2632

Surveillance of human viral contamination and

physicochemical profiles in a surface water lagoon
G. Fongaro, M. A Nascimento, A. Viancelli, D. Tonetta, M. M. Petrucio
and C. R. M. Barardi

ABSTRACT

The present study evaluated the contamination of a surface water lagoon (Peri Lagoon) in G. Fongaro
M. A. Nascimento
Florianópolis, Santa Catarina, Brazil, by human adenovirus (HAdV), polyomavirus JC (JCPyV), hepatitis A. Viancelli
C. R. M. Barardi (corresponding author)
A virus (HAV) and rotavirus species A (RVA). Efforts were driven to determine the correlation between Laöoratorio de virologia Aplicada.
Departamento de Microbiologia,
viral presence and the physicochemical parameters of the lagoon and measure the distribution of Imunologia e Parasitologia. Universidade Federal
these viruses throughout the year (June 2010 to May 2011). A total of 48 samples were collected, de Santa Catarina.
88040-900 Florianopoîis. Santa Catarina State.
concentrated and analyzed by qPCR (quantitative polymerase chain reaction). Approximately 96% of Brazil
E-mail: ceiia.barardi@ufsc.br
the samples were positive for HAdV (46/48), 65% were positive for RVA (31/48), 21% were positive for
D. Tonetta
JCPyV (10/48) and 12% were positive tor HAV (6/48). The presence of JCPyV was positively correlated M. M. Petrucio
Laboratorio de Ecologia de Aguas Continentais.
with that of NOjN, and also there was a positive correlation between the presence of each one of the
Departamento de Ecologia e Zoologia -
viruses (HAdV, HAV and RVA) in winter. Samples from water dedicated for human consumption and Universidade Federal de Santa Catarina.
88040-900, Florianópolis.
recreation tested positive for HAdV by qPCR. These samples were also subjected to viral integrity and Brazil

viability assays: 83% (10/12) contained intact viral particles and 66% (8/12) contained infectious
particles. Our results demonstrate the release of human waste into water sources, justifying the
urgent need to add viral parameters to water quality surveillance.
Key words | environmental monitoring, integrity and viability of HAdV, water lagoon

INTRODUCTION

Water resources have been strongly affected by the incorrect
discharge of effluents from human activities, which compro-
mises water quality and the health of people and animals.
Many human diseases are transmitted by the ingestion of
contaminated food and water, and these diseases can be
fatal (Figueras & Borrego 2010). Gastroenteritis, diarrhea
and other clinical manifestations can be caused by viruses
like human adenovirus (HAdV), rotavirus species A
(RVA), hepatitis A virus (HAV) and polyomavirus strain JC
(JCPyV). HAdV, HAV and RVA replicate in the gastrointes-
tinal tract and are excreted in high concentrations in the
feces of infected humans (lO^-lO'^^ particles/g of feces)
(Bosch et al 2008). JCPyV is excreted in the urine by
approximately 80% of all adults worldwide (Boothpur &
Brennan 2010). These viruses are resistant to water and
sewage treatment because they are able to quickly adsorb
to solid particles and thereby protect themselves from inac-
tivating factors (Hernroth et al. 2002). These viruses had
been indicated as potential markers of environmental con-
tamination surveillance, mainly in water used for human
consumption (Fong & Lipp 2005).
Enteric viruses in environmental samples can be detected
using molecular techniques such as polymerase chain reac-
tion (PCR). These techniques alone do not allow
determination of viral infectivity (ability to replicate in per-
missive cells) or viral integrity (viral particles with protected
genomes by intact/undamaged capsids) (Girones et al
2010). The plaque assay test can be used to attest the capacity
of the viruses present in the environmental sample to infect
and cause lysis in a cell monolayer (Herzog et al. 2008).
The enzymatic assay test can be used to attest the viral
capsid integrity by using DNase or RNase enzymes: genetic
material which is not protected by the viral capsid will be
degraded by nucleases (Girones et al. 2010).
In the city of Florianópolis, Santa Catarina, Brazil, the
largest source of drinking water is the Peri Lagoon (Figure 1).

