diversity of dna and rna viruses in indas assessed via...

Diversity of DNA and RNA Viruses in Indoor Air As Assessed viaMetagenomic SequencingKaryna 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 communitiesinhabit human-occupied buildings and circulate in indoor air;however, viral diversity in these man-made environmentsremains largely unknown. Here we investigated DNA and RNAviruses circulating in the air of 12 university dormitory roomsby analyzing dust accumulated over a one-year period onheating, ventilation, and air conditioning (HVAC) filters. Ametagenomic sequencing approach was used to determine theidentity and diversity of viral particles extracted from theHVAC filters. We detected a broad diversity of virusesassociated with a range of hosts, including animals, arthropods,bacteria, fungi, humans, plants, and protists, suggesting thatdisparate organisms can contribute to indoor airborne viralcommunities. Viral community composition and the distribu-tion of human-infecting papillomaviruses and polyomaviruses were distinct in the different dormitory rooms, indicating thatairborne viral communities are variable in human-occupied spaces and appear to reflect differential rates of viral shedding fromroom occupants. This work significantly expands the known airborne viral diversity found indoors, enabling the design ofsensitive and quantitative assays to further investigate specific viruses of interest and providing new insight into the likely sourcesof viruses found in indoor air.

■ INTRODUCTIONMicrobes are abundant in indoor air. Human-occupiedbuildings, including residential, work, and public spaces, canharbor more than 105 bacterial and fungal cells per cubic meterof indoor air.1−3 Hundreds to thousands of bacterial and fungaltaxa are found indoors, highlighting the diverse nature of thesemicrobial communities.4,5 This realization, combined with thefact that we spend >90% of our time indoors,6 has fueledcoordinated efforts to investigate the microbiome of the builtenvironment (e.g., microBEnet7). Extensive research onbacterial and fungal communities has shown high variabilityin community composition across buildings, or even locationswithin a given building, due to differences in occupants,environmental conditions, geographic location, and many otherfactors.8−10 However, despite decades of research on thebacteria and fungi that humans are exposed to indoors, there islittle information regarding the diversity of viruses found inbuilt environments.Most of the research investigating viruses indoors has

focused on selected human viral pathogens found on surfacesand in air.11−14 However, known human pathogens are unlikelyto be the only viruses we encounter indoors. The application of

viral metagenomic techniques, where viral particles are purifiedfrom a given sample prior to extraction and shotgun sequencingof nucleic acids,15,16 can be used to expand the known diversityof viruses found indoors. An advantage of viral metagenomics isthat this technique can be used to survey viral diversity withouta priori knowledge of the viruses present in a given sample,resulting in the detection of novel viruses as well as thecharacterization of viral communities (i.e., viromes) in a widerange of environments,17 including outdoor air18 and indoorair.19,20 However, the fairly low viral biomass in indoor air(approximately 105 virus-like particles per cubic meter21)presents a challenge for viral metagenomic studies.22 Thoughmost of these viral particles are unlikely to be pathogens, oreven human-associated viruses, a more complete assessment ofthe viral diversity circulating in indoor air is broadly relevant tounderstanding buildings as microbial ecosystems and thefactors that shape indoor exposure to viruses.

Received: August 15, 2017Revised: December 5, 2017Accepted: January 3, 2018Published: January 3, 2018

Article

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

© XXXX American Chemical Society A DOI: 10.1021/acs.est.7b04203Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Heating, ventilation, and air conditioning (HVAC) filters,which serve as passive, long-term, high volume air samplers inindoor spaces, have been used successfully to describe thediversity of airborne bacteria and fungi circulating insidebuildings.23−26 Because HVAC filters can be used for integratedsampling of indoor airborne bacterial and fungal commun-ities,23 here we evaluated if metagenomic sequencing of virusescould also be performed from these filters. For this purpose,viral purification strategies were combined with high-throughput sequencing on a subset of HVAC filters previouslyused to investigate bacterial and fungal communities inside aUniversity of Colorado dormitory building.24 By using areplicated room design, this study aimed to determine whattypes of viruses circulate in indoor air, how viral communitiesvary across rooms within a single building, and how thesedifferences relate to the room’s occupants. To our knowledge,this study represents the most comprehensive investigation ofthe DNA and RNA viral diversity found inside buildings.Although many studies have focused on identifying human viralpathogens in indoor air, this study reveals that indoor airharbors a diversity of viruses associated with a wide range ofpotential hosts.

■ MATERIALS AND METHODSSample Collection and Processing. This study utilized a

subset of HVAC fan coil unit filters collected for a previousproject investigating bacterial and fungal diversity in dormitoryrooms.24 The filters were typical residential filters with a MERV8 rating, which are on average 70−80% efficient at collecting3−10 μm particles.27 The HVAC filters were installed 12months prior to collection within each dormitory room housingundergraduate students on the University of Colorado campusin Boulder, Colorado, USA. Each room was equipped with a fancoil unit that heated or cooled recirculated room air (no outsideair) at a constant volumetric flow rate. The filters from each fancoil unit only filtered the air from an individual room, allowingfor a time-integrated aerosol sample from each room. Becauseeach wing of the dormitory building had a separate HVACsystem, filters for viral metagenomic analysis were collectedfrom the East wing to minimize variability due to HVACsystem differences. Selected HVAC filters originated from sixmale and six female double-occupancy rooms. In addition tothese 12 filters from individual rooms, a pooled sample of eightfilters from the North and West wings of the building wasprocessed to detect potential viruses present in other wingswith separate HVAC systems. HVAC filters were stored at −20°C within 5 days of collection until further processing.Virus particles were partially purified from HVAC filters and

