﻿JOURNAL OF VIROLOGY, May 2009, p. 4205­4215 Vol. 83, No. 9
0022-538X/09/$08.000 doi:10.1128/JVI.02403-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Isolation and Genetic Characterization of H5N2 Influenza Viruses
from Pigs in Korea
Jun Han Lee,1
Philippe Noriel Q. Pascua,1
Min-Suk Song,1
Yun Hee Baek,1
Chul-Joong Kim,2
Hwan-Woon Choi,2,3
Moon-Hee Sung,4
Richard J. Webby,5
Robert G. Webster,5
Haryoung Poo,6
and Young Ki Choi1
*
College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763,
Republic of Korea1
; College of Veterinary Medicine, Chungnam National University, 220 Gung-Dong, Yuseoung-Gu, DaeJeon 305-764,
Republic of Korea2
; Choongang Vaccine Laboratory, Daejeon 305-348, Republic of Korea3
; Bioleaders Corporation,
Daejeon, Republic of Korea4
; Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital,
262 Danny Thomas Place, Memphis, Tennessee 381055
; and Korean Research Institute of Bioscience and
Biotechnology, Daejeon, Republic of Korea6
Received 20 November 2008/Accepted 3 February 2009
Due to dual susceptibility to both human and avian influenza A viruses, pigs are believed to be effective
intermediate hosts for the spread and production of new viruses with pandemic potential. In early 2008, two swine
H5N2 viruses were isolated from our routine swine surveillance in Korea. The sequencing and phylogenetic analysis
of surface proteins revealed that the Sw/Korea/C12/08 and Sw/Korea/C13/08 viruses were derived from avian
influenza viruses of the Eurasian lineage. However, although the Sw/Korea/C12/08 isolate is an entirely avian-like
virus, the Sw/Korea/C13/08 isolate is an avian-swine-like reassortant with the PB2, PA, NP, and M genes coming
from a 2006 Korean swine H3N1-like virus. The molecular characterization of the two viruses indicated an absence
of significant mutations that could be associated with virulence or binding affinity. However, animal experiments
showed that the reassortant Sw/Korea/C13/08 virus was more adapted and was more readily transmitted than the
purely avian-like virus in a swine experimental model but not in ferrets. Furthermore, seroprevalence in swine sera
from 2006 to 2008 suggested that avian H5 viruses have been infecting swine since 2006. Although there are no
known potential clinical implications of the avian-swine reassortant virus for pathogenicity in pigs or other species,
including humans, at present, the efficient transmissibility of the swine-adapted H5N2 virus could facilitate virus
spread and could be a potential model for pandemic, highly pathogenic avian influenza (e.g., H5N1 and H7N7) virus
outbreaks or a pandemic strain itself.
The infection of swine with influenza A viruses is of signif-
icant importance to the swine industry and to the epidemiology
of human influenza. The severity of the clinical symptoms
appears to be dependent upon the infecting virus strain, the
age and immune status of the animal, and the presence of
concomitant pathogens or environmental stress factors. Swine
influenza viruses (SIVs) also cause respiratory diseases in hu-
mans, and several instances of the zoonotic transmission of
SIV from pigs have been reported (13, 22, 36, 43). The ability
of the influenza virus to cross between animal species is con-
trolled by the viral genes, and the prevalence of transmission
depends on the species involved.
Domesticated pig species are known to allow the productive
replication of both avian and human influenza viruses. This
susceptibility is due to the presence of both -2,3­ and 2,6­
galactose-sialic acid (sialyloligosaccharide) linkages of cellular
receptors lining the pig trachea (22). Successful transmission
between animal species can follow genetic reassortment in
which a progeny virus containing a specific gene constellation
has the ability to replicate in the new host. Under experimental
conditions, pigs are susceptible to infection with a range of
avian and human influenza viruses (29), and though not often
documented, the interspecies transmission of avian viruses to
pigs in nature has been reported. Pigs in Europe and China
have been infected with avian H1N1 viruses (14, 37), and a
purely avian H4N6 influenza virus caused a disease outbreak in
pigs in Canada (27). In China, Peiris et al. reported on swine
infections with avian H9N2 influenza virus, and since then,
frequent exposure to this subtype has been documented (34,
41). Lately, reassortant viruses between avian H5N1 and H9N2
subtypes caused pig diseases and death in some parts of China
(6, 7, 57), and in the United States, reassortant H2N3 viruses
(between American avian-like and contemporary swine triple
reassortant-like) recently have been identified (32).
In Korea, four subtypes (H1N1, H1N2, H3N1, and H3N2) of
SIVs have been reported in the pig population (23­25, 49).
Phylogenetic analyses have indicated that the Korean isolates
were closely related to the triple-reassortant SIVs recently
isolated from pigs in the United States containing internal
segments derived from human (PB1), swine (NP, M, and NS),
and avian (PB2 and PA) sources (23, 24, 47, 50). More re-
cently, Pascua et al. reported that under vaccine pressure, a
new serogroup of H3N2 SIVs had genetically evolved among
Korean swine (39).
We now describe the isolation and characterization of two
novel H5N2 swine influenza viruses, A/Swine/Korea/C12/08
* Corresponding author. Mailing address: College of Medicine
and Medical Research Institute, Chungbuk National University, 12
Gaeshin-Dong, Heungduk-Ku, Cheongju 361-763, Republic of Ko-
rea. Phone: 82-43-261-3384. Fax: 82-43-272-1603. E-mail: choiki55
@chungbuk.ac.kr.

