×106 PFU/ml) was significantly higher than in W-Aag2 cells (2.6×103 PFU/ml, P<0.05, Mann Whitney test) at 5 day post-transfection (Figure 3.3.C). Those results indicate that Wolbachia infection inhibits DENV replication. Wolbachia infection regulates expression of DENV binding proteins In mosquitoes, several DENV binding proteins have been described (Colpitts et al., 2011, Munoz et al., 2013, Kuadkitkan et al., 2010). To better understand the Wolbachia-induced 55 DENV inhibition, we performed qRT-PCR to measure the expression profiles of 19 reported DENV binding proteins in W-Aag2 and R-Aag2 cells. Using ΔΔCt based fold change calculations with change >2 or <2 fold as cut off levels (Schmittgen and Livak, 2008), we found 6 genes were down-regulated and 5 genes were up-regulated in W-Aag2 cells (Table 3.1.). Among those differentially expressed proteins, beta tubulin ,cadherin and actin have been shown to associated with viral entry (Munoz et al., 2013); histone, enlogation factor, actin and beta are thought to involved in viral transcription and translation. 56 Table 3.1: Differentially expressed DENV binding proteins in W-Aag2 and R-Aag2 cells. qRT-PCR was used to assess the expression of DENV binding proteins. RPS6 was used as an internal reference control. 2−ΔΔCT method was used to calculate fold change for each gene. A total of 11 genes were differentially expressed between Wolbachia-infected and uninfected cells with a fold change <-2 or >2. 57 Figure 3.1: DENV infection of W-Aag2 and R-Aag2 cells. Time course of DENV RNA level in Ae. aegypti W-Aag2 and R-Aag2 cells. The DENV genome copies were measured by qRTPCR. RPS6 was used as a host gene to normalize the data. Error bars are standard errors of the mean of three biological replicates. 58 **** **** * **** * **** Figure 3.2: Wolbachia infection inhibits DENV binding to mosquito cells. (A) Binding of DENV to W-Aag2 and R-Aag2 cells at 4 °C. W-Aag2 and R-Aag2 were incubated with DENV at 10 MOI and 1 MOI for 60 min at 4 °C. Virus-cell binding was measured qRT-PCR analysis, and normalized by RPS6. (B) Unbound of DENV to W-Aag2 and R- Aag2 cells at 4 °C. After incubation, took the DENV incubation medium out, followed by plaque assay to check the virus titer of unbound virus. (C) W-Aag2 and R-Aag2 were incubated with DENV at 10 MOI and 1 MOI for 60 min at 25 °C. Viral genomic copies were measured by qRT-PCR, and normalized by RPS6. Line indicate the mean of 8 replicates *, P<0.05 ; ***, P<0.001; ****, p<0.0001in Student's t-Test. 59 B A C * Figure 3.3: Wolbachia infection inhibits DENV replication in mosquito cells. (A) The relative copies of DENV-2 RNA in W-Aag2 and R-Aag2 cells at 4 hours post-transfection. (B) DENV-2 negative strand RNA levels in W-Aag2 and R-Aag2 cells after DENV-2 RNA transfection. Equivalent amount of purified DENV-2 RNA were transfected into cells, cells were sampled at 4 hour, 1 day, 3 day and 7 day post RNA transfection. Negative strand RNA were measured by Tag-PCR. *, P<0.05 in Student's t-Test. (C) DENV-2 titers in W-Aag2 and R-Aag2 cells supernatant at 5 days post DENV-2 RNA transfection. *, P<0.05 in Mann Whitney test. 60 Discussion DENV is the most critical arboviral pathogen, over 2.5 billion people are at risk of DENV infection. Currently, no effective vaccines and antiviral therapies are available, making the development of novel DENV control approaches an important global public health priority. Wolbachia-based dengue control has emerged as a promising strategy because it provide a solution that is sustainable, inexpensive and green. Understanding the mechanism underlying the Wolbachia-mediated DENV resistance will facilitate the use of Wolbachia-based strategies to block the transmission of dengue viruses by mosquitoes. We have previously reported Wolbachia can induce resistance to DENV in Ae. aegypti in a density-dependent manner, and in this report, we further expand this research to investigate the effect of Wolbachia infection in DENV replication cycle. Here we show that Wolbachia is able to inhibit the intracellular accumulation of DENV genomic RNA in Ae. aegypti mosquito cells. We also demonstrate that Wolbachia infection inhibits DENV binding and lowers the negative strand RNA level. The first step of DENV infection is binding. Direct binding assays were carried out at low temperature (4°C ) to avoid viral entry (Salas-Benito and Del, 1997). We show that there are less DENV particles attached to Wolbachia infected W-Aag2 than to uninfected R-Aag2 at both high and low MOI. The amount of DENV bound to W-Aag2 is 3.34 times lower than to R-Aag2 at 10 MOI. Similar results were observed when cells incubated with DENV at 1 MOI, the amount of DENV bound to W-Aag2 is 3.18 times lower than to R-Aag2. Both results indicate Wolbachia infection inhibits DENV attachment to mosquito cells. Inhibition of viral binding might be caused by regulation of DENV receptors expression(Breiner et al., 2001), modification of DENV cellular receptors ( e.g. Glycosylation; Phosphorylation; Alkylation, etc)(Lee et al., 1999, Lok et 61 al., 2008) or pre-saturation of DENV receptors (Chin et al., 2007) by Wolbachia-related small molecules. The early phase of viral RNA synthesis begins in 30 minutes after infection. We also assayed the DENV binding at 25°C for 60 minutes to investigate whether Wolbachia inhibits viral early events and RNA synthesis. We found the viral genomic RNA levels in W-Aag2 cells were significantly lower than in R-Aag2 cells at both 10 MOI and 1 MOI (6.36 fold and 6.51 