ROLE OF VIRUSES IN NATURAL ECOSYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006818
• Project Start Date: Jul 1, 2015
• Project End Date: Jun 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717

Performing Department
Plant Sciences and Plant Pathology

Non Technical Summary
The traditional view is that viruses act solely as pathogens in which the host tries to eliminate viral infection. Hence, viruses are thought to control community composition by negatively impacting the fitness of infected hosts including plants. This research investigates an alternative hypothesis that chronic viral infections contribute positively to host fitness, increasing the success of the virus-host pair. This hypothesis holds that in highly competitive microbial communities, chronic virus infection protects hosts from infection by more pathogenic viruses. Under this model, the competitive advantage provided by viruses plays a direct role in maintaining microbial biodiversity. This research lies at the intersection of three levels of biodiversity: (i) the taxonomic & phylogenetic level, by examining the evolution and coexistence of distinct viral and host taxa, (ii) the genetic level, by identifying specific microevolutionary changes emerging during host-virus coevolution, and (iii) the functional level, by characterizing how variation in host and viral life-history traits affect community structure and stability. This study will use a tractable Yellowstone hot spring model system that provides a unique opportunity to (i) link temporal changes in virus abundance and diversity to host genetic and taxonomic diversity, (ii) identify chronic viruses, and quantify the fitness consequences of chronic viral infections using a laboratory based system, (iii) assess the effects of removing viruses in laboratory and in situ experiments, and (iv) develop a theoretical and computational model of eco-evolutionary, host-viral interactions that includes the costs and benefits of chronic infections. It is anticipated that this research will provide new insights into how viruses influence not only microbial biodiversity, but also biodiversity of plants and animals.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	4030	1101	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1101 - Virology;

Keywords

virus detection

host-virus interactions

crispr

viral metagenomics

virus population dynamics

Goals / Objectives
This proposal examines how the dynamic interplay between organisms and their viruses influences the generation and maintenance of biodiversity. All organisms are subject to viral infection, moreover, genomes from all domains of life are replete with chronic proviral infections. Established models of host-virus interactions assume viruses are antagonistic, acting as predators or parasites that decrease host fitness. Our goal is to examine an emerging, alternative hypothesis that chronic viral infections contribute positively to host fitness, increasing the success of the virus-host pair as an evolutionary unit. We hypothesize that these benefits are realized in a highly competitive community context where chronic viruses protect their hosts from future infection, and increase their competitive advantage when they act antagonistically against susceptible competitors. How does broadening of the functional role of viral symbiosis change predictions about the evolution of biodiversity in natural systems? Our objective is to extend our understanding of virus-host dynamics to include the benefits of chronic viral infection and account for the corresponding co-evolutionary consequences to host defenses when chronic viruses are beneficial. Our long-term goal is to provide a comprehensive understanding of the role of viruses in natural ecosystems by including their ecological role in multiple forms of symbiosis. Our integrated approach is well positioned to address three core dimensions of diversity: (i) taxonomic & phylogenetic, by studying the evolution and coexistence of distinct viral and archaeal taxa, (ii) genetic, by identifying specific microevolutionary changes emerging in coevolution, (iii) functional, by characterizing variation in interactions and life history traits of viruses. Because viruses infect all organisms, we expect this work to provide transformative understanding of virus-host interactions that is broadly applicable across ecosystems. The Specific Objectives (SO) of this proposal are to:SO1: Link temporal changes in virus abundance and diversity to host genetic and taxonomic diversity in the natural geothermal environment.SO2: Identify chronic viruses, and quantify the fitness consequences of chronic viral infections.SO3: Assess the effects of removing free virus particles in laboratory and in situ experiments.SO4: Develop a theoretical and computational eco-evolutionary model of host-viral interactions that includes the costs and benefits of chronic infections.

