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Current Projects
Discovery and Characterization of Novel Pathogenic Viruses from Animals Diseases amongst organisms are emerging at an increasing rate, but the causative agents of these diseases are largely unknown. Current methods for diagnosing viral infections in marine animals use PCR, immunologic assays, or replication in cell culture to test for specific viruses. The problem with these techniques is that they require prior knowledge and assumptions about the type of virus expected (i.e., you find only what you look for). While current methods are good at diagnosing known viruses, they are very ineffective for discovering new viruses. Diagnosing novel viral infections is difficult due to our inability to culture many viruses on cell lines in the laboratory, the small size and low nucleic acid content of viruses, and the lack of a conserved genetic element that is found in all viral genomes that can be used for PCR-based analyses. The limitations of virus discovery can be overcome through the use of viral metagenomics. First, virus particles are purified from a sample of interest by the selection for viral particles based on size, density, and nuclease resistance. Viral purification is then followed by sequence-independent amplification and shotgun sequencing. We have developed this technique to discover new animal viruses in blood and many different types of tissues, such as brain, lungs, skin and tumors. We are currently collaborating with pathologists and veterinarians throughout the United States to investigate new and unknown viral infections in animals. Once novel viruses are identified from the viral metagenome, PCR primers are designed to rapidly screen a large number of samples in order to determine the prevalence, abundance, and geographical distribution of the novel viruses, as well as epidemiological links between infected animals. By examining healthy animals and the surrounding environment for specific viral pathogens, it will be possible to determine if the apparent increase in diseases amongst marine organisms is due to the introduction of new viruses, or if the viruses are newly associated with disease due to stress or other environmental factors. Using viral metagenomics we have discovered novel viruses in sea turtles and sea lions using this method, and efforts to characterize viruses in other marine animals are ongoing. This approach towards conservation medicine has implications for management of protected species, veterinary diagnosis, treatment of infectious diseases, and our understanding of molecular evolution of viruses. Our results were presented in the International Association for Aquatic Animal Medicine (IAAAM) Conference in 2007 and 2008, in the American Society of Microbiology Conference in 2008, in the Florida Marine Mammal Health conference in 2008. If you are a veterinarian or pathologist treating an animal with an unknown viral infection and are interested in this technique, please contact Mya by e-mail: mya@marine.usf.edu
----------------------------------------------------------------------------------------------------------------------------------- Molecular Analysis of Bacterial Communities Associated with Corals During Culture and Transplantation
Corals are known to harbor abundant microbial communities, with approximately 100 million bacteria per square centimeter of coral surface. Corals rely on their bacteria-laden mucus layer as a protective barrier against pathogens, and can acquire water column microbes to better adapt to changing environmental conditions. Bacterial communities within coral mucus are species-specific associations that are maintained over time and space. Disease and bleaching in corals are often associated with changes in composition or activity of the associated microbial community. The deteriorating health of many coral reefs worldwide has prompted researchers to develop alternate strategies for coral conservation. Our collaborators at The Florida Aquarium, Florida Keys National Marine Sanctuary, Mote Marine Laboratory, and the University of Florida Tropical Aquaculture Laboratory have developed a project to fragment Caribbean scleractinian corals, grow/maintain them in culture, and then transplant them back to the wild. Maintaining corals in culture and then transplanting them into the wild has raised issues about the susceptibility of these corals to bleaching and disease, as it is unknown what effect this will have on the beneficial microbes normally found in their protective mucus layer. Identification of various coral diseases and syndromes has classically been limited to visual characterization, but by the time changes are evident the coral is usually already compromised. The advent of molecular profiling techniques allows for the detection of changes in the coral-associated bacterial community, before and after symptoms present. Monitoring the coral-associated microbial community composition in the natural environment, under various culture conditions, and following transplantation back into the Florida Keys will provide insight into the stability of coral-bacterial associations, determine if transplanted coral represent a source of new bacteria to the environment, and provide preliminary information on the condition of corals prior to transplantation for restoration projects. My project applies both culture work and the molecular community profiling technique, ARISA (Automated Ribosomal Intergenic Spacer Analysis), to track bacterial communities in several species of corals from initial fragmentation, throughout the culturing process, and after transplantation back into the environment. This study will provide insight into the stability of bacterial communities associated with corals, and determine if culture conditions lead to changes in the composition of coral-associated bacterial communities that may affect the health and survival of both the transplanted corals and those on the surrounding reefs. Funding is provided by Project AWARE and the Florida’s Wildlife Legacy Initiative.
