﻿Molecular and Antigenic Characterization of Reassortant
H3N2 Viruses from Turkeys with a Unique Constellation
of Pandemic H1N1 Internal Genes
Yohannes Berhane1
, Helen Kehler1
, Katherine Handel1
, Tamiko Hisanaga1
, Wanhong Xu1
, Davor Ojkic2
,
John Pasick1
*
1 National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Manitoba, Canada, 2 Animal Health Laboratory, University of Guelph, Guelph,
Ontario, Canada
Abstract
Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this
study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production.
The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage.
However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA,
nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR
H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR
H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the
outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from
FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity
between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95%
while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C,
D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions
respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera
collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro.
Citation: Berhane Y, Kehler H, Handel K, Hisanaga T, Xu W, et al. (2012) Molecular and Antigenic Characterization of Reassortant H3N2 Viruses from Turkeys with a
Unique Constellation of Pandemic H1N1 Internal Genes. PLoS ONE 7(3): e32858. doi:10.1371/journal.pone.0032858
Editor: Leo L. M. Poon, University of Hong Kong, Hong Kong
Received November 21, 2011; Accepted January 31, 2012; Published March 21, 2012
Copyright: ß 2012 Berhane et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was funded by the Canadian Food Inspection Agency (CFIA) and by the Poultry Industry Council of Canada (http://www.
poultryindustrycouncil.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: john.pasick@inspection.gc.ca
Introduction
Type A influenza viruses belong to the family Orthomyxoviridae
and have a segmented genome composed of 8 single-stranded
RNAs of negative sense [1]. Although the natural reservoir of
influenza A viruses are wild aquatic and shore birds, these viruses
have been isolated from humans and a wide variety of other
animal species including domestic poultry, swine, horses, minks,
whales, cats and dogs [2,3]. Influenza A viruses are classified on
the basis of their hemagglutinin (HA) and neuraminidase (NA)
surface glycoproteins of which 16 and 9 subtypes respectively have
been identified to date. The antigenic characteristics of these two
surface glycoproteins are important in eliciting protective antibody
responses by the host [1,2].
Although the HA protein is an important target of the host
immune response and subtype specific anti-HA antibodies usually
provide protection against infection with viruses of same HA subtype
[4], new antigenic variants that result from the accumulation of point
mutations (antigenic drift) within antigenic sites, frequently emerge
in response to host immune pressure. This results in the appearance
of antigenic variants within the same subtype that are capable of
evading the host's immune response [5,6]. In addition, new
antigenic variants can also emerge by reassortment when influenza
A viruses with different HA and NA subtypes co-infect the same
animal (antigenic shift). This process leads to the appearance of new
subtypes with dramatic changes in antigenicity [5,7].
As a result of antigenic shift or drift, influenza viruses with novel
combinations of gene segments or point mutations have been
isolated from various animal species [1,5,8,9]. In April 2009 a
novel H1N1 influenza virus reassortant that contained genes from
North American and Eurasian influenza viruses began infecting
people in Mexico. This new influenza virus quickly spread into the
USA and Canada and subsequently worldwide [10]. Soon after
the initial human reports, this pandemic H1N1 (pH1N1) virus was
isolated from a swine herd in Alberta, Canada in May 2009 [9,11].
The first report of pH1N1 virus infection of turkeys came from
Chile in August 2009 [12] and later from Ontario, Canada in
October 2009 [13]. Involvement of these livestock species further
complicated public health and veterinary regulatory responses due
to the unknown roles that pigs, poultry and other domestic animals
might play in the evolution of this virus. Consequently, emergence
of such novel viruses with unique gene constellations not only
poses a threat to human health, but might also have implications
for animal health and international trade.
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Swine influenza viruses have received increased attention in
recent years from both the veterinary and public health authorities
because of pigs being viewed as potential ``mixing vessels'' for the
generation of novel viruses. In North America, the viruses that
have been responsible for outbreaks in swine since 1998 have
changed dramatically from the viruses that were responsible for
outbreaks in the previous 70 years [14]. Prior to 1998 swine
influenza was almost exclusively caused by viruses of the classical
swine H1N1 lineage (cH1N1) which was first identified in North
America in 1930 [15]. This H1N1 virus is also related to the 1918
Spanish influenza virus.
This situation changed in 1998 when a severe outbreak of swine
influenza occurred in North Carolina followed by additional
outbreaks in Minnesota, Iowa and Texas [14,16]. The viruses
responsible for the Minnesota, Iowa and Texas outbreaks were
triple reassortant (TR) H3N2 viruses which contained human
(HA, NA, PB1), swine (NS, NP, M) and avian (PB2, PA) influenza
A virus genes [16,17]. By the end of 1999 these TR H3N2 viruses
were widespread in the US swine population and were first
reported in Canadian pigs and turkeys in 2005 [6].
Approximately 45% of the turkey production in Canada is
located in Ontario, often in areas that are densely populated with
both swine and turkey farms. Since 2005, drops in egg production
in breeder turkeys attributed to and coinciding with the circulation
of TR H3N2 viruses in adjacent pig farms [6] has plagued turkey
producers in Southern Ontario. Others have reported similar
reproductive problems in female turkeys in association with TR
H3N2 infection [18,19,20,21]. As was previously suggested for the
situation in the US [18], the close proximity of swine and turkey
farms appears to play a significant role in the epidemiology of TR
H3N2 influenza virus infection of turkeys.
