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Arch Virol

DOI 10.1007/s00705-011-0940-0


Emergence of novel reassortant H3N2 influenza viruses
among ducks in China
Hongbo Zhou • Anding Zhang • Huanchun Chen •

Meilin Jin

Received: 22 November 2010 / Accepted: 24 January 2011
Ó Springer-Verlag 2011

Abstract During 2006-2009 influenza virus surveillance, northern China [6, 7]. However, little is known about the
three H3N2 viruses were isolated from ducks in Central prevalence of H3N2 influenza virus infection in avian
China. Sequence and phylogenetic analyses revealed that populations in China. In this study, we describe the isola-
most segments of these three isolates had high identity with tion and genetic characterization of H3N2 influenza viruses
H3N2 swine isolates in South China. However, for M, the from domestic ducks in China.
three viruses, along with H1N1 swine isolates of North To learn more about the prevalence of influenza virus
America, formed a cluster; for PB2, two of these isolates among avian populations in China, influenza virus sur-
fell into the cluster of the H5N1 duck isolates, indicating a veillance of avian species including chicken, duck, goose,
reassortment among H3N2, H1N1 swine viruses and H5N1 heron, magpie, mallard, pigeon and widgeon in provinces
avian virus. The emergence of H3N2 virus with incorpo- of Central and Eastern China, including Henan, Hubei,
ration of an H5N1 virus gene raises new concerns about the Hunan, Jiangxi, Anhui and Jiangsu, was carried out from
generation of novel viruses that could affect humans. 2006 to 2009. Over 150 influenza strains, including H3N2,
H4N6, H5N1, H5N2 and H9N2 subtypes, were isolated in
embryonated SPF chicken eggs and stored at -80°C for
At present, H3N2 is the most commonly detected influenza further characterization. Interestingly, three H3N2 viruses
virus in humans and swine [2], but it is sporadic in birds. In isolated from cloaca swabs from ducks, A/Duck/HuBei/06/
1998, members of a new lineage of swine influenza viruses, 2009 (H3N2), A/Duck/HuBei/09/2009 (H3N2) and A/Duck/
H3N2 triple reassortants (TR) with genes derived from HuBei/10/2009 (H3N2), were found to be different from the
human (HA, NA, and PB1), swine (M, NS, and NP), and H3N2 influenza viruses circulating among humans and swine
avian viruses (PA and PB2) were first isolated from pigs in in the world.
the United States [3, 10, 13]. In 2003 and 2004, similar RT-PCR was used to amplify the full-length genomes of
viruses (H3N2 TR) were isolated from turkeys in two the three H3N2 viruses for sequence analysis as described
different locations in the U.S. [1, 9]. Previous studies previously [12]. First, viral RNA was extracted from
demonstrated serologic evidence for H3 avian influenza 250 ll of allantoic fluid using Trizol Reagent (GIBCO-
virus infection in chicken populations in several locations BRL), and reverse transcription was performed using
throughout China and also reported the isolation of H3N8 a universal oligonucleotide primer for influenza virus:
influenza viruses from domestic ducks during 2004-2005 in 5’-AGC AAA AGC AGG-3’. Then, a series of gene-specific
primers were designed to amplify the eight genomic seg-
ments for sequencing. Finally, sequence data were edited and
analyzed using the software DNAStar. Phylogenetic trees
H. Zhou  A. Zhang  H. Chen  M. Jin (&) were generated by the neighbor-joining algorithm.
State Key Laboratory of Agricultural Microbiology, The sequence homologies of the three H3N2 isolates
College of Veterinary Medicine, Huazhong Agricultural
were determined by comparison with sequences available
University, Shizishan Street, Hongshan district,
Wuhan 430070, P.R. China in GenBank (Table 1). The isolates were found to be
e-mail: similar to recent H3N2/Guangdong swine influenza viruses

H. Zhou et al.

A/Swine/Guangdong/110/2002 /H3N2 (99.9%)
A/Swine/Guangdong/110/2002 /H3N2 (99.8%)
Table 1 List of viruses with the highest sequence identity to each gene from A/Duck/HuBei/06/2009, A/Duck/HuBei/09/2009 and A/Duck/HuBei/10/2009 using reference virus sequences
in the PB1, PA, HA, NP, NA gene segments, with

A/Swine/GuangDong/110/2002/H3N2 (99.7%)

A/Swine/Guangdong/110/2002/H3N2 (99.8%)
A/swine/Guangdong/106/2002/H3N2 (99.7%)

A/swine/Guangdong/106/2002/H3N2 (100%)
A/swine/Tennessee/25/1977/H1N1 (99.7%)
homologies ranging from 99.6% to 100%.

A/Swine/Colorado/1/1977/H3N2 (99.7%)
The NS segments of the three isolates possessed the
highest identity with the NS of H3N2 swine influenza virus
A/Swine/Colorado/1/1977, with homologies ranging from
99.7% to 99.9%. However, the H3N2 isolates from ducks
were closely related to early swine H1N1 influenza viruses

(A/swine/Tennessee/25/1977) in the M gene segment, and
the PB2 gene segments of two of the three isolates
(A/Duck/HuBei/06/2009 and A/Duck/HuBei/09/2009)
were closely related to the recent H5N1 avian influenza
viruses (A/Duck/Hunan/8/2008), with 99.7% homology.
In the phylogenetic trees of the HA (Fig. 1), PB1, PA, NP
and NA genes (data not shown), three H3N2 duck isolates
along with the H3N2 swine isolates formed a cluster (swine
lineage) that was separate from the H3N2 human and avian
lineages. Interestingly, however, the M genes of the three

