Proc. R. Soc. B (2012) 279, 3967–3975

*Author† PresenInstitute

Electron10.1098

doi:10.1098/rspb.2012.1271

Published online 1 August 2012

ReceivedAccepted

Reassortant influenza A viruses in wild duckpopulations: effects on viral shedding and

persistence in waterCamille Lebarbenchon1,*, Srinand Sreevatsan2, Thierry Lefevre3,

My Yang2, Muthannan A. Ramakrishnan2,†, Justin D. Brown1

and David E. Stallknecht1

1Department of Population Health, College of Veterinary Medicine, Southeastern Cooperative Wildlife

Disease Study, University of Georgia, Athens, GA 30602, USA2Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul,

MN 55108, USA3Biology Department, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA

Wild ducks of the genus Anas represent the natural hosts for a large genetic diversity of influenza A viruses.

In these hosts, co-infections with different virus genotypes are frequent and result in high rates of genetic

reassortment. Recent genomic data have provided information regarding the pattern and frequency of these

reassortant viruses in duck populations; however, potential consequences on viral shedding and mainten-

ance in the environment have not been investigated. On the basis of full-genome sequencing, we identified

five virus genotypes, in a wild duck population in northwestern Minnesota (USA), that naturally arose from

genetic reassortments. We investigated the effects of influenza A virus genotype on the viral shedding pat-

tern in Mallards (Anas platyrhynchos) and the duration of infectivity in water, under different temperature

regimens. Overall, we found that variation in the viral genome composition of these isolates had limited

effects on duration, extent and pattern of viral shedding, as well as on the reduction of infectivity in

water over time. These results support that, in wild ducks, functionally equivalent gene segments could

be maintained in virus populations with no fitness costs when genetic reassortments occur.

Keywords: avian influenza virus; genetic reassortments; wild birds; mallards; water-borne transmission;

experimental infections

1. INTRODUCTIONInfluenza A viruses (IAV) represent a major threat to

human and veterinary health. The emergence of the

highly pathogenic (HP) H5N1 virus in domestic birds

in southeastern Asia [1] and the introduction of the

swine-origin H1N1 virus in human populations [2] have

shown IAV ability to spread beyond species barriers and

adapt rapidly to new hosts and environmental conditions.

Genetic reassortment between viruses co-infecting the

same host is a key process for the emergence of IAV in

humans [3]. For instance, the genomic characterization

of the swine-origin H1N1 virus has revealed that succes-

sive reassortment between virus genes circulating in

swine, human and wild birds, occurred several years

before its detection in 2009 [4]. Although it is recognized

that genetic reassortment is of primary importance for

host shifts and adaptation to humans, swines and dom-

estic birds, its significance in wild birds remains unclear.

Wild ducks of the genus Anas represent the natural

hosts for a large genetic diversity of low pathogenic (LP)

IAV, with no evidence for species-specific associations

for correspondence (camille.lebarbenchon@univ-reunion.fr).t address: Division of Virology, Indian Veterinary Research, Mukteswar, Uttarakhand 263138, India.

ic supplementary material is available at http://dx.doi.org//rspb.2012.1271 or via http://rspb.royalsocietypublishing.org.

2 June 201211 July 2012 3967

[5,6]. In wild duck populations, host co-infections with

different virus genotypes are frequent [7] and result in

high rates of genetic reassortment, with no clear pattern

of gene segment association [8,9]. It has been suggested

that a large pool of functionally equivalent gene segments

could co-circulate in duck populations, without strong

selective pressure to be maintained as linked genomes

[9]. The increasing genomic data provide novel infor-

mation regarding the pattern and frequency of genome

reassortment in wild ducks, however, its consequences

on viral shedding and maintenance in the environment

have not been investigated.

Shedding pattern and persistence in water represent

two key components of LP IAV transmission dynamics

and dispersal in wild duck populations [10–15]. Viral

replication mainly occurs in the epithelial cells of the intes-

tinal tract, resulting in high virus concentration in faeces

[16,17]. Infected birds contaminate aquatic environments

in which IAV could persist for extended periods of time,

depending on water temperature and physico-chemical

characteristics [18–20]. In addition to LP IAV, it has

been suggested that patterns of viral excretion and environ-

mental persistence could also represent important factors

for the spread of domestic bird-adapted HP viruses by

migratory waterfowl [14,15,21–24].

