﻿Published Ahead of Print 13 October 2010.
2010, 84(24):12636. DOI: 10.1128/JVI.01350-10.
J. Virol.
Parrish
Stephanie Janeczko, Edward C. Holmes and Colin R.
Jessica J. Hayward, Edward J. Dubovi, Janet M. Scarlett,
the United States
Shelters and Its Molecular Epidemiology in
Microevolution of Canine Influenza Virus in
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JOURNAL OF VIROLOGY, Dec. 2010, p. 12636­12645 Vol. 84, No. 24
0022-538X/10/$12.00 doi:10.1128/JVI.01350-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Microevolution of Canine Influenza Virus in Shelters and Its
Molecular Epidemiology in the United States

Jessica J. Hayward,1
Edward J. Dubovi,2
Janet M. Scarlett,2
Stephanie Janeczko,2
Edward C. Holmes,3,4
and Colin R. Parrish1
*
Baker Institute for Animal Health1
and Department of Population Medicine,2
College of Veterinary Medicine, Cornell University,
Ithaca, New York 14853; Center for Infectious Disease Dynamics, Department of Biology, the Pennsylvania State University,
University Park, Pennsylvania 168023
; and Fogarty International Center, National Institutes of Health, Bethesda, Maryland 208924
Received 24 June 2010/Accepted 4 October 2010
Canine influenza virus (CIV) emerged around 2000 when an equine influenza virus (EIV) was transmitted
to dogs in Florida. After 2003, the canine virus was carried by infected greyhounds to various parts of the
United States and then became established in several large animal shelters, where it has continued to circulate.
To better understand the evolution of CIV since its emergence, and particularly its microevolution in spatially
restricted populations, we examined multiple gene segments of CIV from dogs resident in two large animal
shelters in New York City during the period 2006 to 2009. In particular, we focused on viruses circulating in
the two shelters in 2008 and 2009, which we found shared a common ancestor. While viruses in each shelter
were generally monophyletic, we observed some gene flow between them. These shelter sequences were com-
pared to earlier CIV isolates. The shelter viruses differed in 1 to 6 amino acids in each gene segment compared
to viruses isolated in Florida between 2003 and 2005 and in Colorado in 2006 and 2008. A comparison of the
sequences of equine and canine viruses revealed amino acid replacements that distinguished the viruses from
the two hosts, but no clear evidence of positive selection indicative of host adaptation was detected, suggesting
that any host range adaptation in CIV occurred early in the emergence of this virus or even before it transferred
to dogs.
Influenza A viruses are naturally maintained in aquatic
birds, occasionally transfer to mammals to cause individual
infections or outbreaks of disease, and sometimes go on to
cause epidemics and pandemics in their new hosts (25, 37).
Influenza viruses can also transfer between different mamma-
lian host species, as has recently been observed in the case of
swine-origin pandemic H1N1 influenza A virus that emerged
in 2009 in humans (16, 35). However, the determinants of host
range and of interhost transmission of influenza viruses are
often poorly understood. Key questions include the nature of
any host barriers to viral transfer; whether host-adaptive mu-
tants are required for initial infection of new hosts; and the
role, if any, of posttransfer adaptation in determining contin-
ued transmission in the new host species. There are also con-
siderable uncertainties about the epidemiological processes
involved in host switching, including the role played by unusu-
ally dense populations of susceptible hosts, where viruses with
lower transmission efficiencies may be maintained after initial
transfers.
The A/H3N8 canine influenza virus (CIV) is a new pathogen
of dogs (Canis familiaris), which resulted from the transfer of
an intact A/H3N8 equine influenza virus (EIV) (7, 17, 38). CIV
was initially recognized in greyhounds in a Florida training
facility in 2004 and was then spread around the United States
by infected greyhounds in 2004 and 2005, being recognized in
11 states during that period (7). Serological evidence has
shown that the virus was infecting dogs in Florida in 2000 (7).
Although CIV clearly originated from EIV, several differences
between previously circulating equine viruses and the emerged
canine viruses have been documented, including 8 amino acid
replacements in the hemagglutinin (HA) segment (7, 38).
Equine influenza virus A/H3N8 was first recognized in
horses (Equus caballus) in the early 1960s, with the prototype
virus assigned as A/equine/Miami/1/1963 (51). EIV subse-
quently spread to most regions of the globe, with exceptions
including New Zealand (22) and Iceland (49), and with recent
outbreaks in otherwise EIV-free Australia in 2007 (2) and in
South Africa in 2003 that were controlled. EIV has experi-
enced significant evolution during its spread in horses in the
47 years since it emerged, including the emergence of two
antigenically distinct sublineages that were first recognized in
Florida (Florida 1 and 2) but which have subsequently spread
to other regions of the United States and the world (4, 10, 26,
31). Dogs are clearly susceptible to recent strains of A/H3N8
and other influenza A viruses. Sporadic infections were re-
ported for the A/H3N8 EIV during the Australian outbreak
(24), and small outbreaks have been reported in foxhounds in
the United Kingdom (9). In addition, dogs in South Korea
have recently experienced widespread infections by an
A/H3N2 avian influenza virus (28, 46), and canine infections by
the new A/H1N1/09 swine-origin influenza virus have also been
described (13). The A/H3N8 CIV that emerged in the United
States was closely related to EIV strains circulating in horses
around the same period and was clearly derived from a Florida
* Corresponding author. Mailing address: Baker Institute for Ani-
mal Health, College of Veterinary Medicine, Cornell University,
Ithaca, NY 14853. Phone: (607) 256-5649. Fax: (607) 256-5608 E-mail:
crp3@cornell.edu.
