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Viral persistence in water as evaluated
from a tropical/temperate
cross-incubation
YVAN BETTAREL1*, THIERRY BOUVIER2 AND MARC BOUVY1
1
5119 ECOLAG, UNIVERSITÉ MONTPELLIER II, 34095 MONTPELLIER CEDEX 5, FRANCE AND 2CNRS, UMR 5119 ECOLAG,
34095 MONTPELLIER CEDEX 5, FRANCE
IRD, UMR
II,
UNIVERSITÉ MONTPELLIER
*CORRESPONDING AUTHOR: [email protected]
Received February 24, 2009; accepted in principle May 13, 2009; accepted for publication May 18, 2009; published online 9 June, 2009
Corresponding editor: William Li
Virucidal properties of sunlight and temperature have been identified for a long time. However, it is
less well established if virioplankton communities are evenly susceptible or geographically adapted
to these environmental factors. Transplant experiments were conducted between a tropical (Senegal)
and temperate (France) site to examine the effect of natural daylight conditions and temperature on
the persistence of free viruses in water. Fresh- and seawater viruses from both countries were simultaneously subjected to 12 h of full sunlight and dark exposure under their native and “transplanted” latitudes. Consistently, viruses decayed much faster when incubated abroad, regardless of
origin (latitude and/or water type). However, viral losses, in most cases, were not significantly
different between sunlight exposed and dark samples, implying that ambient radiation does not
exert strong negative effects on virioplankton particles. Rather, temperature clearly emerged as the
principal determinant of viral survival in all samples. This suggests that viruses, under local temperature, can adapt through an evolutionary process to survive for longer periods of time thus increasing their chances to encounter their hosts.
I N T RO D U C T I O N
Planktonic viruses are of major importance in aquatic
biomes with considerable ecological and biogeochemical implications for the functioning of aquatic microbial
food webs (Weinbauer, 2004; Suttle, 2005). Viruses are
obligate parasites, and thus cannot survive for extended
periods in ambient water. The conditions for persistence
of those “survivors” before they encounter their hosts
are still challenging for microbiologists. Viruses are
mostly comprised of protein and nucleic acids and their
fate is thus strongly dependent on various environmental factors: (i) exposure to ultra-violet radiation
(UVR) (Murray and Jackson, 1993; Jeffrey et al., 2000;
Maranger et al., 2002), (ii) grazing (Gonzàlez and Suttle,
1993; Bettarel et al., 2005), (iii) temperature (Yates et al.,
1985; Gantzer et al., 1998; Garza and Suttle, 1998), (iii)
adsorption on particulate material (Bitton and Mitchell,
1974; Suttle and Chen, 1992) and (iv) salinity
(Stallknecht et al., 1990; Sinton et al., 2002; Cissoko
et al., 2008). However, their susceptibility to the different
factors may vary profoundly within the viriosphere as
viruses are capable of developing resistance mechanisms
to survive in harsh habitats including hypersaline waters
(Guixa-Boixareu et al., 1996; Bettarel et al., 2006), hot
springs or hydrothermal vents (Geslin et al., 2003;
Breitbart et al., 2004), highly irradiated environments
like the Sahara desert (Prigent et al., 2005) or the superficial sea microlayer (Joux et al., 2006).
Since infectivity is the mechanism through which
viruses exert their ecological and biogeochemical influence, this parameter is generally believed to provide
more relevant estimates of virus decay than disappearance of viral particles (Noble and Fuhrman, 1997;
Wilhelm et al., 1998; Weinbauer et al., 1999). However,
doi:10.1093/plankt/fbp041, available online at www.plankt.oxfordjournals.org
# The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
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the assessment of virus persistence is also of interest
because infectivity cannot be determined via natural
population studies but only from isolates. In addition,
viral disintegration may fuel the viral loop by supplying
additional dissolved organic matter in ambient water
(Middelboe and Lyck, 2002).