In this study, we investigated the effects of LP IAV genome

reassortment on the viral shedding pattern in ducks and

This journal is q 2012 The Royal Society

3968 C. Lebarbenchon et al. Influenza A virus reassortments

persistence in water. We hypothesized that reassortment

could generate genotype-related differences, favouring the

selection for particular viruses, potentially leading to a tem-

poral and spatial heterogeneity in the prevalence and

diversity of virus genotypes, as commonly reported in wild

duck populations [25–27]. Alternatively, non-significant

differences among virus genotypes would support the main-

tenance of functionally equivalent gene segments, with no

fitness costs associated with reassortments, as suggested by

recent genomic studies [9,28].

To test these hypotheses, we focused on wild bird-

origin viruses co-circulating in a duck population in

northwestern Minnesota, USA. First, we performed the

full-genome sequencing of five viruses identified as poten-

tial reassortants (based on the virus subtype; i.e.

haemagglutinin and neuraminidase combination). For

each virus genotype, we then experimentally character-

ized the viral shedding pattern (duration, viral load and

excretion route) based on infections of mallards (Anas

platyrhynchos). Finally, the reduction of infectivity in

water over time was assessed under different temperature

regimens to identify genotype-related differences in the

persistence in aquatic habitats.

2. MATERIAL AND METHODS(a) Virus selection

Isolates were obtained from an ongoing long-term surveillance

of IAV circulation in wild birds in Minnesota, USA [27]. In this

study, five viruses isolated from dabbling duck (adults and

juveniles) during the summer of 2007 (between 14 and

16 September), at Roseau River Wildlife Management Area

(48858039.7700 N, 96800032.0800 W) were selected: A/Mallard/

MN/Sg-00169/2007 (H3N8); A/Mallard/MN/Sg-00219/

2007 (H4N8); A/Mallard/MN/Sg-00170/2007 (H6N1);

A/Mallard/MN/Sg-00107/2007 (H6N2); A/Green-winged

teal/MN/Sg-00197/2007 (H6N8) (referred to hereafter as

their respective subtype). Relative abundance of the five virus

subtypes (i.e. prevalence+95% confidence intervals) in the

waterfowl population at the sampling site was 22.5+9.7%

(H3N8), 9.9+6.9% (H6N1), 5.6+5.4% (H6N2), 2.8+3.9% (H6N8) and 1.4+2.7% (H4N8) (cf. [27] for details).

Second passages of stock viruses were propagated in 9- to

11-day-old specific pathogen-free (SPF) embryonating chicken

eggs [29]. Endpoint dilutions were performed in SPF

embryonating chicken eggs and Madin–Darby canine kidney

(MDCK) cell line to determine the median embryo infectious

dose (EID50) and the median tissue culture infectious dose

(TCID50) [29,30].

(b) Full-genome sequencing

Sample preparation, RNA extraction and amplification of all

gene segments were performed using a single-step multiplex

PCR [31]. cDNA libraries were prepared from PCR-purified

multiplex amplicons with the QIAquick PCR Purification kit

(Qiagen, Valencia, CA, USA) and subjected to adaptor lig-

ation and whole genome sequencing using 454 FLX

technology [32]. Sequencing reads were aligned with IAV

genomes using BlastN algorithm of NCBI BLAST v. 2.2.16,

assembled in GS De Nova Assembler v. 2.0.00.20 (454 Life

Sciences, Branford, CT, USA) and mapped in GS Reference

Mapper v. 2.0.00.20 (454 Life Sciences), as described pre-

viously [32]. Contigs were reassembled in SEQUENCHER

v. 4.9 (Gene Codes, Ann Arbor, MI, USA) and annotated

Proc. R. Soc. B (2012)

based on BLAST analyses. Nucleotide sequences generated

in this study have been deposited in GenBank under the

accession numbers listed in the electronic supplementary

material, table S1.

Nucleotide and amino acid sequences of each gene

segment were aligned with CLC sequence viewer 6.4 (CLC

bio, Aarhus, Denmark), and similarity matrix was calcula-

ted using the program PHYLIP v. 3.68 [33]. Concatenated

sequences, including the eight gene segments were also

generated to estimate the overall level of similarity between

virus genotypes.

(c) Experimental infections

All work was conducted at an enhanced Biosafety Level 2

facility at the Poultry Diagnostic Research Center, Athens,

GA, USA.

