Spatio-temporal distribution and persistence of Mycobacterium Bovis in a badger population

PhD thesis Clare Benton July 2017Studying the dynamics of pathogen transmission within wildlife populations presents an array of challenges. Where populations are socially structured, this can influence parasite transmission, impacting on the effectiveness of disease management strategies. In this thesis, I focus on a well-studied social mammal, the European badger (Meles meles) which is a key wildlife reservoir of a disease of economic importance; bovine TB (caused by infection with Mycobacterium bovis). The social structuring, characteristic of high density badger populations, is of well-established importance in the transmission of bovine TB and has resulted in unexpected management outcomes. However, little is known about the role of kin structure or host genotype on transmission dynamics. In this thesis, I combine traditional spatial epidemiology and ecological analysis of a well-studied badger population with more novel genetic and genomic approaches. Firstly, I investigate the role of kin structure within badger social groups in determining early life infection risk (Chapter 3). Using host genotype data, I demonstrate that cubs who are related to infected adults experience enhanced infection risks. I then explore the role of badger genotype on outcomes of M. bovis exposure and demonstrate that inbred badgers are more likely to show evidence of progressive infection (Chapter 4). Where the social structure of badgers is stable and unmanaged, this is predicted to result in a stable spatial distribution of M. bovis infection. Motivated by an observation of change in the spatial distribution of M. bovis infection in the study population, in the absence of management, I characterise the attrition of a spatially stable infection distribution (Chapter 5). To explore the drivers of this, I detect changes in the genetic population structure (Chapter 6) and present evidence that the population has experienced a period of demographic flux. Finally, I use a novel dataset generated by whole genome sequencing of M. bovis isolates and present evidence of spatial spread of M. bovis infection across the study population (Chapter 7). To conclude, I discuss how my findings demonstrate how genetic and genomic approaches can complement traditional wildlife epidemiology approaches, how they contribute to our understanding of heterogeneity in transmission dynamics and discuss their implications for wildlife disease management.This PhD studentship was funded by the Animal and Plant Health Agency (APHA) and The University of Exeter. Data from the Woodchester Park study is used in chapters 3 – 7 of this thesis, and is funded by Defra

Bayesian methods for source attribution using HIV deep sequence data

The advent of pathogen deep-sequencing technology provides new opportunities for infec- tious disease surveillance, especially for fast-evolving viruses like human immunodeficiency virus (HIV). In particular, multiple reads per host contain detailed information on viral within- host diversity. This information allows the reconstruction of partial directed transmission networks, where estimates of who is source and who is recipient are directly available from the phylogenetic ordering of the viruses of any two individuals. This is a new approach for phylodynamics, and the topic of my thesis. In this thesis, I present updates to the bioinformatics pipeline used by the Phylogenetics And Networks for Generalised Epidemics in Africa consortium for processing HIV deep sequence data and running the phyloscanner program. I then present a semi-parametric Bayesian Poisson model for inferring infectious disease transmission flows and the sources of infection at the population level. The framework is computationally scalable in high- dimensional flow spaces thanks to Hilbert Space Gaussian process approximations, allows for sampling bias adjustments, and estimation of gender- and age-specific transmission flows at a finer resolution than previously possible. In this sense, the methods that I developed enable us to overcome some problems which have been unable to be solved by conventional phylodynamic approaches. We apply the approach to densely sampled, population-based HIV deep-sequence data from Rakai, Uganda. I focus on characterising age-specific transmission dynamics, and examining the sources of HIV infections in adolescent and young women in particular.Open Acces

Exploring the integration of traditional and molecular epidemiological methods for infectious disease outbreaks

BACKGROUND: Understanding the transmission dynamics of infectious pathogens is critical to developing effective public health strategies. Traditionally, time consuming epidemiological methods were used, often limited by incomplete or inaccurate datasets. Novel phylogenetic techniques can determine transmission events, but have rarely been used in real-time outbreak settings to inform interventions and limit the impact of outbreaks. METHODS: I undertook a series of novel studies to explore the utility of combining phylogenetics with traditional epidemiological analysis to enhance the understanding of transmission dynamics. I investigated HIV in an endemic South African setting and Ebola in an acute outbreak in Sierra Leone. The strengths and limitations of this combined approach are explored, ethical issues investigated and recommendations made regarding the implications of this work for public health. RESULTS: Phylogenetics provides an exciting and synergistic tool to epidemiological analysis in outbreak investigation and control. These combined methods enable a more detailed understanding than is possible through either discipline alone. My key findings include: • Identification of infection source: Phylogenetics gives new insight into the role of external introductions (e.g. migrators) in driving and sustaining the high incidence of HIV. • Earlier identification of new emerging clusters: I identified a new cluster of HIV from around a mining community. This is one of the first examples of molecular methods detecting a previously unknown outbreak. • Identification of novel mechanisms of transmission: This work suggests that children may have been infected by playing in puddles contaminated with Ebola, a previously unrecognised route of transmission. CONCLUSION: The integration of these two methods facilitate sophisticated real-time techniques to maximise understanding of transmission dynamics, allowing faster and more effectively targeted interventions. Moving forwards, sequence data should be incorporated into standard outbreak investigation. This is critical at a time when infectious disease outbreaks have led to the some of the most significant global health threats of the recent past