Published ahead of print on 18 February 2009.
4205
(Sw/Kor/C12/08) and A/Swine/Korea/C13/08 (Sw/Kor/C13/08),
which were isolated from pigs in South Korea. Genetic char-
acterization showed that these viruses have high homology to
avian H5N2 influenza viruses recently circulating among wild
birds in Korea (our unpublished data) with the exception of the
NP, M, PA, and PB2 genes of the Sw/Kor/C13/08 isolate, which
have high homologies to those of the H3N1-like virus that
circulated in pigs in Korea in 2006. To investigate the patho-
genic potential of the novel H5N2 porcine viruses in mamma-
lian hosts, we conducted animal challenge experiments in pig
and ferret models. Furthermore, seroprevalence in swine sera
from 2006 to 2008 suggested that the avian H5N2-like H5
viruses have been infecting swine since 2006, even though there
was no large-scale outbreak in swine herds in South Korea.
MATERIALS AND METHODS
Viruses. The viruses used in this study were obtained from 7- to 8-week-old
pigs showing typical clinical symptoms of influenza-like illness from the Chung-
nam province of South Korea. The two swine influenza viruses, A/Swine/Korea/
C12/08 (Sw/Kor/C12/08) and A/Swine/Korea/C13/08 (Sw/Kor/C13/08), were iso-
lated from nasal swabs or lung specimens by the inoculation of supernatants and
tissue homogenates into monolayers of Madin-Darby canine kidney (MDCK)
cells. The subtypes of Sw/Kor/C12/08 and Sw/Kor/C13/08 were determined by
two multiplex reverse transcription-PCR (RT-PCR) assays and confirmed by
sequencing as previously described (3).
Sequencing and phylogenetic analysis. Viral RNA was extracted from cell
culture isolates using a QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA).
RT-PCR was performed under standard conditions using influenza-specific
primers (4, 19). The nucleotide sequencing of the amplified products was done
using a DNA sequencer (Model 377; Applied Biosystems, Perkin-Elmer, Foster
City, CA) and a Taq Dye Deoxy Terminator cycle sequencing kit (Applied
Biosystems, Foster City, CA). The sequences were resolved using the ABI
PRISM collection program (Perkin-Elmer, Foster City, CA). The DNA se-
quences were compiled and edited using the Lasergene sequence analysis soft-
ware package (DNASTAR, Madison, WI). Multiple sequence alignments were
made using Clustal_X (1, 53). The rooted phylograms were prepared using the
neighbor-joining (NJ) algorithm and then plotted using the program NJ plot
(42). The trees presented in Fig. 2a to h are based on the full-length nucleotide
sequences of each gene segment.
Pig serologic tests. A hemagglutination inhibition (HI) assay was performed to
determine the seroprevalence of the novel H5N2 viruses from 2006 to May 2008.
A total of 4,108 sera collected from major swine production provinces were
tested for the presence of H5 antibody. All sera were heat inactivated at 56°C for
30 min and pretreated with receptor-destroying enzyme (RDE) from Vibrio
cholerae (Denka Seiken, Tokyo, Japan) to remove nonspecific serum inhibitors.
The sera then were tested for H5 antibody by the HI technique with 0.5%
chicken red blood cells (38). A neutralization test was done on selected HI-
positive sera to confirm results, as described previously (18). Neutralizing anti-
body titers were expressed as the reciprocal of the highest dilution of the sample
that completely inhibited hemagglutination. Hemagglutination assays were per-
formed according to WHO/World Organization for Animal Health recommen-
dations.
Western blotting. Western immunoblotting was done using H5 viral antigen
derived from the formalin-inactivated H5N2 (Sw/Korea/C13/07) isolate that had
been purified by ultracentrifugation with a CaCl2 cushion at 112,600  g for 3 h
at 4°C (5). Thirty micrograms of H5 protein per lane were separated on a 10%
sodium dodecyl sulfate-polyacrylamide gel and immunoblotted with pig sera at a
dilution of 1:200. All pig sera that gave positive reactions in the H5N2 HI and
virus-neutralizing antibody tests were tested together with a random selection of
at least five sera giving negative reactions (Fig. 1).
Replication and transmission of swine isolate. The experimental infection of
pigs and ferrets with the H5N2 viruses was done in biosafety level 2 (BSL 2) and
3 (BSL 3) containment facilities, respectively. Yorkshire white weanling pigs
(approximately 5 to 6 weeks old) that were found to be free of detectable
influenza virus by serologic testing were inoculated intranasally with 3.3  106
50% egg infective dose (EID50) of virus in a volume of 1.0 ml (divided between
two plastic syringes for the separate inoculation of each nostril). The swine H5N2
viruses were inoculated into two groups, and three uninoculated littermates were
housed in an isolator with two inoculated pigs to test for the pig-to-pig trans-
mission of virus. The pigs' temperatures and food consumption were recorded
daily beginning 2 days before inoculation and ending 14 days after inoculation
(the end of study). Each nostril was swabbed 2, 5, 7, and 9 days after inoculation,
and virus subsequently was titrated in embryonated chicken eggs. The same
procedure was done in 17-week-old male ferrets to study the transmissibility of
the viruses in this host. Two experimental groups (each comprised two inoculated
and two naïve-contact littermate ferrets) were inoculated intranasally with 3.3 
106
EID50 of virus in 0.5 ml sterile phosphate-buffered saline under isoflurane
anesthesia. To monitor virus shedding, nasal washes were collected from all
ferrets every other day for 14 days and were titrated in embryonated chicken
eggs.
