Interdisciplinary Team-Building Pilot Grants

2024 CeZAP Interdisciplinary Pilot Grant Awards

Supported by Fralin Life Sciences Institute and Virginia Agriculture Experiment Station. CeZAP received a large number of high-quality proposals for the program this year. Thank you to all who submitted proposals and congratulations to the following who received awards.

Michael Klemba (PI, CALS), Andrew Lowell (Co-PI, COS), James Weger (Co-PI, VMCVM), Anne Brown (Co-PI, CALS)
Title: Developing pleuromutilin analogs into anti-malarial lead compounds
Summary: Malaria is responsible for over half a million deaths annually. New drugs are needed to combat emerging parasite resistance. We have recently discovered novel analogs of the natural product pleuromutilin with potent anti-malarial activity. The objective of this project is to advance them into lead compounds for pre-clinical testing. We will accomplish this by optimizing their potency and therapeutic index and by characterizing their anti-malarial activity and mode of action.
 
James Weger-Lucarelli (PI, VMCVM), Jake Tu (Co-PI, CALS)
Title: Development of single-cell CRISPR (scCRISPR), a CRISPR screen with a single-cell read-out in the Asian tiger mosquito, Aedes albopictus
Summary: Single-cell CRISPR (scCRISPR) couples CRISPR screening with single-cell sequencing to simultaneously determine the sgRNA that induces the perturbation and the perturbed transcriptome in single cells. In aggregate, the perturbed transcriptome profiles of cells with sgRNAs targeting the same gene can serve as the readout. We propose to develop a scCRISPR screen for mosquitoes and other vectors to facilitate the rapid discovery of genes that control basic biological pathways and vector-pathogen-host interactions.
 
Alasdair Cohen (PI, VMCVM), Leigh-Anne Krometis (Co-PI, CALS), Md Rasheduzzaman (Co-PI, ETSU)
Title: Expanding Access to Safe Drinking Water to Improve Health Outcomes in Rural Regions: A Pilot Intervention Trial in Central Appalachia
Summary: Pathogens in drinking water are known to cause or contribute to negative health outcomes. However, for many rural and lower-income populations in the United States, and in rural regions of Appalachia in particular, we know relatively little about exposures to unsafe drinking water or associated impacts on health and wellbeing. This study will evaluate how drinking water filtration may improve health outcomes for people living in lower-income rural households with well and spring water in Central Appalachia.
 
Sweta Agrawal (PI, COS), Clement Vinauger (Co-PI, CALS)
Title: Disrupted mechanosensing as a novel control strategy for the disease vector, Aedes aegypti
Summary: This proposal integrates cellular neurophysiology, behavior, and pesticide
application to develop a novel and innovative strategy for mosquito control. Mosquitoes, like most animals, rely on somatosensory feedback to coordinate their behavior1. We hypothesize that disrupting somatosensation will impair epidemiologically relevant behaviors, thereby reducing disease transmission. To test this, we will first establish how mosquitoes rely on feedback from somatosensory chordotonal organ (ChO) neurons. We will then define how pesticides targeting ChOs interfere with somatosensory function.
 
Esra Buyuktahakin Toy (PI,COE), Luis Escobar (Co-PI,CNRE), Michael Robert (Co-PI, COS)
Title: Integrating Multi-Scale Machine Learning and Mathematical Modeling for the Optimal Prediction, Surveillance, and Intervention Planning for Dengue Fever
Summary: This research integrates machine learning (ML) and optimization to enhance dengue fever surveillance and management in South America. It advances epidemic logistics planning by integrating an ML-driven prediction mechanism into a spatio-temporal optimal control model. Involving experts from epidemic modeling, optimization, and infectious disease ecology from three VT colleges, this approach will improve disease spread prediction, resource allocation, and intervention strategies, reducing the dengue burden. The findings will advance infectious disease modeling and global dengue fever management.
 
Willandia Chaves (PI, CNRE), Luis Escobar (Co-PI, CNRE), Roberta Marques (Co- PI, Mexico), Daniel Jimenez-Garcia (Co-PI, Mexico),
Title: Leveraging the use of roadkill biobanks toward wildlife pathogen detection through a metagenomics approach
Summary: Wildlife are one of the main sources of emerging infectious diseases, but their pathogen surveillance is challenging. We propose to analyze samples from roadkill across 371 localities from 14 states of Brazil using novel genomic techniques to assess the pathobiome of free-range wildlife. Data will then be used to model pathogen prevalence, distribution, and transmission dynamics in wildlife to identify factors associated with disease emergence and spread.
 
