in march 2014 , an unprecedented 
 outbreak of ebola virus disease 
 ( evd ) began in western africa that as of july 2015 is ongoing and 
 has claimed more than 11000 lives .
the 
 current outbreak is caused by viruses belonging to the species of 
 the zaire ebolavirus , a member of the filoviridae family .
filoviridae are enveloped , filamentous viruses with lengths that 
 may reach > 1000 nm .
ebola virus 
 can be excreted in bodily fluids , including vomit , stool , blood , saliva , 
 semen , and breast milk .
ebola virus loads of up to 10 genome 
 copies ml have been reported in blood , 10 genome copies ml in stool , and 10 genome copies ml in urine ; however , the conversion between genome copies 
 and infectious units is unknown .
once infected , individuals may produce up to 9 l of liquid waste 
 per day , primarily watery diarrhea .
ebola virus is considered a potential bioterrorism 
 agent . in response to the evd epidemic , both 
 the world health organization 
 ( who ) and the u.s .
centers for disease control and prevention advised 
 direct disposal of ebola - contaminated liquid waste into sewage systems 
 ( wastewater collection and treatment systems ) and latrines without 
 disinfection .
initial recommendations 
 were made on the basis of an expected limited persistence of ebola 
 virus in the environment , as ebola virus is an enveloped virus , and 
 a lack of strong evidence for a waterborne transmission route . as 
 stated by a who guidance document , 
ebola virus is likely to 
 inactivate significantly faster in the environment than enteric viruses 
 with known waterborne transmission ( e.g. , norovirus , hepatitis a virus )  .
however , as has been noted in a recent review , 
 the persistence of enveloped viruses in the water environment varies 
 by > 2 orders of magnitude .
recommendations 
 for ebola virus - contaminated wastewater disposal were met with debate 
 ( e.g. , refs ( 1618 ) ) because of uncertainty 
 about ebola virus persistence within wastewater matrices and the lack 
 of a risk - based analysis for waste handling .
wastewater handling recommendations 
 have since been revised to recognize uncertainty in this area and 
 to recommend disinfection of latrines and holding of wastewater prior 
 to handling to allow ebola virus inactivation .
additionally , 
 some facilities have opted to provide additional disinfection prior 
 to disposal of liquid waste into sewer systems .
recent research found both ethanol and hypochlorite to 
 be effective disinfectants for ebola virus dried on surfaces ; however , the disinfection kinetics of ebola 
 virus within liquid matrices remains unknown .
various wastewater disinfection 
 approaches have been recently suggested for pathogen control in an 
 outbreak setting .
currently , no 
 data on ebola virus persistence in wastewater exist , 
 hindering risk estimation and examination of potential environmental 
 exposure routes .
the necessity of evaluating ebola virus persistence 
 in wastewater matrices has previously been highlighted , as wastewater 
 in ebola virus outbreak settings may be temporarily held in open containers 
 or disposed of in open sewers .
historically , 
 the transmission of ebola virus via environmental routes ( droplets , 
 aerosols , or fomites ) has been thought to be unlikely due to epidemiological 
 evidence and environmental sampling .
the 
 primary ebola virus transmission route is via direct contact with 
 bodily fluids .
transmission has previously 
 occurred without known direct contact with infected individuals , providing supporting evidence that ebola virus 
 transmission may be possible via large droplets .
the potential for transmission of ebola virus via wastewater 
 is currently unknown . to address uncertainties regarding ebola 
 virus persistence in wastewater
, 
 we have conducted an initial evaluation of ebola virus persistence 
 within wastewater to address uncertainty and inform ongoing risk assessments . 
 a current ebola virus outbreak strain from guinea ( makona - wpgc07 ) 

was spiked to two end concentrations ( 10 and 10 tcid50 ml ) into a domestic wastewater 
 ( untreated sewage ) sample .
the ebola virus - containing wastewater was 
 sampled for 8 days , and the viability of ebola virus was determined 
 in these samples .
study results are presented , and study limitations 
 and implications are discussed , as well as recommendations for an 
 ongoing research agenda .
untreated wastewater was 
 collected from an anonymous regional wastewater 
 treatment facility in western pennsylvania that receives wastewater 
 from seven communities ( combined population of approximately 60000 ) . 

