Influenza A Virus Surveillance in Live-Bird Markets: First Report of Influenza A Virus Subtype H4N6, H4N9, and H10N3 in Thailand

Abstract

SUMMARY. A one-year influenza A survey was conducted in 10 live bird markets (LBMs) in H5N1 high-risk areas in Thailand from January to December 2009. The result from the survey showed that the occurrence of influenza A virus (IAV) in LBMs was 0.36% (19/5304). Three influenza A subtypes recovered from LBMs were H4N6 (n 5 2), H4N9 (n 5 1), and H10N3 (n 5 16) from Muscovy ducks housed in one LBM in Bangkok. These influenza subtypes had never been reported in Thailand, and therefore such genetic diversity raises concern about potential genetic reassortment of the viruses in avian species in a particular setting. Two influenza A subtypes (H4N6 and H4N9) were isolated from oropharyngeal and cloacal swabs of the same duck, suggesting co- infection with two influenza subtypes and possible genetic reassortment in the bird. In addition, H10N3 infection in ducks housed in the same LBM was observed. These findings further support that LBMs are a potential source of IAV transmission and genetic reassortment.

Full text

Influenza A Virus Surveillance in Live-Bird Markets: First Report of Influenza A Virus
Subtype H4N6, H4N9, and H10N3 in Thailand
Trong Wisedchanwet,
A
Manoosak Wongpatcharachai,
B
Supanat Boonyapisitsopa,
A
Napawan Bunpapong,
A
Waleemas Jairak,
A
Pravina Kitikoon,
C
Jiroj Sasipreeyajun,
D
and Alongkorn Amonsin
ABE
A
Emerging and Re-emerging Infectious Diseases in Animals, Research Unit, Faculty of Veterinary Science, Chulalongkorn University,
Henri-Dunant Road, Patumwan, Bangkok, Thailand
B
Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Road,
Patumwan, Bangkok, Thailand
C
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Road, Patumwan, Bangkok, Thailand
D
Department of Medicine, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Road, Patumwan, Bangkok, Thailand
Received 11 February 2011; Accepted and published ahead of print 28 June 2011
SUMMARY. A one-year influenza A survey was conducted in 10 live bird markets (LBMs) in H5N1 high-risk areas in Thailand
from January to December 2009. The result from the survey showed that the occurrence of influenza A virus (IAV) in LBMs was
0.36% (19/5304). Three influenza A subtypes recovered from LBMs were H4N6 (n52), H4N9 (n51), and H10N3 (n516)
from Muscovy ducks housed in one LBM in Bangkok. These influenza subtypes had never been reported in Thailand, and therefore
such genetic diversity raises concern about potential genetic reassortment of the viruses in avian species in a particular setting. Two
influenza A subtypes (H4N6 and H4N9) were isolated from oropharyngeal and cloacal swabs of the same duck, suggesting co-
infection with two influenza subtypes and possible genetic reassortment in the bird. In addition, H10N3 infection in ducks housed
in the same LBM was observed. These findings further support that LBMs are a potential source of IAV transmission and genetic
reassortment.
RESUMEN. Muestreo de vigilancia del virus de la influenza A en mercados de aves vivas: Primer reporte del virus de la
influenza A subtipos H4N6, H4N9, y H10N3 en Tailandia.
Se realizo
´durante un an
˜o un muestreo para la influenza aviar A en Tailandia en 10 mercados de aves vivas en zonas de alto riesgo
por el subtipo H5N1 desde enero a diciembre del 2009. Los resultados del muestreo mostraron que la presentacio
´n del virus de
influenza A en mercados de aves vivas fue de 0.36% (19/5304). Se recuperaron tres subtipos de influenza A en estos mercados que
incluyeron H4N6 (n52), H4N9 (n51), y H10N3 (n516) a partir de patos reales alojados en un mercado de aves vivas en
Bangkok. Estos subtipos de influenza aviar nunca habı
´an sido reportados en Tailandia, y por lo tanto, esta diversidad gene
´tica
plantea la preocupacio
´n por el potencial reacomodo gene
´tico de estos virus en especies aviares dentro de un entorno particular. Dos
subtipos de influenza A (H4N6 y H4N9) fueron aislados de hisopos orofarı
´ngeos y cloacales del mismo pato, lo que sugiere la co-
infeccio
´n con dos subtipos de la influenza y el posible reacomodo gene
´tico en las aves. Adema
´s, se observo
´la infeccio
´n por el
subtipo H10N3 en patos alojados en el mismo mercado de aves vivas. Estos resultados apoyan las observaciones de que los
mercados de aves vivas son una fuente potencial de transmisio
´n y reacomodo gene
´tico de los virus de influenza aviar.