doi: 10.2166/wst,2012.504
 2683 G. Fongaro et al. \ IHuman virai detection and physicochemicai data in water iagoon
0 420 840
Figure 1 I Location of the Peri Lagoon, Florianópolls, Santa Catarina, Brazii.
This lagoon is located in a protected area of the Peri Lagoon
Municipal Park and has a surface area of 5.7 km^. This
water is cached by the Water Plant Company and, after regu-
lar treatment (coagulation, flocculation, filtration, chlorine
disinfection, and pH adjustment), is distributed to the
inhabitants of Florianopolis. Recreational activities are
allowed in the lagoon, which receives more than 2,000 visi-
tors during the spring and summer seasons (Hennemann &
Petrucio 2on).
The aim of this study was to determine the presence
of HAdV, JCPyV, HAV and RVA in conjunction with
physicochemicai parameters in the water lagoon and to
evaluate the seasonal distribution of these viruses, as
well as the integrity and infectivity of HAdV in the ana-
lyzed samples.
METHODS
Water samples
Two liters of surface water samples were collected from
four sites located at Peri Lagoon, and immediately pro-
cessed as described in the "Virus concentration" section.
Monthly collections were taken at each of the points for
one year (June 2010 to May 2011). The following sampling
water Science & Technoiogy | 66.12 | 2012
Atlantic Ocean
•Meters
1.680 2.520 3.360
sites were selected (Figure 1): (1) central part of the lagoon,
(2) outfall of the Cachoeira Grande Stream, which is con-
sidered to be a preserved environment, (3) outfall of the
Ribeiräo Grande Stream, which aggregates in home mar-
gins and is considered to be a degraded environment, and
(4) a site used by Water Plant Company for human con-
sumption as well as for recreational activities.
Physicochemicai analysis
Using a multiparameter probe (YSI-85), the following
physicochemicai parameter were measured in situ: water
temperature (WT), conductivity (Cond.), pH and dissolved
oxygen (DO). Nitrite (NO2N), nitrate (NO3N) and
ammoniacal nitrogen (NH4 N) were determined in filtered
water samples using a Millipore AP40-47 mm glass fiber
(APHA et al. 2005).
Virus concentration
The concentration of the samples was performed using the
method described by Katayama et al. (2002), which consists
of filtration, concentration and elution of water samples
through a negatively-charged membrane and reconcentrated
Centriprep* YM50 (Millipore) device.
 2684 G. Fongaro eí al. | Human viral detection and physicochemicai data in water lagoon
Extraction of viral nucleic acid and reverse transcription
Nucleic acid extraction was performed using a QIAmp
MinElute Virus Spin Kit (Qiagen) following tbe manufac-
turer's instructions. The nucleic acid was eluted in 60|xL
elution buffer and stored at -80 °C until further analysis.
The reverse transcriptase reaction (RT) was performed to
generate cDNA for RVA and HAV, using a RT enzyme
and random primers (Sensiscript RT Kit - QIAGEN*).
Real time quantitative PCR
Quantitative PCR (qPCR) was performed as described by
Hernrotb et al. (2002), Pal et al. (2006), Jotbikumar et al.
(2005) and Zeng et al. (2008) for HAdV, JCPyV, HAV and
RVA, respectively, using the TaqMan assay. All amplifica-
tions were performed in a StepOne Plus® Real-Time PCR
System (Applied Biosystems). Each sample was analyzed
in duplicate. For each plate, four serial dilutions of standard
were run in triplicate for each assay and the genome copies
(gc) were measured. Ultrapure water was used as the non-
template control for each assay.
Enzymatic test
For this assay, only samples from site 4 (consumption and
recreational activities) that were positive for HAdV by
qPCR were examined. Samples were treated with DNase I
to detect undamaged (potentially infectious) virus particles,
as described by Viancelli et al (2012). To cbeck for potential
enzyme inhibitors in tbe sample matrix, a control was gene-
rated by adding 2.0 x 10^ plaque-forming units (PFU) of
human HAdV-2, previously inactivated for 1 h at 99 C and
30 min under UV light, to a sample of water from the Peri
Lagoon concentrate (negative for HAdV) and nuclease-free
water. The enzyme reactions were performed in triplicate,
using 1 U DNase I (Sigma®). Nucleic acids were extracted
as described in sections on "Extraction of viral nucleic acid
and reverse transcription" and "Real time quantitative PCR".
Plaque assay
The samples used for this test were the same as those used
for the enzymatic test. These water samples were treated
with 10 U/mL penicillin, 10 |xg/mL streptomycin and
0.025 Ug/mL amphotericin B, and 0.25 mL were inoculated
in triplicate in A549 (permissive cells derived from lung
human carcinoma) for tbe plaque assay, as described by
Cromeans et al. (2008) with minor modifications (using
Water Science a Technology | 66.12 | 2012
0.6% Bacto-agar). Afterward, the cells were stained with
20% Gram's crystal violet, and plaques were counted.
Statistical analyses
A nonparametric analysis of variance (Kruskal-Wallis) was
used to evaluate whether the differences in the physico-
chemical parameter obtained from tbese sites were
statistically significant (P < 0.05). Tbese analyses were per-
formed using Statistic 7.0.
The correlation between the analyzed physicochemical
parameter and the presence of the virus was analyzed
using the Pearson correlation test. To assess tbe inñuence
of seasonality on viral detection, analysis of variance
(ANOVA) (P < 0.05) was used. Analyses were performed
using GrapbPad (Prism 5.0, EUA).
RESULTS AND DISCUSSION
Occurrence of enteric viruses and their correlation
with environmental parameters
Tbe measured physicochemical parameters are presented in
Table 1. None of the analyzed parameters showed any sig-
nificant spatial or temporal variation, except tbe WT
which varied temporally from 18.8 to 28.7 C (P < 0.05).
The analyses of vims presence showed that HAdV was
present in 96% (46/48) of the samples; the number of
genome copies (gc)/L for this virus ranged from 1.73 x 10^
to 2.41x10*^. The RVA was present in 65% (31/48) of
sample, ranging from 1.20x10^ to 2.50 x 10^ gc/L. Tbe
JCPyV was present in 21% (10/48) of sample, ranging
from 4.10x10'' to 6.30x lO^gc/L. Tbe HAV was present
in 12% (31/48) of sample, ranging from 7.81x10' to
1.33x lO^gc/L. The overall averages of viruses found in
the Peri Lagoon are shown in Figure 2(a).
The protected environment site was negative for HAV
and JCPyV; the center of the lagoon was also negative for
HAV. The samples from the site of water collected for
buman consumption and recreational activities were 100%
positive for HAdV (12/12); 83% were positive for RVA
(10/12); 33% were positive for JCPyV (4/12), and 33%
were positive for HAV (4/12).
Tbe bigb prevalence of HAdV corroborates with other
studies that demonstrated HAdV as the most frequent
virus in various water matrixes, as it is very resistant to
environmental oscillations and UV radiation (Haramoto
et al. 2010; Rigotto et al. 2010). The HAdV is distributed
 2685 G. Fongaro et al. \ Human virai detection and physicochemical data in water iagoon Water Science & rechnology | 66.12 | 2012