fold, respectively), suggesting Wolbachia may also interfere with other viral early events (entry, nuclocapsid relase, and uncoating) and/or viral replication. The presence of negative-sense RNA is a hallmark of DENV replication within mosquito cells, but detection and quantization of negative strand RNA is complicated by false-priming in regular qRT RCR. Tag qRT-PCR is a strand-specific amplification method which can accurately reflect amounts of a specific RNA strand(Tuiskunen et al., 2010). Using DENV-2 infectious RNA transfection, the viral binding ,entry and uncoating were bypassed. Since the time for de novo DENV-2 production in Aag-2 cells is 1 day(Sim and Dimopoulos, 2010), the negative strand RNA at 1 day post-transfection should come only from the initial round of infection. The relative negative sense RNA level in R-Aag2 cells (0.069 DENV/RPS6) was 2.65 times higher than in W-Aag2 cells (0.026DENV/RPS6) at 1 day post-transfection. Also, DENV titer in the supernatant of R-Aag2 cells (6×106 PFU/ml) was 2300 times higher than in W-Aag2 cells (2.6×103 PFU/ml). Because the difference between R-Aag2 and W-Aag2 cell line appears to be much more pronounced in viral titers than negative strand RNA levels, it is possible that Wolbachia interferes with downstream steps of RNA accumulation (for example, viral translation, assembly and trans-Golgi transport) as well. 62 After viral RNA replication, DENV RNA must go through translation, assembly, and trans-Golgi transport to generate new infectious virions. Viral translation, assembly and maturation occur in ER and Golgi apparatus(Yacoub et al., 2013). To facilitate replication DENV modifies ER membranes (Welsch et al., 2009). Interestingly, Wolbachia has also been found in the ER and Golgi-related vesicles (Cho et al., 2011, Voronin et al., 2004), which also suggests that Wolbachia could interfere with DENV translation, assembly and maturation by competition for space and host resources. Several DENV binding proteins have been identified by protein-protein interaction assays. We measured the expression profile of 19 previously reported DENV binding proteins (Colpitts et al., 2011, Munoz et al., 2013), and found 11genes were differentially expressed in mosquito cells in response to Wolbachia infection. Among those genes, histone 4, histone 2A, beta tubulin, elongation factor-1 alpha, and actin were down-regulated by Wolbachia infection. Histone 4 is the most significantly down-regulated gene ( -24.5 fold). DENV capsid protein has been found to bind and co-localize with histones, though the reasons for this are not clear(Colpitts et al., 2011). It has been hypothesized that DENV capsid protein may bind to histone proteins, preventing the inhibition of viral transcription and aiding in viral replication(Colpitts et al., 2011). Wolbachia infection also down-regulates elongation factor 1-alpha (-4.23 fold), which binds viral RNA and proteins( E, NS2A and NS4B) to aid in viral replication or translation (Blackwell and Brinton, 1997). In addition, two cytoskeleton genes ( actin and beta tubulin) are downregulated by Wolbachia infection. Actin has been shown to associated with viral endocytosis and replication (Acosta et al., 2008). It is reported that tubulin-like protein binding dengue envelope protein in mosquito cells, indicating tubulin could be a viral receptor for DENV entry (Chee and AbuBakar, 2004). Moreover, tubulin is required for Sendai virus and hepatitis C virus replication 63 (Lai et al., 2008, Ogino et al., 2001). We also found that the expression of PI3 kinase, cadherin, myosin, kinectin, and enolase were up-regulated in response to Wolbachia infection. It is likely that Wolbachia may up-regulate some host genes, which are down-regulated during DENV infection. For example, DENV infection down-regulates the expression of cadherin to increase permeability of human primary endothelial cells (Ong et al., 2013). In summary, we have shown that Wolbachia can inhibit DENV binding at both high and low MOI. Moreover, we have also provided evidence that Wolbachia is able to lower negative strand viral RNA levels. Further studies will focus on identifying DENV cellular receptors associated with Wolbachia infection and the mechanism underlying Wolbachia-mediated DENV binding inhibition. 64 REFERENCES 65 REFERENCES ACOSTA, E. G., CASTILLA, V. & DAMONTE, E. B. (2008) Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrinmediated endocytosis. 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Curr Opin Infect Dis, 26, 284-9. 69 Chapter 4: Identification of Wolbachia-mediated Host Factors Involved in Resistance to Dengue Virus Infection Introduction With more than one-third of the world’s population living in areas at risk for transmission, dengue infection is among the most important reemerging infectious diseases in the tropics and subtropics. WHO estimates that 50 to 100 million cases with 22,000 deaths (mostly children) occur annually (WHO, 2014). Neither vaccine nor effective chemotherapy is currently available for dengue infection in humans; prevention of human cases relies almost exclusively on control of the mosquito vectors(WHO, 2012). The causative agent of dengue infection is the Dengue Virus (DENV), a member in the family Flaviviridae. DENV is an enveloped positive-sense RNA virus. The viral genomic RNA is approximate 11 kb in length. Wolbachia is a maternally transmitted intracellular symbiotic bacterium belonging to the α-proteobacteria. Wolbachia is believed to the most pervasive symbiont; it has been detected in most orders