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Science of the Total Environment 530–531 (2015) 314–322
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Behaviour and recovery of human adenovirus from tropical sediment
under simulated conditions
Hugo Delleon Silva a,b, Marco Aurélio Pessoa-de-Souza c, Gislaine Fongaro d, Carlos E. Anunciação e,
Elisângela de P. Silveira-Lacerda f, Célia Regina Monte Barardi d, Marco Tulio Antonio Garcia-Zapata a,⁎
a
Núcleo de Pesquisas em Agente Emergentes e Re-emegentes, Instituto de Patologia e Saúde Pública, Universidade Federal de Goiás, Brazil
Instituto Brasil de Ciência e Tecnologia, Anápolis, Brazil
Departamento de Zootecnia, Pontifícia Universidade Católica de Goiás, Campus II, Goiânia, Brazil
d
Laboratório de Virologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Brazil
e
Laboratório de Diagnóstico Genético e Molecular, Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas II, Universidade Federal de Goiás, Brazil
f
Laboratório de Genética Molecular e Citogenética, Departamento de Biologia Geral, Instituto de Ciências Biológicas I, Universidade Federal de Goiás, Brazil
b
c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Tropical solids decreased genome copy
numbers and viral infectivity.
• Organic matter did not influence
genome copy numbers but
decreased viral infectivity of HAdV-5.
• Acidic pH hinders viral inactivation.
a r t i c l e
i n f o
Article history:
Received 23 February 2015
Received in revised form 12 May 2015
Accepted 18 May 2015
Available online xxxx
Keywords:
Human adenovirus
Infectivity
Organic matter
pH
a b s t r a c t
This study assessed the contributions of pH and organic matter (OM) on the recovery of infectious human adenovirus 5 (HAdV-5) and genome copies (GCs) in waters that were artificially contaminated with tropical soil.
The use of a mathematical equation was proposed based on the flocculation index of clay to assess the recovery
of total GCs in these controlled assays. The results suggest that solids in the water reduced the viral genome copy
loads per millilitre (GC·mL−1) and viral infectivity. OM did not influence the GC·mL−1 recovery rate (p N 0.05)
but led to a 99% (2 log10) reduction in plaque-forming unit counts per millilitre (PFU/mL), which indicates that
infectivity and gene integrity were non-related parameters. Our findings also suggest that acidic pH levels hinder
viral inactivation and that clay is the main factor responsible for the interactions of HAdV-5 with soil. These
findings may be useful for future eco-epidemiological investigations and studies of viral inactivation or even as
parameters for future research into water quality analysis and water treatment.
© 2015 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: Núcleo de Pesquisas em Agente Emergentes e Re-Emegentes, Instituto de Patologia e Saúde Pública, Universidade Federal de Goiás, 74643-970 Goiânia, Goiás
State, Brazil.
E-mail address: [email protected] (M.T.A. Garcia-Zapata).
http://dx.doi.org/10.1016/j.scitotenv.2015.05.075
0048-9697/© 2015 Elsevier B.V. All rights reserved.
H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
1. Introduction
Human adenoviruses (HAdVs) belong to the family Adenoviridae,
genus Mastadenovirus, which contains 57 different serotypes distributed across seven species (A–G) (ICTV, 2013). They are icosahedral,
nonenveloped viruses containing a double-stranded linear DNA
genome (Enriquez, 2002).
These viruses are of substantial public health importance (Silva et al.,
2010), as they are excreted in faeces, urine, and respiratory droplets
(Metcalf et al., 1995; Jiang et al., 2001), and can cause a series of disease
states in infected individuals by the respiratory and faecal–oral routes.
Examples include upper respiratory tract infections (pharyngitis and tonsillitis), lower respiratory tract infections (bronchiolitis and pneumonia),
conjunctivitis, cystitis, and gastroenteritis (Mena and Gerba, 2008).
HAdVs are stable in the environment and resistant to water treatment
methods (Thompson et al., 2003), particularly to UV irradiation (Liden
et al., 2007). Furthermore, they are ubiquitous in the environment yearround (Katayama et al., 2008).
These pathogens are prevalent in both treated and untreated water
(Jiang et al., 2001; Silva et al., 2010; Fongaro et al., 2013) and are often
detected in higher concentrations than other enteric viruses (Wong
et al., 2010). Thus, HAdVs are indicated for use as viral biomarkers of
environmental water and drinking water quality (Wyn-Jones et al.,
2011; Silva et al., 2011).
Detection of HAdVs in water destined for human consumption can
be accomplished either by molecular techniques alone (Silva et al.,
2011) or in combination of these techniques with cell cultures
(Wyn-Jones et al., 2011; Garcia et al., 2012) that allows access infectivity
(ability of the virus to replicate in permissive cells) (Herzog et al., 2008).
Despite the great sensitivity of PCR, the main limitation is the lack of the
correlation between the detected viral genome and viral infectivity,
which limits conclusions about the significance for public health
(Hamza et al., 2011).
Despite advancements in the detection of HAdVs in different water
sources, information is lacking on the relationship of these viruses
with the solids, sediments, or suspended solids in the waters in which
they are present.