concentrated to increase the probability of detecting viralsequences through high-throughput sequencing by adaptingmethods that have been used to investigate viromes fromvarious environmental matrices including soil, water, and air aswell as methods from the biomedical field.14,28−30 To do this,eight pieces (8 × 8 cm) were cut from each individual filterusing a sterile scalpel, to yield a total area of 64 × 64 cm. Forthe pooled sample, a single 8 × 8 cm piece from each of theeight filters was processed. Filter sections were shredded intosmaller pieces using scissors, which were cleaned between filtersusing 10% bleach followed by ethanol flame sterilization. Thefilter pieces were suspended in 225 mL of sterilized, coldsuspension medium (SM) buffer [100 mM NaCl, 8 mMMgSO4·7H2O, 50 mM Tris-Cl (pH = 7.5), 0.01% (w/v)gelatin] and placed on ice for at least 5 min before sonication in

a 8891 Ultrasonic Bath Sonicator, 42 kHz frequency (Cole-Parmer) for a total of 5 min in 1 to 2 min intervals followed byvigorous shaking to detach viral particles from the filter anddisrupt aggregates. To separate desorbed viral particles fromcells and debris after sonication, samples were filtered through a0.22 μm Sterivex filter (Millipore).To concentrate virus particles, filtrates were subjected to two

rounds of ultracentrifugation, which pelleted most virus-likeparticles as assessed through SYBR Gold staining andepifluorescence microscopy.31,32 For this purpose, five 35 mLaliquots of filtrate per sample were placed into 25 × 89 mmultracentrifuge tubes (Beckman Coulter, Inc.) and ultra-centrifuged at 28,000 rpm in a SW28Ti swinging bucketrotor (Beckman Coulter, Inc.) for 4 h at 4 °C. Supernatantswere directly decanted into a second set of ultracentrifuge tubesand centrifuged once more at 28,000 rpm for 3 h at 4 °C.Resulting viral pellets in each of the tubes after the first roundof ultracentrifugation were resuspended with 100 μL of SMbuffer and soaked overnight at 4 °C. The SM buffer containingthe resuspended viral pellets from the first round wassubsequently used to resuspend the pellets from the secondultracentrifuge round by soaking for at least 6 h at 4 °C.Approximately 500 μL of total resuspended viral pellets persample after two rounds of ultracentrifugation were pooled.Free nucleic acids were removed from the viral concentrates byincubating resuspended pellets at 37 °C for 2 h with a nucleasecocktail consisting of 1X Turbo DNase Buffer (Ambion), 21Uof Turbo DNase (Ambion), 4.5U of Baseline-ZERO DNase(Epicenter), 112.5U Benzonase (EMD Millipore), and10 μg/mL RNase A (Sigma-Aldrich).33,34 Nucleases wereinactivated with 20 mM EDTA prior to nucleic acid extractionfrom viral particles.Because a diversity of contaminants, including viruses, can be

found in laboratory reagents and commercially available nucleicacid extraction and amplification kits,35−38 a negative controlsample containing SM buffer alone was included. This negativecontrol was processed alongside the filter samples from thesonication step onward to control for unforeseen contami-nation in laboratory reagents that can potentially impactmetagenomic analyses of low biomass samples. Unfortunately, ablank HVAC filter from the original batch installed in thedormitory rooms was not available to control for contaminantspresent in the filter material. However, any viral contaminationinitially present in the filters should be obscured after collectinga passive, long-term (1 year), integrated air sample.

Nucleic Acid Extraction and Metagenomic LibraryPreparation. Viral DNA and RNA were extracted from 200μL of purified viral particles using the QIAmp MinElute VirusSpin (Qiagen) and RNeasy (Qiagen) kits, respectively,following manufacturer’s protocols. For viral RNA extraction,the on-column DNase digestion was performed according tomanufacturer’s recommendations. RNA was reverse-transcribedusing the SuperScript III First Strand Synthesis System(Invitrogen) with random hexamers. Second-strand synthesiswas performed on the resulting cDNA using the KlenowFragment DNA polymerase (New England Biolabs) followingmanufacturer’s instructions and the resulting products werecleaned using the AMPure XP Purification system (BeckmanCoulter). DNA and cDNA samples were fragmented to 300 bpusing a Covaris M220 instrument. Next-generation sequencing(NGS) library construction was performed with the Accel-NGS1S Plus DNA Library Kit for Illumina Platforms (SwiftBiosciences), which allows low DNA/cDNA inputs to

Environmental Science & Technology Article

DOI: 10.1021/acs.est.7b04203Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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circumvent the introduction of biases while trying to obtainsufficient template for NGS.39 The Accel-NGS 1S Plus kit canalso recover single-stranded DNA (ssDNA) templates,40

eliminating the need for multiple displacement amplification,which is strongly biased toward small, circular templates.41

