﻿Human Infection
from Avian-like
Influenza A (H1N1)
Viruses in Pigs,
China
Huanliang Yang,1
Chuanling Qiao,1
Xu Tang,
Yan Chen, Xiaoguang Xin, and Hualan Chen
In investigating influenza in an immunodeficient child in
China, in December 2010, we found that the influenza virus
showed high sequence identity to that of swine. Serologic
evidence indicated that viral persistence in pigs was the
source of infection. Continued surveillance of pigs and
systemic analysis of swine influenza isolates are needed.
Humans have been infected with avian-like swine
influenza A (H1N1) viruses (SIVs) several times
since the first case was diagnosed in Switzerland in 1986
(1). These cases generally occur in persons who have direct
exposure to pigs (2­4). On December 31, 2010, a 3-year-old
boy in rural Jiangsu Province, People's Republic of China,
who had chronic renal disease (for which he was given
long-term steroid treatment), sought care with influenza-
like symptoms. Laboratory tests at the Chinese Center for
Disease Control and Prevention yielded a positive result
for European avian-like A (H1N1) SIV, indicating that the
European avian-like SIV also caused human infection in
the Asia-Pacific region.
The Study
After notification of the boy's infection from the
Ministry of Health, we performed active public health
surveillance to locate the origin of the infection. A total
of 60 nasal swab specimens were collected from pigs at
the patient's family farm and a local slaughterhouse.
Each swab was placed in 2 mL of minimal essential
medium supplemented with penicillin (2,000 U/mL)
and streptomycin (2,000 U/mL). Virus was isolated by
using 10-day-old specific pathogen-free embryonated
chicken eggs. Hemagglutinin (HA) and neuraminidase
(NA) subtypes were determined as described (5). Three
A (H1N1) SIVs were obtained, including 2 isolates from
pigs in the slaughterhouse and 1 from a pig raised at the
family's farm. Viral RNA was extracted and reverse
transcribed under standard conditions by using the Uni12
(5-AGCAAAAGCAGG-3) primer. The viral genomes
were amplified by PCR and sequenced by using segment-
specific primers (sequences available on request). Genomic
sequencing ultimately showed that the 3 isolates were
virtually identical, and the sequence of the entire genome
of the representative strain A/swine/Jiangsu/40/2011
(Sw/JS/40/11) is available in GenBank (accession nos.
JQ319645­JQ319652). Unrooted phylogenetic trees were
generated by using MEGA5 software (www.megasoftware.
net). The A (H1N1) viruses isolated in this study fell
into the European avian-like swine A (H1N1) lineage
(Figure 1). The homology of the polymerase basic protein
(PB) 2, PB1, polymerase acidic protein, HA, nucleocapsid
protein, NA, matrix (M), and nonstructural protein genes
between the Sw/JS/40/11 virus and the A/Jiangsu/1/2011
(JS/1/11) virus, which was isolated from the child, were
99.3%, 99.3%, 99.3%, 99.7%, 99.7%, 99.4%, 99.6%, and
99.1%, respectively, indicating that they might have been
derived from the same ancestor.
The receptor-binding property of the HA protein is
a major molecular determinant of host range. The amino
acids at sites 190 and 225 of HA are major determinants of
the receptor-binding specificity of the A (H1N1) virus, and
the mutations E190D and D225E in HA switch the virus
receptor-binding specificity from -2,3­linked sialosides to
-2,6­linked sialosides (6). The Sw/JS/40/11 and JS/1/11
isolates have the amino acids D at site 190 and E at site
225 within the HA protein, which implies that these viruses
might preferentially bind to -2,6­linked sialosides.
Potential glycosylation sites (PGSs) also have a major
effect on the antigenic and receptor-binding properties of
influenza A viruses. Molecular analysis showed that the 2
Jiangsu strains had 5 PGSs in their HA1 proteins, 4 of which
were the same as those of the A/Netherlands/386/1986 virus
(the cause of the first avian-like SIV infection in a human).