315
Viral adsorption to solids is known to be an extremely complex
process (Schijven and Hassanizadeh, 2000), and variations can be
observed even among different serotypes of the same virus (Singh
et al., 1986). Studies that report viral adsorption behaviour to solids
usually employ phages as models (Schijven and Hassanizadeh, 2000).
These studies are valid, but only as predictive models, and cannot
infer the true permanence of infectious viruses, such as HAdVs. This is
due to variations in the isoelectric point (pI), virion size, hydrophobicity,
and capsid proteins (Schijven and Hassanizadeh, 2000), among other
factors that have not yet been discovered.
HAdVs are more prevalent than norovirus, enterovirus, hepatitis A
virus, and human poliovirus in biosolids (Wong et al., 2010). Fong
et al. (2010) found that 100% of sewage and effluent samples were
infectious with HAdV. These findings suggest that matrix solids may
play a crucial role in the gene integrity and infectivity of HAdVs.
The interactions of HAdVs in soil solutions were recently analysed in
two studies. In the first, Wong et al. (2012) investigated the influence of
different concentrations of inorganic ions on the aggregation and
deposition behaviours of HAdVs in sandy soil. In the second, Wong
et al. (2013) evaluated the role of soil organic carbon (SOC) and
solution-phase dissolved organic carbon (DOC) on sorption capacity
and reversibility of organic carbon on adsorption of HAdVs. These two
studies assessed adsorption behaviour using real-time quantitative PCR
(qPCR) but did not evaluate the influence of solids and total organic
matter on HAdV infectivity.
qPCR is a highly sensitive technique, capable of detecting a small
number of microorganisms (Botes et al., 2013), but when used alone, it
is unable to distinguish between infectious and non-infectious viral
particles. Furthermore, viral interactions with solid particles may lead to
viral inactivation by loss of capsid integrity, with the consequent release
of genetic material (Schijven and Hassanizadeh, 2000), which may be
identified by qPCR either as an infectious or non-infectious particle.
Therefore, the present study sought to (i) assess the recovery of
infectious HAdV-5 and genome copies from simulated solutions
containing tropical solids under controlled pH values in the presence
or absence of organic matter; and (ii) define a mathematical equation
to assess the recovery rate from clay soils under simulated conditions.
2. Material and methods
The U.S. Environmental Protection Agency (USEPA, 2011) defines the acceptable level of total dissolved solids (TDS) in drinking water as 500 and
1000 mg·L−1 and at pH ranges of 6.0–9.5 (Brazil, 2005) and 6.5–8.5 (USEPA, 2011). Thus, the experiments were conducted to simulate maximum
levels of solids in water contaminated with infectious HAdV-5, using the reference pH values of 6.0 and 8.0.
2.1. Soil characterisation and preparation
Gleysoil (hydromorphic soil) is a typical soil of riverbanks in tropical environments (Rosolen et al., 2014). This type of soil is associated with poor
land management (use and occupation) and intense precipitation runoff from the landscape. In these conditions, organic matter and minerals from
these soils can reach the rivers (FAO, 2006; Reatto et al., 1998) and can be found during the water treatment process. Thus, gleysoil samples were
collected at a depth of up to 15 cm from a native palm swamp area in the municipality of Bela Vista de Goiás, the central portion of the State of
Goiás, Brazil in the Cerrado ecoregion (17° 00′S 48° 47′W). This type of soil is typically found at river and lake borders and wetlands (Rosolen
et al., 2014) and is the most common sediment carried inside rivers and lakes (FAO, 2006; Reatto et al., 1998). The samples were air-dried, passed
through a 2-mm mesh sieve, and any excess plant debris were removed with the aid of tweezers and a magnifier to yield the fine earth fraction
described by Gee and Bauder (1986).
The soil was subdivided into two portions: (i) soil with organic matter (WOM) and (ii) soil without organic matter (OM consumed by H2O2 –
LOM – less organic matter/OM-free). The latter was treated with hydrogen peroxide (Whittig and Allardice, 1986) and autoclaved at 121 °C and
0.105 MPa for 3 h, three times, with 24-h intervals between each treatment (Zhao et al., 2008), to intentionally remove the soil organic matter. To
ensure maximal homogeneity, the soil samples were crushed and stored at room temperature. The results of physicochemical and instrumental analyses of WOM and LOM soil samples are shown in Table 1.
The clay fraction was determined by X-ray diffraction (XRD). The silt, clay (iron-free fraction and saturated with K, Mg, Mg + glycerol, K + 350 °C,
and K + 550 °C), and sand fractions were also separated. Preparation for XRD and XRD itself was performed as described by Whittig and Allardice
(1986) and Resende et al. (2005). Fig. 1 shows the X-ray diffractogram obtained.
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H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
Table 1
Physicochemical and instrumental analyses of soil samples containing organic matter
(WOM) and without organic matter (LOM).