Histopathology. Trachea, serum, lung, liver, intestine, spleen, and kidney from
pigs and ferrets were collected on day 5 after inoculation. To prevent cross-
contamination, different sterile instruments were used for collecting each tissue
(5). Collected tissues were fixed in 10% neutral-buffered formalin. Immunohis-
tochemistry was performed to examine the distribution of SIV antigens in the
lung and other organs in infected animals using a goat polyclonal antibody
against type A influenza virus nucleoprotein (Abcam, Cambridge, MA).
Nucleotide sequence accession numbers. The GenBank accession numbers
assigned to the sequences determined in this study are FJ461592 to FJ461607.
RESULTS
Analysis of clinical samples. Nasal swabs and lung samples
were submitted to the Chungbuk National University between
2004 and 2008 for the diagnosis of SIV infections. Average
swine farms contain approximately 200 sows and 2,100 fatten-
ing pigs, according to the Korean Swine Association, and most
have adopted farrow-to-finish operations (26). The Sw/Kor/
C12/08 swine influenza virus was isolated from nasal swabs
obtained from a 6-week-old Yorkshire white weanling pig from
the Chungnam province through routine virus surveillance in
FIG. 1. Immunoblotting using H5 antigen derived from the Sw/Korea/C13/07 (H5N2) virus as the primary antibody and purified by ultracen-
trifugation with a CaCl2 cushion at 112,600  g for 3 h at 4°C (Beckman). Thirty micrograms of H5 protein per lane was separated on a 10% sodium
dodecyl sulfate-polyacrylamide gel and immunoblotted with pig sera at a dilution of 1:200. All of the sera giving a positive result in the HI and
virus-neutralizing antibody tests were subjected to immunoblotting together with a random selection of sera giving negative HI results. A
representative result indicating the different HI titers obtained for each respective serum is shown. M, 75-kDa protein marker (Bio-Rad); C,
positive control; C, negative control.
4206 LEE ET AL. J. VIROL.
January 2008. In the same province in February 2008, the Sw/
Kor/C13/08 swine influenza virus was isolated from lung tissues
obtained from an 8-week-old Yorkshire white weanling pig with
typical influenza-like symptoms, such as high fever, nasal dis-
charge, coughing, and decreased food consumption. The subtypes
of Sw/Kor/C12/08 and Sw/Kor/C13/08 were found to belong to
the H5N2 family by two multiplex RT-PCR assays and sequenc-
ing as previously described (3). No other subtypes of swine influ-
enza viruses were detected from the specimens submitted on the
same days.
Sequence and phylogenetic analysis. Except for the PB2,
PA, NP, and M genes of Sw/Kor/C13/08, sequence analysis
revealed that each of the RNA segments of the two porcine
H5N2 viruses have high nucleotide homology to avian viruses
of the Eurasian lineage (Table 1). The nucleotide analysis of
the PCR products indicated that Sw/Kor/C12/08 and Sw/Kor/
C13/08 shared 99.2% nucleotide similarities in the HA, NA,
PB1, and NS genes. This high genomic identity in surface genes
and in some internal genes suggests that the same avian virus
is the progenitor of the two isolates. Interestingly though, gene
segments 1 (PB2), 3 (PA), 5 (NP), and 7 (M) of the Sw/Kor/
C13/08 isolate have high homology with Sw/Kor/CN22/06-like
(H3N1) swine influenza viruses, indicating that reassortment
has occurred.
To understand the evolutionary relationship and origin of
the Sw/Kor/C12/08 and the Sw/Kor/C13/08 viruses in detail, the
phylogenetic alignment of the full-length gene sequences was
performed (Fig. 2a to h). Phylogenetic analysis showed that the
HA genes of both swine H5N2 isolates clustered together and
had high nucleotide identities with low-pathogenicity avian
influenza (LPAI) H5 viruses from China, specifically Ga/San
Jiang/160/06 (99%), rather than to recent highly pathogenic
avian influenza H5N1 viruses present among domestic poultry
(represented by Ck/Kor/IS/06; 88.6%) and migratory birds
(represented by Em/Kor/W150/06; 89.3%) from Korea. The
phylogeny of the NA genes paralleled what was observed for
their HA gene comparisons, in which the two swine isolates
still belonged to the same avian Ga/San Jiang/160/06-like lin-
eage (99% sequence homologies) (Fig. 2a and b). Appar-
ently, Korean avian H5N2 influenza viruses isolated from wild
birds in 2006 were also descendants of the Ga/San Jiang/160/
06-like lineage (our unpublished data), indicating a common
avian H5N2 virus precursor.
Phylogenetic analysis of the internal genes clearly showed
the differences between the two porcine influenza virus iso-
lates, supporting the sequence analysis. The phylogenetic anal-
ysis revealed that each of the internal genes of Sw/Kor/C12/08
had a close relationship with different subtypes of LPAI iso-
lates that are entirely of Eurasian phylogenetic lineage, indi-
cating that these segments were derived from multiple virus
sources. For the internal genes of the Sw/Kor/C13/08 virus,
only the PB1 and NS genes were derived from avian origin,
clustering together with the Sw/Kor/C12/08 virus (Fig. 2g and
h). Phylograms of segments 1, 3, 5, and 7 of the virus diverged
from avian to swine lineages and strongly indicated that they
were derived from Sw/Kor/CN22/06-like (H3N1) swine influ-
enza viruses, which were reportedly circulating in Korean pigs
in 2006 (Fig. 2a to f) (47). These results show that the Sw/Kor/
C12/08 isolate is a wholly avian virus, whereas Sw/Kor/C13/08
is an avian-swine reassortant from a possible common pre-
cursor. The analysis of Sw/Kor/C13/08 suggested that some
of the avian H5N2 viruses that infected Korean pigs had
undergone genetic reassortment with a swine influenza vi-
rus, probably due to the coinfection of the two influenza
virus strains in the same pig.
