Journal of Virological Methods 237 (2016) 150153

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Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

A one-step centrifugal ultraltration method to concentrate enteric

viruses from wastewater
Yuanyuan Qiu a, , Bonita E. Lee b , Norma J. Ruecker c , Norman Neumann d,e ,
Nicholas Ashbolt d , Xiaoli Pang a,e
a
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada
b
Department of Pediatrics, University of Alberta, Edmonton, Canada
c
City of Calgary, Water Resources, Calgary, Canada
d
School of Public Health, University of Alberta, Edmonton, Canada
e
Provincial Laboratory for Public Health, Edmonton, Canada

a b s t r a c t

Article history: A one-step centrifugal ultraltration method was developed to enhance rapid detection of human enteric
Received 22 August 2016 viruses and co-occurring viruses in wastewater. Samples were collected pre- and post-UV treatment at
Received in revised form two full-scale tertiary municipal wastewater treatment plants in Calgary, Canada. Viruses were con-
10 September 2016
centrated from 100 mL wastewater samples through direct centrifugation using the Centricon Plus-70
Accepted 10 September 2016
Available online 11 September 2016
ultralter. Seven viruses, including norovirus, rotavirus, sapovirus, astrovirus, enterovirus, adenovirus
and JC virus, were tested using real-time quantitative PCR (rt-qPCR) and cell culture. All of the viruses
were detected in pre- and post-UV samples by rt-qPCR, with rotavirus the most numerous (6.6 log10
Keywords:
Ultraltration GE copies/L). Infectious viruses, by cell culture, were found in all tested pre-UV samples but only in one
Enteric viruses post-UV sample. The results were comparable and consistent to that obtained using virus adsorption-
Wastewater elution method, indicating that the centrifugal ultraltration method is adequate to retain the viruses
Real-time PCR and maintain their infectivity during processing. As a simple, rapid and cost-effective method to screen
Cell culture wastewater viruses, this one-step centrifugal ultraltration method may serve as an effective approach
to assess virus removal and gain knowledge of human virus activity during wastewater treatment.
2016 Elsevier B.V. All rights reserved.

Human viruses shed via the gastrointestinal and urinary tracts
are frequently detected in raw sewage and treated wastewater.
Some of these viruses are major waterborne pathogens (Bosch et al.,
2008; Qiu et al., 2015). Classic enteric viruses include norovirus
(NoV), rotavirus (RV), sapovirus (SaV), astrovirus (AsV), and ade-
novirus (AdV) (Fong and Lipp, 2005). Due to their low infectious
dose, these viruses are very contagious, especially NoV, which is a
major cause of global gastroenteritis (Ahmed et al., 2014). Further-
more, the U.S. EPA (U.S. Environmental Protection Agency, 2012)
has recognized enteric viruses as the likely major hazard group
in sewage-polluted receiving waters, and is seeking appropriate
methods to assess the efcacy of virus removal during wastewater
treatment to reduce environmental pollution.
Due to the typically low virus levels in water matrices, the rst
and most critical step to detect virus in environmental water is to
Corresponding author at: Department of Laboratory Medicine and Pathology,
University of Alberta, WMC 2B2.21, 8440-112 Street, Edmonton, AB, T6G 2J2, Canada.
E-mail address: yuanyuan@ualberta.ca (Y. Qiu).
concentrate virus to enhance detection. Currently, the most com-
mon concentration method is virus adsorption-elution (VIRADEL)
using electropositive or electronegative microporous membrane
lters, such as Virosorb 1MDS and NanoCeram lters (Ikner et al.,
2012; Pang et al., 2012; Cashdollar and Wymer, 2013). The pro-
cess involves the absorption of viral particles to the membrane
through ionic interaction followed by elution with pH adjustment.
However, VIRADEL is time consuming, labor intensive and needs
large sample volume that may require a secondary concentra-
tion procedure to reduce the volume of the eluate to enhance the
sensitivity for detection. Ultracentrifugation is another concentra-
tion technique to pellet macromolecules and viruses from water
(Ammersbach and Bienzle, 2011). It has been used to recover RV
and AdV in wastewater and recreational water (Prata et al., 2012).
As an alternative approach for the concentration of microbes from
water, ultraltration was rst reported for environmental samples
in Australia (Grohmann et al., 1993). This method, which is based
on the size exclusion, has since been increasingly used in the last
decade (Rhodes et al., 2016). The pore size of the ultraltration
ranges from 5 nm to 0.1 m, which can retain a broad range of virus