Modelling the prevalence of wildlife diseases using simulated diagnostic test data

Bayesian Latent Class Models (BLCMs) are algorithms that are used to infer disease prevalence when true disease statuses and gold-standard diagnostic tests are not available. However, limited attention has been given to the specification and validation of BLCMs, which are necessary if credible estimates of diagnostic test performance and disease prevalence are to result. Across six technical chapters, this thesis investigates the fundamental principles of specification and validation via a series of experiments that apply BLCMs to ante-mortem diagnostic test data. To achieve this, simulated arrays of diagnostic test data are generated to reflect the reality of the imperfect trapping and testing efforts that take place in nature. Moreover, the classic Hui-Walter algorithm is generalised within a Bayesian framework to unlock the capability of BLCMs to handle both varying prior information and varying hypotheses simultaneously. Methods to validate BLCMs are developed and then scaled up across a wide range of possible diagnostic testing scenarios via the creation of procedures to explore high-dimensional parameter spaces. For the first time, it is demonstrated that the credibility of BLCM inferences is in fact predictable. Among the key findings discovered are dependence structures that are critical to the identifiability of BLCMs; these structures are uncovered at the limits of parameter spaces, and between the means and variances of the inferred statistics. Accordingly, methods are explored to mitigate for these structures as a further prerequisite to obtaining credible estimates. Attention then turns to testing the core assumptions used to specify the generalised Hui-Walter algorithm. The assumptions about where the true values of diagnostic test performance and disease prevalence exist are removed, and the resulting sensitivity analyses provide confirmation that the findings reported throughout the thesis are indeed generalisable, even to unusual testing scenarios. With a rigorous validation protocol in place, a novel class of time-dependent BLCMs is specified, and then provided with data from one of the world’s longest running wildlife studies. New and rigorously validated inferences of disease prevalence are revealed, and anecdotal trends are corroborated, highlighting the real-world applications of this thesis

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Coronaviruses Research in BRICS Countries

SARS-CoV-2 has infected more than 105 million people worldwide. During this pandemic, researchers and clinicians have been working to understand the molecular mechanisms that underpin viral pathogenesis by studying viral–host interactions. Now, with the global rollout of various COVID-19 vaccines—based on the neutralization of the spike protein using different technologies—viral immunology and cell-based immunity are being investigated. Researchers are also studying how various SARS-CoV-2 genetic mutations will impact the efficacy of these COVID-19 vaccines. At the same time, various antiviral drugs have been identified or repurposed that have potential as anti-SARS-CoV-2 treatments. BRICS (Brazil, Russia, India, China, and South Africa) is the acronym used to associate five major emerging national economies. The BRICS countries are known for their significant influence on regional affairs, including being leaders in scientific and clinical research and innovation. This Special Issue includes researchers from BRICS countries, in particular South Africa, involved in the study of SARS-CoV-2 and COVID-19. Original articles, as well as new perspectives or reviews on the matter, were welcomed. Research in the fields of vaccine studies, pathogenesis, genetic mutations, viral immunology, and antiviral drugs were especially encouraged