Assay
Unit
WOM
LOM
Organic matter (OM)a
Al (aluminium)b
Si (silicon)b
K (potassium)b
Ti (titanium)b
Fe (iron)b
Iron — Fedc
Iron — Feod
Sande
Silte
Claye
Degree of flocculationf
Point of zero charge (PZC)g
Densityh
dag·dm−3
% (wt)
% (wt)
% (wt)
% (wt)
% (wt)
g kg−1
g kg−1
dag·dm−3
dag·dm−3
dag.dm−3
%
3.07
42.06
47.72
0.69
2.54
11.99
131.1
4.0
36.79
24.2
39
27
5.41
1.60
ND
41.72
46.70
0.47
1.97
9.14
20.2
3.0
ND
ND
ND
ND
4.20
ND
i
i
a
Measured by loss on ignition (Ball, 1964).
Scanning electron microscopy/energy-dispersive spectroscopy (semiquantitative data).
c
Iron content of the clay fraction by the oxalate method (Whittig and Allardice, 1986).
d
Iron content of the clay fraction by the dithionite method (Whittig and Allardice,
1986).
e
Dispersion and pipette method (Embrapa, 1997).
f
Ratio of natural clay to total clay (Vettori, 1969, Embrapa, 1997).
g
PZC — potentiometric titration method (Embrapa, 1997).
h
Particle density (Embrapa, 1997).
i
Dimensionless quantity.
b
Fig. 1. Overlay of X-ray diffractograms of original soil (clay with OM) and OM-free soil
(clay without MO), showing the presence of 1:1 (kaolinite) clay as well as 2:1 (vermiculite
and ilite) clays. This composition provided evidence of the degree of soil weathering, particularly by the presence of kaolinite and gibbsite, which are characteristic of highly
weathered soils.
2.2. Preparation of viral inoculum
A549 cells were cultured in 75-cm2 cell culture flasks containing Dulbecco's Modified Eagle's Medium (DMEM — High Glucose 1×) supplemented
with 10% foetal bovine serum (FBS) and 1% PSA (penicillin G, streptomycin, and amphotericin B at final concentrations of 100 U·mL−1, 100 μg·mL−1,
and 0.25 μg·mL−1, respectively). The cells were infected with human adenovirus 5 (species C, serotype 5 — HAdV-5) and cultured in a 5% CO2
incubator at 37 °C until cytopathic effects were observed under an inverted microscope. The cell monolayers were frozen and thawed three times,
and any cellular debris was removed by centrifuging at 5000 rpm at 4 °C for 10 min. The viral inoculum consisted of 1-mL aliquots of the resulting
supernatant.
2.3. HAdV inoculation
Fig. 2 provides a scheme of the experimental study design. The method consisted of separately adding 5, 25, and 50 mg of original soil and OM-free
soil to 50-mL sterile polypropylene tubes and adding 40 mL of ultrapure water, followed by brief manual agitation, to obtain suspended and settled
solids.
The pH of sediment-containing water solutions was adjusted to 6.0 and 8.0 (a different pH level for each soil type and concentration) with 0.1 M
HCl. A 1-mL aliquot of viral inoculum was then added and ultrapure water was added to a final volume of 50 mL, to obtain TDS concentrations of 100,
500, and 1000 mg·L−1 for each test assay (TA). One control assay (CA — without addition of soil), which consisted solely of the viral inoculum in a
50-mL final volume of ultrapure water, was performed for each pH level. These control tubes were processed identically to the test tubes. The same
experiment performed with original soil samples was run simultaneously with LOM soil. All concentrations were run in duplicate with repeats in
triplicate.
Tubes containing the virus and sediment solution were agitated at 150 rpm for 1 h at a temperature of 24 °C (Schijven and Hassanizadeh, 2000;
Gantzer et al., 1994). After agitation, the tubes were rested for 1 h at 24 °C for precipitation of settleable solids.
Two clear phases were observed: (i) the supernatant and (ii) the sediment. Briefly, 200-μL aliquots of the supernatant were collected from each
tube and stored at −80 °C until processing. The remaining supernatant was removed, leaving only the pellet representative of settleable material, to
which 2 mL of ASL buffer from the QIAamp DNA Stool Mini Kit (QIAGEN) was immediately added, following the manufacturer's instructions for
nucleic acid extraction. Nucleic acids were also extracted from the 200-μL supernatant aliquots, using the same kit. All extracted samples were
immediately stored at −80 °C.
2.4. Quantitative real-time PCR (qPCR)
DNA samples were diluted 1:10 to avoid inhibition, and qPCR was performed as described by Hernroth et al. (2002). These specific primers anneal
with the conserved region of the hexon gene of the HAdVs. All amplifications were run in duplicate in a StepOnePlus™ Real-Time PCR System
(Applied Biosystems), using as standard a pBR322 plasmid containing part of the adenovirus hexon gene, to generate a standard curve (R N 0.98).
The results were analysed to measure genomic copy numbers of HAdV-5 (GC·mL−1) in water and in sediment, and the mean GC·mL−1 values
were compared to the mean results obtained in each CA.