Key words: influenza A, live-bird markets, H4N6, H4N9, H10N3
Abbreviations: AI 5avian influenza; HA 5hemagglutinin; HPAI 5highly pathogenic avian influenza; IAV 5influenza A virus;
LBM 5live-bird market; LPAI 5low pathogenic avian influenza; NA 5neuraminidase; OIE 5Office International des
E
´pizooties; RT-PCR 5reverse transcriptase–polymerase chain reaction; VTM 5viral transport media
Influenza A virus (IAV) is an enveloped, single-stranded RNA
virus of the family Orthomyxoviridae. The virus is categorized by its
surface proteins, hemagglutinin (HA) and neuraminidase (NA), into
16 HA and 9 NA subtypes (11). Some influenza A subtypes are very
important and can cause a severe, fatal disease such as avian influenza
subtype H5N1 in humans, cats, dogs, tigers, and several avian
species (1,4,22,23,27,29). The Office International des E
´pizooties
(OIE) has included avian influenza (AI) among the OIE-listed
infectious agents that are characterized by causing severe disease, are
fast spreading, and present serious threats to economies and public
health worldwide.
Live-bird markets (LBMs) are known as a potential source of
avian influenza and have been the origin of IAV outbreaks around
the world (7,15,33). A LBM is usually a place for live poultry to be
sold and slaughtered for fresh poultry meat. Various species of birds
at different ages from different sources are usually housed together in
small wire stack cages. These environments are suitable for IAV
transmission both intra- and interspecies (32,36). Direct contact
and/or indirect contact (aerosol or fecal contact) can occur in LBMs
(36) and can cause influenza infection not only in animals but also in
humans.
Surveys of IAV in many countries have documented that multiple
influenza subtypes are found in LBMs: both highly pathogenic avian
influenza (HPAI) and low pathogenic avian influenza (LPAI)
(8,16,18,20,25). Multiple subtypes circulating in such environments
increase the chance of genetic reassortment of the viruses (17,21) and
thus might lead to the emergence of a new pandemic virus. For
example, ‘‘Asian influenza H2N2’’ in 1957 and ‘‘Hong Kong
influenza H3N2’’ in 1968 resulted from genetic reassortment
between avian and human strains and became pandemic viruses that
spread throughout the world (34).
In Thailand, three types of LBMs can be discerned: A)
conventional LBMs, B) LBMs in local food markets, and C) LBMs
in pet bird markets. Conventional LBMs are usually found in rural
areas as well as urban settings. However, since HPAI H5N1
E
Corresponding author. E-mail: Alongkorn.A@chula.ac.th
AVIAN DISEASES 55:593–602, 2011
593

outbreaks occurred in 2004, some LBMs have been reduced to small-
scale LBMs and are mostly located outside the city centers. Many
conventional small-scale LBMs in rural areas are mobile LBMs.
Transportation of the birds by these mobile LBMs poses a threat of
IAV dissemination. The second type comprises LBMs in local food
markets. This type of LBM includes several LBM vendors in a food
market. Some vendors may have a slaughter area in their own space to
process the meat for customers. The third type is LBMs in pet bird
markets. This type of LBM usually houses many poultry species in the
market, fair, or festival along with other pets. Birds sold in this type of
LBM are pet birds such as song birds, wild birds, chicken, and ducks.
Fighting cocks are sometimes included in this type of LBM.
In Thailand, HPAI H5N1 presented a major threat to public
health and the poultry industry (3,26). A survey of HPAI H5N1 in
LBMs and local food markets in Thailand was reported in 2006.
The study showed that HPAI H5N1 viruses were found in the
markets during H5N1 outbreaks. The results supported the belief
that movement of birds during the outbreaks and LBMs could play
an important role in emergence or re-emergence of HPAI H5N1 (2).
However, information on LPAI in LBMs in the country has
remained limited. Thus, active surveillance in the LBM setting can
provide information on genetic diversity and evolution of LPAI. In
this study we have reported a one-year survey of both LPAI and
HPAI in LBMs in Thailand. Our results demonstrated multiple
influenza A subtypes circulating in one LBM.
