Anti-viral immunity in Anopheles gambiae mosquitoes

Mosquito transmitted viruses (arboviruses) cause significant burden in much of the developing world. Little is known about mosquito responses to viral infection, and how these responses could be utilised to prevent spread of viral disease. Anopheles gambiae, the principal vector of human malaria, unusually transmits virtually no arboviruses, with one known exception - O’nyony-nyong Virus (ONNV). In this thesis the interactions between ONNV and the A. gambiae immune system were studied. Initially ONNV infection in A. gambiae mosquitoes and an A. gambiae derived cell line were characterised. The in vivo transcriptional responses of A. gambiae to viral infection were profiled using full genome microarrays, describing the global response to ONNV infection. This thesis demonstrates that the A. gambiae immune system does respond to viral infection, with genes covering all aspects of immunity being differentially regulated, from pathogen recognition to modulation of immune signalling, complement-mediated lysis/opsonisation and immune effector mechanisms. Furthermore, this study identified four immune genes (a galectin, an MD2-like receptor and two lysozymes) regulated by ONNV infection that are capable of limiting virus during infection. These genes have novel roles in anti-viral immunity, and suggest previously uncharacterised mechanisms for targeting viral infection. Additionally, it is shown that A. gambiae uses a combination of core conserved anti-viral mechanisms, including RNAi, but does not utilise some signalling pathways reported to be anti-viral in other insects. This indicates that species specific mechanisms target viral infection. Finally this study demonstrates that foreign RNA acts as a Pathogen Associated Molecular Pattern (PAMP) in A. gambiae derived cells, and triggers transcriptional responses that dramatically reduce viral infection. In conclusion the data presented in this thesis demonstrate that A. gambiae responds to and is capable of limiting viral infection through conserved and novel immune mechanisms triggered by recognition of viral infection and foreign RNA

Climate, Environmental and Socio-Economic Change: Weighing Up the Balance in Vector-Borne Disease Transmission

Arguably one of the most important effects of climate change is the potential impact on human health. While this is likely to take many forms, the implications for future transmission of vector-borne diseases (VBDs), given their ongoing contribution to global disease burden, are both extremely important and highly uncertain. In part, this is owing not only to data limitations and methodological challenges when integrating climate-driven VBD models and climate change projections, but also, perhaps most crucially, to the multitude of epidemiological, ecological and socio-economic factors that drive VBD transmission, and this complexity has generated considerable debate over the past 10-15 years. In this review, we seek to elucidate current knowledge around this topic, identify key themes and uncertainties, evaluate ongoing challenges and open research questions and, crucially, offer some solutions for the field. Although many of these challenges are ubiquitous across multiple VBDs, more specific issues also arise in different vector-pathogen systems

Progression of Plasmodium berghei through Anopheles stephensi Is Density-Dependent

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete–oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species

Assay harmonization and use of biological standards to improve the reproducibility of the hemagglutination inhibition assay: A FLUCOP collaborative study

The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of antihemagglutinin antibodies. Improved reproducibility of this assay is required to provide meaningful data across different testing laboratories. This study assessed the impact of harmonizing the HAI assay protocol/reagents and using standards on interlaboratory variability. Human pre- and postvaccination sera from individuals (n = 30) vaccinated against influenza were tested across six laboratories. We used a design of experiment (DOE) method to evaluate the impact of assay parameters on interlaboratory HAI assay variability. Statistical and mathematical approaches were used for data analysis. We developed a consensus protocol and assessed its performance against in-house HAI testing. We additionally tested the performance of several potential biological standards. In-house testing with four reassortant viruses showed considerable interlaboratory variation (geometric coefficient of variation [GCV] range of 50% to 117%). The age, concentration of turkey red blood cells, incubation duration, and temperature were key assay parameters affecting variability. Use of a consensus protocol with common reagents, including viruses, significantly reduced GCV between laboratories to 22% to 54%. Pooled postvaccination human sera from different vaccination campaigns were effective as biological standards. Our results demonstrate that the harmonization of protocols and critical reagents is effective in reducing interlaboratory variability in HAI assay results and that pools of postvaccination human sera have potential as biological standards that can be used over multiple vaccination campaigns. Moreover, the use of standards together with in-house protocols is as potent as the use of common protocols and reagents in reducing interlaboratory variability.publishedVersio