H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
317
Fig. 2. HAdV-5 copy numbers and infectious HAdV-5 counts in simulated aqueous solutions of solids obtained from tropical soil samples, under controlled pH and organic matter
conditions.
2.4.1. Mathematical equation to infer the total copy number
The GC·mL−1 values were used to construct a series of mathematical equations to infer the total copy number recovered in relation to the
concentration of clay, both in the suspension (to simulate suspended solids in drinking water) and in the precipitate (to simulate solids that may
settle as water is transported through pipes or containers). For the test assay (TA) of each soil treatment, the total HAdV genome copy numbers in
the supernatant (Nsup) and in the precipitate (Nprec) were calculated according to the following equations, respectively:
Nsup ¼
.
h
ðClay% V cm3 Þ
100
Nprec ¼ ðV cm3 Þ−
hh
i
Floc GC mL−1
V cm3 Clay%
.
100
i
i
Floc GC mL−1
ð1Þ
ð2Þ
where V, Clay% and Floc are, respectively, the volume of sediment (cm3) in the tube, the clay content (%) of the matrix, and its degree of flocculation.
Volume is the direct relationship between mass (g) and density (g·cm−3), where 1 cm3 is equal to 1 mL. The clay content is determined by soil texture analysis, and the degree of flocculation is the difference between total clay and natural clay after dispersion (Table 1).
The total genome copy number for the TA (Nta) was obtained by compiling Eqs. (1) and (2) and deriving Eq. (3) below:
Nta ¼ Nsup þ Nprec :
ð3Þ
The overall recovery rate (Rr), expressed in the number of copies, was calculated by Eq. (4):
Rr ð % Þ ¼
ðNca −Nta Þ
100
Nca
ð4Þ
318
H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
where Nca is the total number of GCs in the CA. Eq. (4) may be rewritten as follows (Eq. (5)):
Rr ð % Þ ¼
.
hh 8
ðClay% V cm3 Þ
<Nca −
:
100
.
i
i h
h
Floc CG mL−1 þ ðV cm3 Þ− ðV cm3 Clay% Þ
100
i
i9
Floc CG mL−1 =
;
N ca
100
ð5Þ
2.5. Cytotoxicity assay
A549 cells were seeded in 24-well plates, at a concentration of 1.5 × 105 cells per well, containing 1× DEMEM with 5% FBS and 1% PSA, and set
aside.
Aliquots of supernatant from original soil samples were treated with 2% PSA and diluted in 1× MEM, at a range of ratios (1:2 to 1:7). A 100-μL
inoculum of each dilution was then placed in contact with the seeded cells for 1 h at 37 °C in a 5% CO2 atmosphere under gentle agitation every
15 min, in triplicate. The inoculum was then removed and 1 mL of 1× DMEM with 5% FBS and 1% PSA was added to the cells. Three cell controls,
containing only cells and maintenance medium, were maintained. The plates were incubated at 37 °C in 5% CO2 and the cell monolayer was
monitored at 24, 48, 72, and 96 h. Cells exposed to inoculated test samples were compared to the cell controls under an inverted microscope. Fixation
and staining with Amido Black (0.1% Amido Black, 5% acetic acid, and pH 2.3–2.4) were then performed sequentially. The stained cell layer was
analysed and the first non-cytotoxic dilution established for use in later assays (Fongaro et al., 2013).
2.6. HAdV quantification by plaque assay method
To infer the presence of infectious HAdVs, triplicate samples of supernatant from each treatment, including cell controls, were treated with 1%
PSA, and 250-μL aliquots (at the 1:6 non-cytotoxic dilution) were inoculated onto six-well plates containing a confluent monolayer of A549 cells
for plaque assay, as described by Cromeans et al. (2008) and adapted by Rigotto et al. (2011). Briefly, the cells were incubated for 1 h at 37 °C
with gentle agitation every 15 min. The inoculum was then removed, and the monolayer was washed twice with phosphate-buffered saline
(PBS), pH 7.2. The wells were overlaid with 2.5 mL of prewarmed 0.6% Bacto Agar mixed 1:1 with 2 × High Glucose DMEM containing 4% FBS,
0.1 mM sodium pyruvate, 2% PSA, and 25 mM MgCl2. The plates were incubated at 37 °C in 5% CO2 for 7 days. All experiments were carried out at
least in duplicate.
The number of plaque-forming units per millilitre (PFU·mL−1) of infectious HAdV-5 in each supernatant was calculated. Using the CA as a reference standard, we calculated the loss of HAdV-5 (difference between CA and TA) and the percent loss. Lost adenoviruses were presumed to have
interacted with the sediment (infectious or non-infectious form: with preserved capsid integrity or as free DNA) or to be present in the supernatant
in a non-infectious form.
To assess the detection limits, serial log dilutions of the supernatant of each sample (including control assays) were prepared and plaque assays
were performed.
2.7. Statistical analysis
Data were tabulated in Microsoft® Excel 2007, and statistical analyses were performed in SPSS® for Windows® 16.0.