One-day-old mallards were purchased from a commercial

source (Murray McMurray Hatchery, Webster City, IA,

USA) and raised for one month under indoor confinement

until the beginning of the experiments. Three days before

inoculation, 30 ducks were randomly assigned to one of six

infection groups (H3N8, H4N8, H6N1, H6N2, H6N8 and

a positive control: A/Mallard/MN/199106/1999 H3N8

(second passage of stock virus), referred to as H3N8-C here-

after) and transferred to isolation units ventilated under

negative pressure with high-efficiency particulate air filters.

Ducks were inoculated in the choanal cleft and the trachea

with a volume of 0.2 ml (split evenly between the two routes)

containing an infectious titre of approximately

105.6 EID50 ml21: (based on back titres: 105.5 EID50ml21 for

H6N1 and H6N2, 105.63 EID50ml21 for H4N8 and H6N8,

105.78 EID50ml21 for H3N8, and 105.53 EID50ml21 for

H3N8-C). Oropharyngeal (OP) and cloacal (CL) swabs

were collected from all ducks before inoculation, and at 1, 2,

3, 4, 5, 7, 9, 11 and 14 days post-inoculation. Swab samples

were stored at 2808C. Virus isolations were performed on

CL and OP swab samples using 9- to 11-day-old SPF embry-

onating chicken eggs [29]. For each sample, RNA was

extracted the same day as the samples were inoculated in

eggs, with the MagMAX-96 AI/ND Viral RNA Isolation kit

(Ambion, Austin, TX, USA), using the Thermo Electron

KingFisher magnetic particle processor (Thermo Electron

Corporation, Waltham, MA, USA) [34]. Real-time PCR

(RT-PCR) targeting the Matrix gene was conducted with the

Qiagen OneStep RT-PCR kit (Qiagen, Valencia, CA, USA)

and the Cepheid SmartCycler System (Cepheid, Sunnyvale,

CA, USA) [35].

Blood samples were collected from the right jugular vein,

before inoculation and the last day of the experiment

(14 days post-inoculation), to ensure that ducks successfully

became infected and produced IA-specific antibodies.

Samples were centrifuged for 30 min at 1500 r.p.m and

sera were stored at 2208C until tested with a commercial

blocking ELISA kit (IDEXX FlockCheck IA MultiS-

Screen Antibody Test; IDEXX Laboratories, Westbrook,

ME, USA).

(d) Water-persistence trials

Distilled water buffered with 10 mM HEPES was adjusted

with 1 N solutions of NaOH or HCl to provide a pH of

7.2. For each virus (H6N1, H6N2, H6N8, H3N8 and

H4N8), we tested the effect of five constant (48C, 108C,

178C, 238C, 288C) and two variable (2208C/48C and

178C/238C) temperature regimens, on infectivity over time.

Table 1. Genome constellation of the five influenza A viruses. Different coloured squares represent gene segments with at

least 95% nucleotide sequence similarity between virus genotypes. (For example, for the NP: the nucleotide sequencesimilarity between H3N8 and N6N2, and between H4N8 and H6N1, is higher than 95%).

virus subtype PB2 PB1 PA HA NP NA MP NS

A/Mallard/MN/Sg-00169/2007 H3N8

A/Mallard/MN/Sg-00219/2007 H4N8

A/Mallard/MN/Sg-00170/2007 H6N1

A/Mallard/MN/Sg-00107/2007 H6N2

A/Green-winged teal/MN/Sg-00197/2007 H6N8

Influenza A virus reassortments C. Lebarbenchon et al. 3969

For each virus, infectious amnio–allantoic fluids were diluted

1 : 100 in the distilled water. Inoculated water samples were

divided into 2 ml aliquots in 5 ml polystyrene round-

bottom tubes and placed in incubators set to the appropriate

temperature. For variable temperature regimens, samples

were transferred daily into incubators set to different fixed

temperatures (i.e. 14 h at low temperature: 2208C or

178C; 10 h at high temperature: 48C or 238C).