Library construction and multiplexing was performed using theAccel-NGS 1S Plus kit following manufacturer’s instructions forDNA inputs <1 ng/μL and 20 cycles of indexing PCR. A totalof 28 libraries, including separate DNA and cDNA from airfilters from each of the 12 individual rooms, a pooled sample,and a negative control, were paired-end sequenced (2 × 250bp) on a MiSeq System (Illumina) at the University ofColorado BioFrontiers Next-Gen Sequencing core facility.Sequences are available at the NCBI Sequence Read Archivedatabase (study number SRP113244; accession numbersSRR5853122−SRR5853149).Sequence Analysis. Sequences were processed using

bioinformatic applications (Apps) available through theCyVerse cyberinfrastructure42 and the pipeline suggested by

the iVirus resource for analysis of viromic data.43 Briefly, rawsequences were trimmed for quality and to remove indexingadapters using the Trimmomatic App version 0.35.0 withdefault parameters,44 except for a read head crop of 10 bpinstead of zero. The quality of the trimmed sequences wasverified with the FastQC-plus App version 0.10.1.45 Quality-filtered sequences were then assembled using the SPAdesassembler App version 3.6.0 with default parameters, k-merlength of 55, and the metagenomics assembly option.46 Contigslarger than 100 bp were selected for further analyses.Contaminant contig sequences were removed from each dataset by comparing (BLASTn, e-value < 0.00001) against adatabase containing assembled sequences from the negativecontrol library.After removing sequences with significant matches in the

negative control database, contig sequences from each librarywere compared (BLASTx, e-value < 0.001) against a viralprotein database containing sequences from the NCBIReference Sequence (RefSeq) database (RefSeq Release

Figure 1. Heatmap showing the distribution of viral OTUs identified in rooms occupied by male (M1−M6) and female (F1−F6) students as well asthose identified in the pooled sample (P) according to taxonomic groups. Bar graph on the right shows the percentage of total viral OTUs identifiedin rooms that were classified within a given group. Colors on the left panel highlight DNA (blue) and RNA (orange) viral groups. DNA viruses arefurther 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 indicatestaxonomic groups that were not detected in a given room. “CRESS DNA” refers to circular replication-associated protein encoding single-strandedDNA viruses that form a diverse and evolutionarily related group. “Uncl” stands for unclassified.



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number 79, https://www.ncbi.nlm.nih.gov/refseq/) as well assequences recently reported from RNA viruses in invertebratesand environmental samples.47,48 Contig sequences withsignificant matches in the viral database were then comparedagainst the GenBank nonredundant (nr) database (BLASTx, e-value 0.001; downloaded December 2016) to remove contigsequences that had higher identities with nonviral sequences(i.e., false positives). Final BLAST results were inspected usingMEGAN6 Community Edition.49 To err on the side of caution,contig sequences from the negative control library were alsocompared against the viral and nr databases (BLASTx, e-value0.001) to identify potential viral contaminants. If one of the topthree BLAST matches for a given contig sequence in thesample libraries included a virus identified in the negativecontrol or phylogenetically related viruses, the sequence wasremoved from further analysis (Supplemental File S1).After false positives and potential viral contaminants were

removed, sequences that could be confidently identified as viralbased on best BLAST matches to known viral sequences wereorganized into operational taxonomic units (OTUs). Here, viralOTUs represent either a single species or a group of relatedviruses in cases where BLAST matches could not be confidentlyassigned to a given viral species and/or when sequences

represented a cohesive group, such as human papillomaviruses,to look for general trends at higher taxonomic levels. Sequencessimilar to bacteriophages infecting Actinobacteria from soilwere grouped according to clusters classified within theActinobacteriophage Database50 (http://phagesdb.org/). AnOTU table (summarized in Supplemental File S1) wasconstructed by recording the presence or absence of eachviral OTU in each dormitory room.Sequences similar to human papillomaviruses (HPVs) and

polyomaviruses (HPyVs) were further investigated given theirwidespread nature in the human population. To do so,approximately 1450 and 650 HPV and HPyV genomes,respectively, were downloaded from GenBank in April 2017.Genome sequences were then dereplicated based on a 95%identity cutoff using CD-HIT.51 Quality-filtered forward readsfrom each of the DNA libraries were compared against curateddatabases containing 247 HPV and 28 HPyV genomes(BLASTn, e-value < 0.00001). Sequences with significantmatches to these databases were then compared against theGenBank nucleotide (nt) database (BLASTn, e-value < 0.001)to eliminate false positives. The number of HPV- and HPyV-related sequences as well as the HPV types and HPyV specieswere recorded (Supplemental File S1). Finally, the classification