In a previous study [13], we genetically characterized pandemic
H1N1 2009 viruses isolated from breeder turkeys that had an
associated drop in egg production. In this paper, we antigenically
and genetically characterized three unique TR H3N2 influenza
viruses that were isolated from three breeder turkey flocks at
different geographic locations in Southern Ontario at different
times during 2011.
Materials and Methods
Clinical submissions
This study involved a field outbreak of influenza A virus in
turkeys. The owners of the animals associated with this study have
read and approved this manuscript but wish to remain
anonymous. Virus isolation procedures involving embryonating
chicken eggs were in compliance with Canadian Council for
Animal Care guidelines. The chicken embryos were inoculated at
9 days of gestation and fluids harvested 5 days afterwards.
All three diagnostic submissions were comprised of tracheal and
cloacal swabs. They originated from three geographically separate
breeder turkey farms owned by the same company in Southern
Ontario that had experienced significant drops in egg production.
Two of three flocks were vaccinated with an inactivated A/
Mallard Duck/MN/79/79 (H3N4) oil emulsion vaccine and had
detectable antibody titers before exposure to field virus. The
original samples concerning this study were collected by the
company veterinarian and submitted to the provincial veterinary
diagnostic laboratory (Animal Health Laboratory, University of
Guelph). Diagnostic test results along with the samples were
forwarded to the National Centre for Foreign Animal Disease
(NCFAD) as part of a federally mandated disease surveillance
program. Additional work was carried out at the company's
request to determine the reason for vaccine failure. The owners of
the birds provided NCFAD with the convalescent sera collected
from sick turkeys and the sera collected from H3N4 vaccinated
turkeys prior to field virus as part of a follow up study aimed at
determining the reason for vaccine failure.
Reference antisera to A/Perth/16/2009 (H3N2) produced in
ferrets was carried out under Animal Use Document H-07-13
``Preparation of reference antisera to various strains of live human
influenza virus in ferrets'' approved by The Canadian Science
Centre for Human & Animal Health Animal Care Committee in
compliance with Canadian Council for Animal Care guidelines.
The remaining antisera were prepared under Animal Use
Document C-08-002 ``Production of antisera to avian influenza
viruses and avian paramyxoviruses'' approved by the same Animal
Care Committee.
The chicken red blood cells are obtained on a weekly basis from
the Canadian Food Inspection Agency's Ontario Laboratory
Fallowfield specific-pathogen-free flock under animal use docu-
ment ACC 11-03 ``Blood collection from farm animals'', that was
approved by the institutional Animal Care Committee in
compliance with Canadian Council for Animal Care guidelines.
The turkey red blood cells were purchased from LAMPIRE
Biological Laboratories, Inc. P.O. Box 270 Pipersville, PA, USA.
1st
submission (FAV-003). On February 24, 2011 tracheal
and cloacal swabs from a breeder turkey flock of 15,000 that was
exhibiting a sudden drop in egg production with no other
apparent clinical signs were submitted to the Animal Health
Laboratory (AHL), University of Guelph. Turkey hens from this
flock had not been vaccinated with the inactivated H3N4 vaccine
mentioned above. A H3N2 subtype influenza A virus was
identified by molecular means and on February 25 tracheal and
cloacal swabs were forwarded to the National Centre for Foreign
Animal Disease (NCFAD), Winnipeg for virus isolation and
further characterization. Sixteen convalescent serum samples were
submitted at a later time point as part of the follow up
investigation.
2nd
submission (FAV-009). On June 6, 2011, 34-week-old
turkey hens from a different geographical location exhibited a 10%
drop in egg production with no other apparent clinical signs. A H3
subtype influenza A virus was identified by molecular means at
AHL, University of Guelph and tracheal and cloacal swabs were
forwarded to NCFAD, Winnipeg for confirmation and further
characterization. These turkeys had been previously immunized
with an inactivated H3N4 vaccine. Follow up serum samples were
not obtained from birds in this flock.
3rd
submission (FAV-010). On June 17, 2011, a flock from a
third geographical location exhibited a 20% drop in egg production
with no other clinical signs. These turkeys had received the
inactivated H3N4 vaccine. Tracheal and cloacal swabs were
forwarded to NCFAD on June 20th
for confirmation. Nineteen
serum samples collected before the turkeys exhibited the drop in
egg production were submitted as part of a follow up investigation.
RNA extraction from swab samples
Tracheal and cloacal swab specimens were clarified by
centrifugation, 50 ml of sample was spiked with an exogenous
internal control (for evaluating nucleic acid extraction efficiency
and presence of PCR inhibitors during RT-PCR) and RNA was
extracted with the MagMAXTM
-96 Total RNA Isolation Kit using
the MagMax 96-well robotic system (Applied Biosystems/Ambion,
Austin, Texas).
Real time RT-PCR
Total RNA extracted from the swab specimens were tested using
the M1 gene specific real-time reverse transcription polymerase
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chain reaction (RRT-PCR) assay [22] and the modified version of
this assay developed at NCFAD and described previously [9].
Virus isolation
Virus isolation was carried out by inoculating the allantoic
cavity of 9-day-old specific-pathogen-free (SPF) embryonating
chicken eggs with clarified and antibiotic treated swab samples.
Embryos were monitored daily for mortality. Amnio-allantoic fluid
(AAF) from dead embryos as well as from embryos at the end of 1st
and 2nd
passages were harvested and tested for the presence of
hemagglutinating agents with chicken red blood cells (CRBC).
The AAF were also tested for the presence of influenza A nucleic
acids by a real-time RT-PCR assay as described above to exclude
presence of influenza A viruses that did not hemagglutinate
CRBC. All submissions underwent up to two passages before
being considered negative.