Table 1 I Physicochemicai anaiysis of the Peri Lagoon (n = 12 per site)

Parameter Site 1 Mean ± SD Site 2 Mean ± SD Site 3 Mean + SD Site 4 Mean ± SD

WT( C) 22.6 ± 3.34 22.9 ± 3.36 22.7 ±3.18 22.9 ± 3.36

Cond. (us/cm) 67.2 ± 5.40 66.5 ± 5.32 67.3 ± 4.58 67.4 ± 4.58
pH 7.0 ± 0.60 7.0 ± 0.42 7.0 ± 0.45 7.1 ±0.52
DO (mg/L) 8.1 ± 1.40 8.3 ± 1.07 8.4 ± 0.99 8.3 ± 0.97
NO2N (ng/L) 0.3 ± 0.07 0.3 ± 0.09 0.2 ± 0.08 0.2 ± 0.08
NO3N (^tg/L) 8.5 ± 6.62 6.8 ±3.13 6.3 ± 3.74 7.9 ± 4.46
(ng/L) 16.7 ± 12.32 14.5 ± 8.31 12.2 ±9.17 13.3 ± 8.09

(a) RVA
1.0x10"-
.S o
1.0x10»»
t) Spring • Summer
JCPyV
Figure 2 (a) Average number (gc/L) of HAdV. RVA. JCPyV and HAV (n = 12 per site) and
(b) their seasonai distribution (n = 48).
worldwide and is responsible for causing various diseases
related to consumption or contact with contaminated
water (Fong & Lipp 2005). This viral agent can be found
in rivers and in coastal waters contaminated with a great
number of other enteric viruses that can be detected all
year round (Tavares et al 2005).
The high number of RVA genomes detected suggests
that the water is contaminated by human sewage. RVAs
are excreted in high concentrations in the feces of
infected individuals (up to 10^^° virus/g) and remain
JCPyV • HAV intact in the environment for long periods of time.
Fumian et al (2on) investigated RVA in human sewage
and found 100% positivity, with quantities ranging from
lO'^ to 10^ gc/L.
While RVA is one of the most imporfant causal agents of
morfality in infants worldwide, two live oral rotavirus vac-
cines (Rotarix" and RotaTeq") have been introduced in
more than 100 countries (Fumian et al. 2on). In Brazil, the
vaccine was included in the immunization program in
2006 (Jiang et al. 2010; Fumian et al. 2on). However, the
impact of this vaccine on the circulation of RVA genotypes
H Autumn
is unlcnown and difficult to predict. Continuous genotype
surveillance is needed to identify the effects of the vaccine
(Bosch et al. 2008).
In the present study, JCPyV was found in 21% of the
samples. It was not associated with any of the other viruses
studied but showed a positive correlation with NO2N.
During the spring and summer, JCPyV detection in the
water samples could be a consequence of the presence of
bathers, who can possibly contaminate the water with
inline. Nitrite is an intermediate state of nitrogen between
ammonium and nitrate. It can be present in sewage, which
contains nitrogen mainly in the form of urea. Bacteria can
biologically oxidize urea into ammonia, and then nitrite
and nitrate (Metcalf & Eddy 2003).
HAV was detected in 12% of the samples, the lowest fre-
quency of any of the viruses. The majority of positive
samples were found in the winter. In this study, samples
positive for HAV were also positive for RVA and HAdV,
which is consistent with Rigotto et al (2010), who reporfed
16.6% positivity for HAV in drinking water. Although
HAV was found at a relatively low frequency compared
with other viruses, this virus is known to be the most resist-
ant RNA virus in aquatic environments and to conventional
water treatments for human consumption (Rzezutka &
Cook 2004).
 2686 G. Fongaro eí al. \ Human viral detection and physicochemicai data in water lagoon Water Science & Technology { 66.12 | 2012