http://dx.doi.org/10.1016/j.jviromet.2016.09.010
0166-0934/ 2016 Elsevier B.V. All rights reserved.
 Y. Qiu et al. / Journal of Virological Methods 237 (2016) 150153
particles, including enteric viruses (Ikner et al., 2012; Cashdollar
and Wymer, 2013). Typically, the water sample passes through the
capillaries or hollow bres using tangential ow. Many viruses,
bacteria and parasites have been concentrated and detected in envi-
ronmental waters using ultraltration (Hill et al., 2007, 2009). In
addition to the traditional hollow-bre ultralters, there are small
centrifugal ultralters that have been mainly used for secondary
concentration of viruses. Centricon Plus-70 is one of these centrifu-
gal ultralters with a molecular weight cut off (MWCO) ranging
from 5 to 100 kDa. The Centricon Plus-70 device has been used as
a secondary concentration method for viruses, bacteria and para-
sites in several studies (Hill et al., 2007; Ikner et al., 2011; Kahler
et al., 2015; Kim et al., 2015). However, the possibility of using cen-
trifugal ultralters as the primary concentration method in water
matrices, especially in wastewater, appears to be unexplored.
Pang et al. reported a virus concentration method that used
NanoCeram disc ltration, beef extract elution and occulation
based on VIRADEL (Pang et al., 2012). This method has been used to
concentrate viruses in both wastewater (Qiu et al., 2015) and sur-
face water (Pang et al., 2012). However, there are limitations with
this method, such as a large sample volume to be processed, the loss
of virions due to multiple processing steps, long processing time
and limited sample throughput. These limitations have prompted
us to look for an alternative approach for virus concentration. In
this study, a simple and rapid one-step centrifugal ultraltration
method was developed using the Centricon Plus-70 centrifugal
ultralter (30-kDa MWCO, Millipore) to concentrate human enteric
viruses from wastewater. In order to determine the effectiveness
of the one-step ultraltration method, VIRADEL developed by Pang
et al. (2012) was used in parallel to concentrate virus from the same
water samples. Real-time quantitative PCR (rt-qPCR) and cell cul-
ture were used to quantify the amount of virus/free viral nucleic
acids and infectious virions respectively after each concentration
method.
Wastewater samples were collected monthly from two munic-
ipal wastewater treatment plants (WWTP), Pine Creek (PC) and
Bonnybrook (BB), located in the City of Calgary, Canada from
November, 2014 to April, 2015. A total of 12 samples were collected
by directly grabbing from the wastewater ow before UV treatment
(pre-UV) and post-UV treatment respectively during the study.
Ten liters of grab water samples were processed using VIRADEL as
previously described (Pang et al., 2012; Qiu et al., 2015). The cen-
trifugal ultraltration was used to concentrate virus from 100 mL
wastewater sample using the Centricon Plus-70 lter according
to the Manufacturers instructions, except for the pre-rinse step.
Briey, 70 mL of water sample were added into the lter cup and
centrifuged at 1900 g for 10 min at room temperature. The l-
trate was discarded and the same procedure was repeated for the
rest of the sample. The ltrate collection cup was then removed
and the concentration cup was placed on top of the lter cup. The
whole device was inverted carefully and centrifuged at 800 g for
2 min. The concentrated sample was collected from the concentra-
tion cup and the volume was measured. The processed concentrate
was stored at 70 C until use.
The volume of the concentrated sample after centrifugal ultra-
ltration ranged from 230 L to 955 L with a median of 327 L.
Each concentrated sample was made up to a nal volume of 2 mL
with PBS. Two hundred microliters and 1 mL of the concentrate
were used for nucleic acid extraction and cell culture, respectively.
Virus mixture containing NoV GII and AdV isolated from clinical
stool samples and conrmed by in-house rt-qPCR assay, as well as
cultured coxsackie B virus (CoV, 4.68 104 infectious unit/mL) was
used for recovery test (Pang et al., 2012). The recovery of virus was
determined by spiking 1 mL of the virus mixture into 99 mL and
10 L of the same pre-UV samples for centrifugal ultraltration and
VIRADEL, respectively. One mL of the same aliquot of virus mixture