Antigenic sites in the H1 HAs, i.e., Sa, Sb, Ca1, Ca2, and
Cb, were compared between A/Netherlands/386/1986
and JS/1/11. Amino acid mutations H159N, K238R, and
G239E were observed at the Ca2 site; R187G at the Ca1
site; and T202D, N203S, S207T, and A212N at the Sb
site. Compared with JS/1/11, the unique mutation D204V,
located at the Sb site, which is an antigenic site near the
receptor-binding site in influenza virus (7), occurred
in the HA1 of Sw/JS/40/11 (Figure 2). No oseltamivir
resistance­conferring substitutions (H274Y and N294S)
were observed in the NA proteins of the 2 viruses, which
suggests that they are sensitive to NA inhibitors (8). The
amino acid sequence of the M2 protein of the 2 isolates did
not contain the I27T or S31N substitution, characteristic
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1144 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 18, No. 7, July 2012
1
These authors contributed equally to this article.
Author affiliation: State Key Laboratory of Veterinary Biotechnology­
Harbin Veterinary Research Institute, Harbin, People's Republic of
China
DOI: http://dx.doi.org/10.3201/eid1807.120009
Avian-like Influenza A (H1N1) Viruses
of amantadine resistance in influenza viruses (9,10). The
627K and 701N residues in the PB2 protein contribute to
the replication and transmission of avian influenza viruses
in mammalian hosts (11­14). Similar to most avian-like A
(H1N1) SIVs, both isolates (JS/1/11 and Sw/JS/40/11) had
701N in their PB2 gene.
Chicken antiserum against different subtype H1N1
or H1N2 SIVs were used for antigenic analysis. The Sw/
JS/40/11 virus reacted with the antiserum against the
classical A (H1N1) SIV (A/swine/Guangdong/6/2010), the
triple reassortant A (H1N2) SIV (A/swine/Tianjin/1/2007),
influenza A(H1N1)pdm09 (A/swine/Heilongjiang/44/2009),
and the avian-like SIV (A/swine/Henan/11/2005) (Table),
but not with the antiserum against the human-like A (H1N1)
SIV (A/swine/Hebei/15/2009). Antiserum against Sw/
JS/40/11 reacted only with the avian-like A (H1N1) SIV
and the human-like A (H1N1) SIV, but the HI titers against
the human-like A (H1N1) SIV were 4-fold lower than
those against the avian-like A (H1N1) SIVs. These results
suggest that the H1 subtype SIVs circulating in China differ
antigenically.
We investigated antibody responses in 20 serum
samples from pigs at the patient's family farm and the
local slaughterhouse. Serologic assays showed that the
seroprevalence of antibodies to the avian-like A (H1N1)
SIVs was 55% and to classical A (H1N1) SIVs and
A(H1N1)pdm09 virus were 25% and 30%, respectively.
Furthermore, antibodies against A(H3N2) SIVs were
observed but at the low rate of 10%.
Conclusions
We showed that similar viruses were simultaneously
prevalent in a local pig population when a child was infected
with an avian-like A (H1N1) SIV. Specifically, isolation of
avian-like SIV from a family farm provides direct evidence
for the origin of the human infection. No further spread of
the Sw/JS/40/2011-like swine strain occurred, according
to the limited information available; however, the incident
aroused interest in influenza in animals, especially in pigs.
Antigenic analysis showed that this avian-like A (H1N1)
SIV was antigenically divergent from classical A (H1N1)
and human-like A (H1N1) SIVs currently circulating in
China, which was further reinforced by the heterogeneity
of their genetic relationships. Since early avian-like A
(H1N1) SIV isolates in humans, amino acid mutations in
the antigenic sites and PGS changes might have altered
the antigenic properties in the avian-like A (H1N1)
SIV cluster. Our data highlight the need to characterize
circulating strains antigenically and genetically through
regular influenza virus surveillance.