The role of wild leporids as reservoirs of infectious agents

Tese de doutoramento em Ciências Veterinárias, apresentada à Universidade de Évora, 2017.Orientadora Líbia Zé-Zé e coorientadora Isabel Lopes de Carvalho, Centro de Estudos de Vetores e Doenças Infeciosas (CEVDI), Instituto Nacional de Saúde Doutor Ricardo Jorge, Águas de Moura, Portugal.Acesso em conformidade com o repositório da UE.The European wild rabbit (Oryctolagus cuniculus) and the Iberian hare (Lepus granatensis), are keystones species in various ecosystems of the Iberian Peninsula and on the local game-based economy. This thesis aimed to investigate the rabbit haemorrhagic disease virus 2 (RHDV2), detected in Portugal in 2012 and currently disseminated in the continent and autonomous regions (Azores, Madeira, Berlengas). Given its alarming impact in the wild rabbit populations, the Portuguese Government has recently activated a plan aiming the control of the disease (Despatch 4757/2017 of May 31st). A second objective of this thesis was the epidemiological surveillance of Francisella tularensis zoonotic vector-borne pathogenic bacterium, with potential impact on Human Health. The Iberian hare is considered reservoir and potential sentinel species for this pathogen. Several methodologies were used to carry out the studies presented in this work, including basic pathology, microbiology and molecular methods. Data analysis involved the resource to phylogenetic inference, statistical programs and bioformatics (e.g. R software). This work enabled the development and validation of the first molecular diagnostic method for RHDV2, currently figuring in the OIE manual, and allowed insights into to the virus dynamic evolution in different epidemiologic and geographic contexts, widening the comprehension of RHDV2 phylogenetic relations among the strains that circulate in Portugal from 2012 until 2017. This study also made possible to ascertain the tularaemia epidemiologic situation in Portugal, confirming the role of wild leporids as reservoirs for the agent and enabling preliminary conclusions on Public Health risk in the country. Moreover, this work allowed confirming ticks as the main vectors for Francisella tularensis in Portugal. Globally, this work contributed to the state-of-the-art of both infections and produced relevant information that can be used to adjust the medical and sanitary prophylactic measures of both diseases to the present reality.Os leporídeos silvestres coelho-bravo (Oryctolagus cuniculus) e lebre ibérica (Lepus granatensis), são determinantes no equilíbrio de vários ecossistemas na Península Ibérica e, simultaneamente, espécies cinegéticas de relevante importância económica. Esta tese visou investigar o vírus da doença hemorrágica dos coelhos de tipo 2 (RHDV2), detetado em Portugal desde 2012 e atualmente disseminado no continente e arquipélagos (Açores, Madeira, Berlengas). Dado o impacto alarmante nas populações de coelho-bravo, o Governo Português ativou recentemente um plano para controlo da doença (Despacho 4757/2017 de 31 de Maio). Um segundo objetivo desta tese consistiu na epidemiovigilância da Francisella tularensis, uma bactéria patogénica zoonótica transmitida por vetores, com potencial impacto em Saúde Pública. A lebre ibérica é seu reservatório e potencial espécie sentinela. Os estudos apresentados nesta tese envolveram a utilização de metodologias clássicas de patologia, microbiologia e moleculares. Para a análise de dados produzidos recorremos, entre outros, a inferências filogenéticas, e a programas de análise estatística e bioformática (eg R software). Este trabalho permitiu o desenvolvimento e validação do primeiro método molecular de diagnóstico para RHDV2, presentemente adotado pelo manual da OIE, e contribuiu para uma compreensão da dinâmica de evolução do vírus em cenários epidemiológicos e geográficos distintos, alargando o conhecimento das relações filogenéticas entre as estirpes que circularam em Portugal entre 2012 e 2017. No que diz respeito à tularémia, este estudo permitiu aferir a situação epidemiológica da doença em Portugal, confirmando o papel dos leporideos silvestres como reservatório e possibilitando conclusões preliminares sobre o risco desta zoonose para a Saúde pública no país. Adicionalmente, permitiu confirmar que os ixodídeos são o principal vetor de F. tularensis em Portugal. Globalmente, este trabalho contribuiu para o estado-da-arte das duas infeções, e disponibilizou informação relevante para adequar o diagnóstico e a profilaxia sanitária e médica destas duas doenças à realidade atualN/

Genotypic and phenotypic variation in the human immunodeficiency viruses.

Despite the involvement of multiple genetic variants of HIV in the causation of disease worldwide, most research has focussed on subtype B, the prevalent subtype in the western world. As a consequence, it is currently not clear whether genetically distinct HIV strains have different biological properties that cause differences in in vivo transmission, disease progression, replication capacity or sensitivity to antiretroviral drugs. In order to increase the ease of classification of viral diversity and thus aid studies into its importance, a novel genotyping tool for classifying HIV-1 subtype based on pol sequence, produced routinely during drug resistance monitoring, has been developed in this thesis. A dataset of 187 full-length HIV-1 sequences was used to generate Gag, Pol, Protease-Reverse Transcriptase (PR-RT) and Env protein sequence alignments. Phylogenetic analyses enabled generation of subtype specific alignments and, whilst sequence variation in the PR-RT dataset was low, this variation was adequate for PR-RT subtype assignment. The subtyping tool, named STAR, utilises position specific scoring matrices (PSSMs) derived from these subtype specific multiple sequence alignments and results in highly accurate reclassification of the subtype alignment sequences, with 98.6% of sequences being accurately assigned a subtype. Subsequent to the development of STAR the importance of HIV genetic variation classified as subtype, was addressed. A comparison of the relative growth capacity of HIV-1 primary isolates of subtypes A, B, C, D, F, group O and HIV-2 was performed in two T-cell environments. A novel reporter cell line was developed specifically to facilitate this work. Clear and consistent differences in in vitro growth phenotype in terms of rate and cytopathogenicity were detected, indicative of intrinsic differences between the HIV-1 types and subtypes. This work was extended by the utilisation of microarray technology which offers the possibility to analyse, at any given time point, the transcriptome of a virus-infected cell. A comparison of the transcriptional responses within T-cells to infection with HIV-1 subtype B, Group O and HIV-2 enabled identification of both core and diverging transcriptional response programs. Whilst the core response program provides insight into the most essential interactions between virus and host during HIV infection of T-cells, analysis of the diverging responses provide evidence that genetically divergent strains of HIV may interact differently with the host. It is proposed that these differences may have the potential to influence disease outcome