151
was mixed with 1 mL (total of 2 mL) and 14 mL (total of 15 mL) of
PBS to test in parallel with the spiked wastewater samples as the
baseline control for centrifugal ultraltration and VIRADEL, respec-
tively. The recovery rate (%) was expressed as a ratio of viral load
detected in the respective processed concentrates as compared
to the baseline control (Qiu et al., 2015). Nucleic acid extraction,
reverse transcription and cell culture were performed as previously
described (Qiu et al., 2015). The qPCR reaction was performed in a
total volume of 10 L containing 2 TaqMan Fast Universal Mas-
ter Mix (Applied Biosystems), 900 nM of each primer, 250 nM of
specic probe, and 2.5 L cDNA. Amplication consists of initial
incubation at 95 C for 20 s followed by 45 cycles of 3 s at 95 C, 30 s
at 60 C. An external standard curve was established for quantica-
tion using the 875 bp DNA fragment of NoV GII by 10-fold dilution
from 100 copies to 1.0 106 copies. Seven types of viruses includ-
ing NoV GI/GII, RV, SaV, AsV, AdV, enterovirus (EV) and JC virus
(JCV) were tested by rt-qPCR. The viral load was expressed as log10
genome equivalent (GE) copies/L after correction of the dilution
steps and original volume of the wastewater sample.
The median% recovery (range) was 3% (18%) for NoV, 50%
(475%) for CoV and 3% (24%) for AdV using the centrifugal ultra-
ltration; and 18% (560%) for NoV, 24% (1628%) for CoV and 5%
(412%) for AdV using the VIRADEL. CoV showed better recovery in
both methods compared to NoV and AdV, which may be due to the
source of viruses since NoV and AdV isolated from stool samples
may contain a proportion of free nucleic acids. Previous studies
demonstrated that viral nucleic acids had much lower recovery
rate compared to infectious virions (Haramoto et al., 2007; Li et al.,
2010). The virus level spiked into the water may also affect recov-
ery efciency. Our recent studies showed higher input of the spiked
NoV in wastewater can increase the recovery (data not shown). The
wide range of recovery for each virus may be attributed to the vari-
ation of wastewater sample matrix. The limit of detection (LOD)
for centrifugal ultraltration was 8 GE copies/mL using rt-qPCR.
The concentration of various viruses in pre- and post-UV samples
using both methods are showed in Tables 1 and 2. Four viruses (NoV
GI/GII, RV, AsV and AdV) were detected in all 12 pre- and post-UV
samples, whereas SaV, EV and JCV were occasionally undetectable
by both methods. SaV, EV and JCV were detected in a fewer num-
ber of samples by the one-step centrifugal ultraltration compared
to VIRADEL. Several factors might have contributed to this result,
such as low level of EV and JCV present in the wastewater samples,
the smaller sample volume used for centrifugal ultraltration, and
sampling variations for grab samples.
Using centrifugal ultraltration, the mean viral load ranged from
3.6 (JCV) to 6.5 (RV) log10 GE copies/L in pre-UV samples (Table 1),
and 4.2 (JCV) to 6.6 (RV) log10 GE copies/L in post-UV samples
(Table 2). Using VIRADEL, the mean viral load ranged from 3.5
(EV) to 6.0 (RV) log10 GE copies/L in pre-UV samples and 3.1 (SaV)
to 5.8 (RV) log10 GE copies/L in post-UV samples. It appears that
the viral load detected using the centrifugal ultraltration was
slightly higher than VIRADEL. The lower level of detected virus
using VIRADEL could be due to the multi-step procedures includ-
ing the preconditioning step. The results from this study suggested
that the one-step centrifugal ultraltration has at least as effective
virus recovery and quantication from wastewater to that of the
more commonly used VIRADEL approach.
Among the 24 pre- and post-UV samples, ten concentrated sam-
ples (5 pre-UV and 5 post-UV) were used in cell culture to examine
the infectivity of the viruses in the processed concentrate. Clinical
strains of coxsackievirus B obtained from the Provincial Laboratory
for Public Health, Calgary site (courtesy of Dr. Julie Fox) was used
as positive control. Strong cytopathic effect (CPE, dene as low,
medium and strong using symbols: +, ++ and +++), were observed
in all of the pre-UV samples for both methods, indicating that the
infectivity of the viruses was retained during the concentration
152 Y. Qiu et al. / Journal of Virological Methods 237 (2016) 150153