The Mann–Whitney U test was used to assess infectivity and copy numbers in relation to the variables of interest (solids content, pH, and presence
or absence of OM) among the treatments as well as between the treatments and the control assay. The significance level was set at 5% (p b 0.05).
A potential role of the autochthonous microbiota on the recovery of
genome copies had been hypothesised. However, as there was no
difference in GC·mL−1 values between the different WOM (active
microbiota) and LOM soil (inactivated microbiota) treatments, this
potential role was ruled out (Tables 2 and 3).
The mean GC·mL−1 both at pH 6.0 and at pH 8.0 was 4.75 × 1015
(data not shown) for each CA; this finding suggests that variations in
pH did not influence the rate of genome copy recovery from CAs.
Furthermore, pH was not significantly associated (p N 0.05) with differences in GC·mL−1 between the WOM and LOM soil samples (Tables 2
and 3). Conversely, there were differences among LOM samples at
pH 8.0 (p N 0.028) (data not shown).
Table 2
HAdV-5 GC·mL−1 values in the supernatant of simulated solutions containing soil with
and without organic matter at two pH levels (6.0 and 8.0).
Table 3
HAdV-5 GC·mL−1 values in the sediment of simulated solutions containing soil with and
without organic matter at two pH levels (6.0 and 8.0).
3. Results
Supernatant
Mean
SD
WOM
LOM
Total
4.04 × 1012
2.49 × 1012
3.26 × 1012
pH 8.0 (GC·mL−1)
3.96 × 1012
4.03 × 1011
1.75 × 1012
3.95 × 1011
3.07 × 1012
3.95 × 1011
Min
Max
p
9.26 × 1012
5.04 × 1012
9.26 × 1012
0.300
WOM
LOM
Total
2.66 × 1012
1.69 × 1012
2.18 × 1012
pH 6.0 (GC·mL−1)
1.07 × 1012
1.19 × 1012
1.36 × 1012
1.95 × 1011
1.29 × 1012
1.95 × 1011
3.67 × 1012
3.77 × 1012
3.77 × 1012
0.112
Mann–Whitney U. HAdV-5: species C, serotype 5 human adenovirus. GC·mL−1: genome
copies per millilitre, as measured by quantitative real-time PCR. WOM: soil containing organic matter; LOM: soil without organic matter; SD: standard deviation.
Sediment
Mean
SD
Max
p
WOM
LOM
Total
2.30 × 1013
3.45 × 1013
2.88 × 1013
pH 8.0 (GC·mL−1)
4.03 × 1013
9.50 × 109
3.57 × 1013
1.07 × 109
3.77 × 1013
1.07 × 109
Min
9.50 × 1013
9.50 × 1013
9.50 × 1013
0.469
WOM
LOM
Total
3.20 × 1013
2.01 × 1013
2.61 × 1013
pH 6.0 (GC·mL−1)
3.14 × 1013
4.30 × 108
3.58 × 10 13
6.07 × 105
3.35 × 1013
6.07 × 105
9.50 × 1013
9.50 × 1013
9.50 × 1013
0.397
Mann–Whitney U. HAdV-5: species C, serotype 5 human adenovirus. GC·mL−1: genome
copies per millilitre, as measured by quantitative real-time PCR. WOM: soil containing organic matter; LOM: soil without organic matter; SD: standard deviation.
H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
Table 4
Total HAdV-5 GC·mL−1 values in supernatant (Nsup), precipitate (Nprec), test assay (Cta),
and final recovery rate (Rr) in three different concentrations (mg·L−1) of soil with and
without organic matter and at two different pH levels (6.0 and 8.0), using the mathematical model developed for this study.
pH
6.0
Soil
WOM
LOM
8.0
p
WOM
LOM
p
[]
100
500
1000
100
500
1000
100
500
1000
100
500
1000
Nsup
Nprec
12
3.49 × 10
1.24 × 1012
3.26 × 1012
1.57 × 1012
3.29 × 1012
2.18 × 1011
0.045
2.52 × 1012
9.17 × 1012
4.18 × 1011
2.69 × 1012
4.15 × 1011
4.36 × 1012
0.196
Nta
10
6.96 × 10
3.48 × 1011
1.34 × 107
6.05 × 109
3.72 × 108
2.81 × 106
0.003
9.49 × 1010
1.62 × 107
4.07 × 1011
9.49 × 1010
1.62 × 107
4.07 × 1011
1.000
Rr (%)
12
3.56 × 10
1.50 × 1012
3.26 × 1012
1.58 × 1012
3.29 × 1012
2.18 × 1011
0.004
2.61 × 1012
9.17 × 1012
8.25 × 1011
2.78 × 1012
4.15 × 1011
4.77 × 1012
0.001
99.925
99.996
99.931
99.965
99.923
99.995
0.110
99.945
99.807
99.983
99.941
99.991
99.900
0.287
Mann–Whitney U. HAdV-5: species C, serotype 5 human adenovirus. GC·mL−1: genome
copies per millilitre, as measured by quantitative real-time PCR. WOM: soil containing organic matter; LOM: soil without organic matter.