For each temperature regimen, the virus concentration

was measured at time of inoculation (day 0) and nine to

23 times post-inoculation. Duplicate 0.5 ml inoculated

water samples were diluted 1 : 1 by addition of 0.5 ml of

2� minimal essential medium (MEM). Serial 10-fold

dilutions (from 1021 to 1026) of the samples were made in

1� MEM supplemented with antibiotics (10 000 U ml21

penicillin G, 10 mg ml21 streptomycin and 25 mg ml21

amphotericin) and titrated on MDCK cells [20,30].

(e) Statistical analyses

All analyses were conducted in R v. 2.12.1 [36]. Percentage

agreement and Cohen’s kappa coefficient (k) were calculated

to estimate agreement between virus isolation and RT-PCR

for virus detection in CL and OP swab samples. Kappa coef-

ficients were interpreted as followed: k , 0.2 indicated a

slight agreement, 0.2 , k , 0.4 indicated a fair agreement,

0.4 , k , 0.6 indicated a moderate agreement, 0.6 , k , 0.8

indicated a substantial agreement and k . 0.8 indicated a

perfect agreement [37].

One-way analysis of variance (ANOVA) was used to test

the effect of virus genotype on: (i) the total shedding duration

(number of days from the first to the last day of successful

virus isolation) and (ii) the restricted shedding duration

(number of consecutive days for which viruses were isolated).

The total shedding duration accounts for the intermittent

viral shedding sometimes reported in Mallard [38], whereas

the restricted shedding duration considers only the conti-

nuous excretion. Separate ANOVAs were performed for the

CL and OP shedding durations. Fligner–Killeen tests were

used prior to the ANOVA to check for homogeneity of

variance [39].

A standard curve was generated to estimate the viral shed-

ding load based on the Matrix gene copy number (hereafter

Proc. R. Soc. B (2012)

M-gene copy) in samples tested by RT-PCR (available upon

request). Successive dilutions (from 1021 to 1025) were per-

formed on a Matrix gene transcript containing 9.1 � 1011

gene copies per microlitre (National Veterinary Services

Laboratory, Ames, IA, USA). As the cycle threshold (Ct)

value and the log10(M-gene copy) were significantly corre-

lated (Pearson’s correlation coefficient r ¼ 20.98, d.f. ¼

70, p , 0.001), a simple regression was performed (n ¼ 72;

adjusted r2 ¼ 0.96, p , 0.001) and Ct values were trans-

formed in log10(M-gene copy) for the tested samples.

Linear mixed models (LMMs) were used to investigate the

effect of virus genotype and time on the CL and OP shedding

load. Generalized linear mixed models (GLMMs) with a

binomial error structure and logit link function were used

to examine the effect of virus genotype and time on the prob-

ability of successfully isolating viruses from CL and OP swab

samples. Full models included time and virus genotype as

explanatory variables, and their interaction. Time and virus

genotype were specified as fixed factors. We included duck

identity as a random factor to account for the repeated

viral measurements performed on individual ducks. Terms

significance were estimated using models comparison based

upon the likelihood ratio test (LRT) statistics.

An analysis of covariance was used to test the effects of

time, virus genotype and temperature regimen on virus infec-

tivity in water. Results from duplicate titrations were

averaged and log10 transformed prior to analyses. Linear

regressions were also used to calculate the time required for

a 90 per cent reduction of infectivity titre in water (i.e.

time required for a decrease of the viral titre by 1 log10

TCID50ml21; [18,20]).

3. RESULTS(a) Genomic characterization

Nucleotide sequences comparison between viruses

showed a level of similarity between gene segments ran-

ging from 70.7 per cent to 100 per cent for the internal

gene segments (PB2, PB1, PA, NP, MP and NS), and

from 51.5 per cent to 99.9 per cent for the HA and NA

genes (table 1). The same pattern was observed when

considering amino acid sequences: PB2, PB1, PA, NP

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H3N8 H4N8 H6N1 H6N2 H6N8 H3N8-C H3N8 H4N8 H6N1 H6N2 H6N8 H3N8-C

(a)

(c)

(b)

(d)

Figure 1. (a,b) Total and restricted viral shedding duration in the cloaca and (c,d) in the oropharynx, based on virus isolation.

Each bar represents the mean duration, in days (with s.e. of the mean), for each tested virus genotype.

3970 C. Lebarbenchon et al. Influenza A virus reassortments

and MP exhibited a high level of similarity (97–100%)

when compared with NS (67–100%), HA (43–99%)

and NA (40–100%). When analysing concatenated

nucleotide sequences, the highest genome similarity was

found between H6N2 and H6N8 (91.8%); the lowest

one was observed between H4N8 and H6N1 (82.7%).

