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we tested the stability of ebov on 3 materials commonly found in an ebola treatment unit ( etu ) in west africa : 1 ) utility - grade ( 308 ) stainless steel washers ( mcmaster - carr , atlanta , ga , usa ) ; 2 ) plastic ( teflon [ polytetrafluoroethylene ] ; mcmaster - carr ) ; and 3 ) tyvek ( from the front of a coverall ) .
for each time point , 3 disks ( 4-cm diameter ) of each material were placed individually into wells of a 6-well plate .
five samples ( 10 l / sample ) containing a total dose of 10 50% tissue culture infectious doses ( tcid50s ) of ebov in cell - free medium were evenly distributed on the disks .
the plates were divided into groups , and each group was placed into a plastic hepa - filtered box and placed at 21c , 40% rh , or 27c , 80% rh .
the samples were dried naturally , and virus titers were determined over a 14-day period . in the surface and fluid stability experiments , all samples were stored at 80c until titration ( 1 freeze  thaw cycle of ebov samples that did not change virus titer ) .
the tcid50 per milliliter for each sample at each time point was calculated by using the spearman - karber method ( 11 ) . because viral decay rates often exhibit first - order kinetics ( 12 )
, we log10 transformed our tcid50 calculations to represent virus titer and used a linear regression analysis ( prism version 6.05 ; graphpad , san diego , ca , usa ) to determine the log10 reduction rate of ebov on each surface at both environmental conditions ( figure 1 ; table 1 ) .
we also determined whether linear regression models were significantly different from each other at the p<0.05 level by using an analysis of covariance equivalent test in prism .
linear regression model showing the effect of different environmental conditions and surfaces on survival of ebola virus ( ebov ) .
virus was dried on 3 surfaces found in outbreak settings at a ) 27c , 80% relative humidity ( rh ) ( west african tropical conditions ) and b ) 21c , 40% rh ( climate - controlled hospital conditions ) .
virus concentration was reduced at a significantly slower rate on all surfaces in hospital conditions than in tropical conditions ( p<0.0001 for all surfaces ) .
an analysis of covariance equivalent test was used to compare linear regression models and determine differences in virus reduction rates .
tcid50 , 50% tissue culture infectious dose .   * na , not applicable . 
y , log10 50% tissue culture infectious dose / ml ; x , days . 
in hospital conditions , virus titer on steel was reduced significantly faster than on plastic ( p = 0.004 ) and on tyvek ( p<0.0001 ) , but there was no significant difference in reduction between tyvek and plastic ( p = 0.13 ) . in tropical conditions , there was no significant difference in virus titer reduction on steel and on plastic ( p = 0.78 )
. however , virus decayed more slowly on tyvek than on steel ( p<0.0001 ) and on plastic ( p<0.0001 ) .
there was no significant difference in reduction rate in virus titer in drying human blood in hospital or tropical conditions ( p = 0.92 ) .
virus was reduced significantly faster at 27c than in water at 21c ( p = 0.0001 ) .
the stability of ebov in water was assessed by diluting 150 l virus stock in 2.85 ml of ambion diethylpyrocarbonate  treated water ( thermo fisher scientific , pittsburgh , pa , usa ) and removing residual protein and medium with 1 initial and 2 rinse spins on amicon ultra centrifugal filters 100k mwco ( merck , darmstadt , germany ) .
ebov was more stable in water at 21c and had an 1 log10 reduction / day in water at 27c ( table 1 ; figure 2 , panel a ) .
linear regression model showing stability of ebola virus ( ebov ) in fluids under different environmental conditions .
virus concentration was reduced at a significantly faster rate in 27c water than in 21c water ( p = 0.0001 ) .
b ) stability in drying or liquid ebov - spiked human blood samples at 2 environmental conditions .
virus concentration was reduced at a significantly faster rate by drying than in liquid blood at both conditions ( p<0.0001 for each condition ) .
no significant difference between reduction rates in virus titer in drying human blood at both conditions was found ( p = 0.92 ) .
error bars indicate mean  sem virus titer . dashed line indicates the limit of detection for the assay .
an analysis of covariance equivalent test was used to compare linear regression models and determine differences in virus reduction rates .
the stability of ebov in human blood was assessed by spiking blood samples from a healthy human volunteer to achieve a 10 tcid50/ml virus titer .
the spiked blood was distributed in 1-ml aliquots into closed screw - top vials to maintain a liquid state , spread in 50-l aliquots onto the bottom of a 24-well plate , and dried .
one group of samples was stored at 21c , 40% rh , and the other group was stored at 27c , 80% rh .
ebov stability in drying blood exhibited first - order kinetics and was viable for up to 6 days at tropical conditions ( table 1 ; figure 2 , panel b ) . to approximate the stability of ebov in naturally infected human blood
blood was collected during necropsy from 3 macaques that were previously enrolled in an animal care and use committee  approved ebov pathogenesis study and were euthanized because they exhibited signs of evd and viremia .
blood samples were divided into 2 groups with 2 sets of 150-l aliquots for each time point ; each group was stored at the conditions described above with each set in the liquid or drying state , and virus viability was assessed over a 14-day period . because of variation in calculated virus titer from each of the individual nhps , the log10 reduction rate could not be approximated and only the initial titer and the duration of viability are shown ( table 2 ) . in general , ebov maintained viability for a longer duration in liquid than in drying blood regardless of initial titer or environmental condition .
* tcid50 , 50% tissue culture infectious dose ; nhp , nonhuman primate ; rh , relative humidity .
we found that ebov can persist on surfaces common in an etu , highlighting the need for adherence to thorough disinfection and doffing protocols when exiting the etus and careful handling of medical waste .
in addition , ebov maintains viability for a longer duration in liquid than in dried blood .
ebov in blood of experimentally infected nhps persists for a similar duration as ebov in spiked human blood .
a recent study showed that blood in the body cavity of an nhp contained viable ebov for up to 7 days after death ( 13 ) .
we detected viable ebov in drying blood for up to 5 days at both environmental conditions in human and nhp blood .
therefore , dried and liquid blood from an infected person in their home or etu should be treated as potentially infectious .
the finding that ebov remains viable in water for as long as 3 ( 27c ) or 6 ( 21c ) days at the experimental concentration warrants further investigation into the persistence of the virus in aqueous environments , such as in wastewater or sewage canals .
viable ebov has been isolated from urine ( 14 ) but not from human stool ( 8) .
first , because standard volumes for samples were used , different volumes or matrices could influence the stability of ebov under the tested conditions .
second , blood samples from the nhps might have different immunologic or biochemical conditions , which can potentially influence virus stability .
third , the experimental conditions in the laboratory are sterile , but in disease - endemic areas and etus , bacteria or chemicals could influence ebov viability .
overall , we found that different environmental conditions , fluids , and surfaces influence the persistence of ebov .
these findings demonstrate that such factors are crucial in understanding transmission and improving safety practices .
we evaluated the stability of ebola virus on surfaces and in fluids under simulated environmental conditions for the climate of west africa and for climate - controlled hospitals . 
 this virus remains viable for a longer duration on surfaces in hospital conditions than in african conditions and in liquid than in dried blood .