Benchmarking informatics approaches for virus discovery: caution is needed when combining in silico identification methods.

Understanding the ecological impacts of viruses on natural and engineered ecosystems relies on the accurate identification of viral sequences from community sequencing data. To maximize viral recovery from metagenomes, researchers frequently combine viral identification tools. However, the effectiveness of this strategy is unknown. Here, we benchmarked combinations of six widely used informatics tools for viral identification and analysis (VirSorter, VirSorter2, VIBRANT, DeepVirFinder, CheckV, and Kaiju), called "rulesets." Rulesets were tested against mock metagenomes composed of taxonomically diverse sequence types and diverse aquatic metagenomes to assess the effects of the degree of viral enrichment and habitat on tool performance. We found that six rulesets achieved equivalent accuracy [Matthews Correlation Coefficient (MCC) = 0.77, Padj ≥ 0.05]. Each contained VirSorter2, and five used our "tuning removal" rule designed to remove non-viral contamination. While DeepVirFinder, VIBRANT, and VirSorter were each found once in these high-accuracy rulesets, they were not found in combination with each other: combining tools does not lead to optimal performance. Our validation suggests that the MCC plateau at 0.77 is partly caused by inaccurate labeling within reference sequence databases. In aquatic metagenomes, our highest MCC ruleset identified more viral sequences in virus-enriched (44%-46%) than in cellular metagenomes (7%-19%). While improved algorithms may lead to more accurate viral identification tools, this should be done in tandem with careful curation of sequence databases. We recommend using the VirSorter2 ruleset and our empirically derived tuning removal rule. Our analysis provides insight into methods for in silico viral identification and will enable more robust viral identification from metagenomic data sets. IMPORTANCE: The identification of viruses from environmental metagenomes using informatics tools has offered critical insights in microbial ecology. However, it remains difficult for researchers to know which tools optimize viral recovery for their specific study. In an attempt to recover more viruses, studies are increasingly combining the outputs from multiple tools without validating this approach. After benchmarking combinations of six viral identification tools against mock metagenomes and environmental samples, we found that these tools should only be combined cautiously. Two to four tool combinations maximized viral recovery and minimized non-viral contamination compared with either the single-tool or the five- to six-tool ones. By providing a rigorous overview of the behavior of in silico viral identification strategies and a pipeline to replicate our process, our findings guide the use of existing viral identification tools and offer a blueprint for feature engineering of new tools that will lead to higher-confidence viral discovery in microbiome studies.

A snapshot of the global drinking water virome: Diversity and metabolic potential vary with residual disinfectant use.

Viruses are important drivers of microbial community ecology and evolution, influencing microbial mortality, metabolism, and horizontal gene transfer. However, the effects of viruses remain largely unknown in many environments, including in drinking water systems. Drinking water metagenomic studies have offered a whole community perspective of bacterial impacts on water quality, but have not yet considered the influences of viruses. In this study, we address this gap by mining viral DNA sequences from publicly available drinking water metagenomes from distribution systems in six countries around the world. These datasets provide a snapshot of the taxonomic diversity and metabolic potential of the global drinking water virome; and provide an opportunity to investigate the effects of geography, climate, and drinking water treatment practices on viral diversity. Both environmental conditions and differences in sample processing were found to influence the viral composition. Using free chlorine as the residual disinfectant was associated with clear differences in viral taxonomic diversity and metabolic potential, with significantly fewer viral populations and less even viral community structures than observed in distribution systems without residual disinfectant. Additionally, drinking water viruses carry antibiotic resistance genes (ARGs), as well as genes to survive oxidative stress and nitrogen limitation. Through this study, we have demonstrated that viral communities are diverse across drinking water systems and vary with the use of residual disinfectant. Our findings offer directions for future research to develop a more robust understanding of how virus-bacteria interactions in drinking water distribution systems affect water quality.

DNA Sequence-Based Approach for Classifying the Mold Status of Buildings.

Dampness or water damage in buildings and human exposure to the resultant mold growth is an ever-present public health concern. This study provides quantitative evidence that the airborne fungal ecology of homes with known mold growth ("moldy") differs from the normal airborne fungal ecology of homes with no history of dampness, water damage, or visible mold ("no mold"). Settled dust from indoor air and outdoor air and direct samples from building materials with mold growth were examined in homes from 11 cities across dry, temperate, and continental climate regions within the United States. Community analysis based on the sequence of the internal transcribed spacer region of fungal ribosomal RNA encoding genes demonstrated consistent and quantifiable differences between the fungal ecology of settled dust in homes with inspector-verified water damage and visible mold versus the settled dust of homes with no history of dampness, water damage, or visible mold. These differences include lower community richness (padj = 0.01) in the settled dust of moldy homes versus no mold homes, as well as distinct community taxonomic structures between moldy and no mold homes (ANOSIM, R = 0.15, p = 0.001). We identified 11 Ascomycota taxa that were more highly enriched in moldy homes and 14 taxa from Ascomycota, Basidiomycota, and Zygomycota that were more highly enriched in no mold homes. The indoor air differences between moldy versus no mold homes were significant for all three climate regions considered. These distinct but complex differences between settled dust samples from moldy and no homes were used to train a machine learning-based model to classify the mold status of a home. The model was able to accurately classify 100% of moldy homes and 90% of no mold homes. The integration of DNA-based fungal ecology with advanced computational approaches can be used to accurately classify the presence of mold growth in homes, assist with inspection and remediation decisions, and potentially lead to reduced exposure to hazardous microbes indoors.

