Journal of General Virology (2014), 95, 1430–1435
Short
Communication
DOI 10.1099/vir.0.063438-0
Perpetuation of H5N1 and H9N2 avian influenza
viruses in natural water bodies
Hongbo Zhang,1,23 Yan Li,1,23 Jianjun Chen,1 Quanjiao Chen1
and Ze Chen1,3,4
Correspondence
Ze Chen
[email protected] or
[email protected]
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences,
Wuhan 430071, PR China
2
Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China
3
College of Life Science, Hunan Normal University, Changsha 410081, Hunan, PR China
4
Shanghai Institute of Biological Products, Shanghai 200052, PR China
Received 3 January 2014
Accepted 24 March 2014
Water bodies are an important route for the spread and transmission of avian influenza virus (AIV).
The determining factor for an AIV to transmit through diffusion in water is the term of viability of the
virus in the water body. To better understand the perpetuation of AIV in natural water bodies, and
thus the risks of AIV spread and transmission via such bodies, we systematically studied the
inactivation dynamics of two AIV strains (H5N1 and H9N2) at different temperatures in water
bodies of important migratory bird habitats within China (Dongting Lake, Poyang Lake, the Hubei
segment of the Yangtze River and Qinghai Lake). We also studied the impact of water-borne
micro-organisms on the perpetuation of AIV. Our findings indicated that water is very likely an
important route for the epidemic spread of AIV, especially during the autumn and winter seasons.
In addition, water-borne micro-organisms might antagonize the persistence of AIV.
Wild waterfowl near wetlands or lakes have been considered
a natural reservoir of avian influenza virus (AIV), and 16
haemagglutinin subtypes and nine neuraminidase subtypes
have been isolated from them (Abdelwhab & Hafez, 2011;
Fouchier et al., 2005). Waterfowl in wetlands or lakes often
contaminate water by releasing AIVs through their excreta
such as faeces. As AIVs remain infectious for months in lowtemperature waters and for .1 week even at 22 uC, water
bodies have been considered an important route for the
spread of AIV (Ito et al., 1995). A previous study showed
that persistence of AIV in water bodies of wild waterfowl
habitats may cause reinfection of migrating birds in the
second year when they return to their original habitats (Ito
et al., 1995). Hinshaw et al. (1979) suggested that some AIVs
might be transmitted via waterways. The persistence of AIV
infectivity in water bodies is of great significance for their
dissemination through water-borne transmission, but there
have been few reports on the persistence of AIV infectivity
in natural water bodies. Most previous studies of the
perpetuation of influenza virus have been performed with
models using distilled water or surface water (Brown et al.,
2007; Keeler et al., 2013; Stallknecht et al., 1990), which
could not truthfully reflect the perpetuation of influenza
viruses in natural water bodies. In the present study, we
3These authors contributed equally to this work.
Two supplementary figures are available with the online version of this
paper.
1430
collected natural water samples from four important
habitats for migratory birds in China (Dongting Lake,
Poyang Lake, the Yangtze River and Qinghai Lake) (Fig. 1),
and we systematically studied the persistence of AIV
infectivity in these water bodies before and after filtration.
The wetlands of Dongting Lake and Poyang Lake are
important wintering grounds and stopovers on the migration routes of East-Asian migrating birds. Residents of the
wetlands usually raise poultry, mostly chicken, duck and
goose mixed together, and each autumn tens of thousands of
migratory birds fly to Dongting Lake. We and other research
groups have isolated multiple subtypes of AIVs from both
wild migratory birds and poultry of the Dongting Lake and
Poyang Lake wetlands (Li et al., 2008; Zhang et al., 2011a, b,
2012). In addition, as both Dongting Lake and Poyang Lake
are connected to the Yangtze River, and water exchange
takes place continuously between the waters of the two
lakes and the mainstream of the Yangtze River, studying
the perpetuation of AIVs in waters of Dongting Lake,
Poyang Lake and the Yangtze River is of great significance
for understanding AIV transmission and dissemination in
this region. In addition, Qinghai Lake in Qinghai Province
of China is a major habitat for migratory birds around the
globe, and from April to June 2005, highly pathogenic avian
influenza broke out in the migratory birds of Qinghai
Lake (Liu et al., 2005). The role of water in this outbreak has
been overlooked and the persistence of infectivity of AIVs in
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Perpetuation of AIVs in natural water bodies
Qinghai Lake
Yangtze River
Poyang Lake
Dongting Lake
Fig. 1. Map showing the locations of Dongting Lake, Poyang Lake, the Yangtze River and Qinghai Lake in mainland China.
