the ebola outbreak 
 in 2014 raised new questions about routes of 
 transmission of the disease . experiments 
 with non - human primates have shown that inhalation exposure to ebola 
 virus can lead to fatal infection . patients with ebola virus 
 disease expel large volumes of diarrhea , which may contain 
 up to 10 genome copies of virus per milliliter , and toilets , sewer systems , and wastewater treatment 
 plants are known to produce bioaerosols . the combination of these factors indicates that the potential exists 
 for aerosolization of ebola virus from wastewater systems . sanitation 
 facilities that are common in developed countries , such as pressure - assisted 
 flush toilets and aeration basins , may provide opportunities for aerosolization 
 of the virus that do not exist in western africa , where the vast majority 
 of cases of ebola virus disease have occurred . given that ebola 
 is a high - consequence pathogen , it is critical 
 to consider all possible exposure routes . what we define as the  secondary aerosolization exposure route ( i.e. , aerosolization from sources other 
 than the infected host ) has been established as a concern for other 
 diseases . for example , a combination of epidemiological , experimental , 
 and modeling approaches suggests that aerosolization from toilets 
 and sewer pipes contributed to an outbreak of severe acute respiratory 
 syndrome in an apartment complex in hong kong in 2003 . an important factor in determining the potential 
 for a pathogen to spread via aerosolization from wastewater is partitioning 
 among the aqueous phase , biosolids , which are not as easily aerosolized , 
 and material surfaces . if a pathogen partitions preferentially to 
 biosolids or surfaces , then the potential for aerosolization is reduced . the goal of this research was to assess the partitioning of viruses 
 in fluids and materials characteristic of modern wastewater systems . we measured the partitioning of an unenveloped virus ( ms2 ) and an 
 enveloped virus ( phi6 ) among the liquid phase , solids , and porcelain , 
 polyvinyl chloride ( pvc ) , polypropylene , and concrete surfaces , using 
 both synthetic sludge and real , anaerobically digested sludge as model 
 fluids . results from this work will provide information not only about 
 the potential for aerosolization of viruses but also about their fate 
 in wastewater systems . because surface chemistry 
 affects partitioning , we considered both 
 unenveloped and lipid - enveloped viruses : ms2 and phi6 , respectively . ms2 ( atcc 15597-b1 ) is an icosahedral , single - stranded rna bacteriophage 
 27 nm in diameter and is widely used as a surrogate for enteric 
 viruses in environmental studies . phi6 ( kindly provided by p. turner 
 of yale university , new haven , ct ) is an icosahedral , double - stranded 
 rna bacteriophage 85 nm in diameter and has been proposed 
 as a surrogate for ebola virus , although 
 the structures of the two viruses differ ( i.e. , roughly spherical 
 vs filamentous ) . table s1 of the supporting information compares the structure 
 of ebola virus and the two surrogates . we propagated the bacteriophages 
 using host bacteria , escherichia coli and pseudomonas syringae , and standard culture methods . concentrations 
 in stock suspensions were 1010 plaque - forming 
 units per milliliter ( pfu ml ) . we tested 
 four bowl - shaped materials commonly used in wastewater 
 systems : porcelain , pvc , polypropylene , and concrete . the supporting information provides further details 
 about the containers used in this study . as a surrogate for 
 diarrhea , we tested both synthetic sludge and 
 real sludge collected from a wastewater treatment plant . we considered 
 both types of sludge to balance a well - defined composition of the 
 fluid against real - world conditions . for synthetic sludge we used a strain of bacillus and e. coli isolated from anaerobically digested 
 sludge as model gram - positive and gram - negative microorganisms of 
 enteric relevance , rather than yeast . for real sludge , we collected 
 anaerobically digested sludge from a wastewater treatment plant whose 
 flow is dominated by domestic sources ( > 99% ) . we stored the sludge 
 at 4 c and used it within a few weeks of collection to ensure 
 a robust population of microorganisms . we did not sterilize the sludge out of concern that doing so would 
 alter its properties and affect partitioning results . we seeded both 
