﻿Published Ahead of Print 19 October 2011.
10.1128/JCM.05676-11.
2011, 49(12):4386. DOI:
J. Clin. Microbiol.
Carl A. Gagnon
and
Christian Bellehumeur, J. Grant Spearman, Josée Harel
Donald Tremblay, Véronique Allard, Jean-François Doyon,
Populations, Mink and Swine
Reassortant in Two Canadian Animal
Pandemic (H1N1) 2009 Influenza A Virus
Emergence of a New Swine H3N2 and
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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p. 4386­4390 Vol. 49, No. 12
0095-1137/11/$12.00 doi:10.1128/JCM.05676-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Emergence of a New Swine H3N2 and Pandemic (H1N1) 2009
Influenza A Virus Reassortant in Two Canadian Animal
Populations, Mink and Swine
Donald Tremblay,1
Ve
´ronique Allard,1
Jean-Franc
¸ois Doyon,4
Christian Bellehumeur,2,3
J. Grant Spearman,5
Jose
´e Harel,1,2,3
and Carl A. Gagnon1,2,3
*
Service de Diagnostic,1
Centre de Recherche en Infectiologie Porcine (CRIP),2
and Groupe de Recherche sur les Maladies
Infectieuses du Porc (GREMIP),3
Faculte
´ de Me
´decine Ve
´te
´rinaire, Universite
´ de Montre
´al, St-Hyacinthe,
Que
´bec, Canada; Veterinary Clinic Jean-Franc
¸ois Doyon, Roxton Falls, Que
´bec, Canada4
; and
Nova Scotia Department of Agriculture, Truro, Nova Scotia, Canada5
Received 5 September 2011/Returned for modification 22 September 2011/Accepted 14 October 2011
A swine H3N2 (swH3N2) and pandemic (H1N1) 2009 (pH1N1) influenza A virus reassortant (swH3N2/
pH1N1) was detected in Canadian swine at the end of 2010. Simultaneously, a similar virus was also detected
in Canadian mink based on partial viral genome sequencing. The origin of the new swH3N2/pH1N1 viral genes
was related to the North American swH3N2 triple-reassortant cluster IV (for hemagglutinin [HA] and
neuraminidase [NA] genes) and to pH1N1 for all the other genes (M, NP, NS, PB1, PB2, and PA). Data
indicate that the swH3N2/pH1N1 virus can be found in several pigs that are housed at different locations.
CASE REPORT
In December 2010, an outbreak of respiratory symptoms
occurred in a hog farm located in the province of Quebec,
Canada. At the site, about 10% of the piglets were coughing,
sows had no clinical signs, and several fattening pigs were
coughing with nasal discharge. No increase of death rate was
observed during this swine flu episode compared to the death
rate that was occurring within the herd prior to the outbreak.
Pools of nasal swabs of sick fattening pigs were collected at the
farm and submitted to testing for the presence of swine influ-
enza virus by a real-time reverse transcription-PCR (RT-
PCR), which is able to detect influenza A viruses by targeting
the conserved region of the M gene (matrix [M1] and ion
channel [M2]) (17, 20). In addition, RT-PCR assays that are
specific for the hemagglutinin (HA) and neuraminidase (NA)
most common subtypes found in swine (H3 versus H1 and N2
versus N1) were conducted (2). Even if the general influenza A
virus PCR assay gave a low positive response, the case was
highly positive for the H3 and N2 subtypes [A/swine/Quebec/
1259260/2010(H3N2)]. For the reconciliation of the obtained
results, sequencing of the HA, M, and nonstructural protein
(NS) genes was conducted. Sequence analyses revealed a new
commingling of the HA, M, and NS genes subtypes in a single
swine flu case (data not shown). To further confirm the ap-
pearance in swine population of a new influenza virus reas-
sortant, a small retrospective study was conducted. Thus, the
M genes of all swine H3N2 (swH3N2) PCR-positive cases (n 
13 cases) that were submitted to the Molecular Diagnostic
Laboratory (MDL) of the University of Montreal from Octo-
ber 2010 to February 2011 were sequenced. All of those cases
originated from different herds experiencing a swine flu epi-
sode. Those herds were located at different sites across the
province of Quebec, Canada. For those cases, various clinical
samples were submitted, such as lung tissues and/or nasal
swabs. When sequence analyses revealed that the M genes
were genetically related to pandemic (H1N1) 2009 influenza
virus (pH1N1), then virus isolation was conducted. Overall, the
sequence analyses of the M gene PCR products revealed that
9 cases, over the 13 swH3N2 PCR-positive cases, possessed a
new reassortment of the HA, NA, and M genes (Fig. 1), with
the M gene originating from the pH1N1 strain (Fig. 1). The M
gene of the remaining 4 cases was genetically related to
swH3N2 (data not shown). The earliest confirmed submitted
case of the new influenza virus reassortant was obtained in
October 2010.