Emily Mevers (PI, COS), Dorothea Tholl (Co-PI, COS), Paul Marek (Co-PI, CALS)
Title: Identifying key biosynthetic genes involved in the production of millipede alkaloids
Summary: The proposed study investigates chemical defensive agents (alkaloids) in millipedes
with recently discovered neuromodulating drug and insecticidal properties. Using biochemical,
molecular, and analytical chemistry techniques, our interdisciplinary team will elucidate core steps in the biosynthesis of these agents based on the previous identification of nearly 40 compounds. Successful completion of the project will lay the foundation to develop synthetic biology tools for the production and biological evaluation of these novel bioagents.

Supported by Fralin Life Sciences Institute, and Virginia Agricultural Experiment Station

Florian Schubot (PI, COS), Ann Brown (Co-PI, CALS)
Title: Identifications of Drug-like Modulators of Pseudomonas aeruginosa Signaling Kinase GacS.
Summary: The bacterium Pseudomonas aeruginosa, designated a critical priority pathogen by the World Health Organization, accounts for an estimated 13% of all intensive care unit infections in the United States. Using their complementing expertise in computational modeling and virtual screening on one hand and in biochemistry and microbiology on the other, the research groups of Dr. Brown and Dr. Schubot have joined forces to identify novel inhibitors of the GacS/Rsm signaling pathway. GacS constitutes a promising target because it plays a pivotal role in facilitating P.aeruginosa infections.

Bisqarup Mukhopadhyay (PI, CALS), Liwu Li (Co-PI, COS), Endang Purwantini (Co-PI, CALS)
Title: Training of exhausted momocytes by engineered mycobacteria BCG strains and mycolic acide components
Summary: Acute sepsis due to polymicrobial infections including respiratory viral infections such as COVID19 poses a grave pandemic concern worldwide. While vaccines offer relatively effective prevention of severe sepsis in infected individuals, rapid and prevalent pathogen mutations significantly limit their effectiveness. Here, the proposed project brings a revolutionarily approach of universal applicability. It will provide BCG strains that with selected mycolic acid derivatives will “rewire” exhausted memory monocytes, as a novel treatment regimen for polymicrobial infections.

Azahar Ali, (PI, CALS), XJ Meng (Co-PI, VMCVM), Juhong Cheng (Co-PI, CALS & COE), Dong Sam Ha (Co-PI, COE)
Title:  Sensing of Hepatitis E Virus using a CRISPR-based 3D printed Microelectrode Biosensor in Swine
Summary: Hepatitis E is a liver disease caused by the hepatitis E virus (HEV). There are an estimated 20 million HEV infections (~44 000 deaths) of hepatitis E across the globe. HEV is a major concern for both swine and humans due to its high transmissibility (transmission occurs via the fecal-oral route). We note that the HEV infection is asymptomatic in swine. Early diagnostics of infection is of utmost importance for their isolation, and treatment. There is no available quantitative test for on-site testing of HEV in swine. In this study, we aim to develop a 3D Electrochemical Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a biosensor for on-site and rapid sensing of HEV, and an antibody test due to the viral infection. The major outcome of the project is to develop a novel tool to mitigate the threat of foodborne spread of hepatitis E.

Supported by Fralin Life Sciences Institute, and Virginia Agricultural Experiment Station

Jonathan Auguste (PI, CALS), Luis Escobar (Co-PI, CNRE), Chloe Lahondere (Co-PI, CALS) 
Title: Employing avian and arthropod surveillance to predict pathogen emergence in Virginia.
Summary: Wild birds play a critical role in the transmission dynamics of viruses globally. Virginia is ideally located within dense avian flyways, providing an outstanding opportunity to survey for endemic and imported viral pathogens. We propose to perform statewide field surveys to explore viral diversity of both avian and mosquito populations among various habitat types, and develop ecological niche models and forecast models at multiple scales, to ultimately predict the presence and emergence of viral pathogens.