Hemagglutination and hemagglutination inhibition tests
Hemagglutination and hemagglutination inhibition (HI) tests
were carried out using standard procedures. For the hemagglu-
tination test, AAF was tested for the presence of hemagglutinating
agents using CRBC or turkey red blood cells (TRBC). Antigenic
characterization of the new isolates was performed by HI assay
using a panel of reference antisera prepared against the 16 known
HA subtypes of influenza A viruses. Two fold serial dilutions of
each reference antiserum were mixed with 4 HA units of each
virus, followed by the addition of 0.5% (v/v) suspensions of CRBC
or TRBC. The reciprocal of the highest dilution of serum that
completely inhibited hemagglutination was considered the HI
titre. The reference antiserum that produced the highest HI titer
indicated the HA subtype of the isolate.
Immune plaque reduction virus neutralization assay
Immune plaque reduction virus neutralization (IPRVN) assay
was carried out using MDCK cells grown overnight to confluency
in 96-well tissue culture plates (Corning, USA). Virus neutraliza-
tion was carried out using a constant amount of H3N2 virus (100
plaque forming units) mixed with equal volumes of 2-fold serial
dilutions (starting 1:20) of convalescent sera collected from
diseased turkeys, field sera collected from turkeys immunized with
the H3N4 vaccine prior to exposure, as well as a panel of reference
H3 antisera. After 1 hr of incubation at 37uC, the virus/antisera
mixtures were applied to the MDCK cell monolayers and
incubated for an additional 1 hr at 37uC. The virus/antiserum
mixture was then replaced with DMEM containing 0.2% (w/v)
bovine serum albumin and 1.5% carboxymethyl cellulose (Sigma).
The cells were incubated at 37uC in a humidified atmosphere of
5% CO2 for 48 hrs after which they were fixed in 10% formalin
solution in PBS. Cells were then permeablized with 20% acetone
in PBS, washed with PBS-Tween and then primed with anti-
influenza nucleoprotein monoclonal antibody [9] for 1 hr. After 3
washes with PBS-Tween solution, the cells were allowed to
incubate with HRP-conjugated goat anti-mouse secondary
antibody (Jackson Immunoresearch) for 1 hr. Finally, plaques
were stained with TrueBlue substrate (KPL, Gaithersburg, MD)
and visualized under the microscope and counted.
The following viruses were used in the IPRVN assay: A/
Turkey/ON/FAV-003/2011 (H3N2), A/Turkey/ON/FAV-009/
2011 (H3N2), A/Mallard/QC/2323-66/2006 (H3N2), A/Duck/
BC/7846/2006 (H3N8) and A/Turkey/BC/1529-3/2005 (H3N2)
a TR virus isolated from domestic turkeys. Reference antisera raised
against A/Turkey/BC/1529-3/2005 (H3N2), A/Duck/BC/7846/
2006 (H3N8) and A/Perth/16/2009 (H3N2) (donated by Dr Yan
Li, National Microbiology Laboratory, Public Health Agency of
Canada) along with field serum samples collected from turkeys that
were vaccinated with A/Mallard Duck/MN/79/79 (H3N4) prior
to exposure to wild type H3N2 virus and convalescent serum
samples collected from turkeys exposed to A/Turkey/ON/FAV-
003/2011 (H3N2), but that were not vaccinated with the H3N4
vaccine were assessed by IPRVN assay.
Amplification, cloning and sequencing of full influenza A
gene segments
Viral RNA was extracted from infectious AAF collected from
embryonating chicken eggs. Total RNA was extracted as
described above using the MagMAXTM
-96 Total RNA Isolation
Kit. Full-length influenza A gene segments were RT-PCR
amplified using universal influenza A primers [23], ligated into
the pCR4H-TOPOH cloning vector (Invitrogen) which was then
used to transform One-Shot TOPO10 E. coli (Invitrogen).
Bacterial clones were screened by PCR and plasmids from clones
that contained the genes of interest were used for sequencing as
described previously [24].
Phylogenetic analysis
Phylogenetic analysis was performed as described previously
[24]. Briefly, the full HA and NA nucleotide sequences and the
reference virus sequences retrieved from GenBank were aligned
initially with the Megalign program (DNASTAR, Madison, WI),
using the Clustal V alignment algorithm. Generation of phyloge-
netic trees was performed by using molecular evolutionary genetics
analysis version 4 (MEGA 4). Phylogenetic trees were generated
with the close-neighbor joining and 500 bootstrap replicate
options of the maximum parsimony method.
Crystal structure manipulations
The amino acid sequences of the HA1 subunit of A/Turkey/
ON/FAV-003/2011 (H3N2) and A/Turkey/ON/FAV-009/
2011 (H3N2) were aligned with those of A/Mallard Duck/MN/
1979 (H3N4) and A/Swine/ON/33853/2005 (H3N2) to identify
amino acid substitutions within the five antigenic sites (A, B, C, D
and E). The HA crystal structure of the H3 subtype influenza
virus, A/duck/Ukraine/1963 (PDB 1MQL) [25] was used as
reference. Molecular graphics images were produced using
PyMOL (http://www.pymol.org). Resulting images were import-
ed into Adobe Photoshop and assembled with Adobe Illustrator
(Adobe).
Results
Clinical submission # 1 (FAV-003)
All 5 swab specimens that were submitted to NCFAD on
February 25, 2011 tested positive with the influenza A matrix real
time RT-PCR assay developed by USDA [22]. Samples were
inoculated into embryonating SPF chicken eggs with only one
yielding virus after 2nd
passage which did not hemagglutinate
CRBC. The presence of virus in AAF was confirmed by influenza
A matrix real time RT-PCR assay. Real time RT-PCR and virus
isolation results for this and the other 2 submissions are
summarized in Table 1.
