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Dispatch
Triple Reassortant H3N2 Influenza A Viruses, Canada, 2005
Christopher W. Olsen,* Alexander I. Karasin,* Suzanne
Carman,† Yan Li,‡ Nathalie Bastien,‡ Davor Ojkic,† David Alves,§ George
Charbonneau,¶ Beth M. Henning,# Donald E. Low,** Laura Burton,** and George
Broukhanski**
*University of Wisconsin-Madison, Madison, Wisconsin, USA; †Animal
Health Laboratory, Guelph, Ontario, Canada; ‡Canadian Science Centre for Human
and Animal Health, Winnipeg, Manitoba, Canada; §Ontario Ministry of Agriculture,
Food, and Rural Affairs, Guelph, Ontario, Canada; ¶Swine Services Group,
Stratford, Ontario, Canada; #Ontario Ministry of Health and Long Term Care,
Clinton, Ontario, Canada; and **Ontario Ministry of Health and Long Term Care,
Toronto, Ontario, Canada
Suggested citation for this article
Since January 2005, H3N2 influenza viruses have been
isolated from pigs and turkeys throughout Canada and from a swine farmer and
pigs on the same farm in Ontario. These are human/classical swine/avian
reassortants similar to viruses that emerged in US pigs in 1998 but with a
distinct human-lineage neuraminidase gene.
Influenza viruses of the classical H1N1 lineage were the
dominant cause of influenza among North American pigs for >60 years (1).
However, in 1998, H3N2 viruses emerged and rapidly spread throughout the US
swine population (2–4).
These were unique triple reassortant genotype viruses, with hemagglutinin (HA),
neuraminidase (NA), and RNA polymerase (PB1) genes of human influenza virus
lineage; nucleoprotein (NP), matrix (M), and nonstructural (NS) genes of
classical swine virus lineage; and RNA polymerase (PA and PB2) genes of North
American avian virus lineage. Further reassortment between these viruses and
classical H1N1 swine viruses led to the emergence of reassortant H1N2 and H1N1
viruses among pigs in the United States (1). The
reassortant H3N2 and H1N2 viruses have also been isolated from turkeys and
ducks in the United States (5–8).
Despite geographic proximity and cross-boundary trade in pigs and turkeys
between the United States and Canada (9, D. Harvey, pers. comm.), these reassortant viruses did not initially infect
animals in Canada. However, beginning in approximately January 2005, H3N2
influenza viruses swept rapidly across Canada. We describe the genetic
characterization of reassortant H3N2 viruses from pigs, turkeys, and a swine
farm worker in contact with sick pigs during this outbreak.
The Study
Influenza viruses were isolated in Madin-Darby canine kidney
cells from lung tissue or nasal swab samples from pigs of various ages (young
growers to adults) manifesting influenzalike illness (ILI) in Manitoba in
January (A/Swine/Manitoba/12707/05), Alberta in February
(A/Swine/Alberta/14722/05), British Columbia in May (A/Swine/British
Columbia/28103/05), and Ontario in July (A/Swine/Ontario/33853/05). No clear
epidemiologic links existed between these farms. A/Ontario/RV1273/05 was
isolated in primary rhesus monkey kidney cells from a nasal swab specimen
collected as part of a diagnostic workup from an otherwise healthy farm worker
in Ontario in whom ILI developed 2–3 days after onset of ILI among pigs on his
premises. Fourteen-day courses of oseltamivir therapy were prescribed for this
patient beginning the day he saw his physician and for 11 other potentially
exposed farm workers beginning 2 days later. The patient recovered uneventfully;
no other respiratory viruses were identified from his samples.
A/Turkey/Ontario/31232/05 was isolated in embryonated hen's eggs from a cloacal
swab sample from turkeys showing a severe drop in egg production. The turkey
farm was located across the road from a swine farm at which pigs concurrently
exhibited ILI, although virus isolates were not available from those pigs.
Nucleotide sequences of the full-length coding regions of
all 8 RNA segments from each virus were determined by direct cycle sequencing with
previously described techniques and primers (3,10,11).
Related reference viruses were identified by BLAST (basic local alignment
search tool) analyses, sequence comparisons were conducted by using DNASTAR
software, version 6.3 (DNASTAR Inc., Madison, WI, USA), and phylogenetic
relationships were estimated from the nucleotide sequences by the method of
maximum parsimony (fast heuristic search algorithm, PAUP software, version
4.0b10 [Sinauer Associates, Inc., Sunderland, MA, USA]) with a bootstrap
resampling method (200 replications).
Pairwise nucleotide identities among the 2005 Canadian
swine, turkey, and human isolates range from 94.0%–100% (NA) to 99.9%–100% (M),
and amino acid identities range from 98.9%–100% (NA) to 100% (M, NP). The human
and swine isolates recovered on a single farm in Ontario are 100% identical in
nucleotide sequences across all 8 RNA segments. Phylogenetically, the 2005
Canadian viruses form single clusters on phylograms for each of the 8 viral RNA
segments, confirming that this epizootic was caused by a single lineage of
viruses. All of the viruses share the same human/classical swine/avian triple
reassortant genotype as the H3N2 viruses that emerged in pigs in the United
States in 1998 (2–4).