Bryan Hsu (PI, COS), Lijuan Yuan (Co-PI, VMCVM), Viviana Parreno (Co-PI, VMCVM)
Title: Engineered Phage as an Immunoprophylactic Against Norovirus
Summary: Norovirus gastroenteritis is a major cause of illness with no regulatory-approved vaccines or anti-virals. We have recently developed an engineered lytic bacteriophage that can coexist with cognate bacteria to sustainably produce heterologous proteins in the mammalian gut for multiple weeks. With the CeZAP pilot grant, we propose to use this in situ expression platform to produce anti-norovirus nanobodies in the intestinal mucosa as a microbiome-based immunoprophylactic against infection with potential applications against other infectious diseases.

Brandon Jutras, (PI, CALS), Clay Caswell (Co-PI, VMCVM)
Title: Creating CRISPR platforms to study the sRNA transcriptome of Treponema pallidum, the causative agent of Syphilis
Summary: Syphilis has killed thousands of people for hundreds of years. The causative agent—Treponema pallidum—has been unculturable for the last century. A watershed moment occurred in 2018 when the first in vitro culture system was developed for T. pallidum. We are now one of the only labs in the world that has been continuously culturing the syphilis agent for > 9 months. This breakthrough, coupled with the combined expertise of the Jutras and Caswell labs, we will develop the first CRISPR platform for T. pallidum and use our technology to probe fundamental concepts in both gene regulation and host-pathogen interactions. 

Chloe Lahondere (PI-CALS), James Weger (Co-PI, VMCVM)
Title: The role of Culex territans mosquitoes and their amphibian hosts in the West Nile virus transmission cycle
Summary: West Nile virus (WNV) is a growing public health threat in the United States, primarily transmitted by Culex mosquitoes to birds but with an estimated 7 million human infections since 19991. Unfortunately, no vaccines or medications currently exist to treat infected humans and the success of control efforts direly needs an improved understanding of Culex mosquitoes’ biology. Here, we propose to determine the role of Culex territans and its amphibian hosts in the WNV transmission cycle by combining field surveillance and experimental infection. This knowledge will be critical for accurate risk assessment and outbreaks prediction

Jingqiu Liao (PI-COE), Leigh-Anne Krometis (Co-PI, CALS), Monica Ponder (Co-PI, CALS), Amy Pruden (Co-PI, COE)
Title: Machine learning-enabled accuate prediction of antimicrobial resistance gene acquisition in Listeria monocytogenes using environmental factors
Summary: Listeria monocytogenes, the etiological agent of listeriosis in humans, can rapidly acquire antimicrobial resistance (AMR) genes, posing increasing food safety and public health concerns. We propose to predict and identify environmental factors promoting its AMR acquisition by developing machine learning models based on a nationwide genomic and environmental dataset. This project will generate preliminary data for extramural proposals focused on the surveillance and risk assessment of AMR in L. monocytogenes along the food supply chain.

Rachel Silverman (PI, VMCVM), Carla Finkielstein (Co-PI, COS)
Title: Enhancing evidence-based COVID-19 mitigation strategies by expediting epidemiologic research using data from Southwest Virginia
Summary: Virginia Tech’s Molecular Diagnostics Lab within the Fralin Biomedical Research Institute has partnered with the Virginia Department of Health since April 2020 to provide accurate SARS-CoV-2 molecular diagnostics and genomic sequencing. The accumulated COVID-19 surveillance, outbreak, clinical, diagnostic, and genomic data from this partnership has been untapped for research purposes. We aim to incorporate epidemiologic methods and data analytics to efficiently conduct COVID-19 research to strengthen evidence-based recommendations that proactively respond to the evolving pandemic.

Zhaomin Yang (PI, COS), Webster Santos (Co-PI, COS), Coy Allen (Co-PI, VMCVM), Michael Kelmba (Co-PI, CALS), Florian Schubot (Co-PI, COS)
Title: Antivirulence--new approach to old foes: Discovery and development of anti-T4P compounds against antibiotic resistant bacteria
Summary: Antivirulence is a promising strategy in the fight against the global antibiotic resistance pandemic. The type IV pilus is a well-known virulence factor in numerous bacterial pathogens of significance. It is a validated and high-value target for development of antivirulence chemotherapeutics. Here a multidisciplinary team of scientists with complementary expertise takes a multifaceted approach to the discovery, validation, and optimization of anti-T4P compounds toward the development of antivirulence chemotherapeutics.

