﻿Genome Sequence of a Natural Reassortant H5N2 Avian Influenza
Virus from Domestic Mallard Ducks in Eastern China
Min Gu,a,b
Junqing Huang,a
Yuxin Chen,a
Jian Chen,a
Xiaoquan Wang,a,b
Xiaowen Liu,a,b
and Xiufan Liua,b
Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China,a
and Ministry of Education Key Lab for Avian
Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, Chinab
Here, we report the genomic sequence of a Chinese reassortant H5N2 avian influenza virus which possessed the polybasic motif
PLREKRRK-R/GL at the hemagglutinin cleavage site. Phylogenetic analysis showed that all eight genes were of the Eurasian lin-
eage, five of which were highly homologous to the endemic clade 2.3.4 H5N1 viruses and their H5N5 reassortant descendants.
These data suggested that novel multisubtypic NA reassortants bearing the H5N1 backbone could be generated through genetic
reassortment in H5N1 circulating regions, which will help in understanding the evolution and segment reassortment mecha-
nism of H5 subtype avian influenza viruses.
Avian influenza virus, a member of the family Orthomyxoviri-
dae, is specified into 16 HA (hemagglutinin) subtypes and 9
NA (neuraminidase) subtypes according to the antigenicity of the
two surface glycoproteins HA and NA (7, 8). Among these, the
H5N1 subtype combination of the Gs/GD lineage is of great con-
cern, as it has caused disastrous damage to the poultry industry
and has posed a serious threat to public health (7). In particular,
the clade 2.3.4 Fujian-like H5N1 viruses have gradually became
endemic in China since 2005 (3, 5) and continue to evolve actively
in China, Vietnam, Laos, and neighboring regions (9). Further-
more, they could even provide the backbones to generate a novel
NA subtype of H5 highly pathogenic avian influenza (HPAI) vi-
ruses, such as H5N5, via genetic reassortment (1).
In this study, an H5N2 virus, A/duck/Jiangsu/m234/2012
(m234), was isolated from apparently healthy domestic mallard
ducks in the Jiangsu Province of eastern China in January 2012. To
characterize this strain in detail, we determined the whole genome
sequence by reverse transcription-PCR performed with the uni-
versal primer set (2) and by sequencing using the ABI 3730 DNA
analyzer (Applied Biosystems).
Eight negative-sense RNA segments, including PB2, PB1, PA,
HA, NP, NA, M, and NS genes, constitute the viral genome, with
full lengths of 2,341, 2,341, 2,233, 1,776, 1,565, 1,466, 1,027, and
890 nucleotides, respectively. In contrast to the recently reported
Chinese H5N2 isolates (4, 10), m234 carried the polybasic motif
PLREKRRK-R/GL at the HA cleavage site, which was consistent
with the molecular signature of HPAI virus. However, typical res-
idues Q226 and G228 (H3 numbering) in the receptor binding
pocket of HA protein indicated that m234 retained the avian-like
SA 2,3Gal receptor preference. The 5-amino-acid deletion at po-
sitions 80 to 84 of the NS1 protein, which has been proven to exist
in more and more H5N1 viruses isolated from 2000 onwards (6),
was not detected in m234.
Phylogenetic analysis showed that all eight genes were of the
Eurasian lineage. The PB2, PA, HA, NP, and M genes were highly
homologous (97% to 98%) to the previously identified reassortant
HPAI H5N5 viruses A/duck/Eastern China/031/2009 and
A/duck/Eastern China/008/2008 (1) and their clade 2.3.4 H5N1
progenitors A/duck/Eastern China/108/2008 and A/duck/Eastern
China/909/2009. The PB1 and NA genes shared the greatest DNA
sequence identities (over 98%) with A/duck/Jiangxi/k0701/
2009(H11N2), whereas the NS gene was most closely related (over
95%) to A/duck/Nanchang/1904/1992(H7N1), which was iso-
lated 2 decades ago. Therefore, we speculated that m234 may
probably be reassorted from the above-mentioned duck origin
avian influenza viruses. The isolation of this natural reassortant
H5N2 virus further highlights that the genetic stability between
the HA and NA subtype pairing of the clade 2.3.4 H5N1 HPAI
viruses may have been disequilibrated to generate novel multisub-
typic NA reassortants based on their genetic backbones.
In conclusion, the genome information of A/duck/Jiangsu/
m234/2012(H5N2) will be helpful to study the evolution and seg-
ment reassortment mechanism of H5 subtype avian influenza vi-
rus in areas where the virus is endemic.
Nucleotide sequence accession numbers. The genome se-
quences of A/duck/Jiangsu/m234/2012(H5N2) were deposited in
GenBank under accession numbers JX507352 to JX507359.
ACKNOWLEDGMENTS
This work was supported by the Major State Basic Research Development
Program of China (973 Program) (grant no. 2011CB505003), the Na-
tional Key Technologies R&D Program of China (no. 2010BAD04B01),
the Priority Academic Program Development of Jiangsu Higher Educa-
tion Institutions (PAPD), and the Program for Changjiang Scholars and
Innovative Research Team in University (PCSIRT, IRT0978).
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Received 27 August 2012 Accepted 27 August 2012
Address correspondence to Xiufan Liu, xfliu@yzu.edu.cn.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.02315-12
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