The presence or absence of OM was not significantly associated
(p N 0.05) with differences in HAdV-5 copy numbers in the supernatant
or sediment of TAs (Tables 2 and 3).
Copy numbers were greater in sediment than in supernatant in all
treatments (Tables 2 and 3). Therefore, the virus or its free DNA somehow interacts better with settleable solids than with suspended solids.
Comparison between mean GC·mL− 1 values in supernatant and
sediment of different test samples and the results obtained in CAs
showed a significant difference (p b 0.001), which demonstrates that,
under the conditions of this study, solids in water reduced the viral genome copy numbers both in the supernatant and in the sediment of all
treatments. For the CA vs. TA comparison, the qPCR results for the supernatant and sediment were consistent with recovery rates N99%
(data not shown).
This result is corroborated by the Rr value, which always exceeded
99.8%, with no significant differences (p N 0.05) across the different tested treatments (Table 4). These data demonstrate that, in samples with a
pH of 8.0, there was a significant difference only between the Nta of
WOM samples and that of LOM soil samples (p = 0.001), with higher
total copy numbers measured in the soil samples with original organic
matter. At pH 6.0, genome copy numbers were also higher in WOM
soil samples for Nsup (p = 0.045), Nprec (p = 0.003), and Nta (p =
0.004) (Table 4).
Quantification of infectious HAdV-5 in the supernatant and per cent
loss is shown in Fig. 3. The mean PFU·mL−1 values measured in CAs at
pH 6.0 and pH 8.0 were 3.0 × 109 and 1.0 × 108, respectively. Compared
Fig. 3. Mean infectious HAdV-5 counts (PFU/mL) obtained by the plaque-forming assay
method and percent viral loss (infectious HAdV-5 not detected in supernatant, killed in supernatant, or somehow bound to sediment) in different test assays. E = log.
319
with the values measured in test assays, these initial measurements
suggest that the presence of solids had an inactivating effect on HAdV5 (p b 0.001), with a trend towards higher infectious viral counts in
the supernatant with lower concentrations of solids (Fig. 3).
LOM soil samples were less harmful to the virus (1–3 log10 reduction
in infectivity) than original soil samples (3–4 log10 reduction). Furthermore, pH 6.0 did not influence infectious viral counts in the supernatant
or the rate of loss in PFU·mL−1 in the original soil or LOM soil
treatments (p = 0.082). On the other hand, at pH 8.0, major variability
in recovery rates and, consequently, in the rate of infectivity loss were
observed. Furthermore, pH 6.0 was associated with a lower rate of
PFU·mL−1 loss in LOM TAs than in the other test assays (Fig. 3).
4. Discussion
4.1. Viral counts
The high rate of recovery in GC·mL−1 or Rr between the LOM and
WOM soil treatments (Tables 2–4) strongly suggests that the presence
or absence of OM did not influence the recovery of viral genome copies
and possibly did not interfere with gene integrity. This finding confirms
the high stability of adenovirus genome in sewage samples (Bofill-Mas
et al., 2006).
Wong et al. (2013) reported an increase in adenovirus genome copy
numbers in the liquid phase after the addition of humic acid in polypropylene containers at pH 7.0. As humic acids are components of organic
matter, this finding is in contrast to our own. Our results suggest that
undissolved or dissolved OM weakens the electrostatic bond between
solids and viral particles (Kimura et al., 2008). This assertion makes
sense because the tests were performed both in the presence and absence of total organic matter, and there was no observed difference in
the recovery rates, both in sediments and in the supernatant.
Organic matter is a complex matrix (Wong et al., 2013) composed
basically by humic acids, fulvic acids and humin (Guimarães et al.,
2013). In regions where soils are formed under a tropical climate influence, the fraction more representative is humin, which is a less reactive
organic matter, and it is known that this fraction is mainly composed of
alkaloids (Guimarães et al., 2013). This explains why the organic matter
does not affect the virus recovery in tropical soils. Wong et al. (2013)
described a higher recovery of HAdVs in the liquid supernatant after
the addition of humic acids. However, this is not valid for tropical
soils, which have higher reactivity than humin (Guimarães et al., 2013).
Moreover, because most of organic matter is usually dissolved, it
might favour a greater CG·mL−1 in the sediment. The data presented
here are supported by Yoshimoto et al. (2012), who showed a negative
correlation between viral adsorption on soils and OM. Furthermore, free
DNA is also poorly adsorbed to soil organic matter (Saeki and Sakai,
2009). Thus, there was no significant difference between CG·mL− 1
recovery and HAdVs and OM.
Staggemeier et al. (2015) analysed the presence of HAdV in 55 water
samples and 21 sediment samples from springs, wells, dams and
streams from farms located in the southern region of Brazil. After
analysis, most of these viral particles in the water were reported to be
non-infectious. Infectious HAdV was detected in only 4 samples (8.8%).
On the other hand, 5 sediment samples (25%) gave positive results for
the presence of infectious viral particles. The viral load for HAdV by
qPCR was higher in the sediment samples than the water. The same result
was obtained in our assays, which also used a type of hydromorphic soil.