Solar radiation that reaches the earth’s surface is
greater as the solar angle increases (Holm-Hansen et al.,
1993). Therefore, one may anticipate the existence of a
latitudinal gradient in the degradative and inactivating
effects of UVR and temperature on viruses (Wilhelm
et al., 2003), unless those pathogens may show some
adaptation to local conditions. Thus, several questions
still remain unresolved, for instance are viruses evenly
susceptible to UVR and temperature on a global scale?
Are tropical conditions more damaging for virioplankton than those met at temperate latitudes? Is there any
ecosystem specificity in the viral response to changes in
irradiance and temperature? These questions are fundamental to assess the mechanisms that influence the survival of viral populations, and have never been
experimentally addressed.
To clarify the role of natural conditions of temperature and solar radiation in the preservation of free
viruses in water, a cross-experimental study was conducted simultaneously at temperate (France) and tropical (Senegal) latitudes. We measured the persistence of
fresh- and seawater viral particles without their hosts
when exposed to natural daylight native and foreign
conditions. We tested whether there is a differential susceptibility of virioplankton to irradiance and temperature with regard to their latitudinal position and water
type (fresh versus seawater).
METHOD
Sampling and treatments
Freshwater and marine viruses were collected at both
temperate (France) and tropical (Senegal) latitudes
between 15th and 20th of November 2005. All water
samples were collected with 2-L acid washed glass
bottles 50 cm below the surface. The tropical fresh- and
seawater samples were taken in Bango Reservoir
(SENfresh; 168040 0300 N, 168270 0400 W) and at a coastal
station in Hann Bay (SENsea; 148420 4300 N,
178250 5700 W), respectively. Meanwhile, the temperate
fresh- and seawater sites were sampled in the
Montpellier region in the Crès Lake (FRAfresh;
438390 2000 N, 038550 4700 E) and Carnon Beach, a coastal
station in the Mediterranean Sea (FRAsea; 438330 0900 N,
048000 2600 E), respectively.
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Free viruses were separated from the different
samples by successive filtration of water through 20.0,
5.0 and 0.2 mm polycarbonate membranes (47 mm in
diameter) to remove prokaryotes and larger organisms
(Fig. 1). Although we did not measure it, we presume
that the amount of planktonic viruses that were
retained on 0.2-mm filters was negligible. Each 0.2-mm
filtrate was homogenized and equally distributed into
8 0.25 L
polyethylene
UV-permeable
sterile
Whirl-Packw bags. Bags were then stored at 2208C
until they were processed. Half of the eight frozen subfiltrates generated per sample were placed in polystyrene boxes with dry ice and sent by plane from
France to Senegal and vice versa, so that viral samples
of both origin (i.e. four frozen replicates of the four
different samples types) could be treated in the two
labs (Fig. 1).
Incubation design
At both sites, after defrosting overnight, bags were
placed in large baths (74 32 18 cm) previously
filled with tap water. Two of the four replicate filtrates
were hermetically wrapped with three layers of aluminium foil before incubation to exclude light; these
were used as duplicate “dark” controls (Fig. 1). The two
others were directly exposed to local full sunlight. All
bags were incubated over a period of 12 h, centred at
solar noon from 0700 to 1900 h. By monitoring the
decay of free virions after separating them from their
microbial hosts, we also prevented bias from potential
recovery from photoreactivation host-mediated processes (Weinbauer et al., 1997), or from UV activation of
lyzogenized cells (by triggering SOS response). The
incubation time of 12 h, from sunrise to sunset, was
chosen to evaluate the effects of natural daylight conditions and temperature and to limit the potential repopulation of a small fraction of prokaryotes that may
have passed through the 0.2-mm membrane filters (this
was verified by bacterial enumeration by epifluorescence
microscopy). Care was taken to place the plastic bath in
an open, fully exposed area to minimize the presence of
shadow. Figure 1 summarizes the entire procedure.