of insects and infects more than 60% of all insect species worldwide (Hilgenboecker et al., 2008). Wolbachia is mainly localized in the reproductive tissues of arthropods and can induce host reproductive abnormalities including cytoplasmic incompatibility (CI), parthenogenesis and male-killing. CI between infected males and uninfected females, or females harboring a different Wolbachia type can cause early embryo death, which reduces egg hatching (Zabalou, et al., 2004). Since uninfected males can successfully mate with infected females, Wolbachia can spread quickly in a population. Wolbachia has recently been shown to confer resistance against pathogen infection in insects (Bian et al., 2010, Walker et al., 2011, Bian et al., 2013). Presently, Wolbachia-based control strategies merit great attention, because it could 70 provide a sustainable, economical, and environmentally friendly solution to prevent vector-borne diseases. The underlying mechanisms of Wolbachia-mediated DENV resistance are unclear, however, two mechanisms have been proposed: host immune activation, and metabolic competition. Given both Wolbachia and DENV have relatively compact genomes (Saridaki and Bourtzis, 2010), they probably require a large number of host factors to survive. Host factors participate in most steps in the DENV life cycle, including entry, viral RNA replication, translation, assembly and maturation (Ahlquist et al., 2003). Meanwhile, Wolbachia infection has been shown to have a profound influence on the expression of host genes, including some immunity and reduction-oxidation (redox) pathway genes, which can regulate DENV replication (Hughes et al., 2011, Pan et al., 2012, Brennan et al., 2008). Thus, we hypothesized that Wolbachia-associated changes in expression of DENV host factors also contribute to DENV resistance. Mosquito cell lines have been widely used as an in vitro system to dissect the immune responses and molecular interactions between microbe and host (Brennan et al., 2008, SalasBenito and Del, 1997). Using the Ae. aegypti cell line system could avoid the physiological complexity of whole mosquitoes and provide a convenient and sensitive tool to study the interactions between Wolbachia and dengue virus in the host. In this work, we used a Wolbachia infected W-Aag2 cell line to study the mechanism underlying the Wolbachia-mediated resistance to DENV. A total of 16 DENV host factors have been shown to be up-regulated using microarray and real-time PCR analyses. Functional RNA interference (RNAi) screen revealed that eleven DENV host factors may be involved in Wolbachia-induced resistance to DENV. 71 Materials and Methods Cell-Culture Maintenance and DENV-2 infection The Ae. aegypti Aag2 cell line was cultured as previously described. Those cells were maintained in Schneider's Drosophila Medium (Invitrogen) supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS). Ae. aegypti W-Aag2 cell line was generated by introducing Wolbachia into Aag2 cells using centrifugation enhanced (shell vial) technique as previously described (Lu et al., 2012). W-Aag2 and R-Aag2 cell line (derived from W-Aag2 cells through rifampicin treatment) were maintained at 25 °C in Schneider's Drosophila Medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies), and were passaged at 1:5 dilution every 6-7 days. The Ae. albopictus cell line C6/36 was grown in Eagle's minimal essential medium (MEM) with 10% (vol/vol) heat-inactivated FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 100 μM nonessential amino acids at 32 °C and 5% (vol/vol) CO2. New Guinea C strain (NGC) of DENV-2 was used in this study. DENV-2 was grown in W-Aag2 and R-Aag2 cells as previously described (Sim and Dimopoulos, 2010). Briefly, cells were seeded in 48-well plates to a confluency of 80%. W-Aag2 and R-Aag2 monolayers were infected with DENV-2 at a desired multiplicity of infection (MOI) of DENV-2 in culture medium. Plates were incubated at 25°C for the duration of the experiment. Microarray analysis W-Aag2 and R-Aag2 cells were lysed by the addition of Buffer RLT(Qiagen). Total RNA was extracted using RNeasy Mini Kit (QIAGEN), according to the manufacturer's instruction. Transcription assays were conducted as reported previously (Nene et al., 2007) with 72 an Ae. aegypti full genome Agilent-based microarray platform. Briefly, 2-3 μg total RNA was used for probe synthesis of cy3- and cy5-labeled cRNA. Hybridizations were conducted using an In Situ Hybridization kit (Agilent Technologies) at 65 °C. Hybridization intensities were measured using an Axon GenePix 4000B scanner, and images were analyzed with GenePix software. The expression data were processed and analyzed as described previously (Nene et al., 2007). RNA Extraction, cDNA synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) The total RNA was extracted from cell lines by RNeasy Mini Kit (Qiagen) and then the cDNA transcript was produced using QuantiTect reverse Transcription Kit (Qiagen). Real-time PCR was conducted using the QuantiTect SYBR Green PCR Kit (Qiagen) and ABI Prism 7900HT Sequence Detection System (Applied Biosystems). DENV-2 genomic RNA was measured by qRT-PCR using primers directed to NS5 gene (Molina-Cruz et al., 2005). The ribosomal protein S6 (RPS6) gene was used for normalization of cDNA templates. RNAi-mediated gene silencing The dsRNA was synthesized in vitro using the Megascript T7 high-yield transcription kit (Ambion). Transfection of dsRNA was carried out using Attractene Transfection Reagent (Qiagen) according to manufacturer's instruction. Briefly, W-Aag2 cells were seeded in 48-well plates 24 hours prior to transfection. 