Regarding infectivity, solids were highly damaging to HAdV-5. A
lower concentration of solids was associated with higher infectious
viral counts, particularly in the soil without organic matter at pH 6.0
(Fig. 3), but this inference requires further investigation. Concomitantly,
OM further facilitated loss of viral infectivity (Fig. 3). This result is
explained by the fact that viruses are inactivated by combination with
organic matter (Gerba, 1984). In contact with organic matter, virions
cease to be infectious, even if the capsid integrity is preserved; this
320
H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
phenomenon notwithstanding, the viral particle may undergo lysis, with
consequent release of genetic material (Schijven and Hassanizadeh,
2000). Therefore, qPCR may have quantified both infectious and
non-infectious viral particles, and qPCR may even have quantified the
genetic material in suspension or adsorbed to solids.
Recently, Wong et al. (2012) analysed the adsorption of HAdV-2 to
sands with different salt concentrations using qPCR alone. It is likely
that part of the HAdV-2 detected in this study was not infectious,
despite the capsid integrity, viruses cannot be infectious, and viral
DNA may have been released into the solution and quantified as an
infectious particle. This finding was demonstrated by Fongaro et al.
(2013), who assessed viral capsid integrity in water drawn from sources
in the city of Florianópolis, Brazil, by the addition of DNAse. According to
these authors, undamaged HAdVs were detectable after the DNAse assay,
but this did not ensure that these viral particles were infectious; therefore, the authors performed ICC-RT-qPCR, to detect viruses undergoing
replication. In the present study, qPCR was used solely to assess the rate
of genome copy recovery in samples with different solids and pH profiles.
When clean samples are assayed 1 log10 PFU·mL−1 corresponds to 3
log10 GC·mL−1 (Fongaro et al., 2013). In the present study, the difference between the GC·mL−1 values recovered from the supernatant in
the test assays and PFU/mL values exceeded 10 log10. This was an extreme reduction, which suggests that gene integrity and infectivity
were inversely proportional. These findings are consistent with those
reported by Vijayavel et al. (2010), who showed that a reduction in
infectivity does not necessarily coincide with a reduction in HAdV
genome copy numbers.
Furthermore, OM propitiated a major reduction in viral infectivity.
This result may at least be partly explained by Horswell et al. (2010),
whose data reported a minimal HAdV survival in samples containing a
sewage and soil solution.
Regarding pH analysis, and specifically in LOM sediments, in samples with pH 8.0, it was not possible to establish which factor accounted
for the significant difference in GC·mL−1 values between the treatments. The results suggest that HAdV-5 interactions at this pH may
have become more unstable, thus contributing to the variation in
genome copy numbers (Gerba, 1984).
Regarding the assessment of infectivity, viruses were more abundant in the supernatant of LOM in pH 6.0 test assays. This may be because the acidic pH had a protective effect on the viruses rather than
because there was a greater amount of viruses interacting with the sediment in these samples, as there were no differences in GC·mL−1 values
between the sediments of the tested sample treatments. According to
Rexroad et al. (2006), acidic conditions protect HAdV from inactivation.
This process occurs because the HAdV-5 capsid stability is significantly
increased by reductions in pH.
4.2. Interactions with the matrix
Soil is an extremely complex matrix (Saeki and Sakai, 2009), influenced by variations in a number of factors, including the concentrations
of sand, silt, clay, organic matter, pH, metal, and oxides, all of which may
affect viral infectivity (Zhao et al., 2008). Therefore, soil characterisation
is of the utmost importance in any study that employs this matrix.
In the present study, point of zero charge (PZC) measurement
demonstrated that suspended or settleable solids had an acidic charge.
As HAdV-5, with an isoelectric point of 4.5 (Trilisky and Lenhoff,
2007), is negatively charged under the pH conditions of the present
experiment, and suspended DNA is also negatively charged, HAdV-5
most probably interacted with solids by means of hydrophobic bonds
or Van der Waals interactions. According to Bales et al. (1991), hydrophobic bonds are the most important reason to explain viral adsorption
to solids. These interactions may occur with the virus or between
DNA and organic matter itself, which contains hydrophobic groups
(Schijven and Hassanizadeh, 2000) as well as with oxides.
Therefore, the viruses may have interacted with the aluminium,
silicon, titanium, potassium, and iron oxides present in the sample
(Table 1). The involvement of these compounds was not assessed, but
they may also contribute to viral inactivation mechanisms (Zhao et al.,
2008; Zhang et al., 2010). These results suggest that clay plays a major
role in the interactions of HAdV-5 with solids. Clay usually has a heterogeneous charge distribution, with negatively and positively charged
points (Schijven and Hassanizadeh, 2000; Kimura et al., 2008), which
facilitates viral interactions.
4.3. Mathematical equation
The proposed mathematical equation was developed for the total
quantification of viral genome copy numbers in matrices that were
artificially contaminated with clay-containing soils and not centrifuged
for precipitation of the clay fraction. This was the first mathematical
equation already proposed to consider clay as the main source of viral
interaction with tropical solids.