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.publishedVersio

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.publishedVersio

Anopheles gambiae Antiviral Immune Response to Systemic O'nyong-nyong Infection

Mosquito-borne viral diseases are found across the globe and are responsible for numerous severe human infections. In order to develop novel methods for prevention and treatment of these diseases, detailed understanding of the biology of viral infection and transmission is required. Little is known about invertebrate responses to infection in mosquito hosts. In this study we used a model system of Anopheles gambiae mosquitoes and O'nyong-nyong virus to study mosquito immune responses to infection. We examined the global transcriptional responses of A. gambiae to viral infection of the mosquito blood equivalent (the hemolymph) identifying a number of genes with immune functions that are switched on or off in response to infection, including complement-like proteins that circulate in the mosquito hemolymph. The switching on of these genes combined with co-infection experiments with malaria parasites suggests that viral infection inhibits the melanisation pathway. Through silencing the function of a selection of viral responsive genes, we identified four genes that have roles in A. gambiae anti-viral immunity; two putative recognition receptors (a galectin and an MD2-like receptor); two effector lysozymes. These molecules have previously non-described roles in antiviral immunity, and suggest uncharacterised mechanisms for targeting viral infection in A. gambiae mosquitoes

Anti-viral immunity in Anopheles gambiae mosquitoes

Mosquito transmitted viruses (arboviruses) cause significant burden in much of the developing world. Little is known about mosquito responses to viral infection, and how these responses could be utilised to prevent spread of viral disease. Anopheles gambiae, the principal vector of human malaria, unusually transmits virtually no arboviruses, with one known exception - O’nyony-nyong Virus (ONNV). In this thesis the interactions between ONNV and the A. gambiae immune system were studied. Initially ONNV infection in A. gambiae mosquitoes and an A. gambiae derived cell line were characterised. The in vivo transcriptional responses of A. gambiae to viral infection were profiled using full genome microarrays, describing the global response to ONNV infection. This thesis demonstrates that the A. gambiae immune system does respond to viral infection, with genes covering all aspects of immunity being differentially regulated, from pathogen recognition to modulation of immune signalling, complement-mediated lysis/opsonisation and immune effector mechanisms. Furthermore, this study identified four immune genes (a galectin, an MD2-like receptor and two lysozymes) regulated by ONNV infection that are capable of limiting virus during infection. These genes have novel roles in anti-viral immunity, and suggest previously uncharacterised mechanisms for targeting viral infection. Additionally, it is shown that A. gambiae uses a combination of core conserved anti-viral mechanisms, including RNAi, but does not utilise some signalling pathways reported to be anti-viral in other insects. This indicates that species specific mechanisms target viral infection. Finally this study demonstrates that foreign RNA acts as a Pathogen Associated Molecular Pattern (PAMP) in A. gambiae derived cells, and triggers transcriptional responses that dramatically reduce viral infection. In conclusion the data presented in this thesis demonstrate that A. gambiae responds to and is capable of limiting viral infection through conserved and novel immune mechanisms triggered by recognition of viral infection and foreign RNA.EThOS - Electronic Theses Online ServiceBBSRCGBUnited Kingdo

Transcriptional responses of <i>A. gambiae</i> G3 mosquitoes to 5′ONNVic-eGFP infection.

<p>The transcriptional responses of <i>A. gambiae</i> mosquitoes inoculated with ≈1640 PFU of 5′ONNVic-eGFP infection were profiled using 4X44K Agilent RNA microarrays. Gene lists include only features that pass strict criteria outlined in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001565#s2" target="_blank">materials and methods</a>. Genes included the analysis are 2-fold or greater regulated at a minimum of 1 of the 3 time points, with T-test P values of <0.05. Genes were categorised based on gene ontology.</p