Turkeys in this barn had not been immunized with the
inactivated H3N4 vaccine. Convalescent serum samples (n = 16)
submitted from this farm tested positive on HI assay using a panel
of reference H3 viruses. The results, which are summarized in
Table 2, show that these sera reacted with the Cluster IV TR
H3N2 virus A/Turkey/BC/1529/2005 as well as A/Mallard/
QC/2223-66/2006 which was isolated from a mallard duck in
Quebec in 2006.
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Clinical submission # 2 (FAV-009)
Three tracheal and 3 cloacal swab specimens from this farm
tested negative using the influenza A matrix real time RT-PCR
assay originally described by USDA [22]. When these were re-
tested using a modified version of this assay designed to have an
increased analytic sensitivity for the matrix gene segment found in
pH1N1 2009 [9], 5 out of 6 of the samples tested positive.
However, a virus which hemagglutinated CRBC and TRBC was
isolated from only one of the tracheal swab specimens.
Clinical submission # 3 (FAV-010)
The swab specimens from this submission also tested negative
when the real time RT-PCR assay targeting the matrix gene as
originally described previously [22] was used; however, 5 out of
6 swabs tested positive using the modified version of this assay and
3 out of 6 samples yielded isolates that hemagglutinated CRBC
and TRBC. These turkeys had been immunized with an
inactivated H3N4 vaccine prior to exhibiting a drop in egg
production. Nineteen serum samples that had been collected
following vaccination but prior to the drop in egg production were
tested using a panel of reference H3 viruses. The results, which are
summarized in Table 2, show that the sera from this farm
(submission FAV-010) reacted most strongly with the H3N2 virus
isolated from a mallard duck in 2006. In contrast, low to negative
titers were observed against the viruses that were isolated from
these 3 submissions after the birds exhibited a drop in egg
production as well as against a Cluster IV TR H3N2 virus that was
isolated from turkeys in 2005.
Virus identification and typing
For the single isolate from submission FAV-003, virus HA
subtyping was done using molecular techniques as this virus did
not hemagglutinate CRBC. For this purpose, the full HA gene
segment was amplified, cloned and sequenced. NA subtyping was
also determined by molecular means using the universal primers
described by Hoffman et al. [23]. Sequencing of the full HA and
NA gene segments confirmed this virus to be H3N2. Since isolates
from submissions FAV-009 and FAV-0010 yielded viruses that
hemagglutinated CRBC and TRBC, the HA subtyping was done
by HI assay using a panel of reference antisera developed against
all known 16 HA subtypes. Isolates from both submissions reacted
with rabbit polyclonal serum developed against A/Turkey/BC/
01529/2005 (H3N2). Neuraminidase typing was done by RT-
PCR, followed by sequencing as described above and isolates from
both submissions were typed as N2.
Genetic characterization of H3N2 isolates
The 8 gene segments for the three H3N2 isolates were
amplified, cloned and sequenced. The sequence data for the
H3N2 viruses isolated in this study were deposited in GenBank
(FAV-003 acc # JN683626-33; FAV-009 acc # JN683634-41 and
FAV-0010 acc # JN706697-704). Genetic relatedness of each
gene segment from each of the isolates from the three farms was
compared with other published influenza A sequences using the
basic alignment search tool (BLAST) from the GenBank database.
Based on the BLAST search, we were able to identify two
genetically distinct H3N2 viruses. The single isolate from
submission FAV-003 was identified as a triple reassortant virus
containing gene segments of avian (PB2, PA), human (PB1, HA,
NA) and swine (NP, M, NS) influenza virus origin. The two virus
isolates from submissions FAV-009 and FAV-0010 contained a
constellation of genes that has not been previously described. Gene
segments PB2, PA, NP and M were from pandemic H1N1 2009
while the remaining gene segments (PB1, HA, NA and NS)
originated from the TR H3N2 viruses that were isolated from pigs
in the USA beginning in 1998 and in Canada beginning in 2005.
The percentage of genetic relatedness of the three H3N2 isolates
to other published influenza viruses in the NCBI database are
summarized in Table 3.
Phylogenetic characterization of the H3N2 isolates
Phylogenetic analysis of the HA and NA genes of the virus
isolated from submission FAV-003 showed that they clustered with
other TR H3N2 viruses that were isolated from pigs in Quebec in
2009 (Fig. 1). The HA (Fig. 1A) and NA (Fig. 1B) genes from the
two virus isolates from submissions FAV-009 and FAV-0010
showed a close evolutionary relationship with a TR H3N2 virus
that was isolated from pigs in the province of Quebec in 2010
(Fig. 1).
Antigenic characterization of the newly isolated viruses
We compared the ability of the three newly isolated TR H3N2
viruses to react with different reference H3 antisera as well as field
sera that had been collected from turkeys that were vaccinated
with the inactivated H3N4 vaccine (submission FAV-0010) and
convalescent serum collected from turkeys following H3N2
exposure (submission FAV-003). Polyclonal antisera raised against
the TR H3N2 virus A/Turkey/BC/1529-3/2005 and the human
seasonal H3N2 virus A/Perth/16/2009 were able to neutralize all
three of the newly isolated viruses. In contrast, polyclonal
antiserum raised against A/Duck/BC/7846/2006 (H3N8) poorly
neutralized the TR H3N2 viruses that were isolated from the
turkeys in this study. The sera collected from H3N4 vaccinated
turkeys (submission FAV-0010) showed strong cross-neutralizing
activity against A/Mallard/QC/2323-66/2006 (H3N2), but weak
activity (titer = 1/40) against the three newly isolated TR H3N2
viruses as well as an earlier TR H3N2 virus from turkeys (A/
Turkey/BC/1529-3/2005). The convalescent sera collected from
Table 1. Influenza A matrix gene real time RT-PCR and virus isolation results for the swab specimens received from the three
turkey farms.