MATERIALS AND METHODS
Live bird markets. Two LBMs in Bangkok and eight LBMs from
four provinces in central Thailand were selected for a one-year influenza
A survey (Table 1). Based on epidemiological data of H5N1 outbreaks,
the central provinces of Thailand are known as high-risk areas of H5N1
dissemination (28). LBMs selected in this study were based on high risk
of HAPI areas and their availability in Bangkok and the provinces. Many
wild birds and migratory bird species such as open-billed storks and wild
ducks are easily found in paddy fields in these areas. In addition free-
grazing ducks are raised in the same areas and are well known as a
potential influenza virus reservoir (12,24).
In this study the LBMs were characterized as large- or small-scale
LBMs by using the approximate number of birds housed per day. LBMs
that house more than 500 birds were characterized as large scale, whereas
the small-scale LBMs house fewer birds. Approximately, 25 samples
from different bird species were collected monthly from each market
during January–December 2009. If there were not enough birds in the
market at the time of collection, samples were collected from all birds on
that day. In total 5304 oropharyngeal and cloacal swabs of 2652 birds
were collected from various bird species housed in those 10 markets. It
has been noted that one market (market B) was identified as a pet bird
market, while the others were a combination of LBMs and local food
markets (Fig. 1; Table 1).
Influenza A virus isolation and identification. The samples were
collected using sterile polyester tip swabs in viral transport media (VTM)
and transported to the laboratory at 4 C within 24 hr. The samples were
stored at 280 C immediately upon arrival. Each sample was subjected
for individual virus isolation by using egg inoculation per OIE
recommendations. In brief, the supernatant of VTM suspension was
inoculated in 11-day specific pathogen-free embryonated chicken egg
and incubated for 72 hr. All eggs were checked for embryonic death
every day. A hemagglutination test was performed by using the allantoic
fluid collected after 72-hr incubation. Positive HA samples were then
subjected to reverse transcriptase–polymerase chain reaction (RT-PCR)
for IAV identification. Briefly, RNA extraction was conducted by using
a QIAampHviral RNA mini kit (QiagenH, Hilden, Germany). IAV was
confirmed by using real time RT-PCR. Subtyping primer sets for the
HA and NA genes were used to identify the subtype of virus by RT-PCR
(30,31). After subtype identification, specific primers for whole gene
sequencing were designed by the computer program Primer 3 v. 0.4.0.
The specific primers are provided in Table 2. The RT-PCR products
were then subjected to whole genome sequencing as previously described
(4), and the nucleotide sequences were subsequently submitted to
GenBank database.
Genetic analysis of influenza A virus. To generate a phylogenetic
tree, phylogenetic analysis was performed using the MEGA 4.1 program
(Tempe, AZ) with the neighbor-joining method with Kimura two-
parameter and the Bayesian analysis using the MrBayes V.3.1.2 with 1
million generations. Bootstrap analysis with 1000 replications and
BMCMC analysis with posterior probability were performed for
supporting tree topology. To genetically analyze the viruses, nucleotide
sequences and deduced amino acids of H4 and H10 viruses were aligned
and compared using the MegAlign program (DNASTAR).
RESULTS
Influenza A virus in LBMs. The result from the one-year
influenza A survey in 10 LBMs in Thailand in the course of 2009
showed that the occurrence of IAV was 0.36% (19/5304 samples) or
0.49% (13/2652 animals) (Fig. 2). The result showed that 6 out of
13 animals harbored viruses in both oropharyngeal and cloacal
swabs; therefore, altogether 19 isolates of IAV were isolated from 13
animals (three isolates from two animals in June and 16 isolates from
11 animals in November). Interestingly, all 19 influenza A isolates
were recovered from Muscovy ducks in only one LBM located in
Bangkok (market A). Based on analysis by animal species, we found
that the occurrence of IAV in Muscovy ducks was 1.9% (13/679),
whereas other avian species were negative. Based on analysis by
month, the occurrence of IAV found in market A amounted to 5%
(2/40 animals) in June and 36.67% (11/30 animals) in November.