1µg of dsRNA was incubated with 3.5 µl Attractene Transfection Reagent in 50 µl Schneider's Drosophila Medium for 10-15 minutes at room temperature and then was transferred to each well. 4 days after transfection, W-Aag2 cells were infected with DENV-2 at a multiplicity of infection (MOI) of 5 in infection medium (Schneider's Drosophila Medium with 2% FBS and 1% penicillin/streptomycin). At 12 hours post DENV 73 infection, dsRNA transfection was repeated. 4.5 days later, the supernatant was collected for detecting infectious viral particles by plaque assay, and W-Aag2 cells were harvested for measuring the DENV-2 RNA levels and Wolbachia densities. Plaque assay The DENV-2 titers were measured by plaque assays, as previously reported(Das et al., 2007, Bian et al., 2010). Briefly, C6/36 cells were seeded in 48-well plates at a density of 4-8 × 104 cells/well and maintained for 2-3 days at 32°C in 5% CO2. The virus-containing culture medium was serially diluted and inoculated into C6/36 cell. After incubation for 5 d, plaque forming uinits (PFUs) were measured from the plates by peroxidase immunostaining, using mouse hyperimmune ascitic fluid (specific for DENV-2, CDC) as the primary antibody and a goat anti-mouse horseradish peroxidase(HRP) conjugate as the secondary antibody. Results Differentially expressed profiles in Wolbachia infected and uninfected Aedes aegypti cells To identify differentially expressed genes in mosquito cells in response to Wolbachia infection; we conducted microarray assays to compare transcript repertoires in Wolbachia infected Ae. aegypti Aag2 cells(W-Aag2) with those of corresponding Rifampicin-cured cells RAag2. Functional classification of significantly regulated genes was analyzed (Fig.4.1). Wolbachia infection significantly regulated 1860 genes in the cell line (910 up-regulated and 950 down-regulated). We observed that among the up-regulated genes, the most highly regulated Wolbachia-responsive transcripts were related to redox/stress/mitochondrion (R/S/M) group (128 genes), except for genes belonging to diverse functions (267 genes). Genes within the R/S/M group represented 14.1 percent of all highly regulated genes with predicted functions. The third 74 largest group included 123 genes with unknown function, followed by 114 genes involved in metabolism. Additionally, 56 genes were identified that are involved in immunity function. Among the down-regulated genes, the largest group contained 107 genes involved in replication/transcription/translation (R/T/T), except for genes with unknown or diverse functions. The infection of Wolbachia significantly up-regulated several characterized immunity genes in W-Aag2 cells. Both Toll and IMD pathway genes were activated in W-Aag2 cells, including Gram-negative bacteria binding protein A1 (GNBPA1), Gram-negative binding protein B1 (GNBPB1), Relish-like protein 1A (Rel1A), and IMD pathway signaling NF-kappaB Relishlike transcription factor Rel2. Transcripts of the antimicrobial peptide genes defensin A, defensin D, cecropin E and cecropin N were highly elevated in W-Aag2 cells as well. We also found a number of antioxidant transcripts were highly regulated in our microarray analysis, including glutathione peroxidase (AAEL012069), Cu,Zn-superoxide dismutase (CuZnSOD; AAEL011498), thioredoxin peroxidase (AAEL013528 and AAEL014548) and manganese superoxide dismutase ((MnSOD; AAEL004823). Moreover, both ferritin light chain(AAEL004335, AAEL010396, AAEL007383) and heavy chain(AAEL010393, AAEL007385) genes were up-regulated by Wolbachia in W-Aag2 cells as well. Select candidate Wolbachia-mediated host factors involved in resistance to DENV infection DENV is a positive-strand RNA virus that encodes 10 proteins. The small coding capacity of DENV demands that the virus use the host machinery for most, if not all, steps of its life cycle (Ahlquist et al., 2003). A large number of DENV host factors have been identified by using genomics techniques, computational approaches and protein-based techniques (Bonizzoni et al., 2012, Sim et al., 2012, Sim and Dimopoulos, 2010, Guo et al., 2010, Sessions et al., 2009, Doolittle and Gomez, 2011, Xi et al., 2008). Among those reported DENV host factors, some 75 were also regulated by Wolbachia. In order to further understand the mechanism of Wolbachiamediated Dengue inhibition, we selected 40 DENV host factors, which were significantly upregulated by Wolbachia in the microarray study (Table 4.1.). To verify the results of microarray analysis, the expression profiles of 40 candidate genes in W-Aag2 and R-Aag2 cells were measured by qRT-PCR. Using 2-ΔΔCt method with fold change >2 as cut off level, a total of 16 genes were selected as candidate Wolbachia-mediated host factors involved in resistance to DENV infection (Table 4.1). Those candidate host factors include 5 genes belonging to R/S/M group, 4 genes with diverse functions, 2 immunity genes, 2 metabolism genes, 2 R/T/T genes and a transport gene. Identify Wolbachia-associated DENV host factors by RNAi screen To test whether differentially regulated host factors are involved in Wolbachia-mediated DENV inhibition, we performed RNAi screen in W-Aag2 cells. At 4 days post dsRNA transfection, cells were infected with DENV-2 at a MOI of 5. At day 5 post viral infection, cell supernatants were collected for detecting infectious viral particles by plaque assay, and W-Aag2 cells were harvested for determining the DENV-2 RNA levels and Wolbachia densities (Figure 4.2. A). Gene knock-down efficiency was evaluated using ∆∆CT method (Haimes and Kelley, 2010) to determine relative gene expression from qRT-PCR data with PRS6 as a reference gene. Except silencing of GPI and IMPase with 70.5% and 82.2% konck-down, dsRNA knock-down efficiency of all other genes were above 90%. Comparing to dsGFP treatment, silencing of 10 genes, including 4 diverse function genes (STX4, Ferritin G, AKR and IMPase), 2 immunity genes (GNBPA1, PGRP-lc), 2 R/S/M genes (NADH-UQRP, ERCC),1 R/T/T gene (SFRS) and 1 metabolism gene (GPI), in W-Aag2 cells significantly increased DENV-2 genomic RNA levels (P< 0.05, Student's t-Test) (Figure 4.2. C). 