Virus enumeration
Two millilitre sub-samples were taken from each bag at
the beginning of the experiment (T0h), after 6 (T6h) and
12 h (T12h). Duplicate sub-samples were fixed with
0.02-mm filtered buffered formaldehyde (final concentration 2% [vol/vol]), immediately stained with SYBR
Gold (Molecular Probes Europe, Leiden, The
Netherlands) as described by Chen et al. (Chen et al.,
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Fig. 1. Experimental design of the study. Processing of the different fresh- and seawater viral filtrates (A) and schematic representation of the
incubations conducted in France and Senegal (B). Cross-hatched bags indicate dark treatments. SEN, Senegal; FRA, France.
2001) and filtered (,15-kPa vacuum) through 0.02-mm
pore size Anodisc membrane filters (Whatman,
Maidstone, UK). Virus-like particles (VLPs) were
counted under blue light with an Olympus BX-60 epifluorescence microscope.
For each bag, the decay of free viruses was calculated
from the percent of viral losses at T6h and
T12h ¼([VLP]T6h/12 h 2 [VLP]T0h)/[VLP]T0h)*100.
Measurement of solar radiation and
temperature
Solar global irradiance (305 – 2800 nm) was measured
continuously (1 min bin averaged intervals summed for
total daily amounts) at both sites from dawn to dusk in
a shaded-free area, using a pyranometer Li Cor 200. A
Li Cor 1000 data-logger monitored and stored the data
at 10-min intervals. Hourly measurements of ultraviolet
radiation (UV-B, 280 – 320 nm) were carried out using a
radiometer IT 1400A. Temperature was recorded in
each bath every 2 h, using a Checktemp1 thermometer
(Hanna instruments).
X-ray scanning and freezing controls
Possible deleterious effects of X-rays on viruses during
luggage scanning in the airport automated security
detection systems were checked. Three samples of
0.2-micron filtered Mediterranean seawater (100 mL
each) were frozen at 2208C for 6 days and transferred
into a regular airport baggage handling security systems
at Montpellier Airport. The installation was composed
of three X-ray security systems (SDE 385 and Rx Rap
Visionw and a CTX9000 DSi invisonw). X-ray intensity
varied from 1.0 to 1.5 mSv per inspection. Two passages were performed to simulate luggage checking at
departure and arrival between France and Senegal.
Viruses were then incubated as described above at
in situ temperature in the dark. VLP counts were
carried out before and immediately after X-ray
exposure to evaluate the direct effect of irradiation.
To assess whether freezing the filtrates may alter viral
integrity, a control experiment was conducted by comparing the persistence of tropical virioplankton after
12 h of sunlight exposure in fresh and 6-days frozen
0.2-mm filtrates from Hann Bay and Bango reservoir
water, on 10 January 2006.
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R E S U LT S A N D D I S C U S S I O N
Light and temperature conditions
During the incubations, global solar irradiance was
maximal at both sites at 1300 h with values reaching 183
and 426 mW cm22 in France and Senegal, respectively.
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The recorded values between 0730 and 1900 h were 2 –
29-fold lower in the former than in the latter.
The maximum UV levels were reached between 1300
and 1400 h at both sites and were up to 7-fold higher in
Senegal (max, 136 mW cm22) than in France (max,
21 mW cm22) (Fig. 2). Water temperature in the baths
Fig. 2. Global solar irradiance (A), UV-B radiation (B) and temperature (C) in the baths during the sunrise-to-sunset incubations conducted in
Senegal and France, the 6th of December 2005.
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ranged from 4.3 to 13.78C in France and from 17.1 to
28.08C in Senegal. These relatively important shifts in
temperature in both sites can be explained by the fact
that the samples were incubated in a closed system with
no water recirculation, and such shifts are thus not truly
representative of natural temperature variations.