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 83Propionibacterium acnes phages Siphoviridae Actinobacteria Human 100 67Staphylococcus Sep9-like phages Siphoviridae Firmicutes Human 57−100 67Lactococcus 936 sensu lato phages Siphoviridae Firmicutes Dairy 95−100 58Bacillus phage G Myoviridae Firmicutes Soil 50−88 58Staphylococcus Twortlikevirus phages Myoviridae Firmicutes Sewage 54−100 58Actinobacteriophage cluster A Siphoviridae Actinobacteria Soil 56−80 50Streptococcus group 1 phages Podoviridae Firmicutes Human 50−99 42Lactococcus C2-like phages Siphoviridae Firmicutes Dairy 94−99 42Sinorhizobium phages Myoviridae Alphaproteobacteria Soil 56−70 42Actinobacteriophage cluster BD Siphoviridae Actinobacteria Soil 54−75 42Lactococcus phages (unclassified) Siphoviridae Firmicutes Dairy 57−96 33Actinobacteriophage cluster F Siphoviridae Actinobacteria Soil 59−91 33Actinobacteriophage cluster L Siphoviridae Actinobacteria Soil 54−73 33Clavibacter phages Siphoviridae Actinobacteria Soil 58−75 33Actinobacteriophage cluster C Myoviridae Actinobacteria Soil 49−76 33Enterobacteriaceae T4-like phages Myoviridae Gammaproteobacteria Sewage 59−92 33Bacillus SPO1virus Myoviridae Firmicutes Soil 44−71 33Actinobacteriophage cluster BE Siphoviridae Actinobacteria Soil 55−83 33crAssphage Unclassified dsDNA Bacteroidetes Human 60−100 33Invertebrate iridescent viruses Irridoviridae Invertebrate Arthropod 47−99 67Human papillomavirus Papillomaviridae Human Human 77−100 67Animal feces associated gemycircularvirus Genomoviridae Unknown Animal 52−100 33Sewage associated gemycircularvirus Genomoviridae Unknown Sewage 79−86 33Insect densovirus Parvoviridae Insect Arthropod 46−66 33Plant tymovirus Tymoviridae Plant Plant 64−93 83Human retrovirus Retroviridae Human Human 55−99 83Ryegrass mottle virus Sobemovirus Ryegrass Plant 86−100 67Tobacco mild green mosaic virus Virgaviridae Tobacco Plant 98−100 67Tobacco mosaic virus Virgaviridae Tobacco Plant 99−100 50Insect picorna-like virus Picornavirales Insect Arthropod 47−99 50Crustacean picorna-like virus Picornavirales Crustacean Arthropod 50−78 50Turnip vein-clearing virus Virgaviridae Turnip Plant 98−100 42Aphis glycines virus 1 Picornavirales Insect Arthropod 47−92 42Pepper mild mottle virus Virgaviridae Pepper Plant 98−100 42Clover Potexvirus Alphaf lexiviridae Clover Plant 95−100 33



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of all HPV types detected in dormitory rooms were verifiedusing the Papillomavirus Episteme database.52

■ RESULTS AND DISCUSSIONOverview. Here we investigated viruses circulating in the air

of university dormitory rooms by performing metagenomicsequencing on partially purified VLPs from dust accumulatedon HVAC filters over a one-year period. Specifically, weanalyzed individual HVAC filters from 12 double occupancyrooms housing either female (n = 6) or male (n = 6) studentsin the East wing of the dormitory, as well as a pooled samplefrom eight rooms located in the North and West wings of thebuilding that used different HVAC systems. A total of 215 viralOTUs, the majority of which represented DNA viruses (72%),were detected across all air filters after examining top BLASTxmatches against the GenBank nr database (Supplemental FileS1). Due to low amino acid sequence identities to known viralspecies (as low as 44% in some cases), most OTUs wereclassified to only the family or order level. Up to 79 viral OTUswere detected on individual HVAC filters, with both female-and male-occupied rooms having approximately 40 OTUs perroom. Identified viral OTUs indicate that bacteriophages andeukaryotic viruses spanning several viral families and genometypes circulate in indoor air (Figure 1).Bacteriophages Dominate Airborne Viral Community

Diversity in Dormitory Rooms. The majority (62%) ofidentified viral OTUs in air filters represented bacteriophages.This was not surprising considering that bacteria are abundantin indoor air.2,21 Viral OTUs were dominated by tailedbacteriophages (order Caudovirales) from the Siphoviridae andMyoviridae families, which were the only viral families detectedin every room (Figure 1). Although the dominance of tailedbacteriophages in indoor air has not been previously noted,tailed bacteriophages tend to dominate environmental viralcommunities described from soil53 and water54,55 as wellhuman-associated viromes. In particular, members of theSiphoviridae and Myoviridae families are abundant in human-associated viromes including the gut,56−59 respiratory tract,60

saliva,61 and skin.62,63 However, the abundance of tailedbacteriophages might be skewed by current knowledge ofbacteriophage diversity because >90% of reference bacterio-phage genome sequences in GenBank (https://www.ncbi.nlm.nih.gov/genome/viruses/) belong to the order Caudovirales.Nevertheless, our results indicate that a broad diversity of tailedbacteriophages are ubiquitous in dormitory room air.The putative host phyla identified for the observed

bacteriophage OTUs included Proteobacteria (45%), Actino-bacteria (30%), Firmicutes (23%), Bacteroidetes (0.75%), andAquificae (0.75%) (Supplemental File S1). These putative hostgroups, with the exception of Aquificae, frequently dominateindoor microbiomes and were widely detected in dormitoryrooms.24 The most widespread viral OTUs potentially infectingProteobacteria, including Pseudomonas and Sinorhizobiumbacteriophages, were most similar to bacteriophages isolatedfrom environmental sources (Table 1). In addition, some of themost widespread viral OTUs were similar to bacteriophagesinfecting Actinobacteria, such as Propionibacterium acnesbacteriophages, as well as those infecting Firmicutes (Staph-ylococcus and Streptococcus bacteriophages) and potentiallyBacteroidetes species (crAssphage) (Table 1). Bacterial speciesfrom most of these genera, including Propionibacterium,Staphylococcus, Streptococcus and Bacteroides, are part of thehuman microbiome64,65 and their presence in indoor environ-