----------------------------------------------------------------------------------------------------------------------------------- Diversity and Distribution of Pathogenic Viruses in the Florida Keys The majority of treated wastewater, as well as untreated sewage, drains into the marine environment. Millions of pathogens (i.e. viruses, protists, and bacteria) are excreted in human fecal matter and current methods of sewage treatment do not always effectively remove these organisms. While point sources of fecal pollution (i.e. treated wastewater discharge, combined sewer overflows, municipal storm sewer systems, etc) can have significant impacts to the health of coastal environments, non-point sources of fecal pollution account for ~ half of the beach closures in the U.S. and can pose an even bigger threat due to difficulties in identifying and mitigating the source of pollution. Run-off, farm animals, wildlife, septic systems, swimmers, and faulty sanitary sewer lines are all examples of non-point sources of fecal contamination. Currently, the US EPA mandates the use of bacterial indicators, such as fecal coliforms and enterococci, to assess water quality. Although monitoring of these bacteria is simple and inexpensive, it has been shown that fecal-associated bacteria are not ideal indicators because they can survive in the environment without active fecal contamination and are more susceptible to decay than other types of pathogens. Since concentrations of fecal coliforms and enterococci inadequately detect fecal pollution and therefore inaccurately depict the risks to human health, many have proposed the use of an alternative viral indicator of wastewater contamination. While it is impractical to monitor the presence of all viral pathogens related to wastewater pollution, the development of an accurate viral indicator of sewage contamination is needed for enhanced water quality monitoring. To develop a comprehensive water quality indicator protocol, it is first necessary to establish a broad, baseline understanding of the many pathogenic, eukaryotic viruses in raw sewage and to assess the natural presence of these viruses in the coastal marine environment. PCR was used to detect 10 major viral groups (adenoviruses, herpesviruses, hepatitis B viruses, morbilliviruses, noroviruses, papillomaviruses, pepper mild mottle viruses, picobirnaviruses, reoviruses, and rotaviruses) in raw sewage collected from throughout the United States and from five marine environments ranging in their proximity to dense human populations. This baseline understanding of viruses in raw sewage and the marine environment will enable educated decisions to be made regarding the use of viruses in water quality assessments and identify viruses that can potentially be used to indicate wastewater pollution in coastal environments. Funding for this research is provided by the Environmental Protection Agency.
----------------------------------------------------------------------------------------------------------------------------------- Agricultural irrigation places enormous demands on natural groundwater supplies. With well water for crop production in Florida becoming more and more restricted, finding alternative water sources for irrigation has become a priority. The use of reclaimed water helps decrease the amount of withdrawals from natural aquifers and as such is economically advantageous to growers. The acreage of crops irrigated with reclaimed sewage water is increasing every year. However, the presence of plant pathogens, particularly viruses, in reclaimed water is largely unknown. We have recently shown that plant viruses are abundant in human feces, suggesting that reclaimed water may be a possible reservoir and dissemination mechanism for pathogenic plant viruses. This may also be a hitherto unknown mechanism for the introduction of exotic plant viruses into Florida. As reclaimed water use in crop production becomes more widespread, it is necessary to understand the implications this might have for growers. The purpose of this project is to characterize RNA and DNA viruses present in reclaimed water, with particular emphasis on detecting plant pathogenic viruses that may be detrimental to crop production. During the course of this project, we will use microscopy to compare the total concentration of viruses in reclaimed water with that of groundwater and raw sewage, and use culture-based and molecular methods to identify plant pathogenic viruses present in both raw sewage and reclaimed water. We will identify viruses through metagenomic (whole community genomic) sequencing to characterize the types of viral sequences found in reclaimed water (collaboration with Yijun Ruan and Christina Nilsson at the Genome Institute of Singapore). This approach will give us a good idea of what viruses are present in this alternative water supply without having prior knowledge of their existence. Through a collaboration with Jane Polston at the University of Florida and Scott Adkins from the USDA, the infectious nature of the viruses detected in raw sewage and reclaimed water will be established by inoculation of indicator plants with virus extracts from raw sewage and reclaimed water. Collectively, this information can be used to determine if there are potential risks for those who use reclaimed water for crop irrigation.