The HA genes (Figure 1) of the Canadian viruses are
most closely related to the cluster III group of American viruses that were
first isolated from pigs in 1999 (4) and
subsequently from turkeys (7,8),
though the HA phylogram topography suggests that these Canadian and related US
viruses represent a new, separate cluster (IV) of viruses. The M, NP, NS, and
polymerase genes of the Canadian viruses are also phylogenetically closely
related to reassortant viruses dating back to 1998 in the United States (data
not shown). In contrast, the NA genes (Figure 2) of
the 2005 Canadian viruses, though still clearly of human lineage, are
phylogenetically distinct from most of the US swine and turkey isolates. This
lineage is represented by human H3N2 isolates from Asunción, Paraguay, (2001)
and New York (2003). However, this lineage of NA genes has also been introduced
into animal influenza viruses on 2 previous occasions. The first is represented
by A/Turkey/Ohio/313053/04 (8), which phylogenetically is the
most closely related virus to the Canadian viruses across all 8 RNA segments.
Since this virus was isolated in February 2004 (8),
nearly 1 year before the first isolations of viruses from Canada, one might
conjecture that this or a closely related virus from the United States was the
source of the Canadian viruses. However, this lineage of NA genes was also
already present in human/classical swine reassortant H1N2 viruses
(A/Swine/Ontario/48235/04, A/Swine/Ontario/55383/04) isolated from Canadian
pigs in 2004 (12). Thus, it is neither possible
nor prudent from the phylogenetic data alone to define the specific
epidemiologic source(s) of the 2005 Canadian H3N2 viruses. However, the
appearance of this lineage of NA genes among H3N2 viruses in turkeys in the
United States and Canada and H3N2 and H1N2 viruses in pigs in Canada suggests
that a complicated web of interspecies transmission, reassortment, and
transboundary movement of viruses occurred in a relatively short period of
time.
Conclusions
To our knowledge, this report describes the first isolation
of a human/classical swine/avian triple reassortant H3N2 virus from a human. This
isolation could not have occurred through cross-contamination in a laboratory
since the animal and human virus isolations and sequencing were conducted in
different locations. Hemagglutination-inhibition (HI) and virus neutralization (VN)
assays of acute- and convalescent-phase (11 and 45 days after the acute-phase
sample) sera did not show evidence of seroconversion against the patient's own
isolate, A/Ontario/RV1273/05 (HI titer = 8 and VN titer = 16 on all 3 test
dates). Thus, although this farm worker had a febrile respiratory illness and
no other etiologic agent was identified, we cannot prove that he was actively
infected with the triple reassortant virus; he may have simply been harboring
the virus in his nasal passages. Nonetheless, this isolation shows that
agricultural workers may be exposed to influenza viruses from livestock.
In summary, this report describes the emergence and rapid
spread since January 2005 of reassortant H3N2 influenza A viruses among pigs
and turkeys across Canada and isolation of a related virus from the nasal
passages of a farm worker in Ontario. The 4 swine isolates chosen for our
analyses provide a sampling of viruses from British Columbia to Ontario, but
clinical reports indicate that the outbreak of ILI in pigs was much more
extensive than this limited number of isolates might suggest. For example, H3N2
virus infections were confirmed on 22 swine farms in Ontario between late April
and early July 2005. Likewise, additional infections of turkeys with H3 viruses
in 2005 were reported in British Columbia (on a farm that was near a swine farm
where H3 virus was detected [13]) and in multiple flocks in
Manitoba (A. Hamel and G. Nayar, pers. comm.). When this Canadian epizootic is
considered together with the extensive spread of genotypically similar H3N2 and
H1N2 viruses in pigs and turkeys seen in the United States since 1998, we see
that viruses with this human/classical swine/avian triple reassortant genotype can
efficiently infect both pigs and turkeys.
The GenBank numbers assigned to the gene sequences of
viruses investigated in this study are as follows: A/Ontario/RV1273/05, DQ469955–DQ469962;
A/Swine/Alberta/14722/05, DQ469963–DQ469970; A/Swine/British Columbia/28103/05,
DQ469971–DQ469978; A/Swine/Manitoba/12707/05, DQ469979–DQ469986;
A/Swine/Ontario/33853/05, DQ469987–DQ469994; and A/Turkey/Ontario/31232/05, DQ469995–DQ470002.
Acknowledgments
We thank Gabriele Landolt, Bruce McNab, Paul Innes, Grant
Maxie, Eng-Soon Chan, Theresa Tam, Kerri Watkins, Jean Wilson, and staff
members from the Ontario Ministry of Health and Long-Term Care and the Perth
District Health Unit for helpful discussions; Alireza Eshaghi for excellent
technical support; and Mo Saif for providing sequence information on
A/Turkey/Ohio/313053/04 before submission to GenBank.
Dr Olsen is a professor of public health at the University
of Wisconsin-Madison School of Veterinary Medicine. His professional interests
include understanding infectious diseases at the human-animal interface, in
particular, interspecies transmission of influenza viruses.
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Suggested citation
for this article:
Olsen CW,
Karasin AI, Carman S, Li Y, Bastien N, Ojkic D, et al. Triple reassortant H3N2
influenza A viruses, Canada, 2005. Emerg Infect Dis [serial on the Internet].
2006 Jul [date cited]. Available from
http://www.cdc.gov/ncidod/EID/vol12no07/06-0268.htm
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