Table 1
Detection and quantication of viruses in pre-UV samples at two municipal wastewater treatment plants in Calgary, Pine Creek (PC) and BonnyBrook (BB) (n = 6).

PC, mean SD (no. of positive sample) BB, mean SD (no. of positive sample)

Virus Centrifugal ultraltration VIRADEL Centrifugal ultraltration VIRADEL

NoV GI 4.8 0.4 (6) 4.1 0.4 (6) 5.1 0.6 (6) 4.4 0.5 (6)
NoV GII 5.5 0.4 (6) 5.0 0.3 (6) 5.9 0.4 (6) 5.4 0.3 (6)
RV 6.3 0.7 (6) 5.7 0.6 (6) 6.5 0.8 (6) 6.0 0.6 (6)
SaV 4.9 0.5 (5) 4.3 0.5 (4) 5.0 0.5 (6) 4.5 0.4 (6)
AsV 5.2 0.3 (6) 4.7 0.2 (6) 5.6 0.2 (6) 5.1 0.3 (6)
EV 4.1 0.8 (5) 3.5 0.3 (5) 4.7 0.4 (4) 4.0 0.4 (6)
AdV 5.2 0.3 (6) 4.9 0.2 (6) 5.4 0.3 (6) 4.6 1.0 (6)
JCV 3.6 0.3 (4) 3.8 0.2 (5) 4.3 0.3 (6) 3.9 0.4 (6)

The mean was calculated based on the virus concentration of log10 GE copies/L. Only positive samples were calculated for mean and standard deviation.

Table 2
Detection and quantication of viruses in post-UV samples at two municipal wastewater treatment plants in Calgary, Pine Creek (PC) and BonnyBrook (BB) (n = 6).

PC, mean SD (no. of positive sample) BB, mean SD (no. of positive sample)

Virus Centrifugal ultraltration VIRADEL Centrifugal ultraltration VIRADEL

NoV GI 4.7 0.4 (6) 3.8 0.3 (6) 5.1 0.5 (6) 4.2 0.4 (5)
NoV GII 5.4 0.3 (6) 4.8 0.2 (6) 5.9 0.5 (5) 5.2 0.2 (6)
RV 6.3 0.8 (6) 5.6 0.6 (6) 6.6 0.7 (6) 5.8 0.5 (6)
SaV 4.7 0.5 (4) 3.1 0.7 (5) 5.0 0.5 (6) 4.2 0.4 (6)
AsV 5.1 0.3 (6) 4.6 0.3 (6) 5.7 0.4 (6) 5.0 0.2 (6)
EV 4.4 0.7 (3) 3.4 0.4 (6) 4.6 0.6 (6) 3.7 0.3 (6)
AdV 4.9 0.2 (6) 4.8 0.2 (6) 5.6 0.6 (6) 4.5 0.7 (6)
JCV 4.2 0.2 (4) 3.6 0.4 (6) 4.9 0.7 (6) 3.8 0.3 (6)

The mean was calculated based on the virus concentration of log10 GE copies/L. Only positive samples were calculated for mean and standard deviation.

Table 3 processing such samples. Therefore, the one-step centrifugal ultra-

Comparison of features between the one-step centrifugal ultraltration and
ltration method, which is rapid and practical, easy to perform
VIRADEL methods.
and has reproducible virus recovery, could be used to assess virus
Centrifugal ultraltration VIRADEL activity during wastewater treatment, which may benet future
Sample volume 100 mL 1L10L development of regulations and guidelines on virus removal for
Processing time/sample 30 min1 h 48 h wastewater treatment.
Volume of Eluate 300 L1 mL 1530 mL
Processing multiple samples 4 samples 1 sample
Acknowledgements

procedures. CPE was also observed in 1 post-UV sample concen-
trated using centrifugal ultraltration, and in 4 post-UV samples by
VIRADEL. The higher number of samples with infectious virus using
VIRADEL was most likely due to the higher volume of wastewater
sampled using VIRADEL (100 fold higher).
Compared to the previously published VIRADEL method (Pang
et al., 2012), one-step centrifugal ultraltration has several advan-
tages: the smaller sample input volume (100 times less than
VIRADEL) and ease for sample collection, short processing time,
multiple samples can be processed in parallel and simple proce-
dures (Table 3). The processed concentrate from the water sample
using centrifugal ultraltration can also be used to detect bacte-
ria and protozoa, which would be concentrated with the viruses
because of their relatively larger size. However, there are some
limitations for its application. Due to the small pore size of the
ultralter (30 kDa in this study), the efciency of the centrifu-
gal ultraltration can be affected by several parameters, such as
microbe types, the water source and other water quality issues.
Turbid water samples containing particulates may clog the lter,
requiring a longer processing time, which has been observed when
primary sewage was processed (data not showed). Thus, a preltra-
tion step may be required for water samples with high turbidity. In
this study, only wastewater was processed using centrifugal ultra-
ltration. As wastewater contains relatively high levels of viruses
compared to other water matrices, this method may not be suit-
able for concentrating viruses from environmental waters such as
surface water and groundwater due to the very low level of viruses
expected. In most of the wastewater treatment plants, virus testing
and monitoring is not routinely performed due to the challenges of
This work was supported by a research grant funded by Alberta
Innovate-Energy and Environment Solution (AIEES). We thank Min
Cao for technical support and staff from City of Calgary, Water Ser-
vices and Water Resources for providing the wastewater samples.
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