First report of influenza A subtypes H4N6, H4N9, and
H10N3 in Thailand. Influenza A subtypes H4N6 (n52), H4N9
(n51), and H10N3 (n516) were isolated from Muscovy ducks in
an LBM located in Bangkok (market A). It is interesting that none of
Table 1. Description of LBMs surveyed in this study.
Market Province Region Type of markets LBM scale
A Bangkok Bangkok LBM in local food market Large
B Bangkok Bangkok LBM in pet birds market Large
C1 Nakhon Sawan Central LBM in local food market Small
C2 Nakhon Sawan Central Conventional LBM Small
D1 Pichit Central Conventional LBM Small
D2 Pichit Central Conventional LBM Small
E1 Phitsanulok Central Conventional LBM Large
E2 Phitsanulok Central Conventional LBM Small
F1 Sukhothai Central Conventional LBM Small
F2 Sukhothai Central LBM in local food market Small
594 T. Wisedchanwet et al.

these subtypes had ever been reported in Thailand, which may be
because influenza surveillance activities had previously been focusing
on only HPAI. In addition, our laboratory had never worked with
these subtypes prior to this study; therefore, contamination of these
subtypes in the laboratory was ruled out. Influenza A subtypes
H4N6 and H4N9 were isolated in June, and H10N3 was isolated in
November 2009. Neither H5 nor H7 viruses were isolated from any
LBMs in this study. This finding correlated with the Department of
Livestock Development reports of no HPAI H5N1 outbreaks in
2009. Out of 19 virus isolates, seven isolates (H4 53, H10 54)
were subjected to whole genome sequencing. Nucleotide sequences
of those 7 IAVs were submitted to the GenBank database with
assigned accession numbers (Table 3).
Genetic analysis of influenza A subtypes H4N6, H4N9,
and H10N3. Based on phylogenetic analysis, IAV subtypes H4
and H10 recovered in Thailand were clustered into the Eurasian
lineage of the HA and NA genes (Figs. 3 and 4). Analysis of the H4
gene showed low pathogenic characteristics at the HA cleavage site
and avian characteristics at receptor binding sites (Q226 and G228)
(35). Moreover, four N-linked glycosylation sites were observed that
were different from those of viruses in the previous study (five
glycosylation sites). Analysis of the H10 gene showed that amino
acids at the HA cleavage site were ‘‘PEIIQGR,’’ suggesting low
pathogenic characteristics. The receptor binding sites of H10 were
Q226 and G228, indicating preferential binding to a 2,3 linkage
receptor (avian-like receptor). In H10 IAV, five N-linked
glycosylation sites were conserved (19).
DISCUSSION
In this study, three types of LBMs in Thailand were surveyed. We
observed that housing periods of birds in LBMs are different among
types of LBMs. Conventional LBMs and LBMs in local food
markets usually house the birds for 1–5 days because of the demand
for poultry meat. In particular occasions, some LBMs may keep
birds for a longer time (7–14 days) depending on supply and
demand. LBMs in local food markets usually sell animals that have
been purchased directly from dealers. Dealers buy hundreds of birds
Fig. 1. Map of Thailand; provinces of the locations of LBMs are depicted.
Influenza A virus surveillance in live-bird markets in Thailand 595

Table 2. List of oligonucleotide primers for IAV H10N3, H4N6, and H4N9 subtypes designed in the study.