Overall, these results suggest that these five viruses

arose from a limited gene pool, likely resulting from gen-

etic reassortment between circulating viruses in the

studied duck population.

(b) Viral shedding in mallard

No ducks in any of the groups were shedding virus at the

time of inoculation and all tested negative for IAV anti-

bodies. All ducks seroconverted by the end of the

experiment (14 days post-inoculation), confirming that

all were successfully infected. For one of the five ducks

inoculated with H6N2, virus was not isolated before day

9 post-inoculation in CL swabs. This late excretion pat-

tern contrasted with the rapid shedding observed in all

other ducks. Because this individual may have been

infected from contact transmission within the isolation

unit rather than from the primary inoculation, this duck

was excluded from the statistical analyses.

Viruses were successfully isolated from both CL and

OP swabs collected from all ducks inoculated with

H3N8-C (control group) during the first 5 days post-

infection, consistent with a previous study performed in

one-month-old mallards with the same virus strain [38].

Overall, a substantial agreement was found between

Proc. R. Soc. B (2012)

viruses isolated in embryonating chicken eggs and

detected by RT-PCR (85.8% agreement, k ¼ 0.70), indi-

cating that when a virus was detected by PCR it was also

likely successfully isolated.

Total and restricted CL shedding durations were 8.8+0.4 and 8.1+0.5 days (mean+ s.e.; figure 1a,b), res-

pectively. There was no effect of virus genotype on

shedding durations (total: F5,23 ¼ 1.7, p ¼ 0.18; restricted:

F5,23 ¼ 0.31, p ¼ 0.9). Total and restricted OP shedding

duration were 6.1+0.4 and 4.7+0.3 days (figure 1c,d),

respectively. A marginally non-significant effect of virus

genotype was found on the total shedding duration

(F5,23 ¼ 2.6, p ¼ 0.056) and a significant effect was

found for the restricted shedding duration (F5,23 ¼ 17.5,

p , 0.001). However, this effect was driven by a single

virus genotype: when ducks inoculated with H6N2 were

excluded from the analysis, no significant differences were

observed between virus genotypes (F4,20 ¼ 2.2, p ¼ 0.11).

Finally, although there was no significant relationship

between the total and the restricted OP shedding duration

(F1,27 ¼ 2.9, p ¼ 0.1), a positive association was found

between the total and the restricted CL shedding duration

(F1,27 ¼ 29.5, p , 0.001).

Overall, the viral shedding load was higher in the cloaca

than in the oropharynx of infected ducks (paired Student’s

t-test, t ¼ 7.4, d.f. ¼ 260, p , 0.001; figure 2a,b), and the

CL and OP shedding were positively correlated (LMM,

LRT x2 ¼ 53, p , 0.001). There was a strong effect of

time on the viral shedding in the cloaca (LRT x2 ¼ 81.2,

p , 0.001; figure 2a) with a sharp increase from day 1 to

day 3 post-inoculation followed by a slow and continuous

day post-inoculation108642 12 14 0 108642 12 14

00

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8

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ene

copy

)

day post-inoculation

(a) (b)

Figure 2. (a) Cloacal and (b) oropharyngeal viral shedding load over time. Each colour line represents a LOESS curve (with

s.e. of the mean) fitted to the values of a virus genotype (green, H3N8; red, H4N8; light blue, H6N1; purple, H6N2; dark blue,H6N8; black, H3N8-C).

Influenza A virus reassortments C. Lebarbenchon et al. 3971

decrease until day 14. We also found a significant effect of

virus genotype on the viral shedding load in the cloaca

(LRT x2 ¼ 13.7, p , 0.05; figure 2a) with ducks inocu-

lated with H3N8-C shedding more viruses than ducks

inoculated with the other virus genotypes (daily mean

log10(M-gene copy)+ s.e.: H3N8-C ¼ 4.65+0.31;

H4N8 ¼ 3.69+0.46; H3N8 ¼ 3.38+0.38; H6N8 ¼

3.29+0.41; H6N1 ¼ 3.26+0.41; H6N2 ¼ 3.11+0.43). However, no significant differences were observed

between virus genotypes when H3N8-C was excluded

from the analysis (LRT x2 ¼ 1.4, p ¼ 0.85). Finally, there

was no virus � time interaction (LRT x2 ¼ 5.9, p ¼

0.31), indicating that shedding pattern did not significantly

vary among virus genotypes (i.e. all virus genotypes

displayed a similar temporal shedding dynamics).