the total raw wastewater flow to the treatment facility is < 10 
 mgd ( million gallons per day ) .
the wastewater 
 was frozen at 80 c to minimize compositional changes 
 prior to analysis .
wastewater characteristics ( table 1 ) were determined at an epa - certified analysis 
 facility ( microbac laboratories , marietta , oh ) .
the region from which 
 the sample was collected uses a combined sewer system that experiences 
 significant infiltration , and the determined composition is typical 
 for the region .
ebola virus cultivation experiments 
 were conducted at rocky mountain 
 laboratories under bsl4 conditions .
wastewater samples were shipped 
 to rocky mountain laboratories overnight on ice . upon receipt , samples 
 were sterilized with 5 mrad of -irradiation .
sterilization 
 was performed to limit cell culture death due to wastewater microbial 
 activity leading to a false positive .
stock virus ( ebola virus guinea 
 makona - wpgc07 , 10 tcid50 ml ) was handled as described previously .
ebola virus was diluted in wastewater to achieve 
 two separate virus titers ( 10 and 10 tcid50 ml ) , and both experiments were completed 
 in triplicate .
the ebola virus concentration in sewage has not been 
 previously measured or estimated ; thus , two separate concentrations 
 were utilized to cover possible concentration scenarios .
tcid50 is an end point dilution series that is used to determine 
 at what dilution 50% of the infected wells produce cell death .
an approximation of focus - forming 
 units ( ffus ) can be made from a poisson distribution utilizing the 
 formula tcid50  0.69 , assuming each ffu is formed 
 from a single virus .
spiked wastewater was then distributed into three 
 labeled vials for each concentration , and samples were taken daily 
 for 8 days .
tests were conducted at 20 c and 40% relative humidity . 
 at each time point , including the time zero measurement , 50 l 
 of wastewater from the bulk wastewater vial was added into 450 l 
 of dulbecco s modified eagle s medium ( dmem , sigma ) 
 supplemented with heat - inactivated fetal bovine serum ( fbs , gibco ) 
 to a final concentration of 2% , pen / strep ( gibco ) to a final concentration 
 of 50 units / ml penicillin and 50 g / ml streptomycin , and l - glutamine ( gibco ) to a final concentration of 2 mm in an appropriately 
 labeled 2 ml screw top vial and frozen at 80 c .
negative 
 controls were 50 l of nonspiked wastewater in 450 l 
 of dmem . to perform the titrations ,
a 96-well 1.1 ml well dilution 
 plate ( axygen , corning , ny ) was prepared . the thawed sample ( 400 l ) 

was placed in the top row of the plate and a 10-fold dilution conducted 
 by passing 40 l of sample into 360 l of dmem .
next , 
 100 l from each well of the dilution plate was transferred 
 to a 96-well cell culture plate seeded with vero cells .
the cells 
 were incubated with virus dilutions for 1 h ; then the medium was removed 
 from the two highest concentrations and rinsed two times with pbs , 
 and 200 l of fresh culture medium was added .
fresh culture 
 medium ( 100 l ) was also added to the remaining wells in the 
 plate .
the plates were incubated at 37 c for 7 days , inspected 
 for the cytopathic effect ( cpe ) , and scored .
the natural 
 logarithm of c / co tcid50 was plotted and fit with a linear trendline for estimation 
 of the inactivation constant ( k ) .
a literature review 
 was then performed to compare observed inactivation to other environmental 
 matrices .
ebola virus was 
 spiked into wastewater at two concentrations and assayed for 8 days 
 to determine persistence in a wastewater matrix .
the time zero time 
 point was measured immediately following addition of ebola virus to 
 the wastewater .
no viable ebola virus was recovered from samples spiked 
 with 10 ebola virus tcid50 ml after the initial time zero sampling .
virus 
 viabilities from the initial 10 ebola virus tcid50 ml concentration are shown in figure 1 and detailed in table s1 .