Gene Primer name Size (bp) Primer sequence (59-39)
PB2 PB2-1F 19 agc raa agc agg tca awt a
PB2-1R 18 ccc att gct gcy ttg cat
PB2-2F 21 gga tgg trg aca tyc tta
PB2-2R 22 ggt tca aay tcc atc tta ttg t
PB2-3F 19 caa tga tgt ggg ara tca a
PB2-3R 19 tgg cca tca gta gaa aca a
PB1 (H10) PB1-101F 21 tga atg gat gtc aay ccg act
PB1-101R 19 gat ttg cat tcg ggg tgt t
PB1-102F 22 kca ctg aca ctg aac aca wtg a
PB1-102R 20 aac atg ccc atc mtc att cc
PB1-103F 23 cat gtt cga aag taa gag cat ga
PB1-103R 20 tga gyt ctt caa tgg tgg aa
PB1 (H4) PB1-41F 21 tga atg gat gtc aat ccg act
PB1-41R 21 agc tcc tct tgt tca gcc tct
PB1-42F 24 caa cac att tcc aga gaa aga gaa
PB1-42R 20 tga aca tgc cca tca tca tt
PB1-43F 20 aat acc ggc aga aat gct tg
PB1-43R 20 act att ttt gcc gtc tga gc
PA PA-1F 20 ctt tgt gcg aca atg ctt ca
PA-1R 20 ctg aga gtg cgt ggt gtt gt
PA-2F 20 agg gca agc ttt ctc aaa tg
PA-2R 20 cca cat cgg tgt cat ttc tc
PA-3F 20 cac agc gga agt atc cca tt
PA-3R 20 cag tgc atg tgt gag gaa gg
H10 H10-1F 20 gta ata atc gcg ctcctt gg
H10-1R 20 tct gcc tca gtg ctt ctt ca
H10-2F 20 cac cag ctt gg atc tgc at
H10-2R 20 ccc act gtt ctt ggt gac aa
H10-3F 20 ccc agg tca atg gac aaa gt
H10-3R 20 cag att gtc gat cgc atg tt
H4 H4-1F 20 agc aaa agc agg gga aac aa
H4-1R 20 cgc att tcc gtc tga ctt ta
H4-2F 20 gca aaa tgg gaa atc tgg ag
H4-2R 20 atg ttc ctc atc cct gtt gc
H4-3F 25 aag agt aca att ctg aac act gca a
H4-3R 20 atc tgg cac ctg atg ttt cc
NP NP-1F 22 agc aaa agc agg gta gat aat c
NP-1R 21 cca tcg tcc cga ctc cct tta
NP-2F 21 tga tgc cac ata cca gag aac
NP-2R 23 agt aga aac aag ggt att ttt ct
N3 N3-1F 20 aaa agc agg tgc gag atg aa
N3-1R 22 cca tga ttt aat gga gtc tgt ca
N3-2F 20 tgg aac cat caa aga cag ga
N3-2R 21 tcc att gac gtt act tgg aga
N3-3F 24 gat aca aca ttt gga gga gtg ttc
N3-3R 22 ttt ctg tta ctt ggg cat aaa cc
N6 N6-1F 20 gca aaa gca ggg tga aaa tg
N6-1R 20 agc tcg gaa tgg gct tct at
N6-2F 20 ctg cag gat gtt tgc tct ga
N6-2R 20 cgg tta gga cct ttg agc ac
N6-3F 20 tcc tga aat gat gac cca ca
N6-3R 24 aga aac aag ggt gtt ttt ctt aaa
N9 N9-1F 24 tcc aaa tca gaa gat tct atg cac
N9-1R 26 ttg ttt ggt cct gat ata cat att ga
N9-2F 20 cgc cca cag tat aca aca gc
N9-2R 18 gtc att cgg tcg ggg att
N9-3F 21 gca ggg ctc aaa tag acc ag
N9-3R 21 atc agg cca gtt cca ttg tc
MM-1F 20 agc raa agc agg tag atr tt
M-1R 21 agt aga aac aag gta gtt ttt
NS NS-1F 20 agc raa agc agg gtg aca aa
NS-1R 20 agt aga aac aag ggt gtt tt
596 T. Wisedchanwet et al.

from one source, either from commercial farms or backyards from
nearby cities, and sell the animals directly to LBM vendors. Mixing
of the birds from different sources or shops usually occurs in the
LBMs during these housing periods. In contrast, pet birds in LBMs
are sometimes kept longer than 1 month due to the lower demand.
Sources of these pet birds are either breeders or the shop vendors
themselves.
The occurrence of IAV in LBMs in the course of 2009 was
0.36%, which was lower than reported by a previous study of IAV
H5N1 in LBMs during 2006–2007 (1.3%) (2). As the previous
study’s exclusive aim had been identification of HPAI (H5N1)
during the period of an AI outbreak, the actual prevalence of both
HPAI and LPAI has largely remained unknown. In the course of the
2009 LBM survey, HPAI H5N1 was not detected from any LBM.
This finding correlated with no H5N1 outbreaks having been
reported in Thailand in 2009. Since 2008 no report of HPAI H5N1
in the country has reflected effective disease control measures such as
strict control of animal movements and routine IAV monitoring in
the country. In this study, we were able to identify the three LPAI
subtypes H4N6, H4N9, and H10N3; however, the overall status of
LPAI in Thailand is still unclear. It is noteworthy that H10N3-
infected Muscovy ducks (11/30) were identified from only one
Fig. 2. Detail of samples collected from avian species from January to December 2009.