 types of sludge with ms2 or phi6 to achieve a final bacteriophage 
 concentration of 10 pfu ml . we conducted experiments in triplicate for each combination of 
 bacteriophage , material , and type of sludge ( 2  4  2 = 
 16 combinations ) , as illustrated in figure s1 . for each replicate , we filled a container with 10 ml of sludge 
 seeded with bacteriophage and 40 ml of autoclaved , ultrapure water 
 to achieve a dilution similar to what might occur with diarrhea in 
 a toilet . after 510 min , we transferred the fluid , including 
 suspended solids , to a sterile , 50 ml tube and centrifuged it at 1700 
 rcf for 3 min . we poured excess fluid off the pelleted solids , briefly 
 vortexed them , and denoted these as the  solid  fraction . following established methods for recovery of viruses from surfaces , we collected the  surface  fraction using three sterile 
 cotton swabs ( puritan 22029488 ) in series , each premoistened with 
 sterile lb broth . we swabbed systematically and with constant pressure 
 over the entire exposed surface area and subsequently eluted virus 
 from the three swabs into 1 ml of lb broth by compressing and swirling 
 the swab in a sample tube . the swabbed surface area was 115 
 cm ( 10% ) for all materials . to prepare samples 
 for analysis by quantitative polymerase chain 
 reaction ( qpcr ) , we first converted rna to cdna . immediately upon 
 separating the fractions , we extracted rna from 140 l subsamples 
 of each fraction using a qiagen qiaamp viral rna kit . for calibration 
 standards , we also extracted viral rna from serially diluted stock 
 suspensions of each bacteriophage , whose concentration ( pfu ml ) was determined by a plaque assay , and carried it 
 through the entire preparation and analysis process . we omitted carrier rna from the extraction processes to increase 
 rna extraction efficiency , and we subjected phi6 rna to a postextraction 
 heat shock treatment ( 110 c , 5 min ) . we immediately synthesized cdna using a bio - rad iscript cdna synthesis 
 kit with 5 l of template rna , 4 l of iscript 5x , 1 l 
 of reverse transcriptase , and 10 l of nuclease - free water , 
 using the kit s recommended thermocycler protocol . we used 
 qpcr to quantify virus concentration in each fraction and 
 in the calibration standards in terms of pfu equivalents per milliliter . we analyzed fractions from synthetic sludge in triplicate ( experimental 
 replicates ) by intercalating dye - based qpcr using bio - rad sybr green 
 mastermix with 5 l of sybr green , 400 nm forward and reverse 
 primers , 2.4 l of nuclease - free water , and 
 1 l of cdna template . we analyzed fractions from real sludge 
 in triplicate ( experimental replicates ) by probe - based qpcr using 
 bio - rad iq supermix with 5 l of iq supermix , 400 nm forward 
 and reverse primers , 300 nm probe , 1.72 
 l of nuclease - free water , and 1 l of cdna template . table s3 shows the primers and probes , and table s4 lists the qpcr conditions . for 
 quality control , we employed serial dilutions of genomically 
 sequenced calibration standards , triplicate reactions , negative template 
 controls , and melt curve analysis . the calibration curve covered a 
 minimum of 6 orders of magnitude to equate qpcr amplification of genomic 
 copies to the concentration determined by a plaque assay in standards . we prepared serial dilutions of cdna and analyzed all 
 standards and samples in triplicate ( technical replicates ) on the 
 qpcr machine ( bio - rad cfx96 real - time system and c1000 thermal cycler ) . additional controls are 
 described in the supporting information . of the 144 individual experimental replicates , we discarded four 
 that failed to amplify . we reviewed preliminary qpcr results using 
 the machine s software to verify the calibration curve and 
 melt curve , including quantification cycle results . melt curve analysis 
 showed a single , well - defined peak at the expected melt temperature 
 for the genome . we spiked 
 ms2 , an unenveloped bacteriophage , and phi6 , an enveloped 
 bacteriophage , into diluted synthetic and real sludge in porcelain , 
 concrete , pvc , and polypropylene containers and measured partitioning 
 among the liquid , solids , and the interior surface of the container . figures 1 and 2 show the concentrations of ms2 and phi6 , respectively , in 
 terms of pfu equivalents in three experimental replicates of each 
 fraction derived from diluted synthetic sludge . the three fractions 