Qinghai Lake was studied for the first time in the current
study.
Natural water body samples were taken from Dongting Lake
and Poyang Lake (freshwater lakes), Qinghai Lake (saltwater
lake), and the Yangtze River (Fig. 1). The water samples were
collected in sealable sterile plastic bottles and transported
back to the laboratory under constant temperature in a
portable 4 uC refrigerator. Electrical conductivity, quantity
of micro-organisms [most probable number (MPN)] and
pH value were used to determined the quality of the water
samples (Table 1). In addition, to study the impact of microorganisms on persistence of virus infectivity in the different
waters bodies, aliquots of the water samples (~200 ml) were
filtered with a 0.22 mm membrane filter (Millipore). The
temperatures of each lake/river during different seasons
and the actual water temperature when collecting the water
samples is shown in Table 1.
H5N1 [A/Henan/12/2004(H5N1)] and H9N2 [A/Jiangsu/
11/2002(H9N2)] viruses were added to the collected water
samples. Aliquots of 5 ml of the water samples were added
to 15 ml centrifuge tubes. These virus-containing tubes
were held at 4, 16 or 28 uC and samples were taken for the
determination of virus titre at various time points. All
experiments were performed in triplicate for each water
sample, the TCID50 ml21 was determined at the time of
inoculation (day 0).
The virus infectivity in water samples was determined by
microtitre end-point titration and the results expressed
as TCID50 ml21. The calculation of TCID50 was based
on the Reed–Muench method. A linear regression model
(y5bx+a) was established using the log10TCID50 for each
virus TCID50 measured as dependent variable y, time
elapsed since influenza virus was added to the water sample
as independent variable x, log10TCID50 for the initial virus
Table 1. Water parameters of Dongting Lake, Poyang Lake, the Yangtze River and Qinghai Lake
Parameter
pH
Conductivity (mS cm–1)
MPN
Mean spring temperature (uC)
Mean summer temperature (uC)
Mean autumn temperature (uC)
Mean winter temperature (uC)
Temperature at sample collection (uC)
http://vir.sgmjournals.org
Dongting Lake
Poyang Lake
Yangtze River
Qinghai Lake
6.95
1546
5.96106
9.8–21.2
23.8–28.4
18.3–25.3
4–17.9
12
7.06
651
6.96106
10–20
25–28
12–25
6–8
13
6.93
355
3.46105
6.6–27
22.3–34
7.6–29
0.4–12
16
8.89
28 220
3.26103
–0.5 to 12.5
7.7–17.8
0.6–15.4
0.9–2.6
10
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H. Zhang and others
Table 2. Linear regression equation and days of infectivity persistence for H5N1 and H9N2 viruses in waters from Dongting Lake,
Poyang Lake, Qinghai Lake and the Yangtze River
Location/strain/sample
Dongting Lake
H5N1
Unfiltered
Filtered
H9N2
Unfiltered
Filtered
Poyang Lake
H5N1
Unfiltered
Filtered
H9N2
Unfiltered
Filtered
Yangtze River
H5N1
Unfiltered
Filtered
H9N2
Unfiltered
Filtered
Qinghai Lake
H5N1
Unfiltered
Filtered
H9N2
Unfiltered
1432
Temperature (6C)
Linear regression model
R2
Estimated regression (days)
4
16
28
4
16
28
y52.5–0.07x
y52.4–0.18x
y52.6–0.82x
y52.5–0.05x