Submission Number Number of swabs Matrix real time RT-PCR Isolation in embryonating chicken eggs
Spackman Modified
FAV-003 5 5/5 ND 1/5
FAV-009 6 1/6 6/6 1/6
FAV-0010 7 ND 5/6 3/6
ND = not determined.
doi:10.1371/journal.pone.0032858.t001
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turkeys from submission FAV-003 cross-neutralized all three of the
newly isolated TR H3N2 viruses as well as A/Mallard/QC/2323-
66/2006 (H3N2) and A/Turkey/BC/1529-3/2005 (H3N2). This
indicated that the flock might have been previously exposed to H3
viruses of avian and swine TR H3N2 origin. The virus
neutralization titer was higher (.2560) against an isolate from
the same farm (FAV-003). Results for HI assays (Table 4) show
similar cross-reactivity patterns as were observed with the virus
neutralization assay (Table 5).
Molecular characterization of the hemagglutinin protein
To determine the level of genetic relatedness between the three
H3N2 turkey isolates, the full hemagglutinin protein (HA0) and
the 328 residues of the HA1 subunit were subjected to pairwise
amino acid identity comparisons with the A/Mallard duck/79/79
(H3N4) vaccine strain [18] and a prototype cluster IV TR H3N2
virus A/Swine/ON/33853/2005 [8] using the ALIGN tool
(NCBI sever). Results of this comparison are summarized in
Table 6.
Table 2. Hemagglutination inhibition titres of pre-exposure serum samples submitted from turkeys vaccinated with H3N4 (FAV-
0010) and convalescent sera collected from turkeys exposed to triple reassortant H3N2 field virus (FAV-003).
Serum
A/Tk/BC/01529/
2005(H3N2)
A/Mallard/QC/2323-6/
2006(H3N2)
A/Tk/ON/FAV003/
2011(H3N2)
A/Tk/ON/FAV009/
2011(H3N2)
A/Tk/ON/FAV0010/
2011(H3N2)
FAV-010 Serum Submission1
1 Neg Neg Neg Neg Neg
2 16 256 8 16 32
3 Neg 32 Neg Neg 8
4 16 256 8 8 16
5 Neg 32 Neg Neg 32
6 32 64 8 16 32
7 Neg 256 8 8 32
8 Neg 256 Neg 8 32
9 Neg 32 Neg Neg 4
10 Neg 64 Neg Neg Neg
11 8 256 8 8 32
12 Neg 256 8 8 32
13 Neg 128 Neg Neg 32
14 Neg 512 Neg 8 32
15 Neg 1024 8 16 64
16 8 16 Neg 4 32
17 Neg 32 Neg Neg 8
18 Neg 32 Neg Neg 16
19 Neg 64 Neg Neg 16
FAV-003 Serum submission
20 .8192 64 .8192 256 1024
21 .8192 512 .8192 512 1024
22 .8192 512 .8192 512 4096
23 4096 128 .8192 256 2048
24 2048 64 4096 512 4096
25 .8192 4096 4096 512 2048
26 .8192 4096 4096 1024 4096
27 4096 64 .8192 256 1024
28 .8192 2048 4096 1024 2048
29 .8192 512 1024 512 4096
30 4096 128 .8192 512 2048
31 .8196 256 .8196 512 1024
32 4096 128 4096 512 2048
33 .8192 64 .8192 512 2048
34 .8192 2048 .8192 1024 .8192
35 .8192 128 .8192 512 .8192
1
Sera from submission FAV-010 tested negative for antibodies to pH1N1 by hemagglutination-inhibition assay.
doi:10.1371/journal.pone.0032858.t002
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Table 3. Top BLAST matches of the 8 gene segments from FAV-003 and FAV-009 obtained from NCBI influenza A virus nucleotide
database.