ments has been linked to human occupancy.9,66 Propionibacte-rium and Staphylococcus bacteriophages are among the mostabundant viruses found on healthy human skin,62,63,67 wherethey have been linked to the modulation of P. acnes populationstructure.68 Likewise, the detection of a Propionibacteriumbacteriophage through metagenomic approaches in cleanrooms has been linked to human presence in the rooms.19

CrAssphage, which is suspected to infect a Bacteroides species, iswidespread in human fecal metagenomes.69 Together theseresults highlight that the identified bacteriophages in dormitoryroom HVAC filters are concordant with bacteria that arecommonly found indoors, including environmental and human-associated taxa.

Diversity of Airborne Eukaryotic Viruses Circulatingin Dormitory Rooms. Although bacteriophages dominatedthe viral diversity captured in dormitory rooms, eukaryoticviruses were also readily detected (Figure 1). Eukaryotic viralOTUs that were present in more than 50% of the roomsincluded putative arthropod-infecting viruses from the familyIridoviridae and order Picornavirales, plant pathogens from thegenus Sobemovirus as well as those from the Tymoviridae andVirgaviridae families, and human viruses from the Retroviridaeand Papillomaviridae families (Table 1). Viral OTUs represent-ing members of the family Genomoviridae were also detected inmore than half of the rooms. Because the only culturedrepresentative of the family Genomoviridae was isolated from afungus70 and sequences similar to proteins encoded by theseviruses have been identified within fungal genomes,71,72

members of this family are suspected to infect fungi. However,a definitive host has not been confirmed for most species ofGenomoviridae.73

Most of the eukaryotic viral OTUs represented putativearthropod-infecting viruses from the order Picornavirales andplant viruses from the family Virgaviridae (Figure 1). Themajority of the Virgaviridae-related sequences had high aminoacid level identities (≥98%) with proteins encoded by membersof the genus Tobamovirus. In contrast, sequences similar toputative arthropod-infecting viruses from the order Picornavir-ales had low amino acid sequence identities to recently reportedviral genomes from invertebrates48 (Supplemental File S1).Therefore, viral sequences identified here, including a novel, ∼10 kb genome (will be described in a separate paper) that ismost similar to viruses identified in Drosophila and ants,indicate that RNA viral diversity has yet to be fully explored.Human endogenous retroviruses were the most widespread

group of human-associated viral sequences found in dormitoryrooms (Table 1). It is possible that these endogenous retroviralsequences represent remnant viral sequences integrated intocellular genomes rather than exogenous viruses becauseretroviral sequences are among the transposable elementsthat constitute 50% of the human genome.74,75 However, thedetected retroviral sequences were most similar to humanendogenous retrovirus K (HERV-K) and multiple sclerosis-associated retrovirus (MSRV) and were mainly detected in theRNA libraries. Expression of structural genes and detection ofvirus-like particles has suggested that the recently acquiredHERV-K can become active under certain physiologicalcircumstances.76−79 In addition, there has been debate aboutwhether or not MSRV can be exogenous and persist as anextracellular particle.80 Due to the possibility of expression ofhuman endogenous retroviral sequences combined with lowamino acid identities (as low as 55%) to known retroviralsequences in some cases, this OTU was retained in the



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analyses. Nevertheless, care should be taken when interpretingthe presence of endogenous retroviruses in viral metagenomicstudies as sequences might represent cellular DNA.The detection of diverse eukaryotic viruses in dormitory

rooms suggests that disparate organisms contribute to indoorairborne viral communities. A high diversity of arthropods live

in indoor spaces, including hundreds of species found inindividual homes.81 In addition, dust samples collected fromsurfaces inside homes contain pollen representing a diversity ofplant species82 and outdoor vegetation may affect thecomposition of indoor bacterial communities.83 Moreover,bacterial species potentially associated with plants and insects

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 eachroom 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 becausedetected sequences share low nucleotide identity (<80%) with known types. The color scale on the heatmap represents relative low (dark purple) tohigh (yellow) abundances of HPV types based on the total number of HPV-related sequences identified in a given room. Gray color on the heatmapindicates HPV types that were not detected in a given room. Because only a single human polyomavirus (HPyV) species was identified in positiverooms, identified species in each room are listed at the bottom of the heatmap, including polyomavirus 6 (PyV6), MW polyomavirus (MWPyV), andMerkel cell polyomavirus (MCPyV). ‘X’ indicates rooms that did not have any HPyV-related sequences.