The settled material of TAs essentially contained three constituents:
sand, silt, and non-flocculated sand. Sand is predominantly composed
by quartz and, similar to silt, is uncharged (Klute and Lee, 1994). Therefore, it was proposed that viruses interacted with the precipitate due to
the presence of clay, perhaps with the aid of oxides or traces of OM.
However, the results obtained indicate that OM did not play a leading
role in this interaction; otherwise, the majority of genome copies or
even of infectious viruses would have been detected in the supernatant,
which had a higher OM content.
The mathematical equation developed here took into account the
degree of flocculation of clay (Table 1), which is dispersed in its most reactive form in the supernatant, but in greater quantity in the precipitate.
Clays promote the formation of strong bonds, and may inhibit viral inactivation when viruses are adsorbed to it (Schijven and Hassanizadeh,
2000). According to Gantzer et al. (1994), poliovirus inactivation was
negligible in seawater containing 3–15 mg·L− 1 montmorillonite.
However, at a concentration of 500 mg·L−1, viral inactivation was
significant.
The soil used in the present study was predominantly clay-based,
containing kaolinite, gibbsite, and vermiculite (Fig. 1). Babich and
Stotzky (1980), in a study of phage inactivation by clay, found that
clays were associated with inactivation in the following descending
order: attapulgite, vermiculite, montmorillonite, and kaolinite. As OM
did not influence the GC·mL−1 values, and as clay is highly abundant
and reactive, it was proposed that genome copy losses were attributable
mostly to the presence of clay. As gene integrity was compromised
(lower genome copy recovery rate), clay also interfered with infectivity,
but its contribution to this reduction was not quantified.
Our results show that the degree of genome copy recovery was
similar to that obtained through GC·mL−1 analysis and that clay does
indeed play a key role in HAdV-5 interactions. However, in contrast to
the results of qPCR in GC·mL−1, there was a rise in the increment of
HAdV-5 GCs in the presence of OM in Nta at pH 8.0 and in Nsup, Nprec,
and Nta at pH 6.0. However, analysis of the recovery rate (Rr) both at
pH 6.0 and at pH 8.0 showed no significant difference (p = 0.110 and
p = 0.287, respectively, at pH 6.0 and pH 8.0). Hence, it was considered
that all tests were equal and that no intervention from the parameters of
interest occurred. The mathematical equation developed for this study
can be used to assess the recovery of viral GCs from simulated soilcontaining environments, but as the focus of the mathematical model
was clay rather than OM, this may be a source of bias, and warrants
further evaluation in studies that employ this equation.
The results obtained in this study differ from the classic findings
reported with bacteriophages and other viruses. This outcome was
expected as viral adsorption is highly dependent on virus type
(Kimura et al., 2008). Furthermore, variation may occur even within
the same virus species. Hartmann et al. (2012) analysed the stability
of different HAdV serotypes at 4 °C over 4 weeks and proved that
H.D. Silva et al. / Science of the Total Environment 530–531 (2015) 314–322
serotypes 1, 2, 11, 22, and 41 are more genetically stable than HAdV-5.
Another factor that can influence within-species variation is genome
size. According to Kennedy and Parks (2009), there is a direct relationship between viral particle stability and genome size, whereby viruses
with larger genomes are more stable and hardy.
4.4. Practical applicability
This study conducted a landmark analysis of the role of pH and OM
on the recovery of genome copies and the infectivity of HAdV-5 in
solids-containing solutions.
HAdVs are viruses of major public health importance (Silva et al.,
2010), have been proposed three times to the EPA Contaminant Candidate List (EPA, 2013) and are a promising viral biomarker of water quality. In response to pressures for increasingly microbiologically safe water
(Albinana-Gimenez et al., 2009), research into the physicochemical characteristics that may affect the infectivity or even the genetic integrity of
these pathogens will be invaluable, especially because monitoring for
these pathogens is extensively based on analysis of their genetic material.
Therefore, the results of this study may be of use to ecoepidemiological studies or as parameters for the development of
water quality analysis and water treatment technologies. HAdVs are
able to survive when suspended in water, and although they bind
efficiently to solids, this interaction is not necessarily protective, despite
the important role of pH as demonstrated herein.
Because treated drinking water generally has low dissolved solids
content and a pH of approximately 6.0, HAdVs are potentially more
pathogenic in these treated waters than in environmental waters. This
finding is counter to previous research conducted with bacteriophages
(Straub et al., 1992; Hussein et al., 1994).
5. Conclusions
This study demonstrated that solids in water reduced viral genome
copy numbers and infectivity of HAdV-5. OM did not influence the
genomic recovery rate, but it induces loss of viral infectivity, which indicates that reduction in viral infectivity does not necessarily coincide
with a reduction in genome copy numbers. Acidic pH levels hinder
viral inactivation.
The mathematical model developed here is useful for evaluating the
recovery rate of viral genomic copies in simulated environments with
water and clay solids. However, further studies are necessary to better
assess this application.
Acknowledgements
We thank Olavo André Oliveira de Souza for the assistance in creating
the figures.
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