The probability of isolating a virus in the cloaca over

time was significantly different between virus genotypes

(GLMM with binomial errors; virus � time interaction:

LRT x2 ¼ 18.4, p , 0.01). This interaction was, however,

no longer significant when H3N8-C was excluded from

the analysis (LRT x2 ¼ 3.6, p ¼ 0.47). We found no signifi-

cant effect of virus genotype on the probability of isolating

viruses in the cloaca (LRT x2 ¼ 5.6, p ¼ 0.23), but there

was a strong effect of time (LRT x2 ¼ 34.1, p , 0.001).

OP viral shedding over time differed among virus gen-

otypes (virus genotype � time interaction: LRT x2 ¼

27.1, p , 0.001; figure 2b). However, only ducks infec-

ted with H6N2 displayed peculiar temporal shedding

dynamics and the interaction between virus genotype

and time was no longer significant when H6N2 was

excluded from the analysis (LRT x2 ¼ 4.2, p ¼ 0.4). We

found a significant effect of virus genotype on the OP

shedding load (LRT x2 ¼ 14, p , 0.05) with ducks

inoculated with H4N8 shedding more virus than

ducks inoculated with the other virus genotypes (daily

mean log10(M-gene copy)+ s.e.: H4N8 ¼ 3.2+0.33;

H3N8-C ¼ 2.5+0.34; H3N8 ¼ 2.51+0.31; H6N8 ¼

2.48+0.32; H6N1 ¼ 2.35+0.29; H6N2 ¼ 0.9+

Proc. R. Soc. B (2012)

0.28). Finally, there was a strong effect of time on the viral

shedding in the oropharynx (LRT x2¼ 261, p , 0.001).

The probability of isolating a virus in the oropharynx

over time was significantly different between virus genotypes

(virus � time interaction: LRT x2 ¼ 17.3, p , 0.01).

This interaction was, however, no longer significant when

H6N2 was excluded from the analysis (LRT x2¼ 6.4,

p¼ 0.17). There was no significant effect of virus genotype

on the probability of isolating viruses in the oropharynx

(LRT x2¼ 3.3, p¼ 0.51), but there was a strong effect of

time (LRT x2¼ 156, p , 0.001).

(c) Infectivity in distilled water

Overall, viral infectivity in distilled water decreased over

time (F1,556 ¼ 218, p , 0.001; figure 3). This decrease

strongly varied with water temperature; the warmer the

water, the faster virus infectivity decreased (temperature

regimen � time interaction, F6,556 ¼ 200, p , 0.001;

figure 3 and table 2). Variable temperature regimens

also affected virus infectivity in water (F1,576 ¼ 5.4, p ,

0.05) with viral titres quickly reduced when exposed to

repeated freeze–thaw cycles (2208C/48C) but not when

exposed to a milder temperature variation (17/238C).

The main effect of temperature regimen was also signifi-

cant (F6,556¼ 193, p , 0.001), with reduced persistence

time observed at high temperatures or for the freeze–

thaw variations. We found a significant main effect of

virus genotype on viral persistence in water (F4,556 ¼

711, p , 0.001); however, a note of caution is warranted,

because slightly different initial doses of virus were used

for each virus genotype. There was no virus by tempera-

ture regimen interaction (F24,532¼ 1.5, p¼ 0.08)

but a significant time by virus interaction (F4,556 ¼ 3.5,

p , 0.01) was found, indicating that the decrease in infec-

tivity over time varied among virus genotype. This

interaction was, however, no longer significant when

the 108C temperature regimen was excluded from the

01234567

log 10

(TC

ID50

ml–1

)lo

g 10(T

CID

50 m

l–1)

log 10

(TC

ID50

ml–1

)H3N8 H4N8

01234567 H6N1 H6N2

01234567 H6N8

temperature regimen:4°C

10°C17°C23°C28°C

17/23°C-20/4°C

time (days)0 200100 300

time (days)0 200100 300

Figure 3. The virus infectivity in water over time as a function of temperature regimens. Colour lines represent the least-squaresregression lines for each temperature regimen (solid lines, constant temperature; dashed lines, variable temperature).

