Quantitative Viral Assessments for Improved Biomonitoring

Project Overview

Viruses are the most abundant biological entities on Earth. In soils, viruses play a large but uncharacterized role as regulators of microbial growth dynamics, microbial evolution and antibiotic resistance, and soil carbon cycling. Current research on the diversity, functional capabilities, and host linkages of environmental viruses is expanding near exponentially. This is largely due to their potential importance for biosecurity, strong potential for antimicrobial function, and heretofore unknown effects on environmental biogeochemical cycles. However, current soil-virus methods are fragmented, limiting our ability to make connections important to ecology, pathogenicity, and potential biotechnology applications. The primary goal of this LDRD was to improve biomonitoring of viruses (i.e., detection, characterization, persistence, and infectivity), identify keystone viral and microbial populations, and obtain a better fundamental-ecosystem-process understanding of viruses in soil systems, with an emphasis on carbon and phosphorus cycling.

To reach this goal, we conducted two initiatives. First, we developed a novel, integrated pipeline that detected and allowed characterization of all virus types in a sample. For this pipeline, we conducted a H218O wet-up study and sampled over a three-week time series. We sequenced 192 stable-isotope probing (SIP) metagenomes to identify active microbes and proviruses, 43 SIP and 39 bulk viromes to identify active virions and capture single- and double-stranded DNA viruses, 39 bulk metatranscriptomics to identify RNA viruses and gene expression, 39 environmental DNA (eDNA) surveys to characterize the origin, function, and persistence of eDNA, and 4 long-read viromes to increase detection of mobile genetic elements and auxiliary metabolic genes, resulting in 3.1 Tbp of sequence data across our time-series incubations. Our first-of-their-kind protocols have allowed the first characterization of free DNA and isotopically-labeled virions in soil. Our data shows the amount of eDNA decreased upwards of 50% from dry soil through one week post wet-up, coinciding with a bloom of microbial populations. Next, recovery of active viral populations increased twofold from week 1 through week 2, coinciding with the decrease of virus-linked active microbial populations. Based on genomic similarity, the eDNA is microbial in origin and viral community turnover occurs within a week. These results suggest that eDNA accumulates over the dry summer and, after the first rainfall, the eDNA fuels the bacterial community, which in turn fuels the viral community. To build upon this, in a second initiative we developed a protocol using radioisotope phosphorus to track virus roles in phosphorus cycling and persistence in soils. We incubated soils with radiolabeled phosphoric acid (H332PO4) and tracked beta radiation and found that virus-particle turnover is on the order of days and more than a third of the radioactive phosphorus was detected in the virus and in the lysate fraction.

Mission Impact

This project supported the NNSA goal of expanding and applying our science-and-technology capabilities to deal with broad national-security challenges and to develop science-and-technology tools and capabilities to meet future national-security challenges. By providing an innovative capability to detect and characterize all types of viruses and virus-host activities, measure virion persistence in the environment, and collect data regarding biological pathogens and antibiotic-resistant bacterial pathogens, this research supports the Laboratory's research-and-development challenge of finding chemical and biological countermeasures to support threat reduction of weapons of mass destruction. This projected also supports the climate and energy-resilience mission by characterizing viral roles as regulators of microbial-growth dynamics and microbial ecophysiology via modulation of host metabolism, including a suite of auxiliary metabolic genes and host-cell lysis. Overall, this research enhances the Laboratory's core competencies in bioscience and bioengineering, as well as nuclear, chemical, and isotopic science and technology. It helped establish multiple competitive early-career scientists and recruit an academic graduate appointee who converted to staff in 2022. The completed work directly addresses a key priority for the DOE BER mission, that is, to discover the underlying biology of microbes as they respond to and modify their environments, and informs on a specific knowledge gap in carbon-cycling processes.

Publications, Presentations, and Patents

Mageeney, C.M. et al. 2022. "Improved Mobilome Delineation in Fragmented Genomes." Frontiers in Bioinformatics 2:866850: 35 (2022); doi.org/10.3389/fbinf.2022.866850. LLNL-JRNL-831204.

Sommers P. et al. 2021. "Integrating Viral Metagenomics into an Ecological Framework." Annual Review of Virology, 8 (2021); doi: 10.1146/annurev-virology-010421-053015. LLNL-JRNL-818035.

Wu, R. et al. 2022. "Permafrost as a Potential Pathogen Reservoir." One Earth 5(4): 351-360 (2022); doi.org/10.1016/j.oneear.2022.03.010. LLNL-JRNL-829002.