Methodological considerations
Freezing the filtrates for aircraft shipment may potentially weaken viral integrity. However, the frozen (6
days) and unfrozen tropical samples (fresh and seawater)
exhibited comparable viral decay after 12 h of full sunlight exposure (Table I). The airport X-ray scan represents another possible source of alteration for
planktonic viruses. X-ray exposed and non-irradiated
control samples showed no significant differences in
their viral decay rates, whatever the ecosystem type
(P , 0.01, n ¼ 4, Mann – Whitney rank sum test)
(Table I). This suggests that the passage of viruses in the
security screener is probably too quick to impair the
stability of virion’s capsid. Overall, both control experiments indicate that the freezing, thawing and X-ray
scanning of the samples cannot explain any potential
differences between experimental treatments. However,
one cannot exclude the potential effects of other sources
of bias during sample handling, including storage and
shipment that may also have somewhat altered viral
integrity. In addition, in this experimental design, not
only viruses but the entire 0.2-mm fraction was treated
with UV, including the collodoidal size fraction. As the
colloidal material can destroy viral infectivity
(Weinbauer, 2004), we suspect that the photo-oxidation
of colloids by UV could result in lower infectivity losses
and thus, in an understimulation of UV effects.
Local adaptation of virioplankton
The results indicate that the four different types of viral
samples (i.e. SENfresh, SENsea, FRAfresh, FRAsea)
invariably exhibited much lower disappearance rates
when incubated locally (Fig. 3). This may show that
viruses from freshwater or marine systems can survive
longer and therefore have greater chances to proliferate
in their native environment than under different light
and temperature conditions. In this transplant experiment between France and Senegal, the two varying
environmental forcing factors tested were irradiance
and temperature and are discussed below.
Role of sunlight
During the incubations conducted in Senegal, losses of
French viruses (from fresh- and seawater) were, after
12 h, respectively, 1.6- and 1.5-fold higher than the
local Senegalese viruses (Fig. 3c and g). In a comparable
cross-incubation, Noble and Fuhrman (Noble and
Fuhrman, 1997) reported that viruses from the North
Sea were also more severely altered under Californian
sunlight than Californian viruses. However, in their
study, infectivity, not particle disappearance, was
screened and we know that the loss of infectivity is a
process separate from destruction of particles
(Wommack et al., 1996). Intuitively, tropical phages may
be presumed to be genetically adapted to protect DNA
and capsids against UV damage. The different mechanisms of how phages resist photooxidative damage are
thought to involve the capsid structure (Jacquet and
Bratbak, 2003), or the dimerization in DNA that may
reduce the susceptibility of destructive enzymes
(Weinbauer, 2004). GþC rich viral genomes are also
thought to display a greater potential for combating
high UV exposure (Kellog and Paul, 2002). In addition,
one may also envisage that genome sizes are of importance, since larger genomes could present a larger
target for potential lethal hits (Weinbauer, 2004). On
the other hand, large genomes might be more densely
packed in the viral capsid and may thus be better
protected.
Table I: Viral loss measured after 12 h of sunlight exposure to test the effects of freezing (2208C) and
airport X-ray on viral integrity
Freezing effects
a
Fresh
Freshwater viruses
Seawater viruses
Statisticsc
X-ray effects
Frozen
a
36.3 + 0.9
36.5 + 1.1
32.0 + 1.8
36.4 + 3.8
No significant difference, P , 0.05, n ¼ 4
a
X-rayb
27.5 + 5.3
26.7 + 8.9
59.6 + 9.3
52.9 + 22.5
No significant difference, P , 0.05, n ¼ 4
Collected in tropical Senegalese water: Bango Reservoir and Hann Bay for fresh- and seawater, respectively.
Collected in temperate French water: Lake Crès and Mediterranean Sea for fresh- and seawater, respectively.
c
Analysed using a Mann– Whitney rank sum test (Sigmastat 2.0).
b
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No X-rayb
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Fig. 3. Percentage of viral loss (error bars represent standard deviation for duplicate samples) after 6 and 12 h of cross-incubation from France
(FRA) and Senegal (SEN). Grey and black coloured bars represent sunlight-exposed and dark control samples, respectively.
Nonetheless, and contrary to our predictions, the
transplanted Senegalese viruses were more severely
destroyed under weak light conditions (France) than
under tropical sunlight (Senegal). More interesting was
the absence (with the exception of SENfresh viruses incubated in Senegal, see below) of significant differences in
viral mortality between dark and light treatments
(Fig. 3), clearly implying that light conditions in our
experiment had no strong virucidal properties.