y52.5–0.13x
y52.2–0.68x
0.94
0.96
0.97
0.96
0.95
0.95
43
16
3
60
23
4
4
16
28
4
16
28
y52.1–0.06x
y52.2–0.17x
y52.3–0.70x
y52.0–0.04x
y51.7–0.13x
y52.2–0.60x
0.96
0.89
0.94
0.98
0.85
0.95
50
17
4
75
23
5
4
16
28
4
16
28
y52.4–0.07x
y52.5–0.20x
y52.6–0.84x
y52.3–0.05x
y52.3–0.12x
y52.2–0.66x
0.97
0.98
0.96
0.98
0.96
0.97
43
15
3
60
25
4
4
16
28
4
16
28
y52.1–0.07x
y52.0–0.18x
y52.4–0.72x
y52.0–0.04x
y52.0–0.13x
y52.2–0.56x
0.97
0.97
0.95
0.97
0.92
0.97
42
16
4
75
23
5
4
16
28
4
16
28
y52.3–0.08x
y52.3–0.17x
y52.8–0.86x
y52.4–0.06x
y52.1–0.13x
y52.3–0.68x
0.94
0.98
0.97
0.98
0.97
0.97
37
17
3
50
23
4
4
16
28
4
16
28
y52.1–0.06x
y52.1–0.19x
y52.5–0.72x
y52.0–0.05x
y51.9–0.15x
y52.3–0.59x
0.97
0.93
0.92
0.94
0.95
0.98
50
16
4
60
20
5
4
16
28
4
16
28
y52.7–0.13x
y52.1–0.34x
y52.8–0.86x
y52.7–0.12x
y52.5–0.34x
y52.3–0.84x
0.93
0.95
0.90
0.93
0.99
0.96
23
9
3
25
9
3
4
y52.1–0.09x
0.89
33
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Journal of General Virology 95
Perpetuation of AIVs in natural water bodies
Table 2. cont.
Location/strain/sample
Filtered
Temperature (6C)
Linear regression model
R2
Estimated regression (days)
16
28
4
16
28
y51.8–0.21x
y52.3–0.70x
y52.1–0.09x
y52.1–0.22x
y52.3–0.68x
0.96
0.97
0.93
0.98
0.96
13
4
34
13
4
added to water samples as constant a and the regression
coefficient as b.
The perpetuation of H5N1 and H9N2 viruses in natural
freshwater is outlined in the top three sections of Table 2. In
unfiltered Dongting Lake water at 4 uC, infectivity of H5N1
and H9N2 could be maintained for 43 and 50 days,
respectively; at 16 uC, infectivity of H5N1 and H9N2 could
be maintained for 16 and 17 days, respectively; and at
28 uC, infectivity was quickly lost, lasting only 3 and 4 days
for H5N1 and H9N2, respectively. In filtered Dongting Lake
water (through 0.22 mm membranes) at 4 uC, infectivity of
H5N1 and H9N2 could be maintained for 60 and 75 days,
respectively; at 16 uC, infectivity of both H5N1 and H9N2
could be maintained for 23 days; and at 28 uC, infectivity
was quickly lost, lasting only 4 and 5 days for H5N1 and
H9N2, respectively .
As Poyang Lake and Dongting Lake are similar in terms of
natural environmental conditions and water quality, the
two AIV strains showed roughly the same inactivation
dynamics in water from Poyang Lake as in water from
Dongting Lake. In unfiltered Poyang Lake water at 4 uC,
infectivity of H5N1 and H9N2 could be maintained for 43
and 42 days, respectively; at 16 uC, infectivity of H5N1 and
H9N2 could be maintained for 15 and 16 days, respectively
(Fig. S1A, available in the online Supplementary Material);
and at 28 uC, infectivity was quickly lost, lasting only 3 and
4 days for H5N1 and H9N2, respectively (Fig. S1A). In
filtered Poyang Lake water (through 0.22 mm membranes)
at 4 uC, infectivity of H5N1 and H9N2 could be maintained
for 60 and 75 days, respectively; at 16 uC, infectivity of
H5N1 and H9N2 could be maintained for 23 and 25 days,
respectively; and at 28 uC, infectivity was quickly lost,
lasting only 4 and 5 days for H5N1 and H9N2, respectively
(Fig. S1B).