In many countries, water quality is evaluated according Treated PFU

to bacteriological standards, even though bacterial contami- .2 1.0x10'- -400

nation is not correlated with the presence of human enteric •320

viruses (Fong & Lipp 2005). -240 -0

'S. et a
The seasonal distribution of viruses showed that the 8:: \ -160 t=
\
10=-
presence of HAdV, RVA and HAV was more frequent in
\
fhe winter, while JCPyV was restricted to spring and
summer (Figure 2(b)). The presence of HAdV, RVA and
HAV was positively correlated but only in winter {P <
0.05) These results can be explained by the fact that
viruses are more stable during the winter, when, for Figure 3 I Average number (gc/L) of HAdV detected in samples (bars) with and without
DNase treatment and (line) number of HAdV PFU/L (n = 12).
example, UV radiation is less intense. In several studies,
enteric viruses have been reported to survive for longer
and occur more frequentiy during the winter months in
natural environments (Lipp et al. 2001; Wetz et al. 2004). expressed as gc/L of total and undamaged HAdV are pre-
In our study, RVA and HAV showed a low rate of preva- sented in Figure 3.
lence in the summer. Fong & Lipp (2005) showed that the The plaque assay for assessing HAdV viability/infec-
higher temperatures in summer can damage the viral tivity revealed that 66.6% (8/12) had viable HAdV
capsid. particles (Figure 3). In December 2010 and February,
March, May 2011, there were no infectious particles of
HAdV.
Comparison of integrated DNase-qPCR and plaque
Despite the ability of viruses to associate with particles
assay to detect infectious adenoviruses
in the water and thereby protect themselves from degra-
dation, the reduction in the number of gc of HAdV after
Viral infectivity studies often include experiments to
enzymatic treatment may be associated with increased
demonstrate a cytopathic effect in cell culture, such experi-
degradation and inactivation of capsid by sun UV radiation
ments are laborious, and many viruses do not produce
and temperature, which causes conformational changes in
cytopathic effects (Rodn'guez et al 2009). Recently, Vian-
the capsid (Fong & Lipp 2005).
celli et al. (2012) used an enzymatic assay with a known
The divergence between the total genomic copy
amount of DNase required to degrade all free viral DNA
number, genomic copies of intact virus and the number of
in order to confirm the applicability of this technique to
infectious viruses highlights the importance of including
detect intact and possibly infectious viral particles. In the
molecular and cell culture techniques during the monitoring
current study, as a secondary objective, integrity and infec-
of viruses in the environment.
tivity of the HAdV was assessed (only samples from site 4
(consumption and recreational activities), positive for
HAdV by qPCR).
In the current study, we used an enzymatic assay with CONCLUSIONS
DNase and found that the concentrated water matrix does
not interfere with DNase activity (data not shown). Ten of Surface water sources are being contaminated with human
12 samples (83%) contained undamaged HAdV particles. enteric viruses, demonstrating the lack of health care and
The logarithmic reduction, after the enzymatic treatment emphasizing the urgency of surveillance and implemen-
measured by qPCR, was 3-logs (samples: June and July/ tation of more efficient methods for removing viruses from
2011), 2-logs (sample: August/2011), and 1-log (samples: the water supply.
September, October and November/2011 and January/
2012). Samples December/2010, February/2011 and
April/2011 did not show reduction after the enzymatic ACKNOWLEDGMENTS
treatment, demonstrating the presence of undamaged
viral particles. The total reduction of genomes occurred This work was supported by CAPES, CTAgro/CNPq/n°25/
in the samples collected in March and May/2011, which 2010 and 'LAPAD-UFSC.
therefore did not contain intact viral particles. The data C.R.M. Barardi receives a fellowship from CNPq.
 2687 G. Fongaro eí al. \ Human viral detection and physicochemical data in water lagoon Water Science & Technology | 66.12 | 2012

Jothikumar, N., Cromeans, T. L., Sobsey, M. D. & Robertson, H.

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First received 9 March 2012; accepted in revised form 16 July 2012

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