A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

A new dicistro-like virus from soldier fly, Inopus flavus (Diptera: Stratiomyidae), a pest of sugarcane.

Native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the family Dicistroviridae. The complete genome sequence consists of 9793 nucleotides with two open reading frames. The genome includes two potential internal ribosomal entry sites (IRESs): one within the 5' UTR and the other in the intergenic region (IGR). Virus particles purified from infected larvae and visualised by electron microscopy were found to be icosahedral, non-enveloped, and 30 nm in diameter.

miRNA Modulation of Insect Virus Replication.

The outcome of virus infection in insects is impacted by regulation of both host and virus gene expression. A class of small RNAs called microRNAs (miRNA) have emerged as important regulators of gene expression that can influence the outcome of virus infection. miRNA regulation occurs at a comparatively late stage of gene expression, allowing for rapid control and fine-tuning of gene expression levels. Here we discuss the biogenesis of miRNAs from both host and virus genomes, the interactions that lead to regulation of gene expression, and the miRNA-mRNA interactions that lead to either antivirus or provirus consequences in the course of virus infection in insects.

Wolbachia-mediated antiviral protection is cell-autonomous.

Vector-borne viral diseases pose significant risks to human health. To control the transmission of these viruses, a number of approaches are required. The ability of the intracellular bacteria Wolbachia to limit viral accumulation and transmission in some arthropod hosts, highlights its potential as a biocontrol agent. Whilst Wolbachia can reduce the transmission of several epidemiologically important viruses, protection is not consistent amongst all insects, viruses and strains of Wolbachia, which confounds elucidation of the mechanisms that underly this protection. Evidence of different mechanisms has emerged, but is not always consistent, suggesting the tripartite interaction may be complex. Here we provide evidence that Wolbachia-mediated antiviral protection is dependent on the presence of Wolbachia in individual cells, and cannot be conferred to surrounding cells. Our results suggest that protection is cell-autonomous, and this has several mechanistic implications, which can direct future research.

Contrasting Patterns of Virus Protection and Functional Incompatibility Genes in Two Conspecific Wolbachia Strains from Drosophila pandora.

Wolbachia infections can present different phenotypes in hosts, including different forms of reproductive manipulation and antiviral protection, which may influence infection dynamics within host populations. In populations of Drosophila pandora two distinct Wolbachia strains coexist, each manipulating host reproduction: strain wPanCI causes cytoplasmic incompatibility (CI), whereas strain wPanMK causes male killing (MK). CI occurs when a Wolbachia-infected male mates with a female not infected with a compatible type of Wolbachia, leading to nonviable offspring. wPanMK can rescue wPanCI-induced CI but is unable to induce CI. The antiviral protection phenotypes provided by the wPanCI and wPanMK infections were characterized; the strains showed differential protection phenotypes, whereby cricket paralysis virus (CrPV)-induced mortality was delayed in flies infected with wPanMK but enhanced in flies infected with wPanCI compared to their respective Wolbachia-cured counterparts. Homologs of the cifA and cifB genes involved in CI identified in wPanMK and wPanCI showed a high degree of conservation; however, the CifB protein in wPanMK is truncated and is likely nonfunctional. The presence of a likely functional CifA in wPanMK and wPanMK's ability to rescue wPanCI-induced CI are consistent with the recent confirmation of CifA's involvement in CI rescue, and the absence of a functional CifB protein further supports its involvement as a CI modification factor. Taken together, these findings indicate that wPanCI and wPanMK have different relationships with their hosts in terms of their protective and CI phenotypes. It is therefore likely that different factors influence the prevalence and dynamics of these coinfections in natural Drosophila pandora hosts.IMPORTANCEWolbachia strains are common endosymbionts in insects, with multiple strains often coexisting in the same species. The coexistence of multiple strains is poorly understood but may rely on Wolbachia organisms having diverse phenotypic effects on their hosts. As Wolbachia is increasingly being developed as a tool to control disease transmission and suppress pest populations, it is important to understand the ways in which multiple Wolbachia strains persist in natural populations and how these might then be manipulated. We have therefore investigated viral protection and the molecular basis of cytoplasmic incompatibility in two coexisting Wolbachia strains with contrasting effects on host reproduction.