Project Methods
SO1: Link temporal changes in virus abundance and diversity to host composition in a geothermal hot spring community (NL10).Experimental strategy. Using a combination of culture-independent techniques, we propose to quantify the relative abundance of viral types, infection prevalence, host specificity, immune and resistance profiles and degree of multiple infection that are ongoing in the simple environment of a single geothermal hot spring over time.The aim is to provide the data that will both inform parameters for the mathematical modeling and validate its predictions in natural populations. Briefly outlined, we propose to perform a combination of (i) yearly viral and cellular community metagenomic analysis, (ii) monthly viral microarray-based and qPCR analysis to quantify abundances of free virus particles and the cell-associated virus fraction (separated by in-line filtration through 0.45 um filters). Our custom microarray will contain multiple oligonucleotide probes to the top 100 abundant virus types and cellular phylotypes based on our previous metagenoinc and SCG datasets. Microarray results will be used to guide qPCR analysis of virus and cell types in the free virus and cell-associated environmental fractions. We have previous experience performing all of these proposed experiments and we do not anticipate any major technical difficulties. To link the taxonomic diversity and abundance of viruses to their host's, we propose a combination of microscopy and single cell based screens of viral types resident within cells. Viral host range, infection prevalence, and the presence of multiple virus infections will be assessed by combined vFISH and rFISH assays.In acomplementary approach, vFISH in combination with FACS will be used to count the number of cells infected by a particular virus type. Multiple virus infections within individual cells will be assessed by multiplexing vFISH probes, each with unique fluorophores. To accurately link genetic diversity in viruses and host populations we propose to perform simultaneous single cell genomic (SCG) screens. These screens will consist of two components; PCR directed cloning and sequencing of sorted single cells based on their rDNA type (100 cells per sampling), and single cell genome sequencing (2-10 cells per sampling point). This will directly link immune profile of the host (CRISPR/Cas diversity), identification of resistance loci (identified through experimental protocols SO2), with infection of specific viral genotypes. In addition, it will allow us to explore the genotypic diversity of co-infecting viral types.SO2: Identify chronic viruses, and quantify the fitness consequences of chronic viral infections.Experimental strategy: To better understand the function of different viruses in the NL10 hot spring community we propose to first determine which types exhibit chronic lifestyle on wild hosts. We will isolate and sequence a set of 20 wild host strains from NL10 in Yellowstone National Park. Sequences will reveal the CRISPR-immunity profile of each strain as well whether they are chronically infected by native viruses. In addition, we will isolate and sequence a set of 20 wild viruses from the same hot spring. We will mark each host strain with a broadly applicable neutral marker for future competition assays. For each pair of marked host and wild virus we will perform replicated infection assays in which hosts are co-cultured with viruses for 10 days. Following infections 100 hosts will be isolated as single colonies and screened for viral infection using PCR primers designed based on viral sequences. Virus-host pairs for which infected host colonies grow will be shown to establish chronic viral infections. Once non-lytic viruses capable of chronic infection are identified we can assess the relative prevalence of chronic and lytic infection and the relative diversity of chronic and lytic viruses using the data generated in SO1. This will allow us to test one of our key predictions about diversity and distribution of chronic viruses. For each established chronic virus-host pair we will quantify the consequences of infection for host fitness. We will measure the cost of establishing initial chronic infection by measuring the delay in growth and host viability across the time course of infection for each chronic virus-host pair. To measure the cost of infection to cell growth we will measure the relative fitness of each infected virus-host pair in competition with the unmarked ancestor of each infected host. We will identify the relative costs of infection in strains with and without CRISPR spacer matches and integrate these parameters into the mathematical model (SO4) and test the consequences of these predictions using the CRISPR immune profiles and viral infection prevalence determined in SO1. This will allow us to test one of our key predictions about the impact of immunity on chronic infections.SO3: Quantify the effect of removing free viruses in the laboratory and in situ experiments.Experimental strategy: We will use a field deployable chemostate-like continuous flow reactor (CFR) in which we have engineered a high intensity UV source in-line with the inflow. This UV source allows us to UV inactivate incoming viruses and cells from the hot spring into the CFR pre-seeded with the NL10 cellular and viral community. A comparison of the cellular community dynamics within the CFR, +/- UV treatment will be used as a method to examine the effects of altering free virus inputs into a microbial community. In the field using this CFR, we have demonstrated that the active component of the microbial community sampled from an acid sulfate chloride hot spring, when localized to the CFR, was not statistically different from that sampled directly from the spring (12). We will begin by developing the application of this method to isolated virus and host populations under controlled experimental laboratory conditions. We propose to remove free viruses from competition experiments and examine the consequences on host relative fitness. Innovations will include optimization of UV dosage. The cells will be collected, returned to media and incubated approximately 10-14 days. These cultures, along with controls of cultures containing both free virus and cells, will be sampled daily for up to 14 days. Samples will be analyzed by viral and cellular qPCR analysis, vFISH and rFISH techniques and SCG analysis. After optimization under laboratory conditions, we will extend our studies to the field. This will involve the isolation of native hosts from free viruses in situ in NL10. The cells will be collected, returned to hot spring solution free of virus and cells (accomplished by 0.02 uM filtration), and maintained in situ. These cultures, along with controls of cultures containing both free virus and cells, will be sampled daily for up to 14 days. Samples will be analyzed by viral/cellular microarray analysis, selective viral and cellular qPCR analysis, vFISH and rFISH techniques and SCG analysis.SO4 Develop a theoretical and computational eco-evolutionary model of host-viral interactions that includes the costs and benefits of chronic infections.Modeling strategy: We will extend the computational model we developed for CRISPR-induced coevolution to include the changes in host-viral interactions caused by chronic infection. In the prior model, we considered the eco-evolutionary dynamics of host populations (Ni) and viral populations (Vj)

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologists in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellows in STEM fields.? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project continues to provide direct training of undergraduate students, graduate students, and postdoctoral scholars in the discovery, characterization and detection of viruses.? How have the results been disseminated to communities of interest?Results of this research have been communicated to parties of interest by publications in the scientific literature, interest articles in the general press, public lectures to the general public, invited seminars at universities, and by speeches at national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue our research agenda according to our project plans.?