ebola virus titer was rapidly reduced ( approximately 
 99% ) within the first day of the test , consistent with an inability 
 to identify infectious ebola virus from the initial sample with 10 ebola virus tcid50 ml on day 
 1 .
persistence of an initial ebola virus concentration of 10 tcid50 ml in domestic wastewater 
 ( untreated sewage ) ( a ) including the time zero time point and ( b ) 
 excluding the time zero time point to mitigate potential aggregation 
 effects .
fit inactivation constants ( k ) were determined to be 1.08 when including time 
 zero and 0.35 when excluding time zero .
error bars are 1 
 standard deviation . there was a rapid decrease 
 ( approximately 99% ) in ebola virus titer 
 within the first day of the test .
in addition to inactivation , viral 
 particle aggregation or adsorption to wastewater particles may play 
 a role in the apparent rapid viral decrease and enhanced viral persistence . 
 in this case
first , aggregated 
 particles would not be detected as multiple infectious units , resulting 
 in an apparent rapid decrease .
second , it has been previously recognized 
 that aggregation increases viral persistence and resistance to inactivation 
 stressors .
similarly , viral association with particles 
 has been recognized to provide protection from inactivation , including association of viral particles with 
 wastewater solids .
mechanistically , particle 
 association is believed to protect viral particles from inactivation 
 by shielding them from environmental stressors and is dependent on 
 the organism and particle type . utilizing the current assay , it can not be determined if the initial 
 rapid decrease in viral titer was due to viral inactivation or aggregation . 

aggregation would result in an apparent viral titer decrease as each 
 viral aggregate would function as an infectious unit in the cell culture 
 assay . to address this uncertainty , we plotted two inactivation curves , 
 both including ( figure 1a ) and excluding the measured time zero point ( figure 1b ) .
a linear trendline , as has been previously 
 suggested for viral and ebola virus persistence , showed a lower fit ( r = 0.59 including the time zero time point , and r = 0.67 excluding the time zero time point ) 
 than that previously observed for ebola virus in deionized water ( r > 0.91 ) , but a fit better than that previously 
 observed for ebola virus in human blood ( r < 0.29 ) .
the inactivation constant ( k ) was determined 
 to be 1.08 when including the time zero time point and 0.35 
 when excluding the time zero time point .
on the basis of the model 
 fit , the t90 ( time for 90% inactivation ) 
 would be 2.1 days including the time zero time point and 6.6 days 
 excluding the time zero time point .
the observed ebola virus inactivation 
 in wastewater was slower than that observed for deionized water , which 
 required 1.8 days for 90% inactivation at 21 c .
the observed ebola virus inactivation in wastewater was 
 more rapid than that reported for human blood , which required 20 days 
 for a 90% inactivation , and results are 
 consistent with recent studies that identified viable ebola virus 
 to persist in infected macaque blood for > 8 days . in general , ebola virus was found to be less persistent 
 in wastewater than model enteric viruses . while the t90 for ebola virus in wastewater was found to be < 1 
 day , the t90 for hepatitis a is greater 
 than 17 days and the t90 for enteric adenovirus is 33 days ; however , the t90 for poliovirus 
 is 5 days , which is between the observed t90 values including or excluding the time zero 
 time point .
the results demonstrate a more rapid initial viral titer 
 decrease but overall enhanced persistence of ebola virus in wastewater 
 compared to the proposed enveloped surrogate bacteriophage phi6 .
this 
 study has two primary limitations 
 that may alter the persistence of ebola virus compared to what may 
 be observed in the field .
first , the tested wastewater was more dilute 
 than would be expected in typical latrine waste . in general , interaction with constituents within the wastewater 
 ( e.g. , ammonia ) would be expected to contribute to more rapid inactivation 
 of viruses ; however , the true effect 
 of these constituents on ebola virus persistence is unknown .
second , 
 the wastewater was frozen to minimize compositional changes and disinfected 
 ( -irradiation ) prior to utilization to limit microbial activity 
 resulting in false positive viral cell culture . microbial activity 
 within wastewater matrices would be expected to contribute to more 
 rapid inactivation of infectious viral particles ; however , the true effect of microbial activity on ebola virus persistence 
 is unknown .