76 To determine whether those candidate DENV host factors affected the generation of new infectious virions in W-Aag2, we assessed viral titers in cell medium by plaque assay after RNAi transcript depletion. Trends toward increased viral titers were observed in all RNAi silencing groups as compared with mock control (dsGFP), except for knock-down of STX4 and ATPD. DENV titers were increased significantly by silencing of Ferritin G, IMPase, MRPs-S18A, and ERCC as compared to the mock control (P<0.01, P<0.01, P<0.05 and P<0.05, respectively in Mann-Whitney U test) (Fig 4.2.D). Except for silencing of MRPs-S18A, knock-down of Ferritin G, IMPase, and ERCC resulted in increased viral genomic RNA levels (Fig 4.2.C). DENV inhibition induced by Wolbachia is density-dependent, thus the increased level of DENV infection could be caused by a decrease in Wolbachia density (Lu et al., 2012, Osborne et al., 2012). We also measured Wolbachia density after RNAi screen. As shown in Fig 4.2.E, Wolbachia densities in W-Aag2 cells with silencing of ERCC, NADH-UQOR, HSP and ATPD were significantly decreased (P< 0.05, Student's t-Test) compared with mock-depleted (dsGFP) group, indicating that decreased Wolbachia densities may contribute to increased viral loads by knock-down of ERCC and NADH-UQOR. Taken together, these results revealed eleven potential host factors involved in Wolbachia-induced DENV resistance. Those host factors include 4 diverse function genes (STX4, Ferritin G, AKR and IMPase), 2 immunity genes (GNBPA1, PGRP-lc), 3R/S/M genes (MRPs-S18A,NADH-UQRP and ERCC),1 R/T/T gene (SFRS) and 1 metabolism gene (GPI), 77 AAEL009471 Microarray qRT-PCR Functional average average fold group fold change change 8.55 24.77 D Gene name Reference Bonizzoni et al.,2012 AAEL004335 AAEL004086 2.35 2.09 AAEL004566 AAEL013943 AAEL010119 AAEL010119 2.42 2.04 1.99 1.99 AAEL013338 1.8 -1.41 D AAEL006948 3.91 -2.31 D syntaxin 4 knolle (STX4) secreted ferritin G subunit precursor, aldo-keto reductase (AKR) myo inositol monophosphatase (IMPase) mediator complex, 100kD-subunit ER-derived vesicles protein ERV14 ER-derived vesicles protein ERV14 lethal(2)essential for life protein, l2efl tomosyn AAEL000541 1.83 -2.39 D fasciclin, putative AAEL009149 2.16 -2.97 D kinectin, putative AAEL013830 3.67 -3.43 D AAEL007626 4.11 6.06 I AAEL009474 2.89 3.96 I bmp-induced factor gram-negative bacteria binding protein A1(GNBPA1) peptidoglycan recognition proteinlc isoform (PGRP-lc) 7.44 D 2.04 D 2.01 1.8 -1.23 -1.23 D D D D Sim et al.,2010 Sim et al.,2010 Sim et al.,2010 Sim et al.,2010 Bonizzoni et al.,2012 Bonizzoni et al.,2012 Sim et al.,2010, Bonizzoni et al.,2012 Doolittle et al.,2011 Sim et al.,2010, Bonizzoni et al.,2012 Sim et al.,2010, Bonizzoni et al.,2012 Doolittle et al.,2011 Sim et al.,2010 Sim et al.,2010 Table 4.1: Candidate Wolbachia-mediated host factors involved in resistance to DENV infection. We first selected 40 DENV reported host factors, which were significantly up-regulated by Wolbachia, using gene expression microarray data. After further analysis of relative gene expression using qRT-PCR and 2-ΔΔCt method (fold-change cut-off=2), 16 host molecules (shaded rows) were selected as candidates. Functional group abbreviations: D, diverse functions; I, immunity; M, metabolism; R/S/M, redox, stress and mitochondrion; R/T/T, replication, transcription, and translation; TRP, transport. 78 Table 4.1 (cont'd) AAEL003294 Microarray qRT-PCR Functional average average fold group fold change change 2.31 1.8 I Gene name Reference fibrinogen and fibronectin Bonizzoni et al.,2012 Sim et al.,2010,Sim et al.,2012 Sim et al.,2010 Sim et al.,2010 Sim et al.,2010, Bonizzoni et al.,2012 Sim et al.,2010 AAEL000611 1.82 1.14 I antibacterial peptide, putative AAEL000621 AAEL009384 1.89 5.89 -2.73 I -4.72 I antibacterial peptide, putative fibrinogen and fibronectin AAEL003857 3.06 -4.8 I Defensin AAEL009237 10.07 5.46 M glycoside hydrolases (GHs) glucose-6-phosphate isomerase (GPI) AAEL012994 2.76 3.51 M AAEL002542 2.41 1.56 M triosephosphate isomerase AAEL011233 2.51 1.38 M SM protein G AAEL011356 7.04 -4.02 M AAEL002783 1.86 N/A R/S/M AAEL008601 2.57 2.69 R/S/M AAEL007355 3 2.41 R/S/M alcohol dehydrogenase mitochondrial ribosomal protein, L37 mitochondrial ribosomal protein, L28 (MRPs-L28) mitochondrial ribosomal protein, S18A ( MRPs-S18A) 79 Sim et al.,2010 Sim et al.,2010, Bonizzoni et al.,2012, Xi et al., 2008 Sim et al.,2010, Bonizzoni et al.,2012 , Doolittle et al.,2011 Bonizzoni et al.,2012 Sim et al.,2010 Sim et al.,2010 Sim et al.,2010 Table 4.1 (cont'd) Microarray qRT-PCR average average fold fold change change Functional group Gene name AAEL007355 3 2.41 R/S/M AAEL013350 1.8 2.33 R/S/M AAEL005508 1.95 2.31 R/S/M AAEL013693 2.02 2.14 R/S/M AAEL002523 2.03 1.61 R/S/M AAEL011463 AAEL011752 4.56 5.69 1.1 R/S/M -1.98 R/S/M