Segment A/Tk/ON/FAV003/2011(H3N2) A/Tk/ON/FAV009/2011(H3N2)
PB2 NA 99% A/swine/QC/1840-2/2009(H3N2) 99% A/Ontario/315637/2009(H1N1)
AA 99% A/swine/QC/1840-2/2009(H3N2) 99% A/Australia/24/2009(H1N1)
PB1 NA 99% A/swine/QC/1698-1/2009(H3N2) 99% A/swine/Minnesota/66853/2006(H3N2)
AA 99% A/swine/QC/1840-2/2009(H3N2) 99% A/turkey/BC/1529-3/2005(H3N2)
PA NA 99% A/swine/QC/1698-1/2009(H3N2) 99% A/Ontaio/9739/2009(H1N1)
AA 99% A/swine/QC/1840-2/2009(H3N2) 99% A/Canada-MB/RV2023/2009(H1N1)
HA NA 99% A/swine/QC/1698-2/2009(H3N2) 99% A/swine/QC/1268883/2010(H3N2)
AA 98% A/swine/QC/1840-2/2009(H3N2) 99% A/swine/QC/1268883/2010(H3N2)
NP NA 99% A/swine/QC/1698-1/2009(H3N2) 99% A/swine/Taiwan/CH-1204/2004(H1N1)
AA 99% A/swine/QC/1697-1/2009(H3N2) 98% A/Regensburg/D6/2009(H1N1)
NA NA 99% A/swine/QC/1698-2/2009(H3N2) 98% A/Tk/BC/1529-3/2005(H3N2)
AA 99% A/swine/QC/1698-5/2009(H3N2) 98% A/Ontario/RV1273/2005(H3N2)
M NA 99% A/swine/QC/1698-5/2009(H3N2) 99% A/Taiwan/126/2009(H1N1)
AA 99% A/swine/QC/1840-2/2009(H3N2) 100% A/Ontario/RV1527/2009(H1N1)
NS NA 98% A/swine/QC/1698-1/2009(H3N2) 98% A/Tk/OH/313053/2004(H3N2)
AA 97% A/swine/QC/1698-1/2009(H3N2) 97% A/Sw/N.Carolina/02023/2008(H1N1)
doi:10.1371/journal.pone.0032858.t003
Figure 1. Phylogenetic analysis of the HA and NA genes. The HA (a) and NA (b) gene segments of the unique TR H3N2 viruses isolated from
turkeys were compared with other TR H3N2 viruses from turkey (open circle), quail (open diamond) and pig (open triangles) that were previously
sequenced by our laboratory [24].
doi:10.1371/journal.pone.0032858.g001
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The predicted amino acid identities of the HAl subunit
(excluding the signal peptide) between the H3N4 vaccine strain
and FAV-003 was 79% and between the H3N4 vaccine strain and
FAV-009 or FAV-0010 was 80%. By comparison, the predicted
amino acid identity between a prototype H3N2 (A/Sw/ON/
33853/2005) virus from cluster IV and the three turkey isolates
was 95%. When the turkey isolates were compared with each
other, the predicted amino acid identity decreased to 91%.
To determine whether the amino acid changes occurred in any
of the previously identified [18,26] 5 antigenic sites (A, B, C, D
and E), we aligned the amino acid sequences of the 3 turkey
isolates and compared them with the H3N4 vaccine strain and a
prototype cluster IV H3N2 virus and marked the antigenic sites as
presented in Fig. 2 and Fig. 3. Compared to the vaccine strain,
FAV-003 and FAV-009/10 had 19 and 16 amino acid changes
within the major antigenic sites respectively. The triple reassortant
isolate from the FAV-003 submission contained at least 8 amino
acid differences in HA1 when compared to the swine H3N2 virus
that it was most closely related phylogenetically. In contrast, FAV -
009/10 isolates had 5 amino acid differences in the 328 amino
acid residues of the HA1 subunit when compared with the
phylogenetically related A/Sw/QC/1265553/2010 (H3N2) virus.
The turkey isolates from this study possessed some unique changes
at antigenic site B; in the prototype cluster IV virus, amino acids at
position 155 ­ 160 (HNLDYK) were changed to HNLNYK in the
virus isolated from submission FAV-003 and YHLGHK in the
viruses isolated from submissions FAV-009/10. The changes in
antigenic site A for FAV-009/10 were almost identical to those of
A/Sw/QC/126553/2010 (H3N2), but 132N was substituted with
132D. The receptor binding site (RBS) of the influenza virus HA is
a conserved pocket of amino acids surrounded by antigenically
variable antibody binding sites [27]. The RBS of the viruses
examined in this study were mostly conserved; the turkey viruses
had the amino acids Y98, G134, S136, W153, H183, Y195,G225
and S227 which was the same for the prototype TR H3N2 cluster
IV virus and the H3N4 vaccine strain. Notable changes were
E190D, Q226V and G228S which were found in all 3 turkey
viruses when compared with the H3N4 vaccine strain.
To predict N-linked glycosylation sites (Asn-X-Ser/Thr, where
X is any amino acid except Pro), we used the NetNGlyc 1.0
Table 4. Antigenic characterization of triple reassortant H3N2 viruses isolated from turkeys by hemagglutination-inhibition assay
using turkey red blood cells and various reference H3 antisera.
Polyclonal Antisera Viruses
Dk/BC/
7846/06
Tk/BC/
1529/05
Dk/ON/
05/00
Perth/
16/09
Tk/ON/
FAV003/11
Tk/ON/
FAV009/11
Tk/ON/
FAV010/11
Rabbit 1anti- A/Dk/BC/7846/06
(H3N8)
2048 32 ND ND 32 16 16
Rabbit 2anti- A/Tk/BC/1529/05
(TR H3N2)
ND 4096 32 ND 2048 128 256
Goat 3anti- A/Dk/ON/05/00
(TR H3N2)
ND 32 2048 ND 32 16 16
Ferret 4anti- A/Perth/16/09
(H3N2)
ND 128 ND 640 512 256 128
Negative Rabbit Serum 0 0 0 0 0 0 0
doi:10.1371/journal.pone.0032858.t004
Table 5. Summary of cross neutralization assay results as determined by IPRVN of TR H3N2 viruses isolated from turkeys using
various reference H3 antisera.