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were identified in the dormitory rooms.24 Therefore, plants andarthropods are likely to be significant sources of airborneindoor bacteria and viruses. This study suggests that fungi alsoserve as a source of airborne viruses, as several OTUs mostsimilar to fungal viruses were identified, including RNA viruses(members of the Chrysoviridae, Hypoviridae, and Partitiviridaefamilies as well as unclassified ssRNA and dsRNA viruses) andpotentially ssDNA viruses (family Genomoviridae, see above).Unexpectedly, one of the rooms contained sequences similar tofeline papillomavirus type 2 (FePV2), which infects domesticcats.84 The presence of nonhuman occupants, such as pets, canaffect indoor bacterial communities9 and the detection ofFePV2 suggests that pets may also influence indoor viraldiversity. However, because university policies prohibited petsin these dormitory rooms, it is unknown if the detection ofFePV2 reflects the presence of an illicit cat in the dormitoryroom or resulted from external contact between humanoccupants and cats.Diversity of Airborne Human Papillomaviruses and

Polyomaviruses in Dormitory Rooms. NonenvelopeddsDNA viruses from the Papillomaviridae and Polyomaviridaefamilies, including human papillomaviruses (HPVs) andpolyomaviruses (HPyVs), were widespread in the dormitoryrooms. These two groups of viruses constitute part of thehuman-associated virome in both healthy and disease states.HPVs, which have evolved replication strategies to thrive intheir host’s cutaneous and mucosal epidermal tissue,85 havebeen strongly linked to the development of cervical cancer86−88

as well various other cancers and skin diseases.89−93 HPyVs cancause serious diseases in immunocompromised patientsincluding Merkel cell carcinoma, progressive multifocalleukoencephalopathy, nephropathy, trichodysplasia spinulosaand hemorrhagic cystitis.94−96 However, healthy individuals arealso known to be asymptomatically infected with HPVs andHPyVs, both of which may be acquired early in infancy.97−106

Furthermore, HPVs and HPyVs are the most abundant groupsof human-infecting viruses associated with the healthy humanskin viral flora62,98 and novel species have been discovered fromhealthy individuals.107−109

The diversity of HPV- and HPyV-related sequences wasfurther investigated because the presence of these viruses indormitory rooms is directly linked to human occupancy. Bycomparing unassembled sequences to custom databases, thedetection of HPV- and HPyV-related sequences increased.HPVs, the second most widespread group of human-infectingviruses detected in dormitory rooms (Table 1), were morediverse than HPyVs (Figure 2). A total of 54 HPV types,including 7 unclassified HPVs, were detected, with up to 24HPV types detected in a single room (Figure 2). All of thedetected HPV-related sequences were most similar to speciesclassified within the Alphapapillomavirus, Betapapillomavirus,and Gammapapillomavirus genera, which contain most knownHPV species.110,111 The majority of the HPV-related sequenceswere similar to cutaneous viruses from the Beta- andGammapapillomavirus genera. Alphapapillomaviruses, includingboth genital/mucosal (type 51) and cutaneous (types 3 and125) HPVs, were only detected in three of the dormitoryrooms. The most widespread HPVs included types 23 and 120,which were detected in 33% of the rooms, followed by HPVtype 24, which was detected in 25% of the rooms. Although allof these cutaneous HPV types were originally isolated from skinlesions,110,112 these types have also been detected in the skin ofhealthy individuals.103 In addition, HPV type 51, an

alphapapillomavirus that has been classified as high-risk dueto its oncogenic potential,113,114 was detected in a male-occupied room. This high-risk HPV type has been found at lowprevalence compared to other HPV types in healthy womenand men.103,114,115 These results indicate that a high diversity ofHPVs circulates in indoor air, including HPV types with lowprevalence in the general population that might be of clinicalrelevance.In contrast to HPVs, HPyVs were mainly restricted to male-

occupied rooms and, although coinfection with multiple HPyVsis common,109,116 only a single HPyV species was detected ineach positive room (Figure 2). Three HPyV species, namelyHPyV6, MW polyomavirus (MWPyV), and Merkel cellpolyomavirus (MCPyV), were detected. HPyV6 and MWPyCwere discovered in skin and fecal samples, respectively, fromhealthy individuals,109,117 whereas MCPyV was originallyidentified in Merkel cell carcinoma118 and later established asa widespread cutaneous virus in the human population.94 Weassembled a complete MCPyV genome that shared 99%genome-wide pairwise identity to a genome recovered from theskin of a healthy individual109 (Accession number HM011544)(Supplemental File S2). The prevalence of MWPyV in thegeneral population and environmental samples is low comparedto other HPyVs like MCPyV;97,109,119 however, MWPyVseemed to be more widespread in dormitory room air thanHPyV6 and MCPyV. This suggests that differences in sheddingrates, namely the number of virions released by infected cells atany given time, between HPyV species may contribute todifferences between airborne HPyV distribution and that ofhuman subjects.The higher diversity of airborne HPVs detected in dormitory

rooms compared to HPyVs supports previous reports of lowHPyV genetic diversity on human skin.98 Indeed, to date, morethan 30 HPV species comprising >200 HPV types52,120,121 havebeen described compared to only 12 HPyV species.96,106 HPVdiversity might be driven by a high selective pressure from thehuman immune system.67 Although HPVs and HPyVs arewidespread in the human population and have similarcharacteristics, there could be differences in tissue tropism orvirion shedding rates that might affect their relativerepresentation in indoor air. Moreover, HPyVs were mainlydetected in male rooms, suggesting that shedding rates for thisgroup of viruses differ between male and females. This isplausible considering that female children are more likely toshed MWPyV in feces than male children122 and seropositivityfor certain HPyV species is more common among males.116

Notably, MWPyV was the only HPyV species detected infemale rooms (Figure 2). In addition, because Merkel cellcarcinoma is more prevalent in males,123 some studies have alsoshown that MCPyV is highly prevalent in the skin of healthymales.124,125 Interpersonal variation in HPV and HPyV speciescomposition62,63,98 and potential differences in shedding ratesbetween HPyV species might affect indoor airborne viral loadsof these human-infecting viruses.