----------------------------------------------------------------------------------------------------------------------------------- Abundance, Dynamics, and Diversity of Viruses in the Northwestern Sargasso Sea Viruses are ubiquitous, abundant, and dynamic components of marine communities. The majority of marine viruses are phage (viruses that infect bacteria). Viruses influence global carbon and nutrient cycles, regulate the composition of marine bacterial communities, and play a major role in horizontal gene transfer. Recent studies have revealed an enormous diversity of viruses in the oceans; however, most in-depth studies of viral diversity have focused on a single sample – a snapshot in space and time. Since bacterial and viral communities are dynamic in nature (rapid infection, production, decay), snapshots of diversity are inadequate for describing the microbial ecology of marine systems. This project will examine the abundance, diversity, and dynamics of viruses over a depth profile at Hydrostation S in the northwestern Sargasso Sea. Viral abundance throughout a depth profile will be determined monthly by direct microscopic counts and changes in the composition of the viral community will be monitored using molecular methods (pulsed-field gel electrophoresis, hybridization against microarrays, and PCR for specific phage genes). Rates of virus production and decay, as well as the impact of viruses on the bacterial community, will be determined. Finally, this project will examine the ecology of the marine single-stranded DNA Microphage, a newly described viral group found to be abundant in the Sargasso Sea. The goal of this project is to produce a comprehensive dataset on the spatial and temporal variation of the viral community at this site and determine if there are recurring (and thus predictable) patterns in viral abundance and diversity. This project is a collaboration with Craig Carlson, and is funded by a Microbial Interactions and Processes grant from the National Science Foundation.
----------------------------------------------------------------------------------------------------------------------------------- Isolation and Characterization of Viruses from an Acid Mine Drainage System Acid mine drainage (AMD) is a worldwide environmental problem associated with past mining activity. Within disused mines, extreme environmental conditions may develop that are characterized by high levels of dissolved sulfur, iron, and other metals as well as very low pH (0.5-1.0). While unique communities of bacteria and archaea have been identified in these systems, viruses and interactions with their hosts have yet to be fully characterized. We are working in collaboration with the Banfield lab at the University of California at Berkeley (UCB), who have been studying this system for many years. Methods for viral isolation from the AMD biofilm are being optimized for each sampling location, and viral concentrates are being generated for metagenomic sequencing and proteomics studies in collaboration with Oak Ridge National Laboratory (ORNL) and UCB. Viral genomes will be reconstructed from metagenomic sequences, allowing for estimates of viral community structure. The level of viral diversity will be assessed and compared to bacterial and archaeal diversity in the AMD system in collaboration with UCB. Viral morphology will be characterized using TEM and cryo-electron tomography in collaboration with Lawrence Berkeley National Laboratory (LBNL). This project is funded by a Genomes to Life grant from the Department of Energy.
----------------------------------------------------------------------------------------------------------------------------------- Microbial Ecology of Living Stromatolites in Cuatro Cienegas, Mexico The Cuatro Cienegas Basin (CCB) is a system of hundreds of pools and streams in the Chihuahuan desert of Coahuila, Northern Mexico. The CCB is thought to have the highest level of endemic biodiversity in all of North America, supporting more than 70 endemic species of aquatic invertebrates and vertebrates. The microbial communities (consisting of Bacteria, Archaea, and viruses) in the CCB pools are also extremely abundant and diverse. The pools in the CCB are excellent sites to examine natural microbial and viral diversity, as well as the impacts of increased human activity, since these are geographically isolated areas surrounded by the Chihuahuan desert. One of the most unique features of the CCB is the presence of large, microbialites, which are widespread throughout the fossil record, but only actively forming in a few unique locations. With collaborators David Hollander (USF), Janet Siefert (Rice University), and Valeria Souza (UNAM), we are using genomics and stable isotope analyses to examine the diversity and activity of microbes associated with the microbialites. Bulk analyses have revealed the presence of a diverse, redox-dependent microbial community and current research is examining microbial metabolism on small spatial scales to identify processes directly involved in carbonate precipitation. This project is supported by the National Geographic Society and a New Researcher Grant from the University of South Florida. See videos about this field site by Neilan Kuntz (Plaid Productions)
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