 Supplemental material for this article may be found at http://jvi
.asm.org/.

Published ahead of print on 13 October 2010.
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1 lineage EIV (7). Despite being an important recent example
of cross-species transmission, little is known about the current
spread of CIV among dogs in the United States or its evolution
in its new host. The limited data available suggest that CIV is
currently found mostly in the northeastern United States and
in Colorado; that it is primarily being maintained in animal
facilities with large numbers of incoming susceptible dogs, such
as animal shelters and kennels; and that it has not spread
widely within the household dog population (J. M. Scarlett and
E. J. Dubovi, unpublished data).
The emergence of CIV from EIV provides an unusual op-
portunity to investigate the properties and dynamics of viral
evolution during and shortly after a host transfer event. To this
end, we examined the evolution of the CIV circulating during
2008 and 2009 in two large dog shelters in New York City and
compared the viruses to those collected in some other regions
of the United States since 2003. By determining the genetic
diversity of the viruses within dog shelters, we were also able to
explore the microevolution of CIV in defined populations,
utilizing sampling that was restricted in both time and space.
MATERIALS AND METHODS
Viral samples and endemically transmitted viruses: shelters/epidemiology.
Samples were collected from naturally infected dogs from various regions of the
United States between 2005 and 2009 (Table 1). Six samples were collected from
dogs in New York between 2005 and 2007, two from dogs in Colorado in 2006
and 2008, and one each from Philadelphia in 2008 and Virginia in 2009. Naturally
transmitted CIV samples obtained from two animal shelters that were 8 km
apart in New York City that were controlled by the same organization (shelter A
and shelter B) were examined in more detail. Each shelter accepted 300 to 500
dogs per month. Serological testing of 100 incoming dogs in 2008 indicated that
the great majority of dogs entering the shelter were seronegative for CIV on
arrival, but more than 20% became infected within 5 days of introduction into
the shelter (Scarlett and Dubovi, unpublished).
Sample collection. Many samples examined were submitted as diagnostic speci-
mens, tested for the presence of CIV by reverse transcription-PCR (RT-PCR), and
isolated into embryonated eggs or MDCK tissue culture cells. Other viruses were
obtained from shelters A and B in New York City between November 2008 and
December 2009 (Table 1). These samples were obtained as nasal or pharyngeal
swabs from dogs with respiratory disease or as lung samples from dogs that died.
RNA extraction and reverse transcriptase PCR. RNA was extracted from the
samples by the spin protocol of the QIAamp viral RNA minikit (Qiagen, Va-
lencia, CA). A two-step RT-PCR was used to amplify the gene segments, using
H3N8-specific primers (Table 2). The four largest gene segments (PB1, PB2, PA,
HA) were each amplified as two overlapping fragments. Because not all seg-
ments were sequenced for viruses from every dog, we necessarily focused on the
HA1, M, NS, and NA segments. The HA1 RT-PCR protocol used has been
described previously (20). For the other 11 fragments, the protocol was as
follows. Briefly, cDNA was made with universal primer Uni12a (AGC AAA
AGC AGG), deoxynucleoside triphosphates (dNTPs), Superscript III reverse
transcriptase (Invitrogen, Carlsbad, CA), and RNase inhibitor (New England
Biolabs, Ipswich, MA), with 5 g total RNA, and incubated at 55°C for 60 min
followed by 70°C for 15 min. A 5-l aliquot of the cDNA was included in the final
PCR step, with 1 M each primer, 2.5 mM MgCl2, 0.2 mM dNTPs, and 0.1 U
AmpliTaq Gold (Applied Biosystems, Carlsbad, CA). The reaction cycle con-
sisted of an initial denaturation at 94°C for 10 min, followed by 45 cycles of 94°C
for 20 s, 53°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min.
PCR products were purified using the spin protocol of the Qiagen PCR purifi-
cation kit.
TABLE 1. Origins of CIV samples and isolates examined
Sample Sampling datea
Sample typeb
Location
Dog 1 3/20/09 Nasal swab Shelter A
Dog 2 3/20/09 Trachea/nasal Shelter A
Dog 3 3/20/09 Tracheal wash Shelter A
Dog 4 3/20/09 Tracheal wash Shelter A