 were 46 ml of liquid , 4 ml of wetted solids , and 1 ml of surface extract . ms2 and phi6 partitioned similarly in synthetic sludge ; concentrations 
 were highest in the liquid and solid phases for all materials tested , 
 with the exception of phi6 in porcelain , where phi6 concentrations 
 were comparable in all three fractions . mean concentrations of ms2 
 in each fraction were within 1 order of magnitude of each other , while 
 mean concentrations of phi6 varied by up to 2 orders of magnitude . background concentrations of genomic material were detectable in both 
 types of sludge , particularly for phi6 , but were at least 4 orders 
 of magnitude lower than in the seeded sludge . concentration of ms2 
 bacteriophage , determined by qpcr and reported 
 in pfu equivalents per milliliter , in synthetic sludge diluted 5-fold 
 in water in each of three phases : 46 ml of liquid , 4 ml of wetted 
 solids , and 1 ml of material surface extract for porcelain , concrete , 
 pvc , and polypropylene . concentration of phi6 bacteriophage , determined 
 by qpcr and reported 
 in pfu equivalents per milliliter , in synthetic sludge diluted 5-fold 
 in water in each of three phases : 46 ml of liquid , 4 ml of wetted 
 solids , and 1 ml of material surface extract for porcelain , concrete , 
 pvc , and polypropylene . figures 3 and 4 show the concentrations 
 of ms2 and phi6 , respectively , 
 in terms of pfu equivalents in three experimental replicates of each 
 fraction derived from diluted , real , anaerobically digested sludge . the variability was much larger in real sludge than in synthetic sludge , 
 likely due to heterogeneities in the real sludge . the average concentration 
 of ms2 in the solid fraction was 2 orders of magnitude lower 
 than in the other fractions for all materials except concrete . average 
 concentrations of ms2 were highest in the liquid fraction for concrete , 
 porcelain , and pvc ( figure 3 ) . in polypropylene , the average concentrations of ms2 in the liquid 
 and surface extract from concrete were of a similar order of magnitude ; 
 however , the surface samples exhibited much larger variability . average 
 concentrations of phi6 in real , anaerobically digested sludge were 
 highest in the liquid fraction for all materials . average concentrations 
 were 12 orders of magnitude lower in the surface extract 
 and were 24 orders of magnitude lower in the wetted solids 
 ( figure 4 ) . concentration 
 of ms2 bacteriophage , determined by qpcr and reported 
 in pfu equivalents per milliliter , in anaerobically digested sludge 
 diluted 5-fold in water in each of three phases : 46 ml of liquid , 
 4 ml of wetted solids , and 1 ml of material surface extract for porcelain , 
 concrete , pvc , and polypropylene . concentration of phi6 bacteriophage , determined 
 by qpcr and reported 
 in pfu equivalents per milliliter , in anaerobically digested sludge 
 diluted 5-fold in water in each of three phases : 46 ml of liquid , 
 4 ml of wetted solids , and 1 ml of material surface extract for porcelain , 
 concrete , pvc , and polypropylene . each point represents an experimental 
 replicate , and there are three nearly overlapping points for solids 
 in concrete . a mass balance indicated 
 that the sum of virions recovered ( pfus ) 
 from all three fractions was roughly within 1 order of magnitude of 
 the number of virions initially spiked into the samples ( table s5 ) , so the amount of virus recovered was 
 close to the amount spiked into the samples . one exception was phi6 
 in real sludge , for which the total amount of virus recovered was 
 23 orders of magnitude higher , on average , than the number 
 of virions spiked into the samples for all four material types . a 
 negative control consisting of sludge that was not spiked with virus 
 failed to amplify , it appears that phi6 replicated rapidly in host bacteria present in 
 the sludge during the experiment , and thus , the partitioning results 
 represent both spiked and new virus . figure s2 shows the partitioning of 
 ms2 and phi6 among the three fractions for synthetic and real sludge 
 and all four materials . in all cases , at least 94% of the virions 
 partitioned into the liquid fraction . in synthetic sludge , up to 4% 
 of virions partitioned to the solids , while in real sludge , no more 
 than 0.8% of virions partitioned to the solids . in synthetic sludge , 
 partitioning to the material surface was low , at most 1% except for 
 phi6 in porcelain , where 6% of virions partitioned to the surface . 