As Dongting Lake and Poyang Lake are interconnected with
the Yangtze River, they continuously exchange water with
the mainstream of the Yangtze River. Therefore, the water
quality of these three water bodies is by and large similar.
Our results also showed that persistence of AIV infectivity
was similar in these three waters. In unfiltered Yangtze
river water at 4 uC, infectivity of H5N1 and H9N2 could
be maintained for 37 and 50 days, respectively; at 16 uC,
infectivity of H5N1 and H9N2 could be maintained for
17 and 16 days, respectively; and at 28 uC, infectivity was
quickly lost, lasting only 3 and 4 days for H5N1 and H9N2,
respectively (Fig. S1C). In filtered Yangtze River water at
http://vir.sgmjournals.org
4 uC, infectivity of H5N1 and H9N2 could be maintained
for 50 and 75 days, respectively; at 16 uC, infectivity of
H5N1 and H9N2 could be maintained for 23 and 20 days,
respectively; and at 28 uC, infectivity was quickly lost,
lasting only 4 and 5 days for H5N1 and H9N2, respectively
(Table 2, Fig. S1C).
Our results indicate that temperature is the main determinant of persistence of AIV infectivity in natural freshwater at
lower temperatures, and microbial (bacteria, etc.) activity
might have some impact on AIV survival; at higher
temperatures, the temperature plays a leading role in the
inactivation of AIVs.
Our results also showed that AIVs could survive for quite a
long time at low temperatures in water from the Wuhan
segment of the Yangtze River, i.e. the mainstream of the
Yangtze River, which enables AIVs to spread to a greater
range through water in the mainstream of the Yangtze
River.
The perpetuation of H5N1 and H9N2 viruses in natural
saltwater is outlined in the bottom section of Table 2. In
unfiltered Qinghai Lake water at 4 uC, infectivity of H5N1
and H9N2 could be maintained for 23 and 33 days,
respectively; at 16 uC, infectivity of H5N1 and H9N2 could
be maintained for 9 and 13 days, respectively; and at 28 uC,
infectivity was quickly lost, lasting only 3 and 4 days for
H5N1 and H9N2, respectively (Fig. S1D). In filtered
Qinghai Lake water at 4 uC, infectivity of H5N1 and H9N2
could be maintained for 25 and 34 days, respectively; at
16 uC, infectivity of H5N1 and H9N2 could be maintained
for 9 and 13 days, respectively; and at 28 uC, infectivity was
quickly lost, lasting only 3 and 4 days for H5N1 and H9N2,
respectively (Fig. S1D).
Our results indicated that temperature is still the major
determinant of persistence of AIV infectivity in water from
the largest saltwater lake in China – Qinghai Lake. At lower
temperatures, microbial activity does not have a significant
impact on AIV survival, which might be due to the significantly lower MPN value in Qinghai Lake as compared with
the freshwater lakes (Table 1) and subsequently less
microbial sabotage on AIV infectivity. Our results also
indicated that at each of the temperatures tested, the two
AIV strains had shorter persistence in Qinghai Lake water
than in the freshwater lakes, suggesting high salinity or pH
also destroys infectivity of AIVs. Previous research showed
that the persistence and subsequent transmission would be
greatest in cold freshwater habitats, with pH values ranging
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H. Zhang and others
from 7.4 to 7.8. However, this conclusion was based on
results obtained using distilled water (Stallknecht et al.,
1990). An irreversible conformational change occurs in the
haemagglutinin glycoprotein of AIV at a low pH (Doms et al.,
1985), but there are no reports on the mechanism for viral
deactivation at high pH. In the present study, our results
demonstrated that the AIVs could survive for a few days at
pH 8.9 in Qinghai Lake and the potential mechanism for this
needs to be explored. However, it has to be pointed out that
AIVs could still perpetuate for rather a long time in Qinghai
Lake at low temperatures, which enables AIV transmission
among migrating birds to occur through the water body.