Influenza A virus surveillance in live-bird markets in Thailand 597

market in Bangkok in November. This high occurrence of LPAI in
ducks on one sampling day may suggest an LPAI outbreak.
However, the origin of the infected flocks could not be traced back
to further identify the source of viruses; therefore, the status of
influenza A H10N3 infection at the farm level is unknown. In
addition, as the sources of H10N3 viruses remained unknown, it
could not be concluded that they had either originated from the
same duck farm or had been picked up at the LBM after mingling.
Although LPAI subtypes in LBMs in this study may or may not
represent the LPAI status in other parts of Thailand, these data
provide information on the diversity of LPAI subtypes circulating in
the country.
All influenza A viruses in this study were collected from 1.9% of
Muscovy ducks (13/679) without any clinical signs observed at the
time of collection, while all other avian species were negative for
IAV. It has been known that ducks and other aquatic birds are a
reservoir of IAV and that almost all HA and NA subtypes can be
isolated from wild ducks without any discernible clinical signs (34).
In this study, five markets have housed and sold ducks; however,
only in market A could three subtypes of IAV be isolated. A previous
study has shown that influenza A viruses are more easily isolated
from juvenile ducks than mature ducks (34). However, in our study,
no IAV could be isolated from juvenile ducks. All influenza viruses
were isolated from mature ducks from one market (market A). In the
study, the viruses could be isolated only from ducks in one market
and in two months (June and November). This may be due to the
nature of the market (market A receives poultry from many central
provinces of Thailand) and a seasonal factor.
Both IAV subtypes H4 and H10 isolated in the study were
clustered into the Eurasian lineage of the HA and NA genes. The
result suggested that the origins of the H4 and H10 IAV circulating
in Thailand were either from wild bird habitats in the Eurasian
flyway (but not North American flyway) or from endemic viruses
(but never been reported). Analysis of H4 viruses showed that the
HA cleavage site of the H4 viruses was PEKATR. The amino acid
pattern at the HA cleavage site indicated it was low pathogenic,
characteristic of influenza virus (14). The receptor binding site of
HA viruses (Q226 and G228) was similar to H4 viruses in the
database, suggesting that the viruses preferentially bind to a 2,3-
linked sialic acid receptor (avian-like receptor) (5). The H4 viruses in
Thailand contain four N-linked glycosylation sites that are less than
those of the viruses described in a previous report, suggesting
changes of biological functions or virulence of the viruses (9).
Analysis of H10 viruses showed that the HA cleavage site of the H10
viruses was PEIIQGR, indicating low pathogenic characteristics.
However, pathogenicity of the H10 IAV may not be determined by
only amino acids at the cleavage site (10). To confirm pathogenicity
and specificity of the viruses to the host, an intravenous
pathogenicity index test should be conducted. The receptor binding
site of H10 viruses contains amino acids (Q226 and G228) that
show preferential binding to an avian receptor. It is noted that
mutation of amino acids at these two positions (Q226L and G228S)
may change the binding ability from 2,3 sialic acid linkage to 2,6
sialic acid linkage, which is predominant in mammalian species (5).
In addition, the H10 viruses posed histidine (H117), glutamic acid
(E184), and glutamine (Q216) (H10 numbering), which favor
binding of 2,3 sialic acid linkage as well (19). Therefore, the ability
of H10 viruses to infect mammalian hosts may be less important. In
addition, 5 N-linked glycosylation sites of H10 isolates were
conserved at the position shown in previous findings (19,37).
Analysis of the N3 gene showed that amino acid deletion was not
observed, which was similar to other N3 viruses in the database. It is
noted that 23 amino acids deletion of the N3 gene are usually
displayed in the viruses from terrestrial or domesticated aquatic birds
but are usually absent in wild avian species (6). In this study, however,
the Muscovy ducks that possessed the H10N3 IAV could not be
traced back to the origin; therefore, we do not know whether these
ducks were raised with or without contact from wild avian species.
In June 2009, H4N6 and H4N9 were isolated from oropharyn-
geal and cloacal swabs, respectively, from the same Muscovy duck.