Antigenic analysis and Western blotting. To investigate the
prevalence of swine infection with the H5N2 viruses, swine
sera collected from 2006 to May 2008 from at least four swine
production provinces in Korea were tested. Of the 4,108 pig
serum samples surveyed, 35 (0.85%) were positive for the
swine H5N2 viruses by the HI assay (Table 2). As early as
January 2006, positive serum samples already were obtained,
with titers ranging from 40 to 160 HI units. The prevalence rate
was only about 1.04% in 2006 but was down to around 0.74%
in 2007 and 0.77% in 2008. Collectively though, the serologic
data indicated that viral exposure was substantially higher dur-
ing the winter months.
To further confirm the specificity of the HI test results, virus
neutralization tests were conducted. To test for the presence of
neutralizing antibodies against H5 viruses, selected HI-positive
pig sera were diluted twofold and mixed with 100 50% tissue
culture infective doses of Sw/Kor/C13/08 or Sw/Kor/C12/08. In
addition, an H5N1 virus (EM/Kor/W149/06) also was used to
test the specificity of the antibodies present. Results showed
that neutralizing antibodies against the swine H5N2 viruses
correlated with the titers obtained from the HI assays (Table
2). Similar neutralization titers were obtained using a wild bird
H5N2 virus. In contrast, lower neutralizing antibody titers (a
difference of about two- to fourfold) were observed in the
TABLE 1. Sequence homology of each gene from the two porcine H5N2 viruses with reference virus sequences available in GenBanka
Gene
Sw/Kor/C12/08 Sw/Kor/C13/08
% Identity
Virus with the highest degree of
sequence identity (accession no.)
Subtype
Phylogenetic
lineage
% Identity
Virus with the highest degree of
sequence identity (accession no.)
Subtype
Phylogenetic
lineage
HA 99.7 Ga/SanJiang/160/06 (EF634332) H5N2 Avian 99.8 Ga/SanJiang/160/06 (EF634334) H5N2 Avian
NA 99.8 Ga/SanJiang/160/06 (EF634332) H5N2 Avian 99.5 Ga/SanJiang/160/06 (EF634334) H5N2 Avian
PB2 96.7 Dk/Denmark/65047/04 (DQ251450) H5N2 Avian 99.5 Sw/Kor/CN22/06 (DQ923521) H3N1 Swine
PB1 97.6 Dk/Denmark/65047/04 (DQ251450) H5N2 Avian 97.8 Dk/Denmark/65047/04 (DQ251450) H5N2 Avian
PA 98.4 Ck/Taiwan/165/99 (DQ376806) H6N1 Avian 99.7 Sw/Kor/CN22/06 (DQ923517) H3N1 Swine
NP 98.8 Dk/Hokkaido/Vac-3/07 (AB355930) H5N1 Avian 99.8 Sw/Kor/CN22/06 (DQ923513) H3N1 Swine
M 98.5 Dk/Jiang Xi/6568/04 (EF597301) H4N6 Avian 99.7 Sw/Kor/CN22/06 (DQ923511) H3N1 Swine
NS 99.8 Dk/Hokkaido/120/01 (AB286880) H6N2 Avian 99.4 Dk/Hokkaido/120/01 (AB286880) H6N2 Avian
a
The numbers in parentheses are GenBank accession numbers for the reference virus sequences. Ck, chicken; Dk, duck; Ga, garganey; and Sw, swine.
VOL. 83, 2009 NOVEL LPAI H5N2 VIRUS INFECTION IN SWINE 4207
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swine sera when an avian H5N1 virus was used (data not
shown).
H5 antigen against the Sw/Kor/C13/08 virus, which was pu-
rified by ultracentrifugation at 112,600  g for 3 h at 4°C, was
used in the Western blot analysis. The immunoblotting results
correlated with the results of the HI and virus-neutralizing
tests (Fig. 1 and Table 2). These findings showed that anti-H5
antibodies were present in several pigs and that the H5N2-like
viruses could naturally infect pigs, although the incidence of
such infection is low, as shown by the seroprevalence survey
(Table 2) and the absence of a reported large-scale outbreak in
swine herds due to this subtype.
Animal experiments. Two pigs were infected with H5N2/08
viruses, and three contact pigs were added 1 day later in the
same isolators and housed together until the end of the exper-
iments. Mild cough, nasal discharge, and elevated body tem-
perature were observed on days 2 through 7 in all inoculated
pigs, but no acute respiratory signs were observed, such as
lethargy and dyspnea (data not shown). All of the viruses were
recovered from nasal swabs obtained from the inoculated pigs
after intranasal infection. On day 2 after inoculation, the mean
virus titer of nasal swabs was 2.3 log10 EID50/0.1 ml. Virus
was shed for at least 5 days (Table 3). Virus titers in nasal
swabs peaked on day 5 at 2.5 to 3.33 log10 EID50/0.1 ml. The
infection of the wholly avian-derived virus (Sw/Kor/C12/08)
persisted for as long as 5 days but was not transmitted to any
of the three contact pigs. However, two of the three littermates
of the avian-swine reassortant (Sw/Kor/C13/08) were positive
for virus transmission on day 5, with mean titers of 2.7 and 2.3
log10 EID50/0.1 ml (Table 3). The pigs inoculated with Sw/Kor/
C13/08 showed higher nasal swab titers, persisting for as long
as 7 days, than those inoculated with the wholly avian-derived
isolate (Table 3).
