Diversity of DNA and RNA Viruses in Ind...As Assessed Via Metagenomic

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Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX pubs.acs.org/est

Diversity of DNA and RNA Viruses in Indoor Air As Assessed via Metagenomic Sequencing Karyna Rosario,*,† Noah Fierer,‡,§ Shelly Miller,∥ Julia Luongo,∥ and Mya Breitbart† †College of Marine Science, University of South Florida, Saint Petersburg, Florida 33701, United States ‡Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado 80309, United States §Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado 80309, United States ∥Department of Mechanical Engineering, University of Colorado, Boulder, Colorado 80309, United States *S Supporting Information

ABSTRACT: Diverse bacterial and fungal communities inhabit human-occupied buildings and circulate in indoor air; however, viral diversity in these man-made environments remains largely unknown. Here we investigated DNA and RNA viruses circulating in the air of 12 university dormitory rooms by analyzing dust accumulated over a one-year period on heating, ventilation, and air conditioning (HVAC) filters. A metagenomic sequencing approach was used to determine the identity and diversity of viral particles extracted from the HVAC filters. We detected a broad diversity of viruses associated with a range of hosts, including animals, arthropods, bacteria, fungi, humans, plants, and protists, suggesting that disparate organisms can contribute to indoor airborne viral communities. Viral community composition and the distribution of human-infecting papillomaviruses and polyomaviruses were distinct in the different dormitory rooms, indicating that airborne viral communities are variable in human-occupied spaces and appear to reflect differential rates of viral shedding from room occupants. This work significantly expands the known airborne viral diversity found indoors, enabling the design of sensitive and quantitative assays to further investigate specific viruses of interest and providing new insight into the likely sources of viruses found in indoor air.

■ INTRODUCTION viral metagenomic techniques, where viral particles are purified from a given sample prior to extraction and shotgun sequencing Microbes are abundant in indoor air. Human-occupied 15,16 buildings, including residential, work, and public spaces, can of nucleic acids, can be used to expand the known diversity harbor more than 105 bacterial and fungal cells per cubic meter of viruses found indoors. An advantage of viral metagenomics is of indoor air.1−3 Hundreds to thousands of bacterial and fungal that this technique can be used to survey viral diversity without taxa are found indoors, highlighting the diverse nature of these a priori knowledge of the viruses present in a given sample, microbial communities.4,5 This realization, combined with the resulting in the detection of novel viruses as well as the fact that we spend >90% of our time indoors,6 has fueled characterization of viral communities (i.e., viromes) in a wide ff range of environments,17 including outdoor air18 and indoor coordinated e orts to investigate the microbiome of the built 19,20 environment (e.g., microBEnet7). Extensive research on air. However, the fairly low viral biomass in indoor air 5 21 bacterial and fungal communities has shown high variability (approximately 10 virus-like particles per cubic meter ) 22 in community composition across buildings, or even locations presents a challenge for viral metagenomic studies. Though within a given building, due to differences in occupants, most of these viral particles are unlikely to be pathogens, or environmental conditions, geographic location, and many other even human-associated viruses, a more complete assessment of factors.8−10 However, despite decades of research on the the viral diversity circulating in indoor air is broadly relevant to bacteria and fungi that humans are exposed to indoors, there is understanding buildings as microbial ecosystems and the little information regarding the diversity of viruses found in factors that shape indoor exposure to viruses. built environments. Most of the research investigating viruses indoors has Received: August 15, 2017 focused on selected human viral pathogens found on surfaces Revised: December 5, 2017 and in air.11−14 However, known human pathogens are unlikely Accepted: January 3, 2018 to be the only viruses we encounter indoors. The application of Published: January 3, 2018