Supported by Fralin Life Sciences Institute, and Virginia Agricultural Experiment Station

Coy Allen (PI, VMCVM), Pablo Sobrado (Co-PI, COS), James Weger-Lucarelli (Co-PI, VMCVM)
Title: Quelling the Storm: Attenuating Overzealous Inflammation Following SARS-CoV-D Infection by Modulating Regulatory NOD-like Receptor Signaling
Summary: New therapeutic strategies to combat the COVID-19 associated “cytokine storm” and pediatric cases of Multisystem Inflammatory Syndrome in Children (MIS-C) are direly needed. Preliminary data suggests NLRX1 plays a role in inflammation resolution following SARS-CoV-2 infection. The research team is currently screening the Virginia Tech compound library to identify candidates that either inhibit or activate NLRX1. The pilot studies proposed here will evaluate the top drug screening candidates in mouse models of SARS-CoV-2.

Rana Ashkar (PI, COS), Kevin Edgar (Co-PI, CNRE)
Title: Molecular Design of Robust Biocompatible Liposomal Carriers of mRNA Vaccines
Summary: Development of enhanced liposomal drug carriers is more pressing than ever. With increasing demand for mRNA vaccines, e.g. for coronavirus, it has become clear that shortcomings in liposomal carriers could be detrimental to effective vaccine delivery. Major challenges include 1) rapid liposomal degradation or uptake, and 2) toxic buildup from synthetic stabilizing materials. We address these challenges through an integrated molecular design of sterol-enhanced liposomes functionalized with novel biopolymers for enhanced mechanical stability, biocompatibility, and target specificity.

Juhong Chen (PI, CALS / COE), Lijuan Yuan (Co-PI, VMCVM), Matthew Moore (Co-PI, University of Massachusetts Amherst)
Title: Improving the limit of viral pathogen detection in food matrices using engineering phages to concentrate and purify viral particles
Summary: Viruses pose grave threats to public health and the global economy because of their low infectious dose and difficulty of detection. Many analytical methods exist for the detection of environmentally present viruses, but the upstream concentration is required. However, current viral concentration techniques are not ideal, limiting the ability to realistically detect most food and environmental viruses. Herein, we aim to engineer phages displaying specific virus-binding nanobodies to facilitate the concentration and purification of viruses for easier detection.

Andrew Lowell (PI, COS), Anne M. Brown (Co-PI, CALS)
Title: Pioneering new routes for antibiotic development: Using computational modeling and medicinal chemistry to reconfigure chemotherapeutics as selective antibiotics
Summary: We are using a combination of cutting-edge molecular modeling and medicinal chemistry techniques to convert broadly toxic chemotherapeutic drugs into bacteria-specific antibiotics. This work enhances the field of large-scale biochemical modeling (by developing new tools to better handle large organelles, such as the bacterial ribosome that is our preliminary target) and creates new groups of antibiotics from existing, potent drugs (a novel field of research that will be further developed with medicinal chemistry).

Nick Ruktanonchai (PI, VMCVM), Ryan Calder (Co-PI, VMCVM), Omar Saucedo (Co-PI, COS). 
Title: Understanding food worker decisions during COVID-19 to minimize worker disease and food system disruption from future pandemics
Summary: Food workers have been disproportionately affected by the COVID-19 pandemic due to outbreaks in food facilities and aggravating socioeconomic factors. These workers’ importance to food supply chains makes understanding their response to COVID-19 and future pandemics doubly important for food security and pandemic preparedness more broadly. We use a novel survey-based and modeling methodology to understand drivers of COVID-19 in food facilities and how different policy interventions may protect worker health and food system reliability.

David Schmale (PI, CALS), Peter Vikesland (Co-PI, COE), Nastassja Lewinski (Co-PI, Virginia Commonwealth University)
Title: A Novel Aerosol Collection System to Trap and Detect Obligate Plant Pathogens on Live Plant Cells
Summary: New technologies are required to collect and rapidly detect high risk plant pathogens before they cause plant disease pandemics. Such approaches must accommodate strategies to detect obligate (require a host to survive) and unculturable plant pathogens. We propose to develop a novel aerosol collection system to trap and detect obligate plant pathogens from the atmosphere on living plant cells. Our proposal addresses the CeZAP focus areas of Ecology and Epidemiology Environmental Microbiology.