Airborne Viral Community Variation in DormitoryRooms. Each dormitory room exhibited a different distributionof viral OTUs as well as HPV types, revealing a unique“fingerprint” of airborne viral species (Figures 1 and 2).Although Siphoviridae and Myoviridae members were detectedin all of the samples (Figure 1), the distribution of specificOTUs within these groups varied. It is possible that variation insequencing depth among the samples might affect the detectionof low abundance viral OTUs; however, even the dominant



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viral OTUs varied across individual rooms. This observationsuggests that airborne viral communities indoors are variable inhuman-occupied spaces as has been shown for bacteria.126

The disparate airborne viral communities of individual roomsmay reflect variability in occupant-associated microbiomesbecause individuals can release distinct “microbialclouds”.126,127 Notably, there are marked interpersonal differ-ences in viral communities associated with healthy human skinand there can be viral “blooms” in certain body sites.62,63

Furthermore, individuals with active viral infections may shedmore viral particles compared to healthy individuals, furthercontributing to airborne viral community dynamics. Forexample, the detection of rhinoviruses, which are the mostcommon causative agents of the common cold and other upperrespiratory tract infections,128,129 in one of the rooms mightreflect a symptomatic infection of a room occupant. Althoughasymptomatic rhinovirus infection can be common in youngchildren,130 viral detection and loads are significantly higher insymptomatic adults.131,132

Challenges Associated with Airborne Virus SourceTracking. The potential sources of observed viral OTUs indormitory rooms were examined by classifying the top BLASTmatch of each OTU based on its original source of isolation or,if that information was not available, its host’s niche. Theanalyses suggest that soil is likely the largest contributing sourceto airborne viral communities in dormitory rooms (Figure 3).The bacterial community in dust from floors and other indoorsurfaces has been shown to harbor taxa from human occupantsas well as environmental sources including soil.66,126,133 Dustresuspension, which has been suggested as an important sourceof indoor airborne bacteria,133 might also be a significant sourceof airborne viruses. However, our inability to accurately tracksources of airborne viruses might alter the true contributionsfrom potential sources described here.Due to their small size, virus particles may stay airborne for

significantly longer periods and, in turn, be transported forlonger distances than bacteria and fungi, making it difficult topinpoint their original source.22 This is further complicated byvirion stability, which can allow certain viruses to withstandharsh conditions, including the passage of viruses of dietaryorigin through the gut. For example, bacteriophages infectingLactococcus lactis, a bacterium extensively used in theproduction of dairy products, were widespread in dormitoryrooms (Table 1) and their bacterial host has been identified asone of the most abundant bacterial species in floor dust.66

Moreover, a ∼2 kb L. lactis bacteriophage contig sequence fromthe dormitory rooms had significant matches to metagenomicsequences from human feces134 available through the IMG/VRdatabase.135 Due to the remarkable stability of L. lactisbacteriophages,136 we cannot determine if the detectedbacteriophages originated directly from dairy products,137

from floor dust, or from the feces of room occupants.Estimated virus to bacteria ratios ranging from 0.7 to 1 inindoor environments suggest that bacteriophages in indoor airand surfaces might not be able to replicate due to hostdormancy or low host densities.20,21 These low ratios supportthe idea that bacteriophages might originate from the samesource as their hosts (e.g., dairy products, dust, feces) ratherthan propagating indoors.Virion stability also obscures source tracking of eukaryotic

viruses found indoors. Plants were proportionally the secondlargest source of viral OTUs detected in dormitory rooms(Figure 3). However, the majority of these viral OTUs were

most similar to members of the genus Tobamovirus, which havestable virions that can remain infectious after disinfectiontreatments and persist for long periods outside their planthost.138,139 Although identified tobamoviruses included virusesinfecting edible crops (tomato, cucumber, pepper), tobaccoplants, and grasses (Supplemental File S1), all of these virusesare dominant in human feces140 and raw sewage.141 Due totheir stability and presence in various sources, it is not possibleto determine if plant viruses detected in dormitory room airoriginated from plant material, such as houseplants and foodproducts, or if they indicate fecal material from roomoccupants. Notably, the detection of viruses that have beenproposed as indicators of fecal pollution, including pepper mildmottle virus142 and crAssphage143 (Table 1), suggest thatviruses originating from human feces can become airborne.Therefore, plants might contribute to indoor airborne viraldiversity indirectly through dietary consumption of plant-related products and subsequent aerosolization of fecal matter.Another virus detected in this study that might originate

from plant-related products is cannabis cryptic virus (CCV), anRNA virus that causes persistent infections in hemp (Cannabissativa).144 Because CCV is widespread among hempvarieties,144 the detection of this virus in two of the rooms

Figure 3. Potential sources of viruses detected on dormitory roomHVAC filters. Graphs show (A) the percentage of rooms (n = 12)where viruses from a given source were detected and (B) thepercentage of viral OTUs in each source category.