Humidity and Deposition Solution Play a Critical Role in Virus Inactivation by Heat Treatment of N95 Respirators.

Supply shortages of N95 respirators during the coronavirus disease 2019 (COVID-19) pandemic have motivated institutions to develop feasible and effective N95 respirator reuse strategies. In particular, heat decontamination is a treatment method that scales well and can be implemented in settings with variable or limited resources. Prior studies using multiple inactivation methods, however, have often focused on a single virus under narrowly defined conditions, making it difficult to develop guiding principles for inactivating emerging or difficult-to-culture viruses. We systematically explored how temperature, humidity, and virus deposition solutions impact the inactivation of viruses deposited and dried on N95 respirator coupons. We exposed four virus surrogates across a range of structures and phylogenies, including two bacteriophages (MS2 and phi6), a mouse coronavirus (murine hepatitis virus [MHV]), and a recombinant human influenza A virus subtype H3N2 (IAV), to heat treatment for 30 min in multiple deposition solutions across several temperatures and relative humidities (RHs). We observed that elevated RH was essential for effective heat inactivation of all four viruses tested. For heat treatments between 72°C and 82°C, RHs greater than 50% resulted in a >6-log10 inactivation of bacteriophages, and RHs greater than 25% resulted in a >3.5-log10 inactivation of MHV and IAV. Furthermore, deposition of viruses in host cell culture media greatly enhanced virus inactivation by heat and humidity compared to other deposition solutions, such as phosphate-buffered saline, phosphate-buffered saline with bovine serum albumin, and human saliva. Past and future heat treatment methods must therefore explicitly account for deposition solutions as a factor that will strongly influence observed virus inactivation rates. Overall, our data set can inform the design and validation of effective heat-based decontamination strategies for N95 respirators and other porous surfaces, especially for emerging viruses that may be of immediate and future public health concern.IMPORTANCE Shortages of personal protective equipment, including N95 respirators, during the coronavirus (CoV) disease 2019 (COVID-19) pandemic have highlighted the need to develop effective decontamination strategies for their reuse. This is particularly important in health care settings for reducing exposure to respiratory viruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Although several treatment methods are available, a widely accessible strategy will be necessary to combat shortages on a global scale. We demonstrate that the combination of heat and humidity inactivates a range of RNA viruses, including both viral pathogens and common viral pathogen surrogates, after deposition on N95 respirators and achieves the necessary virus inactivation detailed by the U.S. Food and Drug Administration guidelines to validate N95 respirator decontamination technologies. We further demonstrate that depositing viruses onto surfaces when suspended in culture media can greatly enhance observed inactivation, adding caution to how heat and humidity treatment methods are validated.

Degradation of phthalate esters in floor dust at elevated relative humidity.

Emerging investigator series: Phthalate esters are present at elevated concentrations in floor dust, and resuspension of dust represents a major source for human exposure to chemicals. Biodegradation of phthalates occurs in aquatic systems and soils but has not been demonstrated in house dust. The goal of this study was to quantify indoor phthalate ester degradation through both biotic and abiotic mechanisms. Worn carpet squares were embedded with dust and incubated for one to six weeks at equilibrium relative humidity (ERH) levels of 50, 80, 85, 90, 95, and 100%, and nine phthalates were measured. Removal was observed for DEHP, BBzP, DINP, DiDP, and DMP (p < 0.05) when incubated under elevated relative humidity conditions. Abiotic and biotic losses were examined separately using dust spiked with deuterated di(2-ethylhexyl)phthalate (d-DEHP) that was embedded in carpet and incubated at 100% ERH. Abiotic processes resulted in a 10.1% (±1.1%, standard error) to 69.6% (±4.8%) decrease in total d-DEHP after one week (p = 0.03) and a 27.2% (±1.4%) to 52.0% (±2.1%) decrease after three weeks (p = 0.008). Biodegradation resulted in a decrease in total d-DEHP after one week, ranging from 5.9% (±8.9%) to 8.5% (±1.7%) (p = 0.07) and a 1.7% (±3.9%) to 10.3% (±4.5%) decrease after three weeks (p = 0.044). Metatranscriptomic-based analysis indicates that fungi found in carpet dust express genes capable of degrading phthalate esters via various biochemical processes (including ß-oxidation and hydrolysis). Overall, these results support the hypothesis that phthalate losses in floor dust are due to a combination of abiotic and microbial degradation at ≥80% ERH.

Ten questions concerning the implications of carpet on indoor chemistry and microbiology.

Carpet and rugs currently represent about half of the United States flooring market and offer many benefits as a flooring type. How carpets influence our exposure to both microorganisms and chemicals in indoor environments has important health implications but is not well understood. The goal of this manuscript is to consolidate what is known about how carpet impacts indoor chemistry and microbiology, as well as to identify the important research gaps that remain. After describing the current use of carpet indoors, questions focus on five specific areas: 1) indoor chemistry, 2) indoor microbiology, 3) resuspension and exposure, 4) current practices and future needs, and 5) sustainability. Overall, it is clear that carpet can influence our exposures to particles and volatile compounds in the indoor environment by acting as a direct source, as a reservoir of environmental contaminants, and as a surface supporting chemical and biological transformations. However, the health implications of these processes are not well known, nor how cleaning practices could be optimized to minimize potential negative impacts. Current standards and recommendations focus largely on carpets as a primary source of chemicals and on limiting moisture that would support microbial growth. Future research should consider enhancing knowledge related to the impact of carpet in the indoor environment and how we might improve the design and maintenance of this common material to reduce our exposure to harmful contaminants while retaining the benefits to consumers.