James Weger-Lucarelli (PI, VMCVM), Anne M. Brown (Co-PI, CALS)
Title: Molecularly barcoded SARS-CoV-2 to probe in vivo evolutionary dynamics
Summary: Tools enabling a better understanding and prediction of SARS-CoV-2 transmission are desperately needed to design public health measures aimed at reducing transmission. Here, we will generate a molecularly barcoded SARS-CoV-2, allowing the study of transmission dynamics (e.g., bottlenecks, the compartmentalization of transmitted virus, and the effect of different biological factors, e.g., sex, obesity, age) in vivo. The molecularly barcoded virus will be shared openly with other researchers within CeZAP and throughout the scientific community.

Mike Zhang (PI, CALS / COE), James Weger-Lucarelli (Co-PI, VMCVM)
Title: Conjugation of multiple antigens onto a hubrid nanoparticle and the evaluation of its immunogenic potentials
Summary: Vaccination is one of the most important methods in fighting against infectious diseases. Often, to maximize protection, vaccines that are able to elicit significant immune responses toward more than one pathogen-associated antigen are needed. This project aims to develop a nanoparticle-based platform that can simultaneously display multiple antigens. Three different proteins will be conjugated onto the surface of nanoparticles, and animal tests will be conducted to test the immunological potentials of those nanoparticles.

Supported by Fralin Life Sciences Institute, and Virginia Agricultural Experiment Station

Brandon Jutras (PI, CALS), Coy Allen (Co-PI, VMCVM); Pablo Sobrado, (Co-I, CALS); Rich Helm (Co-I, CALS)
Title: Reprogramming the peptidoglycan cell-wall of Borrelia burgdorferi to understand, treat, and cure chronic Lyme disease
Summary: Late stage manifestations of Lyme disease, such as Lyme arthritis, are complex and poorly understood. We have discovered that unique fragments of peptidoglycan (PG)—an essential component of the bacterial cell wall— are released by Borrelia burgdorferi, the Lyme disease agent, during growth. Virtually all bacteria have PG but, as it turns out, the chemical composition of B. burgdorferi PG is unique and unlike any previously described. B. burgdorferi PG is capable of both persisting in Lyme arthritis patients’ months after infection and causing arthritis in a mouse model. Here, we propose to alter the chemistry of B. burgdorferi PG and determine the impacts on infection, persistence, and pathogenesis.

Mohamed Seleem (PI, VMCVM), Paul R. Carlier (Co-PI, COS)
Title: Optimization of salicylamide analogs for combating multidrug-resistant Neisseria Gonorrhoeae
Summary: Studies proposed in this grant application build upon advances made in collaborative efforts between the Seleem and Carlier laboratories (College of Veterinary Medicine and College of Science, respectively) over the past few months to optimize the repurposed FDA-approved drug, salicylamide (1a) against Neisseria gonorrhoeae. The Carlier lab will carry out analog design and synthesis while the Seleem lab will perform the antibacterial study with the goal to increase potency and efficacy of salicylamide.

Juhong Chen (PI, CALS/COE), Clay Wright (Co-PI, CALS/COE); Lijuan Yuan (Co-PI, VMCVM); Matthew Moore (Co-PI, University of Massachusetts Amherst)
Title: Development of engineered yeasts to concentrate and purify foodborne viruses for easier detection
Summary: Viruses pose grave threats to public health and the global economy because of their low infectious dose and difficulty of detection. Many analytical methods exist for detection of environmentally present viruses, but upstream concentration is required. However, current viral concentration techniques are not ideal, limiting the ability to detect most viruses routinely. We aim to engineer yeast displaying specific virus-binding peptides on their surface proteins to facilitate concentration and purification of viruses for easier detection.

Michael Schulz (PI, COS), Lijuan Yuan (Co-PI, VMCVM)
Title: Developing Antiviral Polymers to Inhibit Norovirus Infection
Summary: Noroviruses (NoVs) are the most prevalent cause of nonbacterial acute gastroenteritis, but no FDA-approved vaccines or anti-viral drugs currently exist for NoV infection. Antiviral polymers can prevent viral infections but have never been investigated for NoV treatment. We will synthesize the first anti-NoV materials and evaluate them in the gnotobiotic (Gn) pig model. This state-of-the-art animal model will enable us to draw strong translational conclusions about the potential clinical efficacy of these novel antiviral materials.