Specific-pathogen-free 10-day-old embryonated chicken
eggs were inoculated via the chorioallantoic sac for virus iso-
lation as previously described (5). The presence of the influ-
enza virus within the allantoic fluid collected at the second
and/or third passage was confirmed by hemagglutination,
transmission electron microscopy, and PCR assays. The viral
RNA was isolated from submitted samples and allantoic fluids
with a commercial kit (QIAamp viral RNA minikit; Qiagen,
Mississauga, Ontario, Canada). The full lengths of the viral
RNA segments were amplified by RT-PCR, the PCR products
were purified (QIAquick PCR purification kit; Qiagen), and
both strands of the purified DNA PCR products were se-
quenced with the same primer sets used in the RT-PCR by
standard automated sequencing methods, as previously de-
scribed (5). The resulting sequences were compared with other
swine influenza virus reference sequences, such as those avail-
able in the Diagnostic Veterinary Virology Laboratory
(DVVL) of the University of Montreal (5) as well as those in
the GenBank database. BioEdit Sequence Alignment Editor
version 7.0.9 (Ibis Therapeutics; Carlsbad, CA) and Geneious
bioinformatics version 5.4.6 (Biomatters, Ltd., Auckland, New
* Corresponding author. Mailing address: Faculte
´ de Me
´decine Ve
´-
te
´rinaire, Universite
´ de Montre
´al, 3200 rue Sicotte, St-Hyacinthe,
Que
´bec, Canada J2S 7C6. Phone: 450-773-8521 (8681). Fax: 450-778-
8108. E-mail: carl.a.gagnon@umontreal.ca.

Published ahead of print on 19 October 2011.
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Zealand) were used for sequence analyses and phylogenic tree
construction, as previously described (5). From the 9 cases
confirmed to possess pH1N1 M gene, 6 influenza virus
strains were isolated: A/swine/Quebec/1265553/2010(H3N2)
(sw/Qc/1265553/10), A/swine/Quebec/1262080/2010(H3N2) (sw/
Qc/1262080/10), A/swine/Quebec/1267568/2010(H3N2) (sw/
Qc/1267568/10), A/swine/Quebec/1257774/2010(H3N2) (sw/Qc/
1257774/10), A/swine/Quebec/1257777/2010(H3N2) (sw/Qc/
1257777/10), and A/swine/Quebec/1267566/2011(H3N2) (sw/
Qc/1267566/11). It is noteworthy that 5 isolates were tested in
cell culture and were confirmed to be able to replicate in the
MDCK (Madin-Darby canine kidney) cell line. Subsequently,
the viral genomes of the 6 isolated strains were sequenced and
analyzed (Fig. 1). Overall, the viral genes of the new influenza
virus reassortant (swH3N2/pH1N1) are genetically related to
swH3N2 for the HA and NA genes and pH1N1 for the M,
nucleoprotein (NP), NS, polymerase basic protein 1 (PB1),
PB2, and polymerase acidic (PA) protein genes (Fig. 1). The
nucleotide sequence identities of the swH3N2/pH1N1 viral
genes compared to their respective swH3N2 [A/swine/Quebec/
4001/2005(H3N2)] and pH1N1 [A/New York/19/2009(H1N1)]
reference strain counterparts varied between 95.6 and 97.5%,
97.2 and 98.4%, 98.7 and 99.8%, 98.8 and 99.5%, 99.2 and
99.7%, 97.3 and 99.7%, 99.3 and 99.7%, and 99.2 and 99.5%
for the HA, NA, M, NS, NP, PB1, PB2, and PA genes, respec-
tively (Fig. 1). As illustrated in Fig. 2, the HA genes of the 6
swH3N2/pH1N1 strains were classified within the swH3N2 tri-
ple-reassortant cluster IV (Fig. 2). Noteworthy, the swH3N2/
pH1N1 strains seem to cluster within two subgroups named
subclusters IVa and IVb (Fig. 2). The HA pairwise nucleotide
identities between the two swH3N2/pH1N1 subclusters were
94.7 to 95.9%, and those within each of the subclusters were
96.4 to 98% and 99.1 to 99.8% for IVa and IVb, respectively.