Trubl, G. "Unraveling the Hidden Potential of Active Microbial and Viral Communities in Soil Ecosystems." Phages for Health and Energy: Bridging National Lab and Academic Research Capabilities Agenda Symposium, Virtual. September 2021. LLNL-PRES-826516.

Trubl, G. "Characterizing Viruses for Ecology, Biotechnology, and Health Benefits." Georgetown Frontiers in Biotechnology. Virtual. September 2021. LLNL-PRES-826018.

Trubl, G. "Unraveling the Hidden Potential of Viruses in Soil Ecosystems Using Stable Isotope Probing Metagenomics." San Francisco State University Virology BIOL420. Virtual. April 2021. LLNL-PRES-821962.

Trubl, G. "Using SIP-Metagenomics to Characterize Soil Viruses." JGI Virus Group Meeting. Virtual. March 2021. LLNL-PRES-820751.

Trubl, G. "Quantitative Viral Assessments for Improved Biomonitoring." Emerson Lab Meeting at UC-Davis. Virtual. March 2021. LLNL-PRES-820392.

Trubl, G. "Combining Stable Isotope Probing and Metagenomic Approaches to Increase Detection of Viruses, Characterize their Activity, and Increase Virus-Host Linkages." VEGA Viral EcoGenomics & Applications Symposium, Berkeley, CA. April 2021. LLNL-PRES-821698.

Trubl, G., et al. "Uncovering the Multi-Faceted Roles of Active Viruses in Soil Ecosystems Following a Simulated Wet-Up." 18th International Society for Microbial Ecology Conference, Lausanne, Switzerland. August 2022. LLNL-POST-838449.

Trubl, G. et al. "Uncovering the Multi-Faceted Roles of Active Viruses in Soil Ecosystems Following a Simulated Wet-Up." Seminar speaker, Environmental Microbial Genomics Group at University of Lyon, France. August 2022. LLNL-PRES-839814.

Trubl, G. "Unraveling the Hidden Potential of Active Microbial and Viral Communities." Invited talk, SCIOMICS Webinar in Association with Pine Biotech. Virtual, January 2022. LLNL-PRES-830593.

Trubl, G. et al. "Uncovering the Multi-Faceted Roles of Active Viruses in Soil Ecosystems Following a Simulated Wet-Up." International Soil Virus Workshop, Holbaek, Denmark. June 2022. LLNL-PRES-836791.

Trubl, G. "Increased Biomonitoring of Active Microbial and Viral Communities Using Stable Isotope Probing Combined with Meta-omics Approaches." AbSciCon 2022, Atlanta, GA. May 2022. LLNL-PRES-835712.

Trubl, G. "Tracking the Activities of Viral Populations in Soil Ecosystems." Invited talk, The Omics Research Symposium. Virtual. August 2022. LLNL-PRES-833391.

Trubl, G. "Characterizing Viruses for Ecology, Biotechnology, and Health Benefits." Guest speaker, Georgetown BCHB-559 Course. Virtual. September 2022. LLNL-PRES-839648.

Trubl, G. et al. "Environmental Conditions Shape Active Viral Community Structure and Virus-Host Dynamics in Soil Ecosystems." DOE Genomic Sciences Meeting. Virtual, February 2022. LLNL-POST-831034.

Trubl, G. "Uncovering the Multi-Faceted Roles of Active Viruses in Soil Ecosystems Following a Simulated Wet-Up." BBTD Mini Symposium, Livermore, CA. September 2022. LLNL-POST-839701.

Trubl, G. "Using SIP-meta-omics to Characterize Soil Viruses" BioSecurity Group Presentation. Livermore, CA. February 2022. LLNL-PRES-829827.

Trubl, G. "Forays into Soil Viromics." LLNL Microbiology/Immunology Group Meeting. Livermore, CA. September. 2022. LLNL-PRES-839813.

Grant, G. et al. "Soil Forensics with DNA Stable Isotope Probing to Characterize Active Viral Populations." BBTD Mini Symposium. Livermore, CA. September. 2022. LLNL-POST-839865.

Trubl, G. "Quantitative Viral Assessments for Improved Biomonitoring" LW-LDRD 3rd-Year Extension Presentation. Virtual. June 2022. LLNL-PRES-835718.

Gogul, G. et al. "Response of Soil Viral Communities to a Changing Climate." LLNL Summer Student SLAM. Virtual. August. 2022. LLNL-PRES-837861-DRAFT.