Migrating birds carrying AIV will release the virus into the
environment at stopover sites on their migration route.
When these birds fly away, abiotic environmental factors
would then play important roles in the spread of AIV;
water bodies have been considered repositories for AIV (Ito
et al., 1995; Lang et al., 2008). Studies have shown that AIV
is most stable in slightly alkaline (pH 7.428.2) environments with a low salt concentration and at temperatures
below 17 uC (Stallknecht et al., 1990). Hinshaw et al.
(1979) also considered waterways as important media for
AIV transmission between poultry and migratory birds.
Most previous studies of influenza virus infectivity perpetuation in water are modelled on distilled water. To better
understand how AIV is inactivated in natural water bodies,
we studied and reported for the first time (to our knowledge)
the perpetuation of H5N1 and H9N2 AIV strains (currently
the two most harmful to the poultry industry and the most
frequently isolated subtypes in poultry markets) in water
from important habitats of migratory birds in mainland
China. Our results indicate that the AIV strains could
maintain their infectivity for quite a long time in freshwater
bodies at low temperatures.
In the high-salinity and high-pH water from Qinghai Lake
(the largest saltwater lake in China), temperature is still
the main determinant of persistence of AIV infectivity. In
addition, although persistence of infectivity for both AIV
strains in Qinghai Lake water was markedly shorter than
in water from the freshwater lakes (Fig. S2), it is noted
that AIV could still persist for quite some time at lower
temperatures, which would enable the epidemic spread of
AIV among migratory birds through water-borne transmission in Qinghai Lake. Qinghai Lake witnesses the highest
density of migratory birds in the spring of each year.
As Qinghai Lake is located at a high altitude where the
temperature is lower, the lake water temperature is relatively
low as well, which undoubtedly provides the possibility
for long-term persistence of AIV in the lake water. In 2005,
highly pathogenic H5N1 avian influenza broke out for the
first time among migratory birds in Qinghai Lake (Liu et al.,
2005), which attracted the attention of influenza researchers
worldwide. However, few researchers studied the role of
the water body as transmission medium in this outbreak.
Our study shows that the virus could spread via the water
body of Qinghai Lake, as the virus could survive in Qinghai
1434
Lake water for ~30 days at 4 uC and for ~14 days even at
16 uC.
For Dongting Lake, Poyang Lake and the Yangtze River,
micro-organisms in the waters might have an inhibiting
effect on AIV stability. When pre- and post-filtration water
samples were compared, the H5N1 and H9N2 viruses
could survive longer in filtered water from the three
freshwater sources at 4 and 16 uC. Fujioka et al. (1980)
found that microbes present in seawater could inactivate
human intestinal viruses. Toranzo et al. (1982) studied
the impact of antibiotic-producing bacteria on virus
stability and found bacteria also had antagonistic effects
on viruses.
Dongting Lake and Poyang Lake wetlands are important
stopover and wintering sites on the migration routes of
migrating birds in East Asia. It is noted that many poultry
farms are located in the Dongting Lake and Poyang Lake
wetlands, and it is commonplace for poultry and migratory
birds to share the same water area. Therefore, it is very
likely that AIV could spread through water-borne transmission among poultry and wild waterfowl. Monitoring
the water at aggregation and breeding sites of migratory
waterfowl is very important for the early detection of
AIV, and it is of greater significance for understanding the
mechanism of virus transmission between domestic fowl
and migratory birds.
Acknowledgements
This study was supported by National 973 Project (2010CB530301)
and the National Natural Science Foundation of China (31070141
and 81172738).
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