Overall, the HA amino acid identities between the swH3N2/
pH1N1 strains were 94.5 to 99.8% (data not shown), which
suggested the existence of possible antigenic variations be-
tween those strains.
To ascertain this hypothesis, a hemagglutination inhibition
(HI) assay was conducted, as previously described (5), using as
antigen the 6 swH3N2/pH1N1 isolated strains. The reference sera
used in the HI assay, ck/150/90 and sw/4001/05, were obtained
following immunization of chicks with a formalin-inactivated
H3N2 swine influenza virus strain that was isolated in Quebec in
1990 or from naturally infected pig with the reference strain
A/Swine/Quebec/4001/05 (H3N2) (5), respectively. In addition,
20 pig sera were tested. These sera had been collected from 6- to
8-week-old pigs originating from the barn where animals experi-
enced the first confirmed flu case related to the swH3N2/pH1N1
reassortant (A/swine/Quebec/1259260/2010, which is a strain clas-
sified within subcluster IVa, as illustrated in Fig. 2). Statistical
analyses were done using GraphPad Prism version 4 software.
The one-way analysis of variance (ANOVA) nonparametric
Friedman test was used to determine if there is a statistically
significant difference between viruses with regard to the HI re-
sults. Interestingly, the antigenic reactivity of the 20 pig sera tested
was significantly higher (P  0.05) against homologous strains of
subcluster IVa than that of heterologous strains of subcluster IVb
(Fig. 3). In addition, the HI titers of the reference sera (ck/150/90
and sw/4001/05) demonstrated the existence of antigenic differ-
ences between IVa and IVb strains (Fig. 3). Overall, the HI
results confirmed the existence of antigenic variations between
strains of subclusters IVa and IVb (Fig. 3), supporting the
genomic results (Fig. 2).
Concomitant with the characterization of the first swine flu
FIG. 1. Viral genome sequence homologies of the new swH3N2/pH1N1 reassortant virus with swH3N2 and pH1N1 reference strains. When two
squares between an swH3N2/pH1N1 strain and a reference strain possess the same color, it indicates that the viral gene is genetically related. The
influenza virus reference strains used for comparison are A/New York/19/2009(H1N1) (GenBank accession no. FJ984388 to FJ984394 and GQ457503)
and the swH3N2 cluster IV Quebec reference strain A/swine/Quebec/4001/2005(H3N2) (GenBank accession no. EU826543 to EU826550). Numbers in
squares indicate the percentages of nucleotide homology compared to the genetically related strain. n.d., not determined.
VOL. 49, 2011 CASE REPORTS 4387
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case, RNA samples extracted from a mink tissue sample sent
by the Provincial Laboratory of Nova Scotia, Canada, were
received for further characterization. The strain (A/mink/Nova
Scotia/1265707/2010) was confirmed to be an H3N2 strain by
PCR and originated from a farm where mink kits were pre-
senting serious coughing but very low mortality. The HA gene
of the new mink strain shared higher nucleotide homologies
with the 6 swH3N2/pH1N1 strains and an swH3N2 reference
strain (95.7% to 100%) than pH1N1 (52.6%) (Fig. 1 and 2).
Further sequencing of the mink strain M gene showed a 99.8%
nucleotide homology to pH1N1 (Fig. 1) and only 88% homol-
ogy compared to swH3N2. Unfortunately, not enough tissue
was available for further characterization and virus isolation.