microbial activity reduces viral persistence through both 
 the production of metabolites detrimental to viral persistence and 
 direct usage of the viral particles as a nutrient source .
additionally , the influence of other environmental 
 characteristics ( e.g. , temperature , ph , and mixing ) on ebola virus 
 persistence is unknown and may contribute to altered environmental 
 behavior . as such
, we believe these results to be an upper bound for 
 ebola virus persistence in wastewater matrices .
subsequently , we caution 
 extrapolation of these results without a holistic assessment of all 
 factors , including wastewater composition , dilution , and potential 
 exposure routes .
the results of this 
 study suggest a potential 
 exposure route to infectious ebola virus via wastewater ; however , 
 any assessment of potential exposure routes must consider the effects 
 of wastewater composition , dilution of contaminated wastewater , and 
 inactivation of ebola virus during treatment and holding .
additionally , 
 the possibility for ebola virus transmission via wastewater and subsequent 
 infection remains unknown .
the who updated guidelines in january 2015 
 to recommend holding of latrine waste for 1 week prior to further 
 handling or transport .
the objective 
 of this holding period is to allow ebola virus die - off . on the basis 
 of these results
, it would be reasonable to approximate a three - log 
 ( i.e. , 99.9% ) removal of ebola virus due to this holding period . the 
 resulting risk would ultimately depend upon the initial ebola virus 
 concentration in wastewater , potential for exposure , and susceptibility 
 to infection via wastewater exposure .
the wastewater travel times 
 for wastewater via a sewer system to a centralized sewage treatment 
 works would typically be < 1 day , depending on system dynamics .
further assessment is necessary to determine 
 ebola inactivation and dilution within this period and potential human 
 exposure routes , including workers within the sewer system and ebola 
 virus persistence within wastewater sludges .
the greatest exposure 
 risk would be expected for persons in contact with contaminated wastewater 
 prior to significant dilution , treatment , or holding .
these 
 results demonstrate a persistence of ebola virus in wastewater greater 
 than what has previously been suggested and the potential of a wastewater 
 exposure route to infectious ebola virus .
while ebola virus was found 
 to be generally less persistent than enteric viruses in wastewater , 
 the identified survival period suggests value in a nuanced evaluation 
 of wastewater exposure risks during an epidemic response .
specifically , 
 these findings highlight the value of a precautionary approach to 
 wastewater handling within an outbreak scenario , in response to both 
 ebola virus and other emerging viruses .
 in the wake of the ongoing 2014/2015 
 ebola virus outbreak , significant 
 questions regarding the appropriate handling of ebola virus - contaminated 
 liquid waste remain , including the persistence of ebola virus in wastewater . 
 
 to address these uncertainties , we evaluated the persistence of ebola 
 virus spiked in sterilized domestic sewage . 
 the viral titer decreased 
 approximately 99% within the first test day from an initial viral 
 titer of 106 tcid50 ml1 ; 
 however , it could not be determined if this initial rapid decrease 
 was due to aggregation or inactivation of the viral particles . 
 the 
 subsequent viral titer decrease was less rapid , and infectious ebola 
 virus particles persisted for all 8 days of the test . 
 the inactivation 
 constant ( k ) was determined to be 1.08 ( 2.1 
 days for a 90% viral titer decrease ) . 
 due to experimental conditions , 
 we believe these results to be an upper bound for ebola virus persistence 
 in wastewater . 
 wastewater composition is inherently heterogeneous ; 
 subsequently , we caution that interpretation of these results should 
 be made within a holistic assessment , including the effects of wastewater 
 composition , dilution , and potential exposure routes within wastewater 
 infrastructure . 
 while it remains unknown if ebola virus may be transmitted 
 via wastewater , these data demonstrate a potential exposure route 
 to infectious ebola virus via wastewater and emphasize the value of 
 a precautionary approach to wastewater handling in an epidemic response . 
Introduction
Methods
Results and Discussion