Polyclonal
Antisera Viruses
A/Tk/BC/15293/05
(TR H3N2)
A/Dk/BC/7846/06
(H3N8)
A/Tk/ON/FAV003/11
(H3N2)
Tk/ON/FAV009/
11(H3N2)
A/Mal/QC/2323-66/06
(H3N2)
A/Tk/BC/1529-3/05
(TR H3N2)
2560 ND 640 640 40
A/Dk/BC/7846/06
(H3N8)
80 2560 40 40 2560
A/Perth/16/09
(H3N2)
1280 ND 640 1280 40
FAV-010 (#1)
H3N4 vaccinated
40 ND 40 40 1280
FAV-010 (#2)
H3N4 vaccinated
40 ND 40 40 2560
Convalescent serum FAV-003 (#1) 2560 ND 2560 2560 1280
Convalescent serum FAV-003 (#2) 1280 ND 1280 1280 2560
ND ­ Not Done.
doi:10.1371/journal.pone.0032858.t005
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Server. Based on this, the H3N4 vaccine strain had 5 glycosylation
sites at positions 8, 22, 38, 165 and 285 as described previously
[18]. However, the three turkey isolates from this study had
6 potential N-linked glycosylation sites at positions 22, 38, 63, 126,
165 and 285 (underlined in Fig. 2). These sites were also the same
for other phylogenetically related triple reassortant viruses, but the
three turkey isolates lacked one additional potential glycosylation
site at residue 246. This site was also present in the prototype
cluster IV virus and other recent and closely related swine isolates
from Quebec.
Discussion
In a previous report, we isolated pandemic H1N1 2009 virus
from a breeder turkey flock that exhibited respiratory illness and a
drop in egg production [13]. In this report, we describe the genetic
Table 6. Percent similarities in the amino acid residues of the HA0 and HA1 of the three H3N2 isolates from turkeys compared to a
prototype cluster IV TR H3N2 strain.
A/Tk/ON/FAV003/2011 A/Tk/ON/FAV010/2011 A/Sw/ON/33853/2005
Isolate HA0 HA1 HA0 HA1 HA0 HA1
A/Tk/ON/FAV009/2011 94% 91% - - - -
A/Sw/ON/33853/2005 97% 95% 96% 95% - -
A/Tk/ON/FAV010/2011 94% 91% 99% 99% 96% 95%
A/M.duck/MN/79/1979 ND 79% ND 80% ND 80%
ND = Not done [full HA sequence of A/Mallard duck/MN/79/1979 (H3N4) was not available].
doi:10.1371/journal.pone.0032858.t006
Figure 2. Locations of the amino acid alterations at the major antigenic sites of HA1 molecule in the H3 crystal structure. (I) Side view
of a HA monomer in cartoon format with major antigenic sites A, B, C and D shown in spheres (II, III, IV, and V??amino acid changes identified at the
major antigenic sites. The locations of changed amino acids are indicated and colored in red. (VI, VII, VIII, IX, and X) Back view of panels I, II, III, IV, and
V, respectively. The view in panels I to V is rotated 180u along Y-axis.TK/ON/FAV-003/2011 vs. DK/MN/1979 (II and VII); TK/ON/FAV-009/2011 vs. DK/
MN/1979 (III and VIII); TK/ON/FAV-003/2011 vs. SW/ON/33853/2005 (IV and IX); TK/ON/FAV-009/2011 vs. SW/ON/33853/2005 (V and X).
doi:10.1371/journal.pone.0032858.g002
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Figure 3. Alignment of the H3 HA1 amino acid sequences (without signal peptide). Amino acids of the HA1 subunit of the three unique
turkey isolates, the duck H3N4 vaccine strain, a prototype cluster IV TR H3N2 virus (A/SW/ON/33853/2005) and phylogenetically related isolates A/Sw/
QC/1265553/2010 (H3N2) and A/SW/QC/1698-1/2009 (H3N2) were aligned. Residues shown in red, green, blue and purple represent previously
identified antigenic sites A, B, C and D respectively. Potential glycosylation sites are underlined.
doi:10.1371/journal.pone.0032858.g003
H3N2 Viruses with Pandemic H1N1 Internal Genes
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and antigenic characterization of unique TR H3N2 viruses
bearing gene segments from 2009 pandemic H1N1 (PB2, PA,
NP and M) and TR H3N2 (PB1, HA, NA and NS) viruses, the
latter of which have been circulating in swine and soon after turkey
populations in the USA since 1998 [16] and in Canada since 2005
[8]. To our knowledge, this is the first report of the isolation of
pH1N1/TR H3N2 reassortant viruses from a domestic poultry
species.
Phylogenetic analysis of the HA and NA genes from FAV-009
and FAV-0010 showed that they are closely related to A/Swine/
QC/126553/2010 (H3N2) and A/Swine/QC/1268883/2010
(H3N2) which were isolated from pigs with respiratory illness in
Quebec. The latter H3N2 isolate from swine also has pandemic
H1N1 internal genes [28], but of a different assemblage than those
found in FAV-009 and FAV-010. The other TR H3N2 virus (FAV-
003) was closely related to other TR H3N2 viruses that were isolated
from pigs in 2009 in Quebec. Although phylogenetic analysis shows
that these viruses were likely introduced to turkeys from pigs, we
were not able to find any epidemiologic links between the farms.
In contrast to the chicken reproductive tract that contains only a2-
3-linked sialic acids receptors, turkeys may contain both a2-3-linked
and a2-6-linked sialic acids receptors in their reproductive tract
which could be involved in the attachment and replication of human
and swine-like influenza viruses [19,20]. Amino acids at position 226
and 228 in the HA1 subunit of H3 subtype influenza viruses were
shown to be necessary for the switch in receptor specificity from a2-
3-linked to a2-6-linked sialic acids and adaptation from avian to
mammalian hosts [29]. All three turkey isolates described here had
mutations in these 2 amino acid positions (Q226V and G228S) that
are associated with the change from a2-3 to a2-6 specificity. The
drop in egg production in these turkeys is most likely associated with
virus replication in their reproductive tracts.