Heating, ventilation, and air conditioning (HVAC) filters, a 8891 Ultrasonic Bath Sonicator, 42 kHz frequency (Cole- which serve as passive, long-term, high volume air samplers in Parmer) for a total of 5 min in 1 to 2 min intervals followed by indoor spaces, have been used successfully to describe the vigorous shaking to detach viral particles from the filter and diversity of airborne bacteria and fungi circulating inside disrupt aggregates. To separate desorbed viral particles from buildings.23−26 Because HVAC filters can be used for integrated cells and debris after sonication, samples were filtered through a sampling of indoor airborne bacterial and fungal commun- 0.22 μm Sterivex filter (Millipore). ities,23 here we evaluated if metagenomic sequencing of viruses To concentrate virus particles, filtrates were subjected to two could also be performed from these filters. For this purpose, rounds of ultracentrifugation, which pelleted most virus-like viral purification strategies were combined with high- particles as assessed through SYBR Gold staining and throughput sequencing on a subset of HVAC filters previously epifluorescence microscopy.31,32 For this purpose, five 35 mL used to investigate bacterial and fungal communities inside a aliquots of filtrate per sample were placed into 25 × 89 mm University of Colorado dormitory building.24 By using a ultracentrifuge tubes (Beckman Coulter, Inc.) and ultra- replicated room design, this study aimed to determine what centrifuged at 28,000 rpm in a SW28Ti swinging bucket types of viruses circulate in indoor air, how viral communities rotor (Beckman Coulter, Inc.) for 4 h at 4 °C. Supernatants vary across rooms within a single building, and how these were directly decanted into a second set of ultracentrifuge tubes differences relate to the room’s occupants. To our knowledge, and centrifuged once more at 28,000 rpm for 3 h at 4 °C. this study represents the most comprehensive investigation of Resulting viral pellets in each of the tubes after the first round the DNA and RNA viral diversity found inside buildings. of ultracentrifugation were resuspended with 100 μL of SM Although many studies have focused on identifying human viral buffer and soaked overnight at 4 °C. The SM buffer containing pathogens in indoor air, this study reveals that indoor air the resuspended viral pellets from the first round was harbors a diversity of viruses associated with a wide range of subsequently used to resuspend the pellets from the second potential hosts. ultracentrifuge round by soaking for at least 6 h at 4 °C. Approximately 500 μL of total resuspended viral pellets per ■ MATERIALS AND METHODS sample after two rounds of ultracentrifugation were pooled. Sample Collection and Processing. This study utilized a Free nucleic acids were removed from the viral concentrates by subset of HVAC fan coil unit filters collected for a previous incubating resuspended pellets at 37 °C for 2 h with a nuclease project investigating bacterial and fungal diversity in dormitory cocktail consisting of 1X Turbo DNase Buffer (Ambion), 21U rooms.24 The filters were typical residential filters with a MERV of Turbo DNase (Ambion), 4.5U of Baseline-ZERO DNase 8 rating, which are on average 70−80% efficient at collecting (Epicenter), 112.5U Benzonase (EMD Millipore), and 33,34 3−10 μm particles.27 The HVAC filters were installed 12 10 μg/mL RNase A (Sigma-Aldrich). Nucleases were months prior to collection within each dormitory room housing inactivated with 20 mM EDTA prior to nucleic acid extraction undergraduate students on the University of Colorado campus from viral particles. in Boulder, Colorado, USA. Each room was equipped with a fan Because a diversity of contaminants, including viruses, can be coil unit that heated or cooled recirculated room air (no outside found in laboratory reagents and commercially available nucleic 35−38 air) at a constant volumetric flow rate. The filters from each fan acid extraction and amplification kits, a negative control coil unit only filtered the air from an individual room, allowing sample containing SM buffer alone was included. This negative for a time-integrated aerosol sample from each room. Because control was processed alongside the filter samples from the each wing of the dormitory building had a separate HVAC sonication step onward to control for unforeseen contami- system, filters for viral metagenomic analysis were collected nation in laboratory reagents that can potentially impact from the East wing to minimize variability due to HVAC metagenomic analyses of low biomass samples. Unfortunately, a system differences. Selected HVAC filters originated from six blank HVAC filter from the original batch installed in the male and six female double-occupancy rooms. In addition to dormitory rooms was not available to control for contaminants these 12 filters from individual rooms, a pooled sample of eight present in the filter material. However, any viral contamination filters from the North and West wings of the building was initially present in the filters should be obscured after collecting processed to detect potential viruses present in other wings a passive, long-term (1 year), integrated air sample. with separate HVAC systems. HVAC filters were stored at −20 Nucleic Acid Extraction and Metagenomic Library °C within 5 days of collection until further processing. Preparation. Viral DNA and RNA were extracted from 200 Virus particles were partially purified from HVAC filters and μL of purified viral particles using the QIAmp MinElute Virus concentrated to increase the probability of detecting viral Spin (Qiagen) and RNeasy (Qiagen) kits, respectively, sequences through high-throughput sequencing by adapting following manufacturer’s protocols. For viral RNA extraction, methods that have been used to investigate viromes from the on-column DNase digestion was performed according to various environmental matrices including soil, water, and air as manufacturer’s recommendations. RNA was reverse-transcribed well as methods from the biomedical field.14,28−30 To do this, using the SuperScript III First Strand Synthesis System eight pieces (8 × 8 cm) were cut from each individual filter (Invitrogen) with random hexamers. Second-strand synthesis using a sterile scalpel, to yield a total area of 64 × 64 cm. For was performed on the resulting cDNA using the Klenow the pooled sample, a single 8 × 8 cm piece from each of the Fragment DNA polymerase (New England Biolabs) following eight filters was processed. Filter sections were shredded into manufacturer’s instructions and the resulting products were smaller pieces using scissors, which were cleaned between filters cleaned using the AMPure XP Purification system (Beckman using 10% bleach followed by ethanol flame sterilization. The Coulter). DNA and cDNA samples were fragmented to 300 bp filter pieces were suspended in 225 mL of sterilized, cold using a Covaris M220 instrument. Next-generation sequencing suspension medium (SM) buffer [100 mM NaCl, 8 mM (NGS) library construction was performed with the Accel-NGS MgSO4·7H2O, 50 mM Tris-Cl (pH = 7.5), 0.01% (w/v) 1S Plus DNA Library Kit for Illumina Platforms (Swift gelatin] and placed on ice for at least 5 min before sonication in Biosciences), which allows low DNA/cDNA inputs to

B DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article

Figure 1. Heatmap showing the distribution of viral OTUs identified in rooms occupied by male (M1−M6) and female (F1−F6) students as well as those identified in the pooled sample (P) according to taxonomic groups. Bar graph on the right shows the percentage of total viral OTUs identified in rooms that were classified within a given group. Colors on the left panel highlight DNA (blue) and RNA (orange) viral groups. DNA viruses are further distinguished by bacteriophage (dark blue) and eukaryotic (light blue) viral groups. The color scale on the heatmap represents relative low (dark purple) to high (yellow) abundances based on the total number of viral OTUs identified in a given room. Gray color on the heatmap indicates taxonomic groups that were not detected in a given room. “CRESS DNA” refers to circular replication-associated protein encoding single-stranded DNA viruses that form a diverse and evolutionarily related group. “Uncl” stands for unclassified. circumvent the introduction of biases while trying to obtain the iVirus resource for analysis of viromic data.43 Briefly, raw sufficient template for NGS.39 The Accel-NGS 1S Plus kit can sequences were trimmed for quality and to remove indexing also recover single-stranded DNA (ssDNA) templates,40 adapters using the Trimmomatic App version 0.35.0 with eliminating the need for multiple displacement amplification, default parameters,44 except for a read head crop of 10 bp which is strongly biased toward small, circular templates.41 instead of zero. The quality of the trimmed sequences was Library construction and multiplexing was performed using the verified with the FastQC-plus App version 0.10.1.45 Quality- Accel-NGS 1S Plus kit following manufacturer’s instructions for filtered sequences were then assembled using the SPAdes DNA inputs <1 ng/μL and 20 cycles of indexing PCR. A total assembler App version 3.6.0 with default parameters, k-mer of 28 libraries, including separate DNA and cDNA from air length of 55, and the metagenomics assembly option.46 Contigs filters from each of the 12 individual rooms, a pooled sample, larger than 100 bp were selected for further analyses. and a negative control, were paired-end sequenced (2 × 250 Contaminant contig sequences were removed from each data bp) on a MiSeq System (Illumina) at the University of set by comparing (BLASTn, e-value < 0.00001) against a Colorado BioFrontiers Next-Gen Sequencing core facility. database containing assembled sequences from the negative Sequences are available at the NCBI Sequence Read Archive control library. database (study number SRP113244; accession numbers After removing sequences with significant matches in the SRR5853122−SRR5853149). negative control database, contig sequences from each library Sequence Analysis. Sequences were processed using were compared (BLASTx, e-value < 0.001) against a viral bioinformatic applications (Apps) available through the protein database containingsequencesfromtheNCBI CyVerse cyberinfrastructure42 and the pipeline suggested by Reference Sequence (RefSeq) database (RefSeq Release

C DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article

Table 1. Speciﬁc Viral OTUs Identiﬁed in at Least One Third of the Dormitory Rooms

BLAST match/OTU group Taxonomic group Host group Source type BLASTx similarity (%) Positive rooms (%) Pseudomonas group 1 phages Myoviridae Gammaproteobacteria Soil 60−93 83 Propionibacterium acnes phages Siphoviridae Actinobacteria Human 100 67 Staphylococcus Sep9-like phages Siphoviridae Firmicutes Human 57−100 67 Lactococcus 936 sensu lato phages Siphoviridae Firmicutes Dairy 95−100 58 Bacillus phage G Myoviridae Firmicutes Soil 50−88 58 Staphylococcus Twortlikevirus phages Myoviridae Firmicutes Sewage 54−100 58 Actinobacteriophage cluster A Siphoviridae Actinobacteria Soil 56−80 50 Streptococcus group 1 phages Podoviridae Firmicutes Human 50−99 42 Lactococcus C2-like phages Siphoviridae Firmicutes Dairy 94−99 42 Sinorhizobium phages Myoviridae Alphaproteobacteria Soil 56−70 42 Actinobacteriophage cluster BD Siphoviridae Actinobacteria Soil 54−75 42 Lactococcus phages (unclassified) Siphoviridae Firmicutes Dairy 57−96 33 Actinobacteriophage cluster F Siphoviridae Actinobacteria Soil 59−91 33 Actinobacteriophage cluster L Siphoviridae Actinobacteria Soil 54−73 33 Clavibacter phages Siphoviridae Actinobacteria Soil 58−75 33 Actinobacteriophage cluster C Myoviridae Actinobacteria Soil 49−76 33 Enterobacteriaceae T4-like phages Myoviridae Gammaproteobacteria Sewage 59−92 33 Bacillus SPO1virus Myoviridae Firmicutes Soil 44−71 33 Actinobacteriophage cluster BE Siphoviridae Actinobacteria Soil 55−83 33 crAssphage Unclassified dsDNA Bacteroidetes Human 60−100 33 Invertebrate iridescent viruses Irridoviridae Invertebrate Arthropod 47−99 67 Human papillomavirus Papillomaviridae Human Human 77−100 67 Animal feces associated gemycircularvirus Genomoviridae Unknown Animal 52−100 33 Sewage associated gemycircularvirus Genomoviridae Unknown Sewage 79−86 33 Insect densovirus Parvoviridae Insect Arthropod 46−66 33 Plant tymovirus Tymoviridae Plant Plant 64−93 83 Human retrovirus Retroviridae Human Human 55−99 83 Ryegrass mottle virus Sobemovirus Ryegrass Plant 86−100 67 Tobacco mild green mosaic virus Virgaviridae Tobacco Plant 98−100 67 Tobacco mosaic virus Virgaviridae Tobacco Plant 99−100 50 Insect picorna-like virus Picornavirales Insect Arthropod 47−99 50 Crustacean picorna-like virus Picornavirales Crustacean Arthropod 50−78 50 Turnip vein-clearing virus Virgaviridae Turnip Plant 98−100 42 Aphis glycines virus 1 Picornavirales Insect Arthropod 47−92 42 Pepper mild mottle virus Virgaviridae Pepper Plant 98−100 42 Clover Potexvirus Alphaflexiviridae Clover Plant 95−100 33 number 79, https://www.ncbi.nlm.nih.gov/refseq/) as well as represented a cohesive group, such as human papillomaviruses, sequences recently reported from RNA viruses in invertebrates to look for general trends at higher taxonomic levels. Sequences and environmental samples.47,48 Contig sequences with similar to bacteriophages infecting Actinobacteria from soil significant matches in the viral database were then compared were grouped according to clusters classified within the against the GenBank nonredundant (nr) database (BLASTx, e- Actinobacteriophage Database50 (http://phagesdb.org/). An value 0.001; downloaded December 2016) to remove contig OTU table (summarized in Supplemental File S1) was sequences that had higher identities with nonviral sequences constructed by recording the presence or absence of each (i.e., false positives). Final BLAST results were inspected using viral OTU in each dormitory room. MEGAN6 Community Edition.49 To err on the side of caution, Sequences similar to human papillomaviruses (HPVs) and contig sequences from the negative control library were also polyomaviruses (HPyVs) were further investigated given their compared against the viral and nr databases (BLASTx, e-value widespread nature in the human population. To do so, 0.001) to identify potential viral contaminants. If one of the top approximately 1450 and 650 HPV and HPyV genomes, three BLAST matches for a given contig sequence in the respectively, were downloaded from GenBank in April 2017. sample libraries included a virus identified in the negative Genome sequences were then dereplicated based on a 95% control or phylogenetically related viruses, the sequence was identity cutoff using CD-HIT.51 Quality-filtered forward reads removed from further analysis (Supplemental File S1). from each of the DNA libraries were compared against curated After false positives and potential viral contaminants were databases containing 247 HPV and 28 HPyV genomes removed, sequences that could be confidently identified as viral (BLASTn, e-value < 0.00001). Sequences with significant based on best BLAST matches to known viral sequences were matches to these databases were then compared against the organized into operational taxonomic units (OTUs). Here, viral GenBank nucleotide (nt) database (BLASTn, e-value < 0.001) OTUs represent either a single species or a group of related to eliminate false positives. The number of HPV- and HPyV- viruses in cases where BLAST matches could not be confidently related sequences as well as the HPV types and HPyV species assigned to a given viral species and/or when sequences were recorded (Supplemental File S1). Finally, the classification

D DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article of all HPV types detected in dormitory rooms were verified ments has been linked to human occupancy.9,66 Propionibacte- using the Papillomavirus Episteme database.52 rium and Staphylococcus bacteriophages are among the most abundant viruses found on healthy human skin,62,63,67 where ■ RESULTS AND DISCUSSION they have been linked to the modulation of P. acnes population 68 Overview. Here we investigated viruses circulating in the air structure. Likewise, the detection of a Propionibacterium bacteriophage through metagenomic approaches in clean of university dormitory rooms by performing metagenomic 19 sequencing on partially purified VLPs from dust accumulated rooms has been linked to human presence in the rooms. fi fi CrAssphage, which is suspected to infect a Bacteroides species, is on HVAC lters over a one-year period. Speci cally, we 69 analyzed individual HVAC filters from 12 double occupancy widespread in human fecal metagenomes. Together these fi rooms housing either female (n = 6) or male (n = 6) students results highlight that the identi ed bacteriophages in dormitory fi in the East wing of the dormitory, as well as a pooled sample room HVAC lters are concordant with bacteria that are from eight rooms located in the North and West wings of the commonly found indoors, including environmental and human- building that used different HVAC systems. A total of 215 viral associated taxa. OTUs, the majority of which represented DNA viruses (72%), Diversity of Airborne Eukaryotic Viruses Circulating were detected across all air filters after examining top BLASTx in Dormitory Rooms. Although bacteriophages dominated matches against the GenBank nr database (Supplemental File the viral diversity captured in dormitory rooms, eukaryotic S1). Due to low amino acid sequence identities to known viral viruses were also readily detected (Figure 1). Eukaryotic viral species (as low as 44% in some cases), most OTUs were OTUs that were present in more than 50% of the rooms classified to only the family or order level. Up to 79 viral OTUs included putative arthropod-infecting viruses from the family were detected on individual HVAC filters, with both female- Iridoviridae and order Picornavirales, plant pathogens from the and male-occupied rooms having approximately 40 OTUs per genus Sobemovirus as well as those from the Tymoviridae and room. Identified viral OTUs indicate that bacteriophages and Virgaviridae families, and human viruses from the Retroviridae eukaryotic viruses spanning several viral families and genome and Papillomaviridae families (Table 1). Viral OTUs represent- types circulate in indoor air (Figure 1). ing members of the family Genomoviridae were also detected in Bacteriophages Dominate Airborne Viral Community more than half of the rooms. Because the only cultured Diversity in Dormitory Rooms. The majority (62%) of representative of the family Genomoviridae was isolated from a 70 identified viral OTUs in air filters represented bacteriophages. fungus and sequences similar to proteins encoded by these 71,72 This was not surprising considering that bacteria are abundant viruses have been identified within fungal genomes, in indoor air.2,21 Viral OTUs were dominated by tailed members of this family are suspected to infect fungi. However, bacteriophages (order Caudovirales) from the Siphoviridae and a definitive host has not been confirmed for most species of 73 Myoviridae families, which were the only viral families detected Genomoviridae. in every room (Figure 1). Although the dominance of tailed Most of the eukaryotic viral OTUs represented putative bacteriophages in indoor air has not been previously noted, arthropod-infecting viruses from the order Picornavirales and tailed bacteriophages tend to dominate environmental viral plant viruses from the family Virgaviridae (Figure 1). The communities described from soil53 and water54,55 as well majority of the Virgaviridae-related sequences had high amino human-associated viromes. In particular, members of the acid level identities (≥98%) with proteins encoded by members Siphoviridae and Myoviridae families are abundant in human- of the genus Tobamovirus. In contrast, sequences similar to associated viromes including the gut,56−59 respiratory tract,60 putative arthropod-infecting viruses from the order Picornavir- saliva,61 and skin.62,63 However, the abundance of tailed ales had low amino acid sequence identities to recently reported bacteriophages might be skewed by current knowledge of viral genomes from invertebrates48 (Supplemental File S1). bacteriophage diversity because >90% of reference bacterio- Therefore, viral sequences identified here, including a novel, ∼ phage genome sequences in GenBank (https://www.ncbi.nlm. 10 kb genome (will be described in a separate paper) that is nih.gov/genome/viruses/) belong to the order Caudovirales. most similar to viruses identified in Drosophila and ants, Nevertheless, our results indicate that a broad diversity of tailed indicate that RNA viral diversity has yet to be fully explored. bacteriophages are ubiquitous in dormitory room air. Human endogenous retroviruses were the most widespread The putative host phyla identified for the observed group of human-associated viral sequences found in dormitory bacteriophage OTUs included Proteobacteria (45%), Actino- rooms (Table 1). It is possible that these endogenous retroviral bacteria (30%), Firmicutes (23%), Bacteroidetes (0.75%), and sequences represent remnant viral sequences integrated into Aquificae (0.75%) (Supplemental File S1). These putative host cellular genomes rather than exogenous viruses because groups, with the exception of Aquificae, frequently dominate retroviral sequences are among the transposable elements indoor microbiomes and were widely detected in dormitory that constitute 50% of the human genome.74,75 However, the rooms.24 The most widespread viral OTUs potentially infecting detected retroviral sequences were most similar to human Proteobacteria, including Pseudomonas and Sinorhizobium endogenous retrovirus K (HERV-K) and multiple sclerosis- bacteriophages, were most similar to bacteriophages isolated associated retrovirus (MSRV) and were mainly detected in the from environmental sources (Table 1). In addition, some of the RNA libraries. Expression of structural genes and detection of most widespread viral OTUs were similar to bacteriophages virus-like particles has suggested that the recently acquired infecting Actinobacteria, such as Propionibacterium acnes HERV-K can become active under certain physiological bacteriophages, as well as those infecting Firmicutes (Staph- circumstances.76−79 In addition, there has been debate about ylococcus and Streptococcus bacteriophages) and potentially whether or not MSRV can be exogenous and persist as an Bacteroidetes species (crAssphage) (Table 1). Bacterial species extracellular particle.80 Due to the possibility of expression of from most of these genera, including Propionibacterium, human endogenous retroviral sequences combined with low Staphylococcus, Streptococcus and Bacteroides, are part of the amino acid identities (as low as 55%) to known retroviral human microbiome64,65 and their presence in indoor environ- sequences in some cases, this OTU was retained in the

E DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article

Figure 2. Distribution of human-infecting viruses from the Papillomaviridae and Polyomaviridae families identified in rooms occupied by male (M1− M6) and female (F1−F6) students as well as those identified in the pooled sample (P). The distribution of human papillomaviruses (HPVs) in each room is shown as a heatmap. Specific HPV types are listed on the left panel and colors highlight different genera, including Alphapapillomavirus (green), Betapapillomavirus (tan), and Gammapapillomavirus (light brown). ** indicates HPV types that cannot be confidently assigned because detected sequences share low nucleotide identity (<80%) with known types. The color scale on the heatmap represents relative low (dark purple) to high (yellow) abundances of HPV types based on the total number of HPV-related sequences identified in a given room. Gray color on the heatmap indicates HPV types that were not detected in a given room. Because only a single human polyomavirus (HPyV) species was identified in positive rooms, identified species in each room are listed at the bottom of the heatmap, including polyomavirus 6 (PyV6), MW polyomavirus (MWPyV), and Merkel cell polyomavirus (MCPyV). ‘X’ indicates rooms that did not have any HPyV-related sequences. analyses. Nevertheless, care should be taken when interpreting in indoor spaces, including hundreds of species found in the presence of endogenous retroviruses in viral metagenomic individual homes.81 In addition, dust samples collected from studies as sequences might represent cellular DNA. surfaces inside homes contain pollen representing a diversity of The detection of diverse eukaryotic viruses in dormitory plant species82 and outdoor vegetation may affect the rooms suggests that disparate organisms contribute to indoor composition of indoor bacterial communities.83 Moreover, airborne viral communities. A high diversity of arthropods live bacterial species potentially associated with plants and insects

F DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article were identified in the dormitory rooms.24 Therefore, plants and alphapapillomavirus that has been classified as high-risk due arthropods are likely to be significant sources of airborne to its oncogenic potential,113,114 was detected in a male- indoor bacteria and viruses. This study suggests that fungi also occupied room. This high-risk HPV type has been found at low serve as a source of airborne viruses, as several OTUs most prevalence compared to other HPV types in healthy women similar to fungal viruses were identified, including RNA viruses and men.103,114,115 These results indicate that a high diversity of (members of the Chrysoviridae, Hypoviridae, and Partitiviridae HPVs circulates in indoor air, including HPV types with low families as well as unclassified ssRNA and dsRNA viruses) and prevalence in the general population that might be of clinical potentially ssDNA viruses (family Genomoviridae, see above). relevance. Unexpectedly, one of the rooms contained sequences similar to In contrast to HPVs, HPyVs were mainly restricted to male- feline papillomavirus type 2 (FePV2), which infects domestic occupied rooms and, although coinfection with multiple HPyVs cats.84 The presence of nonhuman occupants, such as pets, can is common,109,116 only a single HPyV species was detected in affect indoor bacterial communities9 and the detection of each positive room (Figure 2). Three HPyV species, namely FePV2 suggests that pets may also influence indoor viral HPyV6, MW polyomavirus (MWPyV), and Merkel cell diversity. However, because university policies prohibited pets polyomavirus (MCPyV), were detected. HPyV6 and MWPyC in these dormitory rooms, it is unknown if the detection of were discovered in skin and fecal samples, respectively, from FePV2 reflects the presence of an illicit cat in the dormitory healthy individuals,109,117 whereas MCPyV was originally room or resulted from external contact between human identified in Merkel cell carcinoma118 and later established as occupants and cats. a widespread cutaneous virus in the human population.94 We Diversity of Airborne Human Papillomaviruses and assembled a complete MCPyV genome that shared 99% Polyomaviruses in Dormitory Rooms. Nonenveloped genome-wide pairwise identity to a genome recovered from the dsDNA viruses from the Papillomaviridae and Polyomaviridae skin of a healthy individual109 (Accession number HM011544) families, including human papillomaviruses (HPVs) and (Supplemental File S2). The prevalence of MWPyV in the polyomaviruses (HPyVs), were widespread in the dormitory general population and environmental samples is low compared rooms. These two groups of viruses constitute part of the to other HPyVs like MCPyV;97,109,119 however, MWPyV human-associated virome in both healthy and disease states. seemed to be more widespread in dormitory room air than HPVs, which have evolved replication strategies to thrive in HPyV6 and MCPyV. This suggests that differences in shedding their host’s cutaneous and mucosal epidermal tissue,85 have rates, namely the number of virions released by infected cells at been strongly linked to the development of cervical cancer86−88 any given time, between HPyV species may contribute to as well various other cancers and skin diseases.89−93 HPyVs can differences between airborne HPyV distribution and that of cause serious diseases in immunocompromised patients human subjects. including Merkel cell carcinoma, progressive multifocal The higher diversity of airborne HPVs detected in dormitory leukoencephalopathy, nephropathy, trichodysplasia spinulosa rooms compared to HPyVs supports previous reports of low and hemorrhagic cystitis.94−96 However, healthy individuals are HPyV genetic diversity on human skin.98 Indeed, to date, more also known to be asymptomatically infected with HPVs and than 30 HPV species comprising >200 HPV types52,120,121 have HPyVs, both of which may be acquired early in infancy.97−106 been described compared to only 12 HPyV species.96,106 HPV Furthermore, HPVs and HPyVs are the most abundant groups diversity might be driven by a high selective pressure from the of human-infecting viruses associated with the healthy human human immune system.67 Although HPVs and HPyVs are skin viral flora62,98 and novel species have been discovered from widespread in the human population and have similar healthy individuals.107−109 characteristics, there could be differences in tissue tropism or The diversity of HPV- and HPyV-related sequences was virion shedding rates that might affect their relative further investigated because the presence of these viruses in representation in indoor air. Moreover, HPyVs were mainly dormitory rooms is directly linked to human occupancy. By detected in male rooms, suggesting that shedding rates for this comparing unassembled sequences to custom databases, the group of viruses differ between male and females. This is detection of HPV- and HPyV-related sequences increased. plausible considering that female children are more likely to HPVs, the second most widespread group of human-infecting shed MWPyV in feces than male children122 and seropositivity viruses detected in dormitory rooms (Table 1), were more for certain HPyV species is more common among males.116 diverse than HPyVs (Figure 2). A total of 54 HPV types, Notably, MWPyV was the only HPyV species detected in including 7 unclassified HPVs, were detected, with up to 24 female rooms (Figure 2). In addition, because Merkel cell HPV types detected in a single room (Figure 2). All of the carcinoma is more prevalent in males,123 some studies have also detected HPV-related sequences were most similar to species shown that MCPyV is highly prevalent in the skin of healthy classified within the Alphapapillomavirus, Betapapillomavirus, males.124,125 Interpersonal variation in HPV and HPyV species and Gammapapillomavirus genera, which contain most known composition62,63,98 and potential differences in shedding rates HPV species.110,111 The majority of the HPV-related sequences between HPyV species might affect indoor airborne viral loads were similar to cutaneous viruses from the Beta-and of these human-infecting viruses. Gammapapillomavirus genera. Alphapapillomaviruses, including Airborne Viral Community Variation in Dormitory both genital/mucosal (type 51) and cutaneous (types 3 and Rooms. Each dormitory room exhibited a different distribution 125) HPVs, were only detected in three of the dormitory of viral OTUs as well as HPV types, revealing a unique rooms. The most widespread HPVs included types 23 and 120, “fingerprint” of airborne viral species (Figures 1 and 2). which were detected in 33% of the rooms, followed by HPV Although Siphoviridae and Myoviridae members were detected type 24, which was detected in 25% of the rooms. Although all in all of the samples (Figure 1), the distribution of specific of these cutaneous HPV types were originally isolated from skin OTUs within these groups varied. It is possible that variation in lesions,110,112 these types have also been detected in the skin of sequencing depth among the samples might affect the detection healthy individuals.103 In addition, HPV type 51, an of low abundance viral OTUs; however, even the dominant

G DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article viral OTUs varied across individual rooms. This observation suggests that airborne viral communities indoors are variable in human-occupied spaces as has been shown for bacteria.126 The disparate airborne viral communities of individual rooms may reflect variability in occupant-associated microbiomes because individuals can release distinct “microbial clouds”.126,127 Notably, there are marked interpersonal differences in viral communities associated with healthy human skin and there can be viral “blooms” in certain body sites.62,63 Furthermore, individuals with active viral infections may shed more viral particles compared to healthy individuals, further contributing to airborne viral community dynamics. For example, the detection of rhinoviruses, which are the most common causative agents of the common cold and other upper respiratory tract infections,128,129 in one of the rooms might reflect a symptomatic infection of a room occupant. Although asymptomatic rhinovirus infection can be common in young children,130 viral detection and loads are significantly higher in symptomatic adults.131,132 Challenges Associated with Airborne Virus Source Tracking. The potential sources of observed viral OTUs in dormitory rooms were examined by classifying the top BLAST match of each OTU based on its original source of isolation or, if that information was not available, its host’s niche. The analyses suggest that soil is likely the largest contributing source to airborne viral communities in dormitory rooms (Figure 3). The bacterial community in dust from floors and other indoor surfaces has been shown to harbor taxa from human occupants as well as environmental sources including soil.66,126,133 Dust resuspension, which has been suggested as an important source of indoor airborne bacteria,133 might also be a significant source of airborne viruses. However, our inability to accurately track sources of airborne viruses might alter the true contributions from potential sources described here. Due to their small size, virus particles may stay airborne for fi Figure 3. Potential sources of viruses detected on dormitory room signi cantly longer periods and, in turn, be transported for HVAC filters. Graphs show (A) the percentage of rooms (n = 12) longer distances than bacteria and fungi, making it difficult to 22 where viruses from a given source were detected and (B) the pinpoint their original source. This is further complicated by percentage of viral OTUs in each source category. virion stability, which can allow certain viruses to withstand harsh conditions, including the passage of viruses of dietary origin through the gut. For example, bacteriophages infecting most similar to members of the genus Tobamovirus, which have Lactococcus lactis,abacteriumextensivelyusedinthe stable virions that can remain infectious after disinfection production of dairy products, were widespread in dormitory treatments and persist for long periods outside their plant rooms (Table 1) and their bacterial host has been identified as host.138,139 Although identified tobamoviruses included viruses one of the most abundant bacterial species in floor dust.66 infecting edible crops (tomato, cucumber, pepper), tobacco Moreover, a ∼2 kb L. lactis bacteriophage contig sequence from plants, and grasses (Supplemental File S1), all of these viruses the dormitory rooms had significant matches to metagenomic are dominant in human feces140 and raw sewage.141 Due to sequences from human feces134 available through the IMG/VR their stability and presence in various sources, it is not possible database.135 Due to the remarkable stability of L. lactis to determine if plant viruses detected in dormitory room air bacteriophages,136 we cannot determine if the detected originated from plant material, such as houseplants and food bacteriophages originated directly from dairy products,137 products, or if they indicate fecal material from room from floor dust, or from the feces of room occupants. occupants. Notably, the detection of viruses that have been Estimated virus to bacteria ratios ranging from 0.7 to 1 in proposed as indicators of fecal pollution, including pepper mild indoor environments suggest that bacteriophages in indoor air mottle virus142 and crAssphage143 (Table 1), suggest that and surfaces might not be able to replicate due to host viruses originating from human feces can become airborne. dormancy or low host densities.20,21 These low ratios support Therefore, plants might contribute to indoor airborne viral the idea that bacteriophages might originate from the same diversity indirectly through dietary consumption of plant- source as their hosts (e.g., dairy products, dust, feces) rather related products and subsequent aerosolization of fecal matter. than propagating indoors. Another virus detected in this study that might originate Virion stability also obscures source tracking of eukaryotic from plant-related products is cannabis cryptic virus (CCV), an viruses found indoors. Plants were proportionally the second RNA virus that causes persistent infections in hemp (Cannabis largest source of viral OTUs detected in dormitory rooms sativa).144 Because CCV is widespread among hemp (Figure 3). However, the majority of these viral OTUs were varieties,144 the detection of this virus in two of the rooms

H DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX Environmental Science & Technology Article and the pooled sample suggest that hemp-related products sequences most similar to arthropod-infecting viruses indicates might also contribute to indoor viral diversity. Because that species-rich arthropod communities found in indoor industrial hemp can be used for various purposes (e.g., health spaces81 might be a source of unexplored viral diversity. foods, personal care products, and clothing), it remains to be Airborne viruses associated with consumer products can determined how CCV becomes airborne and if it can be found provide information regarding the spread and diversity of in biological samples, such as feces, from consumers of hemp- viruses relevant to the food (e.g., cheese production) and related products. Furthermore, it is unknown if CCV can also natural product (e.g., Cannabis sativa) industries. cause persistent infections in psychotropic varieties of C. sativa, Because it is not currently practical to process a large number which are genetically distinct from industrial hemp.145 of samples using viral metagenomic approaches, quantitative Overlooking the contribution of human feces to the assays for specific viral taxa in larger sample sets might reveal abundance of nonhuman-infecting viruses, including bacter- statistical differences between different demographic groups. iophages and plant viruses, might lead to a gross under- For example, although HPVs and HPyVs are widespread in the estimation of the contribution of room occupants to airborne human population, the data presented here suggest that there viral diversity. Moreover, several of the viral OTUs most closely might be differences in shedding rates of these viruses by a related to sewage-associated viruses might also represent viruses generally healthy cohort of young adults occupying university from human feces because sewage reflects the fecal microbiome dormitory rooms. Quantitative PCR assays targeting these of human populations146 and harbors a diversity of human- viruses in more rooms and different wings of the building are infecting viruses.34,141 Nevertheless, by investigating viral OTUs needed to confirm if airborne HPVs are more abundant than based on source of isolation or host niche, it is clear that indoor HPyVs and if the concentrations of HPyVs are higher in male- airborne viral diversity includes viruses originating from a wide occupied rooms compared to female-occupied rooms. range of organisms and sources. Furthermore, assays could be extended to air filters found in Methodological Approaches and Future Directions. daycare centers and senior living facilities to better understand To our knowledge, this is the first metagenomic analysis of the epidemiology of these human-infecting viruses in other airborne viral communities captured on HVAC filters. Here we population sectors using a noninvasive, passive sampling show that MERV 8-rated filters that are in place for extended approach. Finally, in addition to epidemiological studies, the periods of time can capture enough aerosolized viral biomass to differential detection of certain human-infecting viruses in allow the detection of a diversity of airborne DNA and RNA female- and male-occupied rooms might contribute to micro- 24,150 viruses. The detection of viruses from MERV 8-rated filters is bial forensic efforts. advantageous because these filters are commonly installed in buildings and have been used to investigate airborne bacterial ■ ASSOCIATED CONTENT 24,126 and fungal communities. It has been suggested that HVAC *S Supporting Information filters with MERV ratings ≥12 should be used for viral The Supporting Information is available free of charge on the metagenomic analyses because they are more efficient at ACS Publications website at DOI: 10.1021/acs.est.7b04203. fi collecting particles 1−3 μm in size compared to those lters Supplemental File S1: Excel workbook with details 22 fi with lower MERV ratings. Although MERV 8 lters are most regarding viral OTUs, human papillomaviruses and efficient at capturing particles >3 μm, these filters can also 27 polyomaviruses, and contaminant sequences (XLSX) capture smaller sized particles. In addition, even though Supplemental File S2: Merkel cell polyomavirus genome μ virions generally have diameters <0.2 m, viruses have been assembled from a male-occupied room in fasta format μ detected in air particles with diameters 0.4−10 m indicating (TXT) that they are associated with other particles when airborne.147−149 Our results demonstrate that MERV 8 filters AUTHOR INFORMATION can capture a diversity of airborne viruses containing various ■ virion morphologies (e.g., icosahedral, filamentous) and Corresponding Author genome types (e.g., ssDNA, dsDNA, ssRNA, dsRNA). *K. Rosario. Email: [email protected]. The viral diversity reported here should be viewed as a ORCID conservative estimate of airborne viruses found indoors because Karyna Rosario: 0000-0001-9847-4113 viruses associated with particles <3 μm might not have been Notes captured in the analysis. In addition, it is likely that the strategy The authors declare no competing financial interest. used for classifying viral OTUs led to an underestimation of viral diversity as several viral OTUs represent groups of viruses ■ ACKNOWLEDGMENTS rather than a single viral species (Supplemental File S1) and OTUs only reflect sequences that could be confidently Authors would like to acknowledge Unilever Industries and the fi National Science Foundation (grants DEB-1239976 and IOS- identi ed as viral. For example, prophage sequences in bacterial fi genomes that have not been annotated as viral in the database 1456301) for nancial support. fi would not have been identi ed in the present analysis. REFERENCES Although metagenomic analyses of airborne viral communities ■ collected on HVAC filters might not capture all of the viral (1) Toivola, M.; Nevalainen, A.; Alm, S. 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N DOI: 10.1021/acs.est.7b04203 Environ. Sci. Technol. XXXX, XXX, XXX−XXX