Impacts
What was accomplished under these goals? The accomplishments of the past 12 months have been focused on conducting research and analyzing experimental results in order to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research accomplishments for this year included 1) the discovery and molecular characterization of two new archaeal virus families, (ii) viral and cellular metagenomic analysis of 24 high temperature environments and analysis of their community structure using network analysis, (iii) RNA transcriptomic analysis of 4 high temperature environments and their analysis in order to understand archaeal and viral gene expression and(iv) initial development ofviral-based viral markers useful in predicting the clinical outcomes of treatment of individuals with metabolic syndrome by FMT treatment.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wohlgemuth, R., Littlechild, J., MontI, D., Schnorr, K., van Rossum, T., Siebers, B., Menzel, P., Kublanov, I., Gunn Rike, A., Skretas, G., Szabo, Z., Peng, X., Young, M. Discovery of novel hydrolases from hot environments. Biotechnoloy Advances. 36:2077-2100, 2018. DI: 10.1016/j.biotechadv.2018.09.004
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Munson-McGee, J., Peng, S., Dewerf, S., , Stepanauskas, R., Whitaker,RW., Weitz, J., Young, M. A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments. ISME J. 12:1706-1714, 2018 DI: 10.1038/s41396-018-0071-7
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Munson-McGee, J., Snyder, J., Young, M. Archaeal Viruses from High-Temperature Environments. Genes 9, 2018. DI: 10.3390/genes9030128
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hochstein, R., Bollschweiler, D., Dharmavaram, S., Lintner, N., Plitzko, J., Bruinsma, R., Engelhardt, H., Young, M., Klug, W., Lawrence, C.M. Structural studies of Acidianus tailed spindle virus reveal a structural paradigm used in the assembly of spindle- shaped viruses. PNAS 115:2120-2125, 2018. DI: 10.1073/pnas.1719180115
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Jarett, J., Nayfach, S., Podar, M., Inskeep, W., Ivanova, N., Munson-McGee, J., Schulz, F., Young, M., Jay, Z., Beam, J., Kyrpides, N.,Malmstrom, R., Stepanauskas, R., Woyke, T. Single-cell genomics of co-sorted Nanoarchaeota suggests novel putative host associations and diversification of proteins involved in symbiosis. Mircobiome. 6:161, 2018. DI: 0.1186/s40168-018-0539-8
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Roux, S.,Adriaenssens, E., Dutilh, B., Koonin, E., Kropinski, A., Krupovic, M., Kuhn, J., Lavigne,, R., Brister, J., Varsani, A., Ramy K. Aziz11, Seth R. Bordenstein12, Peer Bork13, Mya Breitbart14, Guy Cochrane15, Rebecca A. Daly16, Christelle Desnues17, Melissa B. Duhaime18, Joanne B. Emerson19, Fran�ois Enault20, Jed A. Fuhrman21, Pascal Hingamp22, Philip Hugenholtz23, Bonnie L. Hurwitz24, Natalia N. Ivanova1, Jessica M. Labonte25, Rex R. Malmstrom1, Manuel Martinez-Garcia26, Ilene Mizrachi4, Hiroyuki Ogata27, David Paez-Espino1, Marie-Agn�s Petit28, Catherine Putonti29,30,31, Thomas Rattei32, Alejandro Reyes33, Francisco Rodriguez-Valera34, Karyna Rosario14, Lynn Schriml35, Frederik Schulz1, Grieg F. Steward36, Matthew B. Sullivan37,38, Shinichi Sunagawa39, Curtis A. Suttle40, Ben Temperton41, Susannah G. Tringe1, Rebecca Vega Thurber42, Nicole S. Webster43,44, Katrine L. Whiteson45, Steven W. Wilhelm46, K. Eric Wommack47, Tanja Woyke1, Kelly Wrighton16, Pelin Yilmaz48, Takashi Yoshida49, Mark J. Young50, Natalya Yutin4, Lisa Zeigler Allen51,52, Nikos C. Kyrpides1, Emiley A .Eloe-Fadrosh. Minimum Information about Uncultivated Virus Genomes (MIUViG). Nature Biotechnology. 2018, in press.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologists in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellows in STEM fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project continues to provide direct training of undergraduate students, graduate students, and postdoctoral scholars in the discovery, characterization and detection of viruses. How have the results been disseminated to communities of interest?Results of this research have been communicated to parties of interest by publications in the scientific literature, interest articles in the general press, public lectures to the general public, invited seminars at universities, and by speeches at national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue our research agenda according to our project plans.