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and the pooled sample suggest that hemp-related productsmight also contribute to indoor viral diversity. Becauseindustrial hemp can be used for various purposes (e.g., healthfoods, personal care products, and clothing), it remains to bedetermined how CCV becomes airborne and if it can be foundin biological samples, such as feces, from consumers of hemp-related products. Furthermore, it is unknown if CCV can alsocause persistent infections in psychotropic varieties of C. sativa,which are genetically distinct from industrial hemp.145

Overlooking the contribution of human feces to theabundance of nonhuman-infecting viruses, including bacter-iophages and plant viruses, might lead to a gross under-estimation of the contribution of room occupants to airborneviral diversity. Moreover, several of the viral OTUs most closelyrelated to sewage-associated viruses might also represent virusesfrom human feces because sewage reflects the fecal microbiomeof human populations146 and harbors a diversity of human-infecting viruses.34,141 Nevertheless, by investigating viral OTUsbased on source of isolation or host niche, it is clear that indoorairborne viral diversity includes viruses originating from a widerange of organisms and sources.Methodological Approaches and Future Directions.

To our knowledge, this is the first metagenomic analysis ofairborne viral communities captured on HVAC filters. Here weshow that MERV 8-rated filters that are in place for extendedperiods of time can capture enough aerosolized viral biomass toallow the detection of a diversity of airborne DNA and RNAviruses. The detection of viruses from MERV 8-rated filters isadvantageous because these filters are commonly installed inbuildings and have been used to investigate airborne bacterialand fungal communities.24,126 It has been suggested that HVACfilters with MERV ratings ≥12 should be used for viralmetagenomic analyses because they are more efficient atcollecting particles 1−3 μm in size compared to those filterswith lower MERV ratings.22 Although MERV 8 filters are mostefficient at capturing particles >3 μm, these filters can alsocapture smaller sized particles.27 In addition, even thoughvirions generally have diameters <0.2 μm, viruses have beendetected in air particles with diameters 0.4−10 μm indicatingthat they are associated with other particles when air-borne.147−149 Our results demonstrate that MERV 8 filterscan capture a diversity of airborne viruses containing variousvirion morphologies (e.g., icosahedral, filamentous) andgenome types (e.g., ssDNA, dsDNA, ssRNA, dsRNA).The viral diversity reported here should be viewed as a

conservative estimate of airborne viruses found indoors becauseviruses associated with particles <3 μm might not have beencaptured in the analysis. In addition, it is likely that the strategyused for classifying viral OTUs led to an underestimation ofviral diversity as several viral OTUs represent groups of virusesrather than a single viral species (Supplemental File S1) andOTUs only reflect sequences that could be confidentlyidentified as viral. For example, prophage sequences in bacterialgenomes that have not been annotated as viral in the databasewould not have been identified in the present analysis.Although metagenomic analyses of airborne viral communitiescollected on HVAC filters might not capture all of the viraldiversity found in indoor air, it provides an important baselinefor abundant and widespread viruses found in a given indoorspace. The data presented here can be used to design targetedassays for groups of viruses that are not typically considered,such as viruses associated with nonhuman occupants andconsumer products. Notably, the detection of novel viral

sequences most similar to arthropod-infecting viruses indicatesthat species-rich arthropod communities found in indoorspaces81 might be a source of unexplored viral diversity.Airborne viruses associated with consumer products canprovide information regarding the spread and diversity ofviruses relevant to the food (e.g., cheese production) andnatural product (e.g., Cannabis sativa) industries.Because it is not currently practical to process a large number

of samples using viral metagenomic approaches, quantitativeassays for specific viral taxa in larger sample sets might revealstatistical differences between different demographic groups.For example, although HPVs and HPyVs are widespread in thehuman population, the data presented here suggest that theremight be differences in shedding rates of these viruses by agenerally healthy cohort of young adults occupying universitydormitory rooms. Quantitative PCR assays targeting theseviruses in more rooms and different wings of the building areneeded to confirm if airborne HPVs are more abundant thanHPyVs and if the concentrations of HPyVs are higher in male-occupied rooms compared to female-occupied rooms.Furthermore, assays could be extended to air filters found indaycare centers and senior living facilities to better understandthe epidemiology of these human-infecting viruses in otherpopulation sectors using a noninvasive, passive samplingapproach. Finally, in addition to epidemiological studies, thedifferential detection of certain human-infecting viruses infemale- and male-occupied rooms might contribute to micro-bial forensic efforts.24,150

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.7b04203.

Supplemental File S1: Excel workbook with detailsregarding viral OTUs, human papillomaviruses andpolyomaviruses, and contaminant sequences (XLSX)Supplemental File S2: Merkel cell polyomavirus genomeassembled from a male-occupied room in fasta format(TXT)

■ AUTHOR INFORMATIONCorresponding Author*K. Rosario. Email: [email protected] Rosario: 0000-0001-9847-4113NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSAuthors would like to acknowledge Unilever Industries and theNational Science Foundation (grants DEB-1239976 and IOS-1456301) for financial support.

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