﻿Pandemic (H1N1)
2009 Virus on
Commercial Swine
Farm, Thailand
Donruethai Sreta, Siriporn Tantawet,
Suparlark N. Na Ayudhya,
Aunyaratana Thontiravong,
Manoosak Wongphatcharachai,
Jiradej Lapkuntod, Napawan Bunpapong,
Ranida Tuanudom, Sanipa Suradhat,
Linda Vimolket, Yong Poovorawan, Roongroje
Thanawongnuwech, Alongkorn Amonsin,
and Pravina Kitikoon
A swine influenza outbreak occurred on a commercial
pig farm in Thailand. Outbreak investigation indicated that
pigs were co-infected with pandemic (H1N1) 2009 virus and
seasonal influenza (H1N1) viruses. No evidence of gene
reassortment or pig-to-human transmission of pandemic
(H1N1) 2009 virus was found during the outbreak.
In April 2009, a novel swine origin influenza A (H1N1)
virus, now referred to as pandemic (H1N1) 2009 virus,
emerged in humans in Mexico and the United States and
spread worldwide (1). In May 2009, pandemic (H1N1)
2009 was confirmed in 2 patients in Thailand who had a
history of travel to Mexico. Shortly after emergence of
this virus, reports of transmission from humans to pigs on
pig farms were documented (2,3). Human-to-pig transmis-
sion of this virus was reported in Thailand on December
17, 2009 (www.dld.go.th/dcontrol/Alert/Ah1n1/H1N1%20
update22_12_2009.pdf). Pigs showed mild respiratory
signs; only 1 pandemic (H1N1) 2009 virus was isolated
from 80 nasal swab specimens.
Swine influenza virus (SIV) was reported in Thailand
in 1981 (4). All 3 subtypes (H1N1, H3N2, and H1N2) of
this virus are circulating in Thailand (5). A recent pathogen-
esis study demonstrated that subtype H1N1 induces typical
SIV-like illness and slightly more severe gross lesions than
illness induced by subtype H3N2 (6). Genetic data indicate
that SIV (H1N1) in Thailand differs from pandemic (H1N1)
2009 virus. SIV (H1N1) in Thailand contains surface pro-
teins of influenza viruses from North America and Eurasia,
which are also found in pandemic (H1N1) 2009 virus; SIV
(H1N1) in Thailand contains internal proteins of viruses
from Eurasia; and pandemic (H1N1) 2009 viruses contain
swine, human, and avian virus gene segments (5,7).
We report an outbreak of infection with pandemic
(H1N1) 2009 virus during November 2009­March 2010 on
a commercial pig farm in Thailand. The outbreak presum-
ably resulted from human-to-pig transmission because 1 of
the workers on this farm had influenza-like clinical signs at
the beginning of the outbreak. Infection in this worker was
not confirmed because he quit his job on the farm after the
start of the outbreak and could not be located.
The Study
In early November 2009, a small commercial pig farm
in central Thailand reported respiratory problems in pigs
(morbidity rate 50%, mortality rate 10%) in nursery pigs.
The farm contained 3,235 pigs (700 sows, 35 boars, 1,000
piglets, 1,000 nursery pigs, and 500 finishing pigs). It has a
conventional open-house production system in which both
sides of the unit have natural air flow ventilation. The farm
also has continuous nursery herd flow in which new pigs
are continuously added when they are old enough. This pro-
cess results in pigs of different ages being in the same unit.
Sick pigs had clinical signs (fever, cough, nasal discharge,
edematous eyelids, and conjunctivitis) of infection.
Nasal swabs from 20 nursery pigs (4­9 weeks of age)
were submitted to Chulalongkorn University Veterinary
Diagnostic Laboratory. All samples were positive for por-
cine circovirus type 2 and porcine reproductive and respi-
ratory syndrome virus (these viruses are major causes of
swine respiratory disease), and 2 samples were positive for
influenza A virus by reverse transcription­PCR (RT-PCR)
with primers for each specific pathogen (8­10).
Because respiratory problems in nursery pigs contin-
ued, nasal swabs specimens from 20 nursery pigs and fin-
ishing pigs, gilts (young females), and sows (10 per group)
with clinical signs were submitted to the diagnostic labo-
ratory by the end of December 2009. Two samples from
nursery pigs were positive for influenza virus A (H1N1) by
multiplex RT-PCR (11). Both samples were subjected to
virus isolation in MDCK cells (12) and designated RA20
and RA29 (Table 1). Genome characterization identified
RA20 as SIV and RA29 as pandemic (H1N1) 2009 virus
(Table 2; Figure). SIV-positive nasal swabs obtained in
November were then characterized. Results showed that
isolates RA4 and RA9 were pandemic (H1N1) 2009 virus,
which indicated that pigs on the farm were infected with
this virus.