AAEL013790 1.77 -9.5 R/S/M AAEL001280 1.94 2.19 R/T/T AAEL006473 2.01 2.05 R/T/T mitochondrial ribosomal protein, S18A ( MRPs-S18A) heat shock protein 26kD, putative (HSP) nadh-ubiquinone oxidoreductase 24 kda subunit (NADH-UQOR) excision repair crosscomplementing 1 ercc1 (ERCC) mitochondrial inner membrane protein translocase cytochrome P450 glutathione-s-transferase theta, gst mitochondrial ribosomal protein, L50 mitochondrial ribosomal protein S15 (MRPs-S15) arginine/serine splicing factor (SFRS) AAEL010823 1.79 2.32 TRP atp synthase delta chain (ATPD) AAEL000435 2.85 -1.24 TRP AAEL014052 3.37 -1.65 TRP AAEL015067 3.32 -4.08 TRP THO complex, putative endoplasmic reticulum protein erp29 multidrug resistance protein 2 80 Reference Sim et al.,2010 Sim et al.,2010, Bonizzoni et al.,2012 Sim et al.,2010, Bonizzoni et al.,2012 Sim et al.,2010 Sim et al.,2010, Bonizzoni et al.,2012 Bonizzoni et al.,2012 Sim et al.,2010 Sim et al.,2010 Sim et al.,2010 Sim et al.,2010, Xi et al., 2008 Sim et al.,2010, Bonizzoni et al.,2012 Sim et al.,2010, Bonizzoni et al.,2012 Bonizzoni et al.,2012 Bonizzoni et al.,2012 Figure 4.1: Functional classification of differentially expressed genes in the Aag2 cell line in response to Wolbachia infection. The graph shows the functional class distributions of genes that are regulated by Wolbachia infection. 81 A B Figure 4.2: Characterizing Wolbachia-regulated DENV host factors by RNA interference (RNAi) screen. (A) Schematic of experimental design. (B) Knock-down efficiency for dsRNA treatment. Cells were sampled at 24 hours post dsRNA transfection. Gene silencing efficiency was assessed by ∆∆CT method with RPS6 as an endogenous reference gene. (C) Relative DENV-2 RNA copies in W-Aag2 cells treated with dsRNAs. W-Aag2 cells were infected DENV-2 at an MOI of 5 at day 4 post dsRNA transfection. Viral RNA was purified 5 days post infection. qRT-PCR was conducted using primers targeting NS5 gene. The DENV genomic RNA copy number was normalized with RPS6. Error bars represent standard error of 6 biological replicates. *, P<0.05; **,P<0.01; ***, P<0.001; ****, p<0.0001 in Student's t-Test comparing to GFP control. (D) Viral titer in the cell supernatant was measured after RNAi depletion of DENV host factors. dsRNA-mediated silencing of MYD88 was served as a positive control. Lines indicate the geometric mean of the four biological replicates.*, P<0.05; **,P<0.01 in Mann-Whitney U test.. (E) Wolbachia density in W-Aag2 cells after silencing of DENV host factors. Error bars represent standard error of 4 biological replicates, *, P<0.05 in Student's tTest comparing to GFP control 82 Figure 4.2 (cont'd) C D 83 Figure 4.2 (cont'd) E 84 Discussion The Wolbachia genome is extremely reduced; consequently, many biosynthetic pathways are absent in Wolbachia (Kondorosi et al., 2013). To facilitate intracellular infection, Wolbachia needs to interact with numerous host proteins. It has been believed that Wolbachia Type IV secretion system (T4SS) secretes various effector macromolecules into the cytoplasm to alter host gene expression and affect the physiology of host cells (Rances et al., 2008). DENV also requires a number of host proteins at multiple stages of its life cycles. The viral replication is coordinated by a complex of viral and host factors. Thus, we hypothesized that Wolbachia may affect DENV host factors that are important in DENV infection. By comparing our microarray and real-time PCR data of mosquito transcripts changes to Wolbachia infection, we selected 16 candidate DENV host factors, which could be targeted by Wolbachia and then involved in Wolbachia-induced DENV inhibition. Furthermore, we identified eleven potential Wolbachiamediated host factors involved in resistance to DENV infection with RNAi screen. Generally, the gene silencing effect could last from 5-7 days. Since W-Aag2 cells need to be grown for 9 days to observe phenotypes (Figure 4.2.A), we conducted dsRNA transfection twice to achieve long-term knock-down of target genes (McManus and Sharp, 2002). DENV has to go through multiple stages, including binding, fusion, translation, transcription, assembly, maturation and budding to reproduce itself and create new infectious virus particles (Rodenhuis-Zybert et al., 2010). As shown in Chapter 3, Wolbachia infection affects multiple steps of DENV life cycle. Interestingly, knock-down of seven genes (STX4, AKR, GNBPA1, PGRP-lc, NADH-UQRP, SFRS and GPI) resulted in significant increased viral RNA levels compared with mock control, but did not induce changes in viral titers. It is likely that these seven host factors act on stages required for the accumulation of viral RNA (for 85 example, binding, fusion, translation, and transcription), and the Wolbachia-induced inhibition to stages downstream of RNA accumulation (for example, inhibition of assembly, maturation, and budding) is powerful enough to mask the effect of elevated DENV genomic copies. In contrast, viral titers were increased significantly by RNAi depletion of MRPs-S18A , but no change was observed in DENV RNA copies with the same treatment, suggesting MRPs-S18A acts at stages downstream of viral RNA accumulation ( for example, assembly, maturation, budding) (Sessions et al., 2009). Mosquitoes, like all insects, use immune mechanisms, including Toll, IMD, autophagy, JAK-STAT, and siRNA pathways, to protect against viral infection (Ding, 2010, Bronkhorst and van Rij, 2014, Costa et al., 2009, Zambon et al., 2005, Lamiable and Imler, 2014). In accordance with our previous microarray