Table 2. Estimated time (days) required for a 90% reduction of infectivity titre in distilled water.

virus subtype

constant temperature variable temperature

48C 108C 178C 238C 288C 2208C/48C 178C/238C

A/Mallard/MN/Sg-00169/2007 H3N8 245 202 83 44 14 9 65A/Mallard/MN/Sg-00219/2007 H4N8 323 242 109 47 13 10 62A/Mallard/MN/Sg-00170/2007 H6N1 346 241 80 45 14 8 69A/Mallard/MN/Sg-00107/2007 H6N2 274 159 61 43 13 13 63

A/Green-winged teal/MN/Sg-00197/2007 H6N8 309 272 72 44 10 9 46

3972 C. Lebarbenchon et al. Influenza A virus reassortments

analysis (F4,443¼ 1.5, p ¼ 0.19). At this temperature, the

H6N2 virus exhibited a significantly lower persistence

than the H4N8, H6N1 and H6N8 virus, but not

H3N8. No differences were found between the other

four virus genotypes. Finally, there was no significant

three-way interaction between time, virus and tempera-

ture regimen (F24,508 ¼ 1.4, p ¼ 0.11) indicating that

the effect of temperature regimen on virus infectivity

over time was similar among the different virus genotypes.

4. DISCUSSIONOn the basis of full-genome sequencing, we identified five

IAV that naturally arose from genetic reassortments in a

wild duck population in northwestern Minnesota, USA.

The genomic characterization revealed variable level of

nucleotide and amino acid sequence similarities, for the

HA and NA genes but also for internal gene segments

(e.g. NS). In this study, we considered that these viruses

differed not only by the subtype (i.e. genetic differences in

Proc. R. Soc. B (2012)

the HA and NA proteins) but that they overall corre-

sponded to different genotypes, although genotypic

variations were largely driven by HA- and NA-related

genetic differences. We further investigated the effects of

the genotype on the viral shedding pattern in mallards

and the infectivity in water over time under different

temperature regimens. We hypothesize that genetic

reassortments could generate genotype-related differ-

ences in these two components of IAV fitness, favouring

the selection for particular viruses.

CL shedding pattern did not significantly differ

between the five viruses studied, suggesting that naturally

occurring genetic reassortments may not affect viral shed-

ding by this route. As our analysis was based on a limited

number of tested viruses, such a finding should be

reinforced with additional studies focusing on other

virus genotypes, at other locations and in different avian

reservoir hosts (e.g. gulls, waders). This result neverthe-

less suggests that CL shedding may not differ among

LP IAV co-circulating in a single duck population.

Influenza A virus reassortments C. Lebarbenchon et al. 3973

The absence of variation between virus genotypes con-

trasts with host-related effects that can significantly

affect the outcome of virus shedding. Recent studies

have for instance highlighted the effect of bird host

species [40], age [38,41], body condition [42] and pre-

exposure to other IAV [43,44], in the CL shedding dur-

ation and viral load. Such host-related variations may

have direct implications in the transmission dynamics of

IAV in wild duck populations. In addition, it may also

affect long-distance virus dispersal during duck

migrations [14,42,45] and be critical for the spread of

HP viruses [21,22,24,46].

Compared with CL shedding, genotype-related differ-

ences may exist for OP shedding, as one of the five virus

genotype we studied exhibited a significantly different

pattern. Although this result suggests that genetic re-

assortment potentially had a significant effect on the

dominant route of viral shedding in the duck host, we

cannot exclude the possibility of mutations in the H6N2

virus responsible for this reduced OP shedding. In dab-

bling duck species, viral load shed in the oropharynx is

usually lower than in the cloaca [40]. In other bird species

such as geese and gulls, the opposite pattern has been

described [40,47]. The route of viral shedding is likely

to be a determinant for virus transmission in wild bird

populations. For instance, faeces represent an important

source of contamination of aquatic habitats and sub-

sequent infection of dabbling ducks. In gulls, however,

respiratory transmission may represent a more adapted

route, particularly during the breeding seasons when

there are very high bird densities on colonies and contact

between adult and juvenile birds. In gallinaceous poultry,

respiratory transmission also is the dominant route of IAV

transmission, particularly under intensive farming con-

ditions [48,49]. Respiratory transmission is unlikely to

be selected for in wild dabbling ducks; however, when

IAV are introduced to other wild and domestic avian

populations, respiratory transmission can represent a

better-adapted route than faecal–oral transmission and

favour the selection of virus genotypes inducing shedding

from the respiratory tract.