Influenza (flu) is a disease affecting several animal species,
including humans. In our recent history, several pandemics
have arisen in the human population, which were estimated to
cause millions of deaths, such as Spanish flu (1918 to 1920),
Asian flu (1957 to 1958), and Hong Kong flu (1968 to 1969).
The etiological agent, influenza A virus, belongs to the virus
family Orthomyxoviridae. Influenza A virus is a single-stranded
RNA virus with a segmented genome composed of eight gene
segments now recognized to code for 12 protein products (21).
These are the PB2 encoded by segment 1; PB1, PB1-F2, and
N40 proteins encoded by segment 2; PA protein encoded by
segment 3; HA protein encoded by segment 4; NP encoded by
segment 5; NA protein encoded by segment 6; M1 and M2
proteins encoded by segment 7; and 2 NS proteins encoded by
segment 8. Sixteen HA and 9 NA antigenic types have been
reported so far and are used to classify viruses into subtypes H1
to H16 and N1 to N9 (4). Genetic diversity in influenza virus
results from a high replication error rate associated with low-
fidelity RNA polymerase and the reassortment of gene seg-
ments among coinfecting strains. In 1998, an H3N2 triple-
reassortant influenza A virus emerged in the U.S. swine
population (10, 19, 22). RNA viral genes were identified as a
mix of classical swine, human, and avian virus lineages. In 2005,
spreading of this virus was seen in the swine and turkey pop-
ulations of Canada and 2 years later was also seen in the mink
populations of Canada (5, 15). Today, the influenza A virus
subtypes that are mostly found in swine in Canada are usually
FIG. 2. Phylogenetic tree of the HA gene nucleotide sequences of the swH3N2/pH1N1 strains. The designated clusters I, II, III, and IV classify
the North American H3N2 triple-reassortant swine viruses as previously described (4). The phylogenetic tree was generated by the neighbor-joining
method using the Geneious v5.4.6 software with a bootstrap resampling method (1,000 replications). Bootstrap confidence levels are indicated at
the nodes of the phylogenetic tree. Bootstrap values lower than 50 are not indicated for clarity. GenBank accession numbers for the sequences of
all viruses are provided after their names. The horizontal scale indicates the distances between strains; a distance of 0.02 means that the strains
possess 98% nucleotide identity. The asterisks and triangle indicate the swine and mink swH3N2/pH1N1 strains, respectively, identified in this
study. Those strains have been classified within subclusters IVa and IVb. The arrow indicates the other swH3N2 and pH1N1 reassortant strain that
has recently been reported in United States by Ducatez et al. (3); its genes are related to swH3N2 (for HA, NA, NS, PB1, and PB2) and pH1N1
(for M, PA, and NP).
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H1N1 and H3N2 (13, 14). Other subtypes, such as H1N2,
H3N3, and H4N6, are also sporadically found (8, 9, 11). In
April 2009, a novel pandemic triple-reassortant H1N1 influ-
enza A virus (pH1N1) was detected and spread globally as the
first influenza pandemic of the 21st century. The role of ani-
mals in the spreading of the pH1N1 is still unknown, but
several reports showed that human-to-animal transmissions of
the virus have occurred (1, 7). Now pH1N1 is frequently found
in the Canadian swine population (14).
The characterization of the swH3N2/pH1N1 reassortant vi-
ruses from swine in the province of Quebec indicates that
reassortment of gene segments had occurred between the
North American swine H3N2 triple-reassortant from cluster
IV and the pH1N1 strains as early as October 2010. Virus
reassortants possess two genes related to swH3N2 (HA and
NA) and all others related to pH1N1. The fact that 6 swH3N2/
pH1N1 strains were isolated in a short period of time from
animals with clinical signs housed at different locations sug-
gests active transmission of this virus reassortant. Since a sim-
ilar strain was also found in a sample recovered from a mink,
this could illustrate the potential of the virus to cross the
species barrier. How and when the pig and mink populations of
Canada acquired the new swH3N2/pH1N1 strain is unknown.
It is noteworthy that Gagnon et al. have reported that minks
are fed with uncooked pork by-products coming from slaugh-
terhouse facilities (i.e., discarded tissues, such as lung, which is
the target of swine influenza virus) (Fig. 1) (5). This could
explain how the cross-species contamination arises. Further
studies will have to be conducted to establish the full nature of
the swH3N2/pH1N1 reassortant in the mink population.
