Infection with a Virulent Strain of Wolbachia Disrupts Genome Wide-Patterns of Cytosine Methylation in the Mosquito Aedes aegypti

BACKGROUND Cytosine methylation is one of several reversible epigenetic modifications of DNA that allow a greater flexibility in the relationship between genotype and phenotype. Methylation in the simplest models dampens gene expression by modifying regions of DNA critical for transcription factor binding. The capacity to methylate DNA is variable in the insects due to diverse histories of gene loss and duplication of DNA methylases. Mosquitoes like Drosophila melanogaster possess only a single methylase, DNMT2. DESCRIPTION Here we characterise the methylome of the mosquito Aedes aegypti and examine its relationship to transcription and test the effects of infection with a virulent strain of the endosymbiont Wolbachia on the stability of methylation patterns. CONCLUSION We see that methylation in the A. aegypti genome is associated with reduced transcription and is most common in the promoters of genes relating to regulation of transcription and metabolism. Similar gene classes are also methylated in aphids and honeybees, suggesting either conservation or convergence of methylation patterns. In addition to this evidence of evolutionary stability, we also show that infection with the virulent wMelPop Wolbachia strain induces additional methylation and demethylation events in the genome. While most of these changes seem random with respect to gene function and have no detected effect on transcription, there does appear to be enrichment of genes associated with membrane function. Given that Wolbachia lives within a membrane-bound vacuole of host origin and retains a large number of genes for transporting host amino acids, inorganic ions and ATP despite a severely reduced genome, these changes might represent an evolved strategy for manipulating the host environments for its own gain. Testing for a direct link between these methylation changes and expression, however, will require study across a broader range of developmental stages and tissues with methods that detect splice variants.This research was supported by The National Health and Medical Research Council of Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The Relative Importance of Innate Immune Priming in Wolbachia-Mediated Dengue Interference

The non-virulent Wolbachia strain wMel and the life-shortening strain wMelPop-CLA, both originally from Drosophila melanogaster, have been stably introduced into the mosquito vector of dengue fever, Aedes aegypti. Each of these Wolbachia strains interferes with viral pathogenicity and/or dissemination in both their natural Drosophila host and in their new mosquito host, and it has been suggested that this virus interference may be due to host immune priming by Wolbachia. In order to identify aspects of the mosquito immune response that might underpin virus interference, we used whole-genome microarrays to analyse the transcriptional response of A. aegypti to the wMel and wMelPop-CLA Wolbachia strains. While wMel affected the transcription of far fewer host genes than wMelPop-CLA, both strains activated the expression of some immune genes including anti-microbial peptides, Toll pathway genes and genes involved in melanization. Because the induction of these immune genes might be associated with the very recent introduction of Wolbachia into the mosquito, we also examined the same Wolbachia strains in their original host D. melanogaster. First we demonstrated that when dengue viruses were injected into D. melanogaster, virus accumulation was significantly reduced in the presence of Wolbachia, just as in A. aegypti. Second, when we carried out transcriptional analyses of the same immune genes up-regulated in the new heterologous mosquito host in response to Wolbachia we found no over-expression of these genes in D. melanogaster, infected with either wMel or wMelPop. These results reinforce the idea that the fundamental mechanism involved in viral interference in Drosophila and Aedes is not dependent on the up-regulation of the immune effectors examined, although it cannot be excluded that immune priming in the heterologous mosquito host might enhance the virus interference trait

CDC Botswana : sharing another partnership success

CDC Botswana, in partnership with the Ministry of Health since 1995--for a safer, healthier Botswana.Publication date from document properties.CDCBotswanaSharesSuccess_19_07_12.pd

Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis

Background Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been declared a global pandemic and urgent treatment and prevention strategies are needed. Nitazoxanide, an anthelmintic drug has been shown to exhibit in vitro activity against SARS‐CoV‐2. The present study used physiologically‐based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported SARS‐CoV‐2 EC90. Methods A whole‐body PBPK model was validated against available pharmacokinetic data for healthy individuals receiving single and multiple doses between 500–4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the EC90 in >90% of the simulated population. PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. Results The PBPK model was successfully validated against the reported human pharmacokinetics. The model predicted optimal doses of 1200 mg QID, 1600 mg TID, 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12h post dose was estimated. Conclusion The PBPK model predicted tizoxanide concentrations within doses of nitazoxanide already given to humans previously. The reported dosing strategies provide a rational basis for design of clinical trials with nitazoxanide for the treatment or prevention of SARS‐CoV‐2 infection. A concordant higher dose of nitazoxanide is now planned for investigation in the seamless phase I/IIa AGILE trial (www.agiletrial.net)

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

<i>Wolbachia</i>-Associated Bacterial Protection in the Mosquito <i>Aedes aegypti</i>

<div><p>Background</p><p><i>Wolbachia</i> infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural <i>Wolbachia</i> infections in <i>Drosophila melanogaster</i> with the mosquito vector <i>Aedes aegypti</i> artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-<i>Wolbachia</i> associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.</p><p>Methodology/Findings</p><p>Here we assess whether the level of innate immune priming induced by different <i>Wolbachia</i> strains in <i>A. aegypti</i> is correlated with the degree of protection conferred against bacterial pathogens. We show that <i>Wolbachia</i> strains <i>w</i>Mel and <i>w</i>MelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The <i>w</i>MelPop strain provides stronger, more broad-based protection than <i>w</i>Mel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that <i>Wolbachia</i> densities themselves decline during pathogen infection, likely as a result of the immune induction.</p><p>Conclusions/Significance</p><p>This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether <i>Wolbachia</i> may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.</p></div

Immune transcript analyses in <i>D. melanogaster</i> infected with <i>w</i>MelPop and <i>w</i>Mel.

<p>Transcripts are ranked by biological process and/or molecular function. Gene identifiers (Gene ID), Description and Symbol were compiled from Flybase. AFC, Absolute Fold Change, ND, No Detection. Asterisks indicate a statistically significant difference (n = 10, Mann-Whitney <i>U</i> test with q-value adjustment, *: q<0.05, **: q<0.01).</p

Dengue blocking in <i>D. melanogaster</i> and <i>A. aegypti</i> infected by <i>Wolbachia</i> strain <i>w</i>Mel.

<p>69 µl of 10⁷ pfu/ml of DENV2 strain 92T (grey circles) and DENV2 strain ET300 (black circles) were injected into flies (<i>w¹¹¹⁸w</i>Mel) and mosquitoes (MGYP2) infected by <i>w</i>Mel and their tetracycline-treated uninfected counterparts (<i>w¹¹¹⁸</i>tet and MGYP2tet). Dengue levels in individual insects were determined 8 days post-infection by RT-PCR using a TaqMan assay specific to dengue in 1 µg of total RNA. The fraction of flies that had detectable dengue infections is shown above each set of data points. (n = 15, Mann-Whitney <i>U</i> test, **: p<0.01, ***:p<0.001, ****:p<0.0001).</p

Median (with interquartile range) relative <i>Wolbachia</i> density after infection in mosquitoes.

<p>Five pairs of individuals were used for <i>E. carotovora</i> (A), <i>B. cepacia</i> (B), <i>S. typhimurium</i> (C) and <i>M. marinum</i> (D). (Mann-Whitney U-test; * P-value&lt;0.05, ** P-value&lt;0.01).</p

Gene Ontology (GO) terms over-represented among gene transcripts significantly up-regulated in <i>w</i>MelPop-CLA-infected <i>A. aegypti</i>.

<p>Adjusted <i>P</i>-values are the <i>P</i>-values generated by the Ontologizer program <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002548#ppat.1002548-Grossmann2" target="_blank">[38]</a>, using the Benjamini-Hochberg method.</p