Wolbachia-mediated protection of Drosophila melanogaster against systemic infection with its natural viral pathogen Drosophila C virus does not involve changes in levels of highly abundant miRNAs.

The presence of Wolbachia confers virus protection to insects. The molecular mechanism underlying Wolbachia-mediated protection in this tripartite host-endosymbiont-virus interaction is not yet fully understood. In the bipartite association between Drosophila melanogaster and Drosophila C virus (DCV), changes in the expression of microRNAs (miRNAs) influence the outcome of viral pathogenesis. Here we examined whether changes in miRNA expression are similarly involved in the Drosophila-Wolbachia-DCV association. The levels of highly abundant miRNAs in D. melanogaster, Wolbachia-mono-infected D. melanogaster, and DCV- and Wolbachia-bi-infected D. melanogaster were quantified using RT-qPCR and compared. The results show that the abundance of the 17 tested D. melanogaster miRNAs is not affected by Wolbachia endosymbiosis or by bi-infection of Wolbachia and DCV. These results suggest that the in vivo protection conferred by Wolbachia to its native host against D. melanogaster's natural pathogen DCV is not likely to be dependent on or associated with changes in the levels of highly expressed miRNAs.

Drosophila microRNA modulates viral replication by targeting a homologue of mammalian cJun.

MicroRNAs (miRNAs) are important regulators of biological processes, including host-virus interaction. This study investigated the involvement of Drosophila melanogaster miR-8-5p in host-virus interaction. Drosophila flies and cells challenged with Drosophila C virus (DCV) were found to have lower miR-8-5p abundance compared to uninfected samples. Lowering miR-8-5p abundance by experimental inhibition of the miRNA led to an increase in viral accumulation, suggesting that the observed decrease in the miR-8-5p abundance during DCV infection enhances viral replication. miR-8-5p putative targets were identified and included dJun, a transcription factor gene whose mammalian homologue cJun is induced by various viruses through kinase activation. Increasing miR-8-5p abundance using miR-8-5p mimics resulted in a decrease in dJun and GFP reporter levels. Furthermore, when the putative target in dJun was mutated, addition of miR-8-5p mimics did not result in the same antagonistic effect on dJun. These results show negative regulation of dJun by miR-8-5p and suggest that an miRNA-mediated pathway is involved in dJun regulation during viral infection. To analyse the role of dJun during DCV infection, dJun was knocked down in cells prior to DCV infection. Knockdown of dJun decreased DCV replication, providing evidence that dJun up-regulation that is concomitant with miR-8-5p down-regulation during DCV infection supports viral replication. These results highlight the role of miRNA in regulating the transcription factor gene dJun and uncover a previously unrecognized mechanism by which dJun is regulated during host-virus interaction.

Impact of ERK activation on fly survival and Wolbachia-mediated protection during virus infection.

Elevated levels of reactive oxygen species (ROS) provide protection against virus-induced mortality in Drosophila. In addition to contributing to oxidative stress, ROS are known to activate a number of signalling pathways including the extracellular signal-regulated kinases (ERK) signalling cascade. It was recently shown that ERK signalling is important for resistance against viral replication and invasion in cultured Drosophila cells and the gut epithelium of adult flies. Here, using a Drosophila loss-of-function ERK (rolled) mutant we demonstrated that ERK is important for fly survival during virus infection. ERK mutant flies subjected to Drosophila C virus (DCV) oral and systemic infection were more susceptible to virus-induced mortality as compared with wild-type flies. We have demonstrated experimentally that ERK activation is important for fly survival during oral and systemic virus infection. Given that elevated ROS correlates with Wolbachia-mediated antiviral protection, we also investigated the involvement of ERK in antiviral protection in flies infected by Wolbachia. The results indicate that ERK activation is increased in the presence of Wolbachia but this does not appear to influence Wolbachia-mediated antiviral protection, at least during systemic infection.

Infectivity of Drosophila C virus following oral delivery in Drosophila larvae.