data from Ae. aegyti mosquitoes (Pan et al., 2012), we also found a number of Wolbachia-responsive transcripts were related to immunity and R/S/M group in our cell microarray. Recently evidence has shown that Wolbachia infection activated mosquito basal immunity, resulting in the suppression of DENV replication (Xi et al., 2008). Moreover, our previous study reported that Wolbachia could induce reactive oxygen species (ROS)-dependent activation of the Toll pathway to control DENV in Ae. aegypti (Pan et al., 2012). In this study, we identified 2 immunity genes (GNBPA1, PGRP-lc), and 3 R/S/M genes (NADH-UQOR, ERCC and MRPs-S18A) as potential Wolbachia-mediated DENV host factors. Stable symbiotic interaction with Wolbachia in mosquito cells involves ROS generation and induction of antioxidant enzymes (Brennan LJ et al., 2008, Pan et al., 2011). ROS suppresses replication of Hepatitis C Virus in human hepatoma cells; whereas antioxidants tend to counter this suppression (Seronello et al., 2011). NADH-ubiquinone oxidoreductase ( NADH-UQOR) is an enzyme of the mitochondrial electron transport chain, and it is a major source of ROS (Galkin 86 and Brandt, 2005). In this work, knock-down of NADH-UQOR resulted in decreased Wolbachia densities and increased DENV RNA levels, suggesting ROS plays an important role in Wolbachia propagation and Wolbachia-mediated DENV inhibition. The female mosquito vector must take a blood meal in order to complete her life cycle. The blood meal provides a high level of iron that is required for egg development(Nichol et al., 2002). Although sufficient iron is present in the blood meal to provoke the formation of toxic free radicals, mosquitoes have developed mechanisms, partly by iron storage inside ferritin, that allow iron utilization while maintaining protection form iron-mediated oxidative stress (Geiser et al., 2006). Ferritin has also been shown to play an important role in host-pathogen interactions (Schaible and Kaufmann, 2004).The expression of ferritin heavy chains is under the control of the NF-kappaB signaling pathway in mammals (Bubici et al., 2006). It has been found recently that iron metabolism was influenced by the presence of Wolbachia in Ae. aegypti cells (Kremer et al., 2009). Iron is an essential element that is in limited supply in the cell, and it is also a highly toxic precursor of Reactive Oxygen Species (ROS). In accordance with previous work (Pan et al., 2011), we found genes involved in the iron/ferritin metabolism pathways were largely regulated by Wolbachia in mosquito cells. Furthermore, silencing of ferritin G protein resulted in elevated DENV RNA levels and viral titers, indicating ferritin plays an important role in Wolbachia-mediated viral resistance. In conclusion, we have shown eleven potential host factors associated with Wolbachiamediated DENV resistance. 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Furthermore, we observed a positive correlation between Wolbachia density and the expression of the antimicrobial peptide Denfesin D, indicating host immunity could contribute to Wolbachia-density dependent viral inhibition We then provided evidence that Wolbachia infection inhibited the intracellular accumulation of DENV genomic RNA in mosquito cells. DENV binding assay revealed that the attachment of virus particles was significantly inhibited by Wolbachia infection at both high and low multiplicity of infection (MOI). We also carried out a strand-specific tagged RT-PCR to compare the levels of the negative-strand RNA of DENV, a hallmark of viral replication, in Wolbachia infected and uninfected cells. The results showed that Wolbachia infection significantly decreased negative RNA levels in mosquito cells. Furthermore, we compared the gene expression profiles of identified DENV binding proteins between cells with and without Wolbachia infection, and found six genes were down-regulated and five genes were up-regulated in response to Wolbachia infection. Further studies should be conducted to study the molecular mechanisms of Wolbachia-induced inhibition to DENV binding and replication. 93 Figure 5.1: A current model of Wolbachia-mediated viral interference in Ae. aegypti. Modified from Pan et al., 2012 94 Microarray assays indicated that Wolbachia infection significantly regulated 1,860 genes in mosquito cells. In order to further understand the underlying mechanisms of Wolbachiamediated Dengue resistance, we selected 16 previously reported DENV host factors, which were up-regulated by Wolbachia infection. Functional RNA interference (RNAi) screen revealed that silencing of eleven DENV host factors resulted in increased DENV viral loads in Wolbachia infected mosquito cells. Those host factors include four genes with diverse function, three genes belonging to redox/stress/mitochondrion group, two immunity genes, a metabolism gene and a gene belonging to replication/transcription/translation group. In our previous work, we proposed a model of Wolbachia-mediated resistance to DENV that is focusing on reactive oxygen species (ROS) and immunity pathways (Pan et al., 2012). Based on the results generated from the above studies, we further improve this model to better interpret Wolbachia-mediated viral interference (Fig. 5.1). Wolbachia infection influences the expression of several DENV host factors in a density-dependent manner. Some of those host factors are directly related to the activation of immunity pathways, the level of ROS and the expression of antioxidant, thus our previous model could be used