With the variable replication kinetics and transmissibility in
pigs of the two H5N2 porcine isolates, we sought to determine
whether such results could also be obtained in mammalian
hosts by using two groups of ferrets (two inoculated and two
contact animals) as experimental models. All 17-week-old fer-
FIG. 2. (a and b) Phylogenetic tree based on the nucleotide sequences of the H5 HA gene (a) and the N2 NA gene (b). The nucleotide
sequences were aligned using Clustal_X (1, 53), and the phylograms were generated by the NJ method using the tree-drawing program NJ plot
(42). The scale represents the number of substitutions per nucleotide. Branch labels record the stability of the branches during 100 bootstrap
replicates. Only bootstrap values 60% are shown in each tree. The isolates in boldface type are the Korean swine H5N2 viruses being
characterized in this study. Ck, chicken; Dk, duck; Ga, garganey; Gs, goose; Ml, mallard; Qa, quail; RT, rudder turnstone; Tk, turkey; Tl, teal; and
Wb, wild bird. Standard abbreviations are used for state names in the United States. (c to h) Phylogenetic tree based on the nucleotide sequences
of the PB2, PA, NP, M, PB1, and NS genes. The nucleotide sequences were aligned using Clustal_X (1, 53), and the phylograms were generated
by the NJ method using the tree-drawing program NJ plot (42). The scale represents the number of substitutions per nucleotide. Branch labels
record the stability of the branches during 100 bootstrap replicates. Only bootstrap values 60% are shown in each tree. The isolates in boldface
type are the Korean swine H5N2 viruses being characterized in this study. Ab, aquatic bird; Pr, parrot; Ps, pheasant; Sb, shorebird; Sl, shoveler;
Sw, swine; and Te, tern. Standard abbreviations are used for state names in the United States.
4210 LEE ET AL. J. VIROL.
rets used for the study were found to be seronegative at day 0
for antibodies against swine influenza H1N1, H1N2, H3N1,
and H3N2 viruses and avian H5N2 viruses by the HI assay. For
the Sw/Kor/C12/08 virus, nasal washes were positive for virus
(2.7 log10 EID50/0.1 ml) infection only at 2 days after inocula-
tion, whereas a higher viral titer was observed 2 days after
inoculation (3.4 log10 EID50/0.1 ml) in Sw/Korea/C13/08-in-
fected ferrets and persisted until day 7 (Table 3). Thus, the
virus titer obtained from an Sw/Kor/C12/08-infected mamma-
lian host possibly were traces of the infection load and were not
due to successful virus replication, but the Sw/Korea/C13/08
virus did actively replicate in infected ferrets. No observable
clinical signs of influenza-like disease were noted in the in-
fected ferrets, and neither the wholly avian (Sw/Kor/C12/08)
nor the avian-swine reassortant (Sw/Kor/C13/08) virus was
transmitted among the contact ferrets. These results, there-
fore, indicate that the Sw/Kor/C13/08 isolate bearing some
swine gene segments (M, NP, PA, and PB2) can readily infect
both animal models and is efficiently transmissible via contact
in swine hosts, as opposed to the purely avian Sw/Kor/C12/08
isolate. However, neither H5N2 virus could be transmitted in
ferrets.
Histopathologic examination. To investigate the tissue dis-
tribution of viruses, we collected samples of trachea, serum,
lung, liver, intestine, spleen, and kidney from an infected pig
and ferret 5 days after inoculation from each group. The lungs
were positive for virus in all infected pigs and those of contact
pigs of the Sw/Kor/C13/08 virus-infected group (Fig. 3). There-
fore, the H5N2 viruses can persist in pigs for at least 5 days
after inoculation. However, the lungs were positive for virus
only in Sw/Kor/C13/08-infected ferrets (data not shown). No
virus was recovered from the liver, intestine, spleen, or kidney
of any pig or ferret. Lung tissue collected on day 5 showed that
Sw/Kor/C12/08 caused less severe lung and tracheal tissue
damage than Sw/Kor/C13/08 (Fig. 3).
Molecular analysis. The deduced amino acid sequences of
the viral proteins were investigated to examine the molecular
characteristics of the viruses in detail and to identify possible
determinants of interspecies transmission from birds to pigs.