The route of pathogen entry can have a major effect on the ability of a virus to induce a prolific infection, but it can also affect the ability of the host organism to induce an immune response to fight the infection. Transmission of arboviruses that cause serious diseases in humans often begin by an insect ingesting a virus, which then disseminates through the internal organs and tissues and ultimately culminates in virus transmission to a human host. Understanding the effect of a natural route of infection on the host-pathogen interaction may facilitate development of approaches to prevent viral dissemination. Drosophila has been a useful model organism for understanding host-virus interactions; however, most studies have achieved infection by artificially injecting the virus into the host. Here, we developed a single-stranded quantitative PCR able to detect only actively replicating Drosophila C virus (DCV) to study the effect of viral feeding at the early stages of larval development. Exposure of newly hatched larvae to DCV led to 20 % of larvae becoming infected within 12 h post-contamination, and caused a 14 % egg-to-adult mortality. This is the first time, to the best of our knowledge, that it has been shown experimentally that DCV is able to establish a prolific infection following larval feeding. Using these newly developed tools, the results suggest that larvae that become infected die before adult eclosion.

Drosophila melanogaster does not exhibit a behavioural fever response when infected with Drosophila C virus.

Behavioural fever is a widely conserved response to infection. The host increases body temperature (Tb) by altering their preferred temperature (Tp), generating a fever and delaying or avoiding pathogen-induced mortality. This response is not ubiquitous in insects, however, although few studies have investigated this response to viral infection. Here, we examined the change in Tp of Drosophila in response to virus infection using a thermal gradient. No difference in Tp was observed. We suggest that the lack of behavioural fever could be due to the increased energy cost of maintaining a higher Tb whilst the immune response is active. To the best of our knowledge, this is the first study to assay for changes in Tp of infected Drosophila.

Dietary cholesterol modulates pathogen blocking by Wolbachia.

The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This "pathogen blocking" could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV), a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2-5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking.

Wolbachia-mediated antiviral protection in Drosophila larvae and adults following oral infection.

Understanding viral dynamics in arthropods is of great importance when designing models to describe how viral spread can influence arthropod populations. The endosymbiotic bacterium Wolbachia spp., which is present in up to 40% of all insect species, has the ability to alter viral dynamics in both Drosophila spp. and mosquitoes, a feature that in mosquitoes may be utilized to limit spread of important arboviruses. To understand the potential effect of Wolbachia on viral dynamics in nature, it is important to consider the impact of natural routes of virus infection on Wolbachia antiviral effects. Using adult Drosophila strains, we show here that Drosophila-Wolbachia associations that have previously been shown to confer antiviral protection following systemic viral infection also confer protection against virus-induced mortality following oral exposure to Drosophila C virus in adults. Interestingly, a different pattern was observed when the same fly lines were challenged with the virus when still larvae. Analysis of the four Drosophila-Wolbachia associations that were protective in adults indicated that only the w1118-wMelPop association conferred protection in larvae following oral delivery of the virus. Analysis of Wolbachia density using quantitative PCR (qPCR) showed that a high Wolbachia density was congruent with antiviral protection in both adults and larvae. This study indicates that Wolbachia-mediated protection may vary between larval and adult stages of a given Wolbachia-host combination and that the variations in susceptibility by life stage correspond with Wolbachia density. The differences in the outcome of virus infection are likely to influence viral dynamics in Wolbachia-infected insect populations in nature and could also have important implications for the transmission of arboviruses in mosquito populations.

Oxidative stress correlates with Wolbachia-mediated antiviral protection in Wolbachia-Drosophila associations.

Wolbachia mediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect, Wolbachia, and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, suggesting that oxidative stress is important for Wolbachia-mediated antiviral protection. However, Wolbachia experimentally introduced into mosquitoes impacts a range of host fitness traits, some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection in Wolbachia-infected insects, we analyzed oxidative stress of five Wolbachia-infected Drosophila lines. In flies infected with protective Wolbachia strains, hydrogen peroxide concentrations were 1.25- to 2-fold higher than those in paired fly lines cured of Wolbachia infection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with nonprotective Wolbachia strains compared to flies cured of Wolbachia infection. Using a Drosophila mutant that produces increased levels of hydrogen peroxide, we investigated whether flies with high levels of endogenous reactive oxygen species had altered responses to virus infection and found that flies with high levels of endogenous hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates with Wolbachia-mediated antiviral protection in natural Drosophila hosts.

Effects of spontaneous mutations on survival and reproduction of Drosophila serrata infected with Drosophila C virus.