Ancestral genes present in the pH1N1 virus suggest that the
virus has been circulating undetected in the swine population
for an undetermined period of time. This event underlines the
need for surveillance of influenza viruses in swine populations
(6). In our diagnostic laboratory, as seen in many others, rou-
tine surveillance of influenza viruses in swine is done by de-
tection of influenza A virus by targeting the M gene. Further
characterization is often performed by typing the HA and NA
genes. Such a procedure is not sufficient to differentiate reas-
sortants, explaining why the new virus has been unnoticed by
the regular surveillance. It is likely that more cases are ex-
pected to be positive as surveillance programs adapt their
testing. Concerns should also be raised about the surveillance
of human seasonal influenza A virus (H3N2). For example,
inoculation in ferrets with a reassortant pH1N1 and seasonal
human influenza A (H3N2) showed increased severity of le-
sions probably related to a higher viral replication (16).
Reassortant viruses originating from pH1N1 different from
the swH3N2/pH1N1 reassortant of the present article have
already been described in 2010 in swine from around the world
(3, 12, 18), raising the concern of a possibly more pathogenic
reassortant. Most of the pH1N1 and swine influenza virus
reassortants that have been reported are H1N2 viruses (3, 12).
Interestingly, one other swine H3N2 strain (sw/MN/239105/
09), which possesses pH1N1-related genes, has recently been
reported in the United States (3). However, the gene profile of
sw/MN/239105/09 is different from that of our swH3N2/pH1N1
strains, suggesting there were at least two reassortment events
over time. In infected ferrets, the virulence of the sw/MN/
239105/09 strain was similar to that of a North American
swH3N2 triple-reassortant reference strain (3). Similarly, clin-
ical observations in the field also indicate that the swH3N2/
pH1N1 strains are not more pathogenic than circulating en-
demic strains. Nonetheless, full genome sequencing of swine
influenza virus should be regularly performed, and the strains'
pathogenicity should be closely monitored.
Nucleotide sequence GenBank accession numbers. The
GenBank accession numbers of the virus strains sequenced in
this study are as follows: A/swine/Quebec/1259260/2010(H3N2),
JF420890, JF420891, and JF411012; A/swine/Quebec/1265553/
2010(H3N2), JF682719 to JF682724, JF703684, and JF703685;
A/swine/Quebec/1262080/2010(H3N2), JN584176 to JN584182
and JN817421; A/swine/Quebec/1267568/2010(H3N2), JN584183
to JN584190; A/swine/Quebec/1257774/2010(H3N2), JN584191 to
JN584198; A/swine/Quebec/1257777/2010(H3N2), JN584199
to JN584206; A/swine/Quebec/1267566/2011(H3N2), JN584207 to
JN584214; and A/mink/Nova Scotia/1265707/2010(H3N2),
JF682717 and JF682718.
This work was supported by a Natural Sciences and Engineering
Research Council of Canada discovery grant.
FIG. 3. Hemagglutination inhibition (HI) titers of convalescent pig
sera toward swH3N2/pH1N1 viruses. Twenty sera collected from 6- to
8-week-old pigs housed with the animal experiencing the first con-
firmed flu case related to the swH3N2/pH1N1 reassortant (A/swine/
Quebec/1259260/2010) were tested. The boxes extend from the 25th
percentile to the 75th percentile, with a line at the median. The error
bars extending below and above each box show the lowest and highest
HI values obtained with the 20 pig sera that were tested. When an HI
result was negative at the lowest serum dilution (1/10) tested, the value
of the HI titer was established to be equal to 0. Labeling of 2 sets of
data with different superscript letters indicates that these 2 sets of data
are statistically different (P  0.05). Below the x axis, a table indicates
the HI values of the reference sera (ck/150/90 and sw/4001/05) against
each of the swine influenza virus strains tested.
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We are grateful to Brigitte Bousquet and Denis St-Martin for tech-
nical assistance. We are also grateful to the Ministe
`re de l'Agriculture,
des Pe
^cheries et de l'Alimentation du Que
´bec, in particular J. H.
Fairbrother, for collaboration and sharing of swine samples.
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