Pigs can serve as intermediate hosts for influenza
viruses to evolve toward efficient replicability in humans.
The classical A (H1N1) SIVs and European avian-like
A (H1N1) SIVs have circulated worldwide in pigs since
1930 and 1979, respectively, and a classical A (H1N1)
SIV emerged in humans as a triple reassortant, causing the
2009 influenza pandemics (15). Although the virulence and
transmissibility of the avian-like A (H1N1) SIVs remain
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 18, No. 7, July 2012 1145
Figure 1. Phylogenetic tree of selected swine, human, and avian
H1 hemagglutinin 1 sequences. An unrooted phylogenetic tree
was generated by the distance-based maximum-likelihood method
by using MEGA5 software (www.megasoftware.net). Bootstrap
values were calculated on the basis of 1,000 replications; A/swine/
Jiangsu/40/2011 is in boldface. Scale bar indicates nucleotide
substitutions per site.
Figure 2. Multiple alignment of
hemagglutinin protein sequences.
Epitopes Sa, Sb, Ca1, Ca2, and Cb
are indicated. Triangle, Sa; circle,
Sb; square, Ca1; hexagon, Ca2;
diamond, Cb. Putative glycosylation
sites are indicated in blue-lined
boxes.
to be evaluated, recurrent human infections with avian-like
A (H1N1) SIVs suggest that after long-term adaptation in
pigs, the avian-like A (H1N1) SIVs already can replicate
in humans. After further whole-genome adaptation to
the human host or reassortment with other viruses, novel
strains bearing the avian-like swine subtype H1N1 HA gene
are highly likely to be generated with pandemic potential.
Continued surveillance of swine and systemic analysis of
swine influenza isolates are clearly needed.
Acknowledgments
We thank Susan Watson for editing the manuscript.
This study was supported by the 973 Program
(2011CB505001, 2010CB534001), the Chinese National Science
Fund for Distinguished Young Scholars (30825032), Harbin
Municipal S&T Plan (2009AA6BN078), and the Scientific
Research Program of the State Key Laboratory of Veterinary
Biotechnology (NKLVSP201013).
Dr Yang is a veterinary microbiologist in the Harbin
Veterinary Research Institute, Chinese Academy of Agricultural
Sciences, China. His research interests are the surveillance and
molecular epidemiology of SIVs.
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Address for correspondence: Hualan Chen, Harbin Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, 427 Maduan St,
Harbin 150001, People's Republic of China; email: hlchen1@yahoo.com
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1146 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 18, No. 7, July 2012
Table. Antigenic analysis of H1 swine influenza viruses, People's Republic of China*
Virus
HI antibody titers of chicken antiserum against
Classical swine
A (H1N1)
Triple-reassortant
A (H1N2)
A (H1N1)
pdm09
Human seasonal
A (H1N1)
Avian-like swine
A (H1N1)
Avian-like swine
A (H1N1)§
Classical swine A (H1N1) 512 512 512 ¶ 8 ¶
Triple-reassortant A (H1N2) 1024 512 1,024 ¶ 8 ¶
A(H1N1)pdm09 512 512 1,024 ¶ 16 ¶
Human seasonal A (H1N1) 16 32 64 32 32 64
Avian-like swine A (H1N1) 128 64 128 ¶ 512 256
Avian-like swine A (H1N1)§ 32 16 64 ¶ 256 256
*Classical swine A (H1N1), A/swine/Guangdong/6/2010; triple-reassortant A (H1N2), A/swine/Tianjin/1/2007; A(H1N1)pdm09,
A/swine/Heilongjiang/44/2009; human seasonal A (H1N1), A/swine/Hebei/15/2009.
Antiserum was generated by inoculating specific pathogen-free chickens with an oil-emulsified inactivated vaccine derived from the indicated viruses.
Homologous titers are shown in boldface.
A/swine/Jiangsu/40/2011.
¶HI titer <2.
§A/swine/Henan/11/2005.