Despite the fact that pigs are normally viewed as ``mixing
vessels'' for the generation of reassortant influenza A viruses, we
cannot exclude the possibility that the reassortment between TR
H3N2 and pH1N1 took place in turkeys, even though sera from
submission FAV-0010 gave negative HI results when using
pandemic H1N1 antigen (data not shown). Nevertheless, the
results described here should be of concern, considering the
reassortment capacity of this virus and the susceptibility of turkeys
to influenza viruses of H1 to H16 subtypes. According to
Nobusawa et al. [30], a point mutation that resulted in a Glu to
Asp substitution at amino acid position 190 (E190D) in the HA
protein of A/Aichi/51/92 was responsible for the loss of the ability
to bind to CRBC. All three isolates described in this study were
tested for their ability to agglutinate CRBC and TRBC. A/Tk/
ON/FAV-003/2011 did not agglutinate CRBC, but could
agglutinate TRBC. The other two isolates had better hemagglu-
tination results with TRBC than CRBC. All 3 isolates had amino
acid change at E190D, but the only isolate that didn't agglutinate
CRBC was from FAV-003. Therefore, multiple amino acid
changes involving the HA protein are likely necessary to change
the receptor binding specificity. Nakajima et al. 2003 [31]
suggested that the effect of hemadsorption activity of an amino
acid change on the HA protein primarily depends on the position
rather than the species of substituted amino acid. They showed
that mutation of the amino acid at position 156 from lysine to
glutamic acid, asparagine, glutamine, or isoleucine was shown to
be associated with the loss of hemadsorption activity. FAV-003 has
asparagine at this position and this might have had the negative
effect on hemagglutination activity, but the other 2 isolates with
hemagglutination activity had histidine at this position.
The hemagglutinin protein of H3 influenza viruses has
accumulated a number of glycosylation sites during its evolution
over the past 40 years [32]. Glycosylation appears to be one way
by which a virus can mask its epitopes and evade detection by the
host's immune system. In addition, some studies have associated
increasing glycosylation with reduced virulence [32,33,34].
Although the three turkey isolates from this study shared
6 potential N-linked glycosylation sites at position 22, 38, 63,
126, 165 and 285 with the closely related cluster IV viruses of
swine, they lacked a potential glycosylation site at position 246.
In Canada, the only approved vaccine for turkeys against H3N2
viruses is the inactivated H3N4 vaccine made from a strain
isolated from a mallard duck in 1979. Although, two of the breeder
turkey flocks in this study were immunized with this vaccine, the
field strains described here were able to infect the turkeys and
cause a loss in egg production. The fact that the sera collected
from H3N4 vaccinated turkeys were not able to neutralize any of
the newly isolated H3N2 viruses described in this study confirms
the poor efficacy of this vaccine. We believe the vaccine failure was
associated with amino acid substitutions in the globular head
domain of the HA1 subunit which contains the immunodominant
antigenic sites. The percent amino acid identity of the HA1
between the H3N4 vaccine strain and FAV-003 was 79% and with
FAV-009 or FAV-0010 ­ 80%.
Previous studies have also shown that mutations in the
5 immunodominant antigenic sites located on the globular head
of the HA1 subunit play a key role in virus escape from host
immune pressure as a result of accumulated conformational
changes [18,30,35]. The amino acid substitutions coupled with
changes associated with the appearance of oligosaccharide side
chains in the globular head region are responsible for the
generation of antigenic variants [36]. The HA1 of the viruses
from FAV-003 and FAV-009/10 had 19 and 16 amino acid
changes respectively at these major antigenic sites when compared
with the H3N4 vaccine strain. In addition, the isolate from the
FAV-003 submission contained at least 8 amino acid differences in
HA1 when compared to the swine H3N2 virus that it was most
closely related to phylogenetically. According to Wilson and Cox
[35], drifting antigenic variants of epidemiologic importance could
emerge if changes in the five antigenic sites involve more than 4
amino acids and the changes are located in more than 2 of the 5
antigenic sites. The major changes in these newly isolated viruses
were associated with antigenic sites A and B (Fig. 2 and Fig. 3),
which are near the receptor binding site and often play a key role
in role in escape from neutralizing antibodies.
In both humans and domestic animals influenza variants
frequently emerge as a result of point mutations in the HA gene,
resulting in new variants that escape the host immune response.
The vaccine failure described in this study is not surprising;
selection of viruses for animal influenza vaccines should be based
on the results of recent epizootologic, virologic and immunologic
surveillance results. Two of the H3N2 isolates characterized in this
study contained a unique combination of genes derived from
pandemic H1N1 (2009) which underscores the need for contin-
uous surveillance and monitoring of the genetic changes of
influenza A viruses circulating in domestic animals to not only aid
in the selection of the most appropriate vaccine strains but to track
the evolution of viruses that might pose new threats to human and
animal health.
Acknowledgments
We would like to thank Dr Soren Alexandersen for critical review of the
manuscript. We also would like to thank Dr Chris Kranendonk for help
with specimen receiving, laboratory staff of the Animal Health Laboratory,
University of Guelph and the Canadian Food Inspection Agency's Guelph
District Office for sending the samples used in this report.
H3N2 Viruses with Pandemic H1N1 Internal Genes
PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e32858
Author Contributions
Conceived and designed the experiments: YB JP. Performed the
experiments: YB HK KH TH WX DO JP. Analyzed the data: YB JP.
Contributed reagents/materials/analysis tools: YB JP. Wrote the paper:
YB JP.
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