Impacts
What was accomplished under these goals? The activity of this program included conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this year included 1) the discovery and molecular characterization of a entirely new virus family, (ii) analysis of cells directly from the environment that showed that most cells are simultaneously infected by multiple viruses, (iii) completion of initial experiments that show that an archaeal virus functions as a non-replicating but stable VLP in the animal gut environment, opening up the possibility for its use as a drug delivery system and (iv) an analysis of the viral community in the human gut that allows one to predict if an individual will be responsive to FMT based therapies.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wagner, C., Reddy, V., Asturias F., MKhoshouer, M., Johnson, J.E., Manrique, P., Munson-McGee, J., Baumeister, W., Lawrence, C.L., Young, M. Isolation and Characterization of Metallosphaera turreted icosahedral virus (MTIV), a founding member of a new family of archaeal viruses. J. Virol. 91 (20), e00925-17, 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fouqueau, T., Blombach, F., Hartman, R., Cheung, A., Young, M., Werner, F. The transcript cleavage factor paralogue TFS4 is a potent RNA polymerase inhibitor. Nat. Comm. 8, 2017, doi 10.1038/s41467-017-02081-3.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Uldahl, K., Walk, S., Olshefsky, S., Young, M., Peng, X. SMV1, an extremely stable thermophilic virus platform for nanoparticel trafficking in the mammalian GI tract. J. Appl Microb. 123:1286-1297, 2017. doi 10.1111/jam.13584.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Manrique, P., Dills, M., Young, M. The human gut phage community and its implications for health and disease. Viruses, 9. 2017. doi 10.3390/v9060141.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Munson-McGee, J., Peng, S., Dewerf, S., , Stepanauskas, R., Whitaker,RW., Weitz, J., Young, M. A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments. ISME

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologists in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellows in STEM fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project continues to provide direct training of undergraduate students, graduate students, and postdoctoral scholars in the discovery, characterization and detection of viruses. How have the results been disseminated to communities of interest?Results have been communicated to parties of interest by publications in the scientific literature, interest articles in the press, invited seminars at universities, and by speeches at national international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue research according to our project plans.

Impacts
What was accomplished under these goals? The activity of this program included conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this year included 1) high resolution temporal sampling of virus and host populations in a model microbial community, (ii) completion of sequencing of single cell host and associated viral DNA, (iii) the discovery of new viruses to science, (iv) and development of comprehensive methods to identify viral-host assoications in a culture independnet process which is applicable to agriculturaly important plant and animal viruses.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Pomwised, R., Intamaso, U., Teintze, M., Young, M., Pincus, S. H. (2016). Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response. Vaccines, 4(2), 15
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hochstein, R., Amenabar, M. J., Munson-McGee, J. H., Boyd, E., Young, M. (2016). Acidianus tailed spindle virus: a new archaeal large tailed spindle virus discovered by culture-independent methods. Journal of Virology. http://jvi.asm.org/content/early/2016/01/08/JVI.03098-15.abstract
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Manrique, P., Bolduc, B., Walk, S., van der Oost, J., de Vos, W. M., Young, M. (2016). Healthy human gut phageome. Proceedings of the National Academy of Sciences, 201601060.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Gudbergsd�ttir, S. R., Menzel, P., Krogh, A., Young, M., Peng, X. (2016). Novel viral genomes identified from six metagenomes reveal wide distribution of archaeal viruses and high viral diversity in terrestrial hot springs. Environmental microbiology.

Progress 07/01/15 to 09/30/15

Outputs
Target Audience:The general target audience for this project is the scientific and higher education student community. The specific scientific audience is research virologists and microbiologists in the US and worldwide. The specific higher education student community is undergraduate students, graduate students, and postdoctoral fellows in STEM fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the following opportunities for training and professional development; (i) The PhD training of 3 graduate students in virology, (ii) the training of 1 postdoctoral fellows in the molecular aspects of virology, and (iii) the training of technicians on the identification of viruses. How have the results been disseminated to communities of interest?Results have been communicated to parties of interest by publications in the scientific literature, interest articles in the press, invited seminars at universities, and by speeches at national international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue research according to our project plans.

Impacts
What was accomplished under these goals? The activity of this program included conducting and analyzing experiments research is to contribute to our understanding of virus diversity, ecology, and evolution. In specific, the research activities for this year included 1) high resolution temporal sampling of virus and host populations in a model microbial community, (ii) completion of sequencing of single cell host and associated viral DNA, (iii) and the discovery of four new viruses to science.?

Publications