The impact of selection on host immune function genes has been widely documented. However, it remains essentially unknown how mutation influences the quantitative immune traits that selection acts on. Applying a classical mutation accumulation (MA) experimental design in Drosophila serrata, we found the mutational variation in susceptibility (median time of death, LT50) to Drosophila C virus (DCV) was of similar magnitude to that reported for intrinsic survival traits. Mean LT50 did not change as mutations accumulated, suggesting no directional bias in mutational effects. Maintenance of genetic variance in immune function is hypothesised to be influenced by pleiotropic effects on immunity and other traits that contribute to fitness. To investigate this, we assayed female reproductive output for a subset of MA lines with relatively long or short survival times under DCV infection. Longer survival time tended to be associated with lower reproductive output, suggesting that mutations affecting susceptibility to DCV had pleiotropic effects on investment in reproductive fitness. Further studies are needed to uncover the general patterns of mutational effect on immune responses and other fitness traits, and to determine how selection might typically act on new mutations via their direct and pleiotropic effects.

Physiological and metabolic consequences of viral infection in Drosophila melanogaster.

An extensively used model system for investigating anti-pathogen defence and innate immunity involves Drosophila C virus (DCV) and Drosophila melanogaster. While there has been a significant effort to understand infection consequences at molecular and genetic levels, an understanding of fundamental higher-level physiology of this system is lacking. Here, we investigate the metabolic rate, locomotory activity, dry mass and water content of adult male flies injected with DCV, measured over the 4 days prior to virus-induced mortality. DCV infection resulted in multiple pathologies, notably the depression of metabolic rate beginning 2 days post-infection as a response to physiological stress. Even in this depressed metabolic state, infected flies did not decrease their activity until 1 day prior to mortality, which further suggests that cellular processes and synthesis are disrupted because of viral infection. Growth rate was also reduced, indicating that energy partitioning is altered as infection progresses. Microbial infection in insects typically results in an increase in excretion; however, water appeared to be retained in DCV-infected flies. We hypothesise that this is due to a fluid intake-output imbalance due to disrupted transport signalling and a reduced rate of metabolic processing. Furthermore, infected flies had a reduced rate of respiration as a consequence of metabolic depression, which minimised water loss, and the excess mass as a result of water retention is concurrent with impaired locomotory ability. These findings contribute to developing a mechanistic understanding of how pathologies accumulate and lead to mortality in infected flies.

The small interfering RNA pathway is not essential for Wolbachia-mediated antiviral protection in Drosophila melanogaster.

Wolbachia pipientis delays RNA virus-induced mortality in Drosophila spp. We investigated whether Wolbachia-mediated protection was dependent on the small interfering RNA (siRNA) pathway, a key antiviral defense. Compared to Wolbachia-free flies, virus-induced mortality was delayed in Wolbachia-infected flies with loss-of-function of siRNA pathway components, indicating that Wolbachia-mediated protection functions in the absence of the canonical siRNA pathway.

Antiviral protection and the importance of Wolbachia density and tissue tropism in Drosophila simulans.

Wolbachia, a maternally transmitted endosymbiont of insects, is increasingly being seen as an effective biological control agent that can interfere with transmission of pathogens, including dengue virus. However, the mechanism of antiviral protection is not well understood. The density and distribution of Wolbachia in host tissues have been implicated as contributing factors by previous studies with both mosquitoes and flies. Drosophila flies infected with five diverse strains of Wolbachia were screened for the ability to mediate antiviral protection. The three protective Wolbachia strains were more closely related and occurred at a higher density within whole flies than the two nonprotective Wolbachia strains. In this study, to further investigate the relationship between whole-fly Wolbachia density and the ability to mediate antiviral protection, tetracycline was used to decrease the abundance of the high-density, protective Wolbachia strain wAu prior to viral challenge. Antiviral protection was lost when the density of the protective Wolbachia strain was decreased to an abundance similar to that of nonprotective Wolbachia strains. We determined the Wolbachia density and distribution in tissues of the same five fly-Wolbachia combinations as used previously. The Wolbachia density within the head, gut, and Malpighian tubules correlated with the ability to mediate antiviral protection. These findings may facilitate the development of Wolbachia biological control strategies and help to predict host-Wolbachia pairings that may interfere with virus-induced pathology.