 in real sludge , partitioning to the material surface was also low , 
 although both ms2 and phi6 partitioned more to the surface of concrete 
 and polypropylene than to the surface of porcelain and pvc this study demonstrates that partitioning of viruses in wastewater 
 among the liquid , biosolids , and material surface does not mitigate 
 the potential for aerosolization of virus , as most of the virus remains 
 in the liquid phase . airborne viruses have been detected at wastewater 
 treatment facilities at concentrations as high as 3  10 genome copies m for adenovirus . previous studies have shown that virus removal efficiency in wastewater 
 treatment plants ranges widely from 0 to 4 logs . in contrast 
 to our results , some of these studies have shown that viruses adsorb 
 well to solids . our results are consistent with the general finding 
 that the extent of removal of bacteria is greater than the extent 
 of removal of virus in conventional treatment plants . with respect 
 to concerns about transmission of ebola virus , one 
 limitation of this study is that the surrogate viruses may not be 
 representative of ebola virus . its filamentous shape may make it more 
 or less prone to adsorb to solids and surfaces . another limitation 
 is that the time allowed for partitioning was only 510 min , 
 which could be representative of the time excreta remains in a toilet 
 before flushing but is much shorter than the residence time in sewers 
 and at wastewater treatment plants . we have identified five 
 potential sources of uncertainty in quantification 
 of virus concentrations . although our qpcr calibration curves had 
 high r values , the differing matrices 
 of the three fractions may have produced varying extraction and amplification 
 efficiencies . it is possible that genomic material from inactivated 
 virus and/or exogenous genomic material was counted as pfu equivalents . inhibitors are a challenge for any pcr analysis and if severe will 
 result in false negatives or underestimates of gene copy levels . in 
 this study , we expect that any effects of inhibitors would have been 
 minimal , as we were operating at the mid to upper end of the calibration 
 curve and did not have difficulty detecting virus in any of the samples . 
 also , the mass balances did not suggest a major loss of virus . because 
 of requirements of the experimental approach , the surface fraction 
 also included any virions present in the residual liquid that remained 
 in the bowls after pouring out the contents ; allowing the bowls to 
 dry out completely prior to swabbing previous studies have shown that the swabbing method recovers 
 758% of viruses from dry environmental surfaces , depending 
 on the type of swab , virus , surface material , and virus assay ( culture 
 vs molecular ) . thus , there is the possibility 
 for both overestimation and underestimation of the amount of virus 
 that partitioned to porcelain , concrete , pvc , and polypropylene in 
 this study . the mass balance ( table s5 ) 
 suggests that large amounts of virus were not lost during the experiment , 
 but even if the actual numbers of virions on surfaces were 10 times 
 higher , the majority of virions would still be associated with the 
 liquid phase .
to gain insight into the potential 
 for aerosolization of viruses 
 in wastewater systems , we investigated the partitioning of ms2 and 
 phi6 bacteriophages in synthetic sludge and anaerobically digested 
 sludge from a wastewater treatment plant . 
 we evaluated partitioning 
 among the liquid , solids , and material surfaces of porcelain , concrete , 
 polyvinyl chloride ( pvc ) , and polypropylene . in all cases , at least 
 94% of the virions partitioned into the liquid fraction . in real sludge , 
 no more than 0.8% of virions partitioned to the solids and no more 
 than 6% to the material surface . 
 both ms2 and phi6 partitioned more 
 to the surface of concrete and polypropylene than to the surface of 
 porcelain or pvc . 
 partitioning of viruses in wastewater among the 
 liquid , biosolids , and material surface does not appear to mitigate 
 the potential for aerosolization of virus , as most of the virus remains 
 in the liquid phase .