This result led to the speculation that co-infection with two subtypes
or genetic reassortment may occur while a bird was housed in an
LBM (17). It has been known that IAV can replicate in the intestinal
tract of ducks for 30 days (13,34), and thus, if different IAV subtype
was introduced, multiple subtypes infection might occur in birds
and result in genetic reassortment. It should be noted that genetic
reassortment might result in a new reassorted virus that could
potentially become a virulent strain, as has been the case with the
reassorted H2N2 virus or ‘‘Asian flu’’ in 1957 and the reasserted
H3N2 virus or ‘‘Hong Kong flu’’ in 1968.
Although the time of infection by IAV isolated in LBMs can not
be determined, there have been two main findings that can reflect
the risks of LBMs associated with IAV spread and evolution. First,
in June, two subtypes were isolated from the same duck at the same
time of collection. However, we could not determine if the duck
had been infected with these two subtypes before or after entering
the LBM. If the infection occurred after entering the LBM, this
would provide evidence that reassortment of IAV can occur in
LBMs, but if the infection occurred before entering the LBM, this
would prove that multiple influenza A subtypes entered the LBM
during the same period, which would create a risk of reassortment
of IAV in LBMs. Second, in November, from 30 animals sampled
during this month, 11 animals were positive for IAV. If the
infection occurred after entering the LBM, this would prove that
LBMs are suitable environments for transmission and spread of
IAV. But if the infection occurred before entering the LBM, this
would indicate that a high rate of infected animals are entering
LBMs and may transmit to other animals in LBMs. Based on these
two findings, LBMs can play an important role in dissemination
and evolution of IAV.
Table 3. Detail description and GenBank accession numbers of IAVs isolated from Muscovy ducks in LBMs.
Taxon name Month and year Subtype Accession no.
A/Muscovy duck/Bangkok/Thailand/CU-LM1973/2009 June 2009 H4N6 CY062545–CY062552
A/Muscovy duck/Bangkok/Thailand/CU-LM1983/2009 June 2009 H4N6 CY062553–CY062560
A/Muscovy duck/Bangkok/Thailand/CU-LM1984/2009 June 2009 H4N9 CY062561–CY062568
A/Muscovy duck/Bangkok/Thailand/CU-LM4754/2009 November 2009 H10N3 CY062569–CY062576
A/Muscovy duck/Bangkok/Thailand/CU-LM4759/2009 November 2009 H10N3 CY062577–CY062584
A/Muscovy duck/Bangkok/Thailand/CU-LM4761/2009 November 2009 H10N3 CY062585–CY062592
A/Muscovy duck/Bangkok/Thailand/CU-LM4775/2009 November 2009 H10N3 CY062593–CY062600
598 T. Wisedchanwet et al.

Based on our results, surveys of IAV in LBMs can help identify
potential sources of influenza transmissions and genetic reassort-
ment. Preventive measures to reduce the risks of IAV introduction
should be considered, such as certified poultry farms, no practices
of mingling ducks and chickens, and avoiding mixing poultry
from different farms. Several subtypes of IAV isolated in the
LBMs can reflect diversity of viruses circulating in Thailand.
Genetic diversity observed in LBMs serves as good evidence of
Fig. 3. Phylogenetic analysis of HA gene of H4 and H10 influenza A viruses. The viruses isolated in the study are depicted with a star symbol.
Influenza A virus surveillance in live-bird markets in Thailand 599

Fig. 4. Phylogenetic analysis of NA gene of N6, N9, and N3 influenza A viruses. The viruses isolated in the study are depicted with a star symbol.
600 T. Wisedchanwet et al.

settings suitable for dissemination and genetic reassortment of
IAV. In conclusion, continuous surveys of IAV in LBMs should
be carried out in order to monitor influenza virus transmission
and evolution in Thailand.
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ACKNOWLEDGMENTS
This work has been funded in whole with federal funds from the
National Institute of Allergy and Infectious Disease, National Institute
of Health, Department of Health and Human Services, under Contract
No. HHSN266200700007c. Its contents are solely the responsibility of
the authors and do not necessarily represent the official views of the
NIH. We also would like to thank the National Research Council of
Thailand (NRCT, Thailand) and the National Research University of
CHE and the Ratchadaphiseksomphot Endowment Fund (HR1155A)
for grant support to Dr. Amonsin. We would like to thank Ms. Petra
Hirsch for reviewing the manuscript.
602 T. Wisedchanwet et al.