The alignment of the amino acid sequences of the surface
proteins revealed that the two Korean swine H5N2 isolates
have 98.2 and 98.9% sequence identity to the reference
avian isolate (Ga/San Jiang/160/06) in HA and NA while shar-
ing about 98.7 and 99.1% homology with each other in these
respective viral proteins. At the HA1-HA2 cleavage site, both
of the porcine isolates had a Q-R-E-T-R motif in the connect-
ing peptides that is typical for LPAI H5 viruses (9), confirming
their low-pathogenicity pathotypes. All of the isolates had a
glutamine at position 222 (position 226 in H3 numbering) and
a glycine residue at position 224 (position 228 in H3 number-
ing), indicating a preferential binding of sialic acids to the
sugar chain by an 2-3 linkage (Table 4). The potential glyco-
sylation sites in the HA1 domain and proposed receptor bind-
ing sites were conserved in both isolates. None of the porcine
TABLE 2. Serologic reactivity to H5N2 influenza viruses for pig
sera collected from Korean swine farmsa
Yr and mo
No. of sera
tested
No. (%) of sera positive
for H5 antibody in HI
tests
HI/NT titer(s) of
positive sera
2006
January 155 4 (2.58) 40/20, 160/80,
160/80, 80/80
February 156 3 (1.92) 80/40, 80/80, 40/40
March 151 1 (0.64) 40/20
April 135 2 (1.48) 160/80, 80/80
May 139 1 (0.71) 80/40
June 129 1 (0.77) 40/20
September 118 0
October 143 0
November 149 1 (0.67) 40/40
December 163 2 (1.22) 40/20, 80/40
2007
January 181 3 (1.65) 80/40, 160/80, 40/40
February 213 2 (0.93) 40/20, 40/40
March 221 5 (2.26) 40/20, 40/20, 80/40,
April 208 0 80/80, 40/20
May 216 2 (0.92) 40/40, 40/20
June 201 0
September 186 0
October 193 0
November 192 1 (0.52) 40/40
December 209 2 (0.95) 40/20, 160/160
2008
January 218 2 (0.91) 40/40, 40/80
February 223 2 (0.89) 160/80, 40/80
March 209 1 (0.47) 80/40
Total 4,108 35 (0.85)
a
The sera giving positive HI test results were confirmed by Western blotting
using H5 antigen (Fig. 1) and neutralization (NT) tests (Table 2).
TABLE 3. Nasal excretion of H5N2 viruses in pigs and ferrets
Day
Swab viral titer (log10 EID50/0.1 ml) froma
:
Swine Ferret
Sw/Kor/C12/08 Sw/Kor/C13/08 Sw/Kor/C12/08 Sw/Kor/C13/08
Inoculated Contact Inoculated Contact Inoculated Contact Inoculated Contact
1 -- -- -- -- -- -- -- --
2 2.5 -- 2.7 -- 2.7 -- 3.4 --
5 2.5 -- 3.3 2.7 -- -- 2.7 --
7 -- -- 1.3 1.3 -- -- 0.7 --
9 -- -- -- -- -- -- -- --
a
All viruses and animal experiments were handled in BSL 2 (swine infections) or BSL 3 (ferret inoculation) containment facilities approved by the Korea Centers
for Disease Control and Prevention, and the research staff wore fitted HEPA filter masks. The dash marks indicate no detectable virus.
VOL. 83, 2009 NOVEL LPAI H5N2 VIRUS INFECTION IN SWINE 4211
influenza isolates in this study had any deletion in the stalk
region of the NA protein, both the catalytic and framework
sites appeared to be conserved, and none contained mutations
at sites known to confer resistance to neuraminidase inhibitors
(15, 30, 52). However, a potential glycosylation site at position
402 (Asn) of the N2 protein was lost (Lys) in Sw/Kor/C12/08.
Compared to the sequence of Ga/San Jiang/160/06, the two
isolates in this study had mutations at sites in the HA1 domain
(R2Q, D155N, K170E, and N183D) and N2 proteins (Q5H
and I241M). Furthermore, additional mutations could be
noted for the surface proteins of Sw/Kor/C12/08 (in the HA1
domain, S107N and A185E; in the NA, N402K) and Sw/Kor/
C13/08 viruses (in the HA1 domain, H103Y, D261Y, and
L318P; in the NA, W403R), but none of these residues was
FIG. 3. Histologic lesions in pigs infected with porcine H5N2 viruses. Lung tissue sections were stained by immunohistochemistry using a
polyclonal antibody against the nucleoprotein gene (anti-NP) of influenza virus to show influenza virus infection. (a) Sw/Kor/C12/08; (b)
Sw/Kor/C13/08; (c) positive contact (Sw/Kor/CC13/08); (d) negative contact. Magnification, 400. The arrows indicate influenza virus-infected
cells.
TABLE 4. Molecular analysis and comparison of amino acid sequences of different gene products of the swine H5N2 viruses with selected
H5N1 isolates
Straina
HA1 sequence at
NA stalk
deletion
M2 sequence
at aa:
NS sequence
PB2 sequence
at aa 627
aa 222
(226)b
aa 224
(228)b
Cleavage site
(aa 323­330)
26 31
Deletion
of aa
80­84
aa 92
HK/483/97 Q G RERRRKKR Yes L S No E K
GS/437-4/99 Q G RERRRKKR No L S No D E
Ck/HK/YU822.2/01 Q G RERRRKKR Yes L S Yes E E
Dk/HK/821/02 Q G IERRRKKR Yes L S Yes E E
Dk/China/E319-2/03 Q G RE-RRRKR Yes L S Yes E E
Ck/Kor/ES03 Q G RE-RRRKR Yes L S Yes E E
Thailand/1/04 Q G RERRRKKR Yes -- -- -- -- E
Vietnam/1196/04 Q G RERRRKKR --c
I N Yes E K
Ck/Kor/IS/06 Q G GERRRKKR Yes L S Yes E K
Qa/KorKJ4/06 Q G GERRRKKR Yes L S Yes E K
Env/Kor/W150/06 Q G GERRRKKR Yes L S No E K
Sw/Kor/C12/08 Q G RE----TR No L S No D E
Sw/Kor/C13/08 Q G RE----TR No L S No D E
a
The isolates in boldface are the Korean swine H5 viruses examined in this study.
b
The number in parentheses is the H3 numbering.
c
--, Sequence data not available.