Pandemic (H1N1) 2009 investigations on the farm
included clinical surveillance and sample collection from
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 16, No. 10, October 2010 1587
Author affiliations: Chulalongkorn University, Bangkok, Thailand
(D. Sreta, S.N. Na Ayudhya, A. Thontiravong, M. Wongphatchara-
chai, J. Lapkuntod, N. Bunpapong, R. Tuanudom, S. Suradhat, L.
Vimolket, Y. Poovorawan, R. Thanawongnuwech, A. Amonsin, P.
Kitikoon); and Mahidol University, Nakhon Pathom, Thailand (S.
Tantawet)
DOI: 10.3201/eid1610.100665
sick and contact pigs and close monitoring of swine work-
ers and farm pets for influenza-like illness. Nasal swab
specimens were obtained from pigs on January 17, 2010,
January 30, 2010, and March 9, 2010. Because initial
laboratory findings indicated that the outbreak involved
the nursery herd, weaned pigs were moved to a separate
site on the farm to control disease in the nursery pigs. Fol-
lowing Food and Agriculture Organization (www.fao.org)
sample collection recommendations, we obtained 20 nasal
swabs from pigs with SIV-like illness. In addition, nasal
swab specimens (n = 10 per group) were collected from
gilts, sows, and finishing pigs to test for pandemic (H1N1)
2009 virus, although no clinical signs were observed in
any pigs from these age groups. All SIV-positive samples
were subjected to virus isolation (12), virus subtyping by
multiplex RT-PCR (11), and whole genome sequencing
of subtype H1N1 viruses (13). Of 175 samples obtained
during December 26, 2009­March 9, 2010, fifteen swab
specimens from nursery pigs with clinical signs were posi-
tive for influenza (H1N1) 2009 virus; 8 viruses were char-
acterized. No other SIV subtypes were found. On March
9, 1 month after implementing the change in handling of
pigs, no pigs showed respiratory signs and 34 nasal swab
specimens were negative for influenza virus.
Gene sequences were compared for correspond-
ing genes of other influenza virus strains obtained from
GenBank by using the MegAlign program (DNASTAR,
Madison, WI, USA). Phylogenetic trees were construct-
ed by using MEGA4 (www.megasoftware.net/) and the
neighbor-joining method with 1,000 bootstrap replicates.
Whole genome analysis showed that contemporary SIV
(H1N1) and pandemic (H1N1) 2009 virus were concur-
rently circulating in the nursery herd (Table 2; Figure).
On the basis of virus hemagglutinin 1 gene grouping
(14), our findings show that newly isolated SIV (H1N1)
from Thailand are grouped in the classical swine cluster
with other SIV (H1N1) isolates (online Appendix Figure,
www.cdc.gov/EID/content/16/10/1587-appF.htm). There
was no evidence of gene reassortment between SIV
(H1N1) and pandemic (H1N1)2009 virus during the in-
vestigation (Table 2).
To test for evidence of pandemic (H1N1) 2009 virus
interspecies transmission, we obtained serum samples on
January 17, 2010, from 40 pigs in 8 age groups (5/group),
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1588 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 16, No. 10, October 2010
Table 1. Influenza (H1N1) viruses studied, Thailand*
Influenza (H1N1) virus isolate Collection date Identification
Study
designation
GenBank accession no.
(gene segment 1­8)
A/sw/Thailand/CU-RA4/2009 2009 Nov 6 Pandemic (H1N1)2009 RA4 CY062305­CY062312
A/sw/Thailand/CU-RA9/2009 2009 Nov 6 Pandemic (H1N1) 2009 RA9 CY062321­CY062328
A/sw/Thailand/CU-RA20/2009 2009 Dec 26 Thai SIV RA20 CY062281­CY062288
A/sw/Thailand/CU-RA29/2009 2009 Dec 26 Pandemic (H1N1)2009 RA29 CY062297­CY062304
A/sw/Thailand/CU-RA114/2010 2010 Jan 17 Pandemic (H1N1)2009 RA114 CY062265­CY062272
A/sw/Thailand/CU-RA204/2010 2010 Jan 17 Thai SIV RA204 CY062289­CY062296
A/sw/Thailand/CU-RA15/2010 2010 Jan 30 Pandemic (H1N1) 2009 RA15 CY062273­CY062280
A/sw/Thailand/CU-RA75/2010 2010 Jan 30 Pandemic (H1N1) 2009 RA75 CY062313­CY062320
*SIV, swine influenza virus.