Song Li (PI, CALS), Juhong Chen (Co-PI, COE/CALS); Boris Vinatzer (Co-PI, CALS)
Title: RACING: Developing a Rapid Assay using CRISPR, artificial Intelligence, and Nanopore meta-Genome sequencing for emerging pathogen detection
Summary: Developing diagnostic assays for emerging pathogens is typically time consuming and cannot meet the need to stop rapidly spreading diseases. Herein, we plan to develop an approach that substantially simplifies the pipeline for diagnostic assay development. Our approach includes: (1) Improve our cutting-edge machine learning algorithms to identify signature nucleotide sequences for pathogens from culture-free metagenomic sequencing data; and (2) optimize a CRISPR-based biosensing system to detect pathogen DNA using these signatures.

Luis Escobar (PI, CNRE), Gill Eastwood (Co-PI, CALS); Mark Ford (Co-PI, CNRE)
Title: Relationship between Lyme disease and land-form variables
Summary: Our goal is to better elucidate the relationship between Lyme disease and other tick-borne diseases and landscape configuration, accounting for microclimate, tick ecology, and other environmental factors. This project is inspired by the research question: Is tick-borne disease transmission predictable in space and time based on quantifiable environmental correlates? The project hypothesis is that predictability of tick occurrence will vary according to the scale studied. We will model the distribution and abundance of ticks responsible of Lyme disease across diverse environmental conditions.

Jonathan Auguste (PI, CALS), Rana Ashkar (Co-PI, COS); Coy Allen (Co-I, VMCVM); Jesse Erasmus (Co-I, HDT Bio Corp) 
Title: Evaluating the safety and efficacy of a novel vaccine strategy for Cache Valley virus
Summary: Cache Valley virus (CVV) is an important zoonotic arthropod-borne virus in North America. CVV can potentially emerge and become a major pathogen of humans, ovine, caprine, bovine, equid, and poultry species, resulting in potentially devastating consequences on our economy, food availability, human and animal health. To circumvent this threat, we will develop an RNA vaccine that demonstrates exceptional immunogenicity and outstanding safety in murine models, and completely protects against all aspects of CVV disease.

Nisha Duggal (PI, VMCVM), Linsey Marr (Co-PI, COE); Stanca Ciupe (Co-PI, COS)
Title: Aerosol transmission potential of SARS-CoV-2 in exhaled breath
Summary: SARS-CoV-2 is a respiratory virus, with transmission occurring via droplets released during coughing and sneezing. SARS-CoV-2 is also detected in air, and asymptomatic individuals may transmit SARS-CoV-2 in aerosols within exhaled breath. Using a hamster model of SARS-CoV-2 transmission, we will measure infectious SARS-CoV-2 in aerosols collected from exhaled breath and quantify the airborne transmission potential of SARS-CoV-2. This work will improve our understanding of SARS-CoV-2 transmission and lead to future work developing interventions.

Clément Vinauger (PI, CALS), Lauren Childs (Co-PI, COS); James Weger-Lucarelli (Co-I, VMCVM)
Title: Effects of altered larval growing conditions on the vectorial capacity of Aedes aegypti mosquitoes
Summary: The environmental growing conditions of mosquito larvae affect their development and, eventually, alter the body size and host-seeking behavior of adult females. Here, we propose a collaborative and multidisciplinary approach to 1) determine the sensory basis of this phenomenon, 2) define its impact on mosquitoes’ ability to transmit viruses, and 3) integrate this new knowledge in a mathematical model linking environmental effects on mosquito traits with their consequences on population dynamics and vectorial capacity.

Gillian Eastwood (PI, CALS), Luis Escobar (Co-PI, CNRE)
Title: The Effect of Forest Degradation on Mosquito Arboviruses
Summary: Landscape changes can influence biodiversity composition and the emergence of outbreaks, triggering spillover flow of pathogenic agents to incidental hosts. We focus on vector-borne disease, and determine vectors of arboviral pathogens in a previously unexplored region of Guatemala which is experiencing habitat destruction of intact pristine forest. By investigating both altered mosquito vector diversity and infection prevalence of mosquitoes in this system, we can define the impact of a deforestation gradient upon disease risk.