4212 LEE ET AL. J. VIROL.
implicated previously for antigenicity or receptor-binding af-
finity.
Neither virus had an E-to-K mutation at position 627 of the
PB2 protein, which was responsible for the high virulence of
A/Hong Kong/483/97 in a mammalian host model (16). The
avian-like PB2/627E present in the swine-like PB2 is common
for all Korean swine influenza isolates and does not represent
a K-to-E mutation in the PB2 genes of the isolates in this study.
Neither of their M2 proteins had the necessary mutations at
residue 31 (Ser to Asn) to confer amantadine resistance (17,
20), nor did they have glutamic acid at position 92 of the NS1
molecule for escaping host antiviral cytokine responses (46).
To determine whether the pig-passaged H5 isolates had un-
dergone further substitutions or mammalian-like adaptations,
viruses recovered from experimentally inoculated pigs (includ-
ing from the contact swine of Sw/Kor/C13/08) and ferrets were
sequenced. Analysis showed that the sequences of viruses ob-
tained from pigs bore the same sequences as the swine farm
isolates. Interestingly, viruses recovered from ferrets incurred
additional sequence differences indicative of continuous ge-
netic evolution (Table 5). In summary, the molecular analysis
of the viral proteins of the swine H5N2 viruses clearly showed
that they maintained a low-pathogenicity form, and no muta-
tions related to virulence, drug resistance, or a shift in host
preference were acquired.
DISCUSSION
During our routine porcine virus surveillance in January and
February 2008, two LPAIs of the H5N2 subtype (designated
Sw/Kor/C12/08 and Sw/Kor/C13/08) were isolated for the first
time from pigs in Korea. Viruses of the same subtype have
been found among avian species in several countries, including
the United States (31), Mexico (12), Italy (8), Nigeria (11),
China (9), and Japan (35). However, there has been no report
yet of infections of this virus subtype among swine. Initial
homology comparisons of individual genes to viruses available
in the GenBank database showed that the two porcine viruses
originally were derived from avian isolates and are almost
identical to each other, except in four internal segments.
The phylogenetic analysis of the surface genes of the two
porcine H5N2 viruses showed that both the HA and NA seg-
ments originated from a 2006 Chinese avian Ga/San Jiang/
160/06 (H5N2)-like virus as a precursor (Fig. 2a and b). On the
other hand, the internal genes might have originated from
several different avian viruses purely belonging to the Eurasian
lineage. These gene cascades of the avian H5N2 viruses that
infected swine further reflect the dynamic influenza virus gene
pool in this region, and new viruses are created by reassort-
ment events that are very likely to occur in the field, exempli-
fied by H5 viruses from southeastern China (9). However,
phylogenetic analyses of the PB2, PA, NP, and M genes of the
Sw/Ko/C13/08 virus revealed that they were descendants of a
Korean H3N1-like virus (at least 99.5% similarities). In 2006,
Shin et al. reported the presence in the Korean swine popula-
tion of influenza viruses of the H3N1 subtype that were reas-
sortants between human-like influenza viruses and recently
circulating swine viruses (47). It is probable that the two por-
cine H5N2 viruses in this study originated from a common
avian ancestor, but a coinfection with a swine H3N1-like virus
in the same pig as a result of dual susceptibility facilitated an
avian-like (HA, NA, PB1, and NS) or swine-like (PB2, PA, NP,
and M) reassortant H5N2 virus.
Duan et al. reported that low-pathogenicity H5 subtype in-
fluenza viruses were isolated predominantly from migratory or
sentinel ducks during the winter, were barely detected in mar-
ket waterfowl, and were not found in terrestrial poultry in
southern China from 2002 to 2005 (9). In our unpublished
data, avian H5N2 viruses also were found in migratory birds in
Korea but were absent in domestic poultry. Hence, the avian
H5N2 viruses that infected Korean pigs may have come di-
rectly from migratory birds and not from domestic poultry
products, as was suggested by the phylogenetic study, though
the manner of such transmission remains to be resolved.
Although it is difficult to estimate when the avian-like H5N2
virus was first introduced into swine, some evidence suggests
that the virus has been circulating for just a couple of years.
First, the two porcine H5N2 isolates shared high amino acid
and nucleotide sequence identities (98.2%) in the HA and
NA sequences with the Ga/San Jiang/160/06-like virus. The
phylogenetic alignment of the surface genes further showed
the close relationship between the isolates. Second, we found
serologic evidence of avian H5N2 influenza virus infection
from 2006 to the first quarter of 2008, but only in a small
proportion of pigs (0.85%). The low seroprevalence and low
swine-to-swine transmission of the purely avian-like H5N2 vi-
rus suggest that it did not spread significantly throughout
South Korea. It also is possible that following transmission,
successive infections of pigs were subclinical. Subsequently to
successful cross-species transmission, spreading within the new
host population usually requires a period of adaptation of the
virus to that new host (55). Third, the PB2, PA, NP, and M
genes of the Sw/Kor/C13/08 isolate had very high sequence
similarities (98 to 99%) with Sw/Kor/CN22/06, as was shown
phylogenetically, very likely indicating a recent reassortment
event.