Our viral shedding results are consistent with previous

experimental trials (cf. [15] for synthesis) with the highest

probability of virus isolation success in the cloaca during

the first 8 days of infection. The potential effect of

sampling time on the probability to detect viruses has

been previously discussed along with aspects related to

sampling technique, sample conservation and detection

method, which all influence IAV detection [50–52].

Our study provided no evidence for differences in IAV

detection among co-circulating viral genotypes, based

on CL swab sampling. This result suggests that sub-

type-related differences in the probability of virus

detection in the field are likely to be driven by sampling

biases [53] and differences in laboratory techniques

rather than genotype-related differences in the CL shed-

ding. When considering OP shedding however, virus

detection in duck populations may be biased by geno-

type-related differences, suggesting that only OP swab

sampling may not represent an appropriate technique to

estimate subtype diversity in virus populations.

LP IAV have been shown to persist for extended

periods of time in distilled water, depending on tempera-

ture and physico-chemical characteristics [18–20].

Proc. R. Soc. B (2012)

Similar patterns have been reported for viral infectivity

in field water samples [16,18,54] and viruses isolated

from surface lake waters [31]. The five virus genotypes

we studied exhibited a similar response to different temp-

erature regimens. Temperature had a strong effect on the

viral infectivity in water over time: at cold temperatures

(48C, 108C) infectivity was maintained for several

months, whereas under high temperature regimens

(238C, 288C) it remained only for a few weeks. These

results suggest that genetic reassortment may not induce

important variations in persistence in aquatic habitats.

Previous studies have proposed that subtype-related vari-

ations may exist, especially at low temperatures [19,20];

these studies however mainly tested viruses isolated in

different host species, locations and time. Differences in

the persistence of IAV in water may be limited when con-

sidering co-circulating viruses in a single duck population;

however, differences observed between sampled locations

may reflect adaptive responses to local water character-

istics and the ability for IAV to rapidly evolve towards

an optimal level of persistence in changing environments.

Persistence at cold temperature and in frozen lakes has

been invoked as a possible mechanism of overwinter per-

sistence of IAV in the environment and as a source of

contamination for migratory ducks in breeding grounds

[55,56]. Although viral RNA has been found in different

compartments of aquatic habitats, only a limited number

of viruses have been isolated from the environment [57]

and the understanding of the mechanisms involved in the

long-time environmental persistence of IAV in high-

latitude habitats or its significance remains limited. Along

with a previous study [31], we suggest that freeze–thaw

cycles could be a limiting factor for the persistence of IAV

in shallow waters that undergo important temperature

changes before and after ice formation in lakes.

In this study, we highlighted that the virus genome

composition in naturally circulating reassortants has lim-

ited effects on the viral shedding in mallards, as well as on

the persistence in water under different temperature regi-

mens. These findings support that functionally equivalent

gene segments could be maintained in IAV populations,

with limited fitness costs when reassortment occurs [9].

Indeed, our results suggest that genetic reassortment

may not affect viral shedding pattern and persistence in

aquatic habitats, although a larger set of host- and

environment-related factors, as well as virus genotypes,

would need to be tested to confirm these findings.

General care was provided in accordance with an animal useprotocol (AUP no. A2010 6-101) approved by theInstitutional Animal Care and Use Committee at theUniversity of Georgia, Athens, GA, USA.

We are grateful for the help of Virginia Goekjian, ShamusKeeler and Rebecca Poulson in assisting with virusisolations and water-persistence trials. We also thank TaianaCosta and Monique Franca for assistance in animal care.This work was funded by the National Institute of Allergyand Infectious Diseases, National Institutes of Health(NIH), Department of Health and Human Services (undercontract no. HHSN266200700007C). C.L. was supportedby a ‘Fondation pour la Recherche Medicale’ post-doctoralfellowship during this study. The funding agencies did nothave any involvement in the study design, implementationor publishing of this study, and the research presentedherein represents the opinions of the authors, but notnecessarily the opinions of the funding agencies.

3974 C. Lebarbenchon et al. Influenza A virus reassortments

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