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Wolbachia is an endosymbiotic bacterium that can restrict the transmission of human pathogenic viruses by Aedes aegypti mosquitoes. Recent field trials have shown that dengue incidence is significantly reduced when Wolbachia is introgressed into the local Ae. aegypti population. Female Ae. aegypti are anautogenous and feed on human blood to produce viable eggs. Herein, we tested whether people who reside on Tri Nguyen Island (TNI), Vietnam developed antibodies to Wolbachia Surface Protein (WSP) following release of Wolbachia-infected Ae. aegypti, as a measure of exposure to Wolbachia. Paired blood samples were collected from 105 participants before and after mosquito releases and anti-WSP titres were measured by ELISA. We determined no change in anti-WSP titres after ~30 weeks of high levels of Wolbachia-Ae. aegypti on TNI. These data suggest that humans are not exposed to the major Wolbachia surface antigen, WSP, following introgression of Wolbachia-infected Ae. aegypti mosquitoes.

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.

The bacterial endosymbiont Wolbachia is a biocontrol tool that inhibits the ability of the Aedes aegypti mosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that when Wolbachia strains wMel or wAlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand how Wolbachia protects mosquitoes from viral infection. Here, we demonstrate that the Wolbachia strain wPip does not inhibit virus infection in Ae. aegypti. We have leveraged this novel finding, and a panel of Ae. aegypti lines carrying virus-inhibitory (wMel and wAlbB) and non-inhibitory (wPip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism of Wolbachia-mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain’s ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define how Wolbachia prevents Ae. aegypti from transmitting viruses., Author summary Dengue virus, transmitted by the Aedes aegypti mosquito, is one of the fastest-growing infectious diseases, causing an estimated 390 million human infections per year worldwide. Vaccines have limited efficacy and there are no approved therapeutics. This has driven the rise of novel vector control programs, in particular those that use the bacterium, Wolbachia, which prevents transmission of dengue and other human pathogenic viruses when stably introduced into Ae. aegypti populations. Although this is proving to be a highly effective method, the details of how this biocontrol tool works are not well understood. Here we characterise a new Wolbachia strain, wPip, and find that Ae. aegypti carrying wPip are still able to transmit dengue similar to mosquitoes that do not carry Wolbachia. This finding has allowed us to begin a rigorous program of comparative studies to determine which features of a Wolbachia strain determine whether it is antiviral. Understanding these mechanisms will enable us to predict the risk of viral resistance arising against Wolbachia and facilitate preparation of second-generation field release lines.

The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment. IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond dynamically to environmental changes.

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic.

Simple Summary Mosquito-borne viral diseases such as dengue, Zika and chikungunya cause a significant global health burden and are currently increasing in outbreak frequency and geographical reach. Wolbachia pipientis, an endosymbiotic bacterium, offers a solution to this. When Wolbachia is introduced into the main mosquito vector of these viruses, Aedes aegypti, it alters the mosquito’s reproductive biology, as well as reducing the ability of the mosquitoes to transmit viruses. These traits can be leveraged to reduce virus transmission within a community by mass releasing Wolbachia-infected mosquitoes. However, Wolbachia has some negative effects on Aedes aegypti fitness, particularly egg longevity, and the reason behind this remains ambiguous. Insect fitness is very important for the success for Wolbachia-biocontrol strategies as they rely on the released insects being competitive with the wild mosquito population. This review summarises the fitness effects of Wolbachia on Aedes aegypti and investigates the possible contribution of Wolbachia as a nutritional parasite in lowering host fitness. It proposes the next stages of research that can be conducted to address nutritional parasitism to aid in the expansion of Wolbachia-based disease management programs worldwide. Abstract The artificial introduction of the endosymbiotic bacterium, Wolbachia pipientis, into Aedes (Ae.) aegypti mosquitoes reduces the ability of mosquitoes to transmit human pathogenic viruses and is now being developed as a biocontrol tool. Successful introgression of Wolbachia-carrying Ae. aegypti into native mosquito populations at field sites in Australia, Indonesia and Malaysia has been associated with reduced disease prevalence in the treated community. In separate field programs, Wolbachia is also being used as a mosquito population suppression tool, where the release of male only Wolbachia-infected Ae. aegypti prevents the native mosquito population from producing viable eggs, subsequently suppressing the wild population. While these technologies show great promise, they require mass rearing of mosquitoes for implementation on a scale that has not previously been done. In addition, Wolbachia induces some negative fitness effects on Ae. aegypti. While these fitness effects differ depending on the Wolbachia strain present, one of the most consistent and significant impacts is the shortened longevity and viability of eggs. This review examines the body of evidence behind Wolbachia’s negative effect on eggs, assesses nutritional parasitism as a key cause and considers how these impacts could be overcome to achieve efficient large-scale rearing of these mosquitoes.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2–5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on “Successfully transitioning from post-doc to lab head”, winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

Dengue virus (DENV) threatens almost 70% of the world&rsquo, s population, with effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) &alpha, /&beta, 1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5&ndash, IMP&alpha, 1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMP&alpha, binding to IMP&beta, 1, but can dissociate preformed IMP&alpha, 1 heterodimer, through targeting the IMP&alpha, armadillo (ARM) repeat domain to impact IMP&alpha, thermal stability and &alpha, helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low &micro, M concentrations against not only DENV-2, but also zika virus and West Nile virus. This work highlights DENV NS5 nuclear targeting as a viable target for anti-flaviviral therapeutics.

Infection by one of the 4 distinct serotypes of dengue virus (DENV) threatens >40% of the world's population, with no efficacious vaccine or antiviral agent currently available. DENV replication through the virus-encoded nonstructural protein (NS) 5 protein occurs in the infected cell cytoplasm, but NS5 from DENV2 has thus far been shown to localize strongly in the nucleus throughout infection. Here we use specific antibodies cross-reactive with NS5 from DENV1-4 to demonstrate nuclear localization of NS5 from all DENV serotypes for the first time in both infected as well as transfected cells, although to differing extents. The small-molecule inhibitor Ivermectin was inhibitory towards both DENV 1 and 2 NS5 interaction with its nuclear transporter importin α/β in vitro, and protected against infection from DENV1-4. Ivermectin thus has potential in the clinical setting as a dengue antiviral.

The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.

Background Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available. Methods We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition. Results We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model. Conclusions 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model.

Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/β (IMPα/β). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMPα/β-recognized nuclear localization signal [Pryor et al. Traffic 8:795-807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8:795-807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMPα/β using an AlphaScreen protein-protein binding assay.

Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/β (IMPα/β). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMPα/β-recognized nuclear localization signal [Pryor et al. Traffic 8:795-807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8:795-807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMPα/β using an AlphaScreen protein-protein binding assay.