Table 2. Gene origin and percent homology of SIV RNA segments compared with pandemic (H1N1) 2009 virus, Thailand*
Influenza (H1N1) virus
PB2
(1­2229)
PA
(1­2153)
NA
(1­1347)
M
(1­982)
HA
(1­1698)
NS
(1­778)
NP
(1­1443)
PB1
(1­2153)
Pandemic (H1N1) 2009 Avian TRIG Eurasian swine Classical swine Human TRIG
SIV from Thailand§ Eurasian swine Classical swine Eurasian swine
RA4 Avian TRIG Eurasian swine Classical swine Human TRIG
RA9 Avian TRIG Eurasian swine Classical swine Human TRIG
RA20¶ Eurasian swine Classical swine Eurasian swine
83.1% 85.1% 89.5% 94.2% 86.4% 90.8% 82.4% 85.1%
RA29 Avian TRIG Eurasian swine Classical swine Human TRIG
RA114 Avian TRIG Eurasian swine Classical swine Human TRIG
RA204¶ Eurasian swine Classical swine Eurasian swine
83.2% 85.2% 89.5% 94.2% 86.8% 90.8% 82.3% 85.1%
RA15 Avian TRIG Eurasian swine Classical swine Human TRIG
RA75 Avian TRIG Eurasian swine Classical swine Human TRIG
*All swine influenza virus (SIV) isolates except RA20 and RA204 have >99% homology with corresponding genes of A/Nonthaburi/102/2009. PB,
polymerase B; PA, polymerase A; NA, neuraminidase; M, matrix; HA, hemagglutinin; NS, nonstructural; NP, nucleoprotein; TRIG, triple reassorted
internal gene.
Nucleotide positions compared.
A/Nonthaburi/102/2009 (H1N1) virus.
§A/sw/Ratchaburi/NIAH1481/2000 (H1N1) virus.
¶Percent homology of compared sequences with those of corresponding genes of A/Nonthaburi/102/2009.
Pandemic (H1N1) 2009 Virus on Swine Farm, Thailand
15 workers, and 4 farm pets (3 dogs and 1 cat). Samples
were subjected to hemagglutination-inhibition (HI) test-
ing with SIV (H1N1) and pandemic (H1N1) 2009 virus
antigens (12).
Control rabbit antibodies against SIV (H1N1) viruses
did not cross-react with pandemic (H1N1) 2009 virus. Se-
rologic results showed that only 2 (9.5%) of 21 test samples
from the nursery group had positive HI titers for pandemic
(H1N1) 2009 virus and 8 (38%) of 21 had positive HI ti-
ters for SIV (H1N1) virus. For pigs in other age groups,
11 (55%) of 20 had antibodies against pandemic (H1N1)
2009 virus and 14 (70%) of 20 had antibodies against SIV
(H1N1) by HI test. No human cases of co-infection were
observed. We found no evidence of pandemic (H1N1)
2009 virus interspecies transmission from pigs to humans
or to farm pets.
Conclusions
Consistent with findings of previous reports (2,3),
our findings demonstrate that young pigs are susceptible
to infection with pandemic (H1N1) 2009 virus. Infection
in pigs substantiates the hypothesis that the clinical out-
come caused by infection with pandemic (H1N1) 2009 vi-
rus differs from that of infection with SIV (H1N1), which
currently circulates in pigs in Thailand. Serologic results
demonstrated that uninfected populations are susceptible to
infection with pandemic (H1N1) 2009 virus. Results of ge-
nome analysis did not show gene reassortment between the
2 different influenza (H1N1) viruses. However, a previous
report showed that reassortment of influenza virus genes
occurs in pigs (15). Continued monitoring, characterization
of SIVs, and serologic surveillance of pigs are necessary
for future influenza pandemic preparedness.
Acknowledgments
We thank Suphattra Jittimanee, Roongtham Kedkovid, and
Na Taya Charoenvisal for assisting with sample collection; and
the Chulalongkorn University Centenary Academic Development
Project for supporting facilities of the Emerging and Reemerg-
ing Infectious Diseases in Animals Research Unit. This study was
conducted at the Faculty of Veterinary Science and Faculty of
Medicine, Chulalongkorn University.
This study was supported by grants from National Research
Council of Thailand, Emerging Health Risk Cluster, Rach-
adapiseksompoch Endowment Fund, and a subcontract to Chula-
longkorn University from the University of Minnesota under the
National Institute of Allergy and Infectious Diseases, National
Institutes of Health (prime contract no. HHSN266200700007c).
Dr Sreta is a member of the Faculty of Veterinary Medicine
at Rajamangala University of Technology Tawanok and a PhD
candidate in the Veterinary Pathobiology Program, Faculty of
Veterinary Science, at Chulalongkorn University, Bangkok, Thai-
land. Her research interests are swine influenza virus surveillance
and genetic characterization.
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Figure. Percentage of pigs with antibodies against pandemic
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Address for correspondence: Pravina Kitikoon, Faculty of Veterinary
Science, Chulalongkorn University, Henri-Dunant Rd, Pathumwan,
Bangkok 10330, Thailand; email: pravina21@gmail.com
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