Solving the Wolbachia paradox: modeling the tripartite interaction between host, Wolbachia, and a natural enemy.

Wolbachia is one of the most common symbionts of arthropods. Its establishment requires lateral transfer to and successful transmission within novel host species. However, Wolbachia performs poorly when introduced into new host species, and models predict that Wolbachia should seldom be able to establish from low initial frequencies. Recently, various symbionts, including Wolbachia, have been shown to protect their hosts from natural enemies. Hence, Wolbachia invasion may be facilitated by the dynamic interaction between it, its host, and a natural enemy. We model such an interaction whereby Wolbachia induces either complete resistance, partial resistance, or tolerance to a host-specific pathogen and also induces the common manipulation phenotype of cytoplasmic incompatibility (CI). We show that the presence of the pathogen greatly facilitates Wolbachia invasion from rare and widens the parameter space in which "imperfect" Wolbachia strains can invade. Furthermore, positive frequency-dependent selection through CI can drive Wolbachia to very high frequencies, potentially excluding the pathogen. These results may explain a poorly understood aspect of Wolbachia biology: it is widespread, despite performing poorly after transfer to new host species. They also support the intriguing possibility that Wolbachia strains that encode both CI and natural-enemy resistance could potentially rid insects, including human disease vectors, of important pathogens.

Variation in antiviral protection mediated by different Wolbachia strains in Drosophila simulans.

Drosophila C virus (DCV) is a natural pathogen of Drosophila and a useful model for studying antiviral defences. The Drosophila host is also commonly infected with the widespread endosymbiotic bacteria Wolbachia pipientis. When DCV coinfects Wolbachia-infected D. melanogaster, virus particles accumulate more slowly and virus induced mortality is substantially delayed. Considering that Wolbachia is estimated to infect up to two-thirds of all insect species, the observed protective effects of Wolbachia may extend to a range of both beneficial and pest insects, including insects that vector important viral diseases of humans, animals and plants. Currently, Wolbachia-mediated antiviral protection has only been described from a limited number of very closely related strains that infect D. melanogaster. We used D. simulans and its naturally occurring Wolbachia infections to test the generality of the Wolbachia-mediated antiviral protection. We generated paired D. simulans lines either uninfected or infected with five different Wolbachia strains. Each paired fly line was challenged with DCV and Flock House virus. Significant antiviral protection was seen for some but not all of the Wolbachia strain-fly line combinations tested. In some cases, protection from virus-induced mortality was associated with a delay in virus accumulation, but some Wolbachia-infected flies were tolerant to high titres of DCV. The Wolbachia strains that did protect occurred at comparatively high density within the flies and were most closely related to the D. melanogaster Wolbachia strain wMel. These results indicate that Wolbachia-mediated antiviral protection is not ubiquitous, a finding that is important for understanding the distribution of Wolbachia and virus in natural insect populations.

Physical and Chemical Barriers in the Larval Midgut Confer Developmental Resistance to Virus Infection in Drosophila.

Insects can become lethally infected by the oral intake of a number of insect-specific viruses. Virus infection commonly occurs in larvae, given their active feeding behaviour; however, older larvae often become resistant to oral viral infections. To investigate mechanisms that contribute to resistance throughout the larval development, we orally challenged Drosophila larvae at different stages of their development with Drosophila C virus (DCV, Dicistroviridae). Here, we showed that DCV-induced mortality is highest when infection initiates early in larval development and decreases the later in development the infection occurs. We then evaluated the peritrophic matrix as an antiviral barrier within the gut using a Crystallin-deficient fly line (Crys-/-), whose PM is weakened and becomes more permeable to DCV-sized particles as the larva ages. This phenotype correlated with increasing mortality the later in development oral challenge occurred. Lastly, we tested in vitro the infectivity of DCV after incubation at pH conditions that may occur in the midgut. DCV virions were stable in a pH range between 3.0 and 10.5, but their infectivity decreased at least 100-fold below (1.0) and above (12.0) this range. We did not observe such acidic conditions in recently hatched larvae. We hypothesise that, in Drosophila larvae, the PM is essential for containing ingested virions separated from the gut epithelium, while highly acidic conditions inactivate the majority of the virions as they transit.