Some LPAI H5 viruses, aside from H7 viruses, could un-
TABLE 5. Amino acid sequence comparison of the H5N2 viruses
isolated after experimental inoculation into pigs and ferretsa
Isolate and gene
Position
(aa)
aa found in:
Farm isolate Pig
Contact
pig
Ferret
Sw/Kor/C12/08
PB2 144 R R K
634 S S F
741 S S F
NP 486 S S Y
NA 314 S S F
M 20 V V L
Sw/Kor/C13/08
PB2 648 L L L P
653 S S S F
695 L L L F
NA 314 S S S F
NS1 112 T T T A
a
The RNA of viruses recovered from inoculated pigs (including the contact
pig infected with Sw/Kor/C13/08) and ferrets was extracted, reverse transcribed,
and sequenced. Full sequences were compared against those of respective farm
isolates using MegAlign of DNAStar package 5.0.
VOL. 83, 2009 NOVEL LPAI H5N2 VIRUS INFECTION IN SWINE 4213
dergo mutational changes to become highly pathogenic in
avian species by several mechanisms and have been well doc-
umented (12, 21, 28, 40, 51). However, molecular analysis
showed that none of the isolates in this study had any indica-
tions that they had acquired genetic mutations that would
allow a shift in sialic acid receptor-binding preference, elevated
pathogenicity, or increased drug resistance. We also could not
predict whether such a virulence shift would occur if the vi-
ruses were allowed to circulate for an extended period of time
in swine populations. Although both porcine viruses caused
modest virus titers in the nasal swabs and showed moderate
clinical signs, animal experiments showed that Sw/Kor/C13/08
was well adapted in swine and was more easily transmitted
than Sw/Kor/C12/08. Between the Korean swine H5N2 viruses,
the only apparent selective advantage of the Sw/Kor/C13/08
virus over the Sw/Kor/C12 virus is the acquisition of the swine-
like segments. Therefore, it is interesting to speculate that the
possession of the Sw/Kor/C13/08 isolate of the swine gene
complements (i.e., the PB2, PA, NP, and M genes) conferred
a selective advantage for transmissibility in swine hosts, as was
shown in this study. Although our animal experiments showed
that the purely avian-like H5N2 isolate was not transmitted to
contact animals, it is not clear whether transmission would
occur under field conditions (with the presence of bacterial
coinfections and environmental stresses).
The surface genes (HA and NA) are the constant target of
neutralizing antibodies, such that they largely define the ge-
netic evolutions of influenza viruses. In contrast, there are no
known selective pressures that could have driven the acquisi-
tion of specific swine-like internal genes. We could only hy-
pothesize that the accumulation of acquired gene mutations on
the avian progenitor of Sw/Kor/C13/08 defined its persistence
in pigs, and that coinfection with the swine progenitor in the
same host generated a reassortant progeny virus with a certain
gene constellation effect facilitating transmission. There is in-
creasing evidence that the viral polymerases play a major role
in host adaptation (10). More recently, Manzoor et al. have
demonstrated that a swine-derived PB2 gene conferred en-
hanced replicative potential to the reassortant virus that pos-
sessed seven genes of avian origin (33). It also should be noted
that several researchers have proposed the NP gene as a de-
terminant of host range that can either restrict or attenuate
virus replication (44, 48, 54), thereby controlling the successful
transmission of virus to a new host (2). Furthermore, the in-
fluenza virus HA gene was shown to have a preferential asso-
ciation with the M gene, and as such the evolution of the M
gene may reflect host-specific adaptation (45, 54, 56). Overall,
it will be interesting to investigate the individual contribution
of these swine H3N1-derived internal viral genes with respect
to transmissibility.
The isolation of two distinguishable H5N2 viruses in Korean
swine in 2008 represents another significant event in the ability
of pigs to act as intermediate hosts for low-pathogenicity H5
viruses. Just as in the cases of swine infections with avian H9N2
and H5N1 from nearby China, the isolated low-pathogenicity
H5N2 viruses in this study may not be disregarded for their
potential to cause human infections in the future. The shift
from a purely avian-like swine virus to an avian-swine reassor-
tant after apparent coinfection in pigs provides further evi-
dence in the accumulating role of pigs as mixing vessels for
influenza viruses, enhancing the genetic evolution that bridges
the gap between animal and human influenza viruses. It should
also be noted that after a single passage to a mammalian host
(ferret), some genes (most specifically the PB2 gene) already
had acquired amino acid base changes. Such features of the
avian-swine H5N2 porcine virus could be considered a poten-
tial model for pandemic highly pathogenic avian influenza
(e.g., H5N1 and H7N7) virus outbreaks, in which viruses that
were previously nontransmissible in a new host (e.g., humans)
could also gain selective advantage by genetic reassortment
with other strains of different lineages due to coinfection and
through accumulated gene mutations. Although there are no
known clinical implications of the avian-swine reassortant virus
for pathogenicity to pigs or other species, including humans, at
present, the efficient transmissibility of the relatively swine-
adapted virus could facilitate virus spread, and association with
disease outbreaks among swine populations could also be ex-
pected. Thus, it raises concerns for continued surveillance of
yet another atypical influenza virus in pigs that may have the
potential to cross host-species barriers.
ACKNOWLEDGMENTS
This work was supported by grant no. RO1-2005-000-10585-0
from the Basic Research Program of the Korea Science & Engi-
neering Foundation and in part by a TBP grant from KRIBB
(KGM3110912), by contract HHSN266200700005C with the National
Institute of Allergy and Infectious Diseases, by Cancer Center support
grant CA21765 from the National Cancer Institute, and by the Amer-
ican Lebanese Syrian Associated Charities (ALSAC).
We thank Taek-Kyu Oh and Eun Ho Lee for technical assistance.
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