Similarly, Wommack et al. (Wommack et al., 1996) and
Noble and Fuhrman (Noble and Fuhrman, 1997) found
no differences in seawater and estuarine phage mortality between darkened and illuminated treatments.
This, however, does not imply that light resistant viruses
are still infective as we know that a substantial proportion of VLPs in surface water is presumed to be
inactive (Suttle and Chen, 1992; Wilhelm et al., 1998,
Bettarel et al., 2008). However, this view is probably
more complex as infectivity can be restored by
host-mediated processes that can repair UV damaged
phage DNA (for mechanisms involved, see Kellog and
Paul, 2002).
In our study, only tropical freshwater viruses incubated locally (Fig. 3c and d) were significantly more
damaged in exposed than protected samples (Mann–
Whitney rank sum test, P , 0.05). We have no clear
explanation for this finding. Nonetheless, one may also
suspect that the incidence of UVR on the fresh- and
marine environment may vary considerably amongst
the different groups within the virioplankton community, as reported for enteric viruses (Sinton et al., 2002;
Lindel et al., 2007), cyanophages (Garza and Suttle,
1998) or algal viruses (Jacquet and Bratbak, 2003).
Overall, subjecting viruses to an increase of up to
seven times the natural light level did not result in significantly different losses between dark and sunlight
treatments. However, we cannot exclude that there
would be a more deleterious effect at higher irradiance
levels. Clearly, future incubations conducted locally
under different UV regimes are needed to acquire
precise information about the threshold levels of particle
disintegration.
If UVR does not explain the viral losses in the different samples and incubation sites, then other processes
must be involved in direct or indirect destruction of
virus particles. Loss of native viruses incubated in the
dark averaged 34.8 + 11.7% after 12 h (Fig. 3d, h, j
and n). Despite the fact that natural degradation of
phage in the dark can be partly explained by the
activity of inhibitory substances or enzymes (Noble and
Fuhrman, 1997), temperature is thus likely to exert a
stronger control on viral persistence in water.
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Fig. 4. Percentage of viral loss for French and Senegalese particles after 12 h of incubation in the dark at both study sites. 13 and 288C
correspond to the maximum temperature recorded at each site.
Role of temperature
AC K N OW L E D G E M E N T S
Temperature is a strong determinant of virus persistence and infectivity; as demonstrated with phage isolates (Giladi et al., 1995). However, the susceptibility of
natural virioplankton to temperature has been investigated only on rare occasions. Garza and Suttle
(Garza and Suttle, 1998) reported a negative effect of
increasing temperature on cyanophage survival. Suttle
and Chen (Suttle and Chen, 1992) and Noble and
Fuhrman (Noble and Fuhrman, 1997) also showed
that temperature notably affects the decay of virus
infectivity. In this study, the inter-site comparison of
viral decay in the dark allows the effect of temperature to be separated (Fig. 4). After 12 h, both freshwater and marine viruses, regardless of origin, were
always less significantly degraded at native than
foreign temperature (Mann – Whitney rank sum test,
P , 0.05) (Fig. 4). Although the severe degradation of
the French viruses incubated in Senegal (Fig. 4b and
f ) was presumably caused by the thermal stimulation
of ambient lyases, the elevated losses of tropical
viruses exposed to low temperature conditions in
France were more unexpected (Fig. 3l and p). The
reason for this intensive degradation in colder water is
uncertain and may result from the presence of
cold-adapted enzymes with high catalytic efficiency
(Goudrieva et al., 2004).
The enhanced deterioration of viral stocks when
incubated abroad may result from the combination of
complex temperature-mediated processes influencing
the stability of the capsid’s proteins together with the
activity of free nucleases or proteases in water. The time
needed for viruses to develop some temperature adaptation remains another challenging question for future
investigations.
The authors wish to thank Emma Rochelle-Newall for
reading of the manuscript. Thanks are also due to
Marie-Françoise Sarr and El hadj Ndour for their technical assistance.
FUNDING
This work has been funded by the French Institute of
Research for Development (IRD) and the Centre
national de la recherche scientifique (CNRS).
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