A/Swine/Guangdong/110/2002 /H3N2 (99.7%)
isolates were not in the H3N2 swine viruses cluster but

A/Swine/GuangDong/110/2002/H3N2 (99.7%)

A/Swine/Guangdong/110/2002/H3N2 (99.8%)
A/swine/Guangdong/106/2002/H3N2 (99.7%)

A/swine/Guangdong/106/2002/H3N2 (100%)
instead in the classical H1N1 swine influenza virus cluster,

A/swine/Tennessee/25/1977/H1N1 (99.8%)
A/Swine/Colorado/1/1977/H3N2 (99.9%)
indicating that reassortment had occurred between H1N1

A/Duck/Hunan/8/2008/H5N1 (99.7%)
and H3N2 swine influenza viruses (Fig. 1). In addition, the
PB2 genes of A/Duck/HuBei/06/2009 and A/Duck/HuBei/
09/2009 also fell into the cluster of H5N1 avian influenza
virus (Fig. 1), indicating a reassortment among H3N2 swine,

classical H1N1 swine and duck-origin H5N1 influenza
viruses. Therefore, in this study, one of the three duck
influenza virus isolates was identified as a reassortant and the
other two were triple-reassortant H3N2 viruses.
A previous study showed that avian-to-pig interspecies
Virus with the highest nucleotide identity (% nucleotide identity)

transmission and coexistence had occurred in H9N2 viru-
ses of ten genotypes isolated from pigs in China, and three
of the ten genotypes possessed the H5-like avian poly-
merase gene [11], which indicated that swine influenza
viruses with H5-like gene segments had been circulating in
the swine population in China. On the other hand, there
A/Swine/Guangdong/110/2002 /H3N2 (99.6%)
A/Swine/Guangdong/110/2002 /H3N2 (99.9%)
A/Swine/GuangDong/110/2002/H3N2 (99.7%)
A/Swine/GuangDong/110/2002/H3N2 (99.7%)
A/swine/Guangdong/106/2002/H3N2 (99.8%)

was also evidence that avian H5 virus had infected pigs in
A/swine/Tennessee/25/1977/H1N1 (98.8%)
A/Swine/Colorado/1/1977/H3N2 (99.9%)

China [4], which indicated the possibility of the emergence
A/Duck/Hunan/8/2008/H5N1 (99.7%)

of reassortants of H5-like avian virus and the swine virus.
H5N1, H5N2, H9N2 have circulated widely among
avian populations in China, but H3N2 viruses had not been
reported until 2009 in China [8]. In this study, the iso-

lates A/Duck/HuBei/06/2009 and A/Duck/HuBei/09/2009,
originating from reassortment of H3N2 swine, classical
H1N1 swine and duck-origin H5N1 influenza viruses, were
found to be viruses of a novel genotype that differed from
H3N2 influenza viruses circulating among avian species in
the world. A previous study showed that novel H3N2
available in GenBank

reassortant virus, which emerged in the United States
swine population in 1998, were generated by interspecies
transmission from pigs to turkeys in two geographically
distant farms in the United States in 2003 [1]. Phylogenetic
analysis of A/Duck/HuBei/10/2009 also indicates inter-





species transmission of H3N2 from pigs to ducks.

Reassortant H3N2 influenza viruses among ducks in China

Fig. 1 Phylogenetic trees for the HA (nucleotides 30-1730 of HA), distances are proportional to the minimum number of nucleotide
PB2 (nucleotides 28-2314 of PB2) and M (nucleotides 26-1007 of M) differences required to join nodes. The underlined viruses are the
genes of the H3N2 influenza A viruses generated with the software H3N2 viruses isolated and sequenced in this study
DNAStar by using the neighbor-joining algorithm. Horizontal

A previous study showed that multiple reassortments are novel reassortants of swine-like H3N2 avian influenza
occurred when pigs were co-infected with the triple reas- virus and duck-origin H5N1 influenza virus. It is especially
sortant H3N2 (Tx/98) and classical H1N1 viruses; how- worrying that the H3N2 virus has been transmitted from
ever, only specific gene constellations were readily pigs to birds, and possibly vice versa. Our studies
transmissible, and certain HA and NA gene pairs, in con- emphasize the constant need for influenza surveillance in
junction with the TRIG cassette, may have a competitive swine and domestic and wild avian populations and genetic
advantage over other combinations for transmission and analysis of the viral genome for early identification of such
maintenance in swine [5]. In this study, the novel reas- reassortment events. Only by doing this can we better
sortant H3N2 influenza viruses contained a modified TRIG understand interspecies transmission and the emergence of
cassette with the M gene from the classical H1N1 SIV and novel influenza viruses with the potential to infect humans.
the PB2 gene from the H5 avian virus. However, the spe-
cific role of each novel gene in pig-to-avian interspecies Acknowledgments The study was supported by China National
Basic Research Program (China ‘‘973’’ Program 2011CB505004 &
transmission of these H3N2 viruses remained unknown and 2010CB534002), National Natural Science Foundation of China (no.
needs further investigation. 31072154 & 30800832), and China National Science and Technology
Repeated introductions of swine influenza viruses to Major Projects (no. 2008ZX10004-013 and 2009ZX10004-109), the
avian species infected with avian influenza viruses may National Transgenic Major Program (2009ZX08009-141B).
lead to coinfection and provide opportunities for the
emergence of novel reassortants with genes adapted for
replication in pigs or even humans [1]. Likewise, the References
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