to explain the viral interference. In addition, infection with Wolbachia also influences the expression of host proteins involved in DENV life cycle. Given its compact genome, DENV is highly dependent on host cell's machinery to propagate (Sessions et al., 2009). Therefore, Wolbachia-induced changes in the expression of host proteins, which are essential for DENV replication, could also contribute to viral inhibition (Fig. 5.1). For example, Wolbachia infection down-regulates the expression elongation factor 1-alpha (-4.23 fold), which binds to the 3'-untranslated region of DENV RNA to aid in viral replication (De Nova-Ocampo et al., 2002). Future Directions 95 Wolbachia is emerging as a potential control agent to decrease transmission of vectorborne pathogens such as DENV(Cook et al., 2008, Moreira et al., 2009, Xi et al., 2005, Walker and Moreira, 2011, Bian et al., 2013) . Nevertheless, very little is known about the underlying mechanism of Wolbachia-mediated viral inhibition, limiting the capability of exploiting Wolbachia for vector control. A better understanding of interaction between Wolbachia and DENV in mosquito host will facilitate the current effort to eliminate dengue through Wolbachiabased strategies. Our results indicate Wolbachia is able to induce resistance to DENV in host genetic background of Ae. albopictus, and highlight the importance of Wolbachia density in Wolbachiamediated viral inhibition. The molecular mechanisms employed by the host to regulate Wolbachia density are not well understood. The within-host density of Wolbachia can be influenced by factors including insect age, temperatures, antibiotic treatment, length of symbiotic relationship, ROS and MicroRNAs (van Opijnen and Breeuwer, 1999, Unckless et al., 2009, Hussain et al., 2011, Bian et al., 2013). To block pathogen transmission to human, novel control strategies can be developed to increase Wolbachia density in mosquitoes carrying this bacterium. Consequently, there is a need to investigate how Wolbachia density is regulated by hosts and how the Wolbachia machinery controls its replication. Since the Drosophila genome-wide RNAi library is available for high-throughput screen (Sessions et al., 2009), the Wolbachia-infected Drosophila cell line can be used a simple model to study how insect hosts regulate Wolbachia density. Mosquito host factors participate in most stages of DENV life cycle (Ahlquist et al., 2003). Wolbachia could interact with those DENV host proteins to influence dengue infection. Knowledge of those DENV host factors affected by Wolbachia not only informs us the 96 molecular mechanisms exploited by Wolbachia to induce viral protection but also provides potential targets that could be pursued for dengue control and antiviral drug development. There are several future directions associated with the Wolbachia-mediated host factors involved in resistance to DENV infection. First, future work should study molecular mechanisms of action of those eleven host factors involved in Wolbachia-mediated DENV resistance. Second, genomewide RNAi screen could be carried out to uncover more host factors associated with this resistance. In addition, Wolbachia may down-regulate host factors required for DENV replication. However, we only investigated DENV host factors up-regulated in response to Wolbachia infection. Therefore, further study should be carried out to determine what are those DENV host factors down-regulated by Wolbachia and involved in Wolbachia-mediated antiviral protection. Wolbachia infection inhibits DENV binding and replication. Further studies should be focused on the molecular mechanisms of Wolbachia-mediated inhibition to viral attachment and replication. Proteomics analysis of virus-receptor interaction can be used to study how Wolbachia inhibits DENV binding. DENV replicon with Renilla luciferase reporter can be used to study the molecular basis of Wolbachia-mediated inhibition to viral replication (AlcarazEstrada et al., 2010). In addition, we only investigated the impact of Wolbachia infection on DENV binding and replication stages, it is important to determine whether Wolbachia inhibits other stages of DENV life cycle, especially translation, assembly and maturation. The DENV reporter viruses will facilitate studies on the impact of Wolbachia infection on DENV life cycle (Schoggins et al., 2012, Mattia et al., 2011). The Wolbachia-mediated antiviral protection varies depending on Wolbachia strain, infection type and host(Rainey et al., 2014). Theoretical modelling indicates that Wolbachia97 mediated protection has the potential to be a selective force on DENV evolution, perhaps resulting in increased virulence of DENV (Jones et al., 2011). Therefore, there is a clear need for studies of the impact of Wolbachia on DENV evolution in mosquito host. Wolbachia infected cell lines could be used as in vitro models for investigating the long-term effect of Wolbachia on DENV virulence. Genetic analysis of Wolbachia has been hindered not only by the challenge in manipulating this obligate intracellular bacterium, but also by the lack of efficient tools for transformation of Wolbachia. Generation of transgenic Wolbachia will not only help us better understand the mechanisms underlying several Wolbachia-induced phenotypes such as cytoplasmic incompatibility and antiviral protection, but also provide an approach to utilize Wolbachia as a gene driver to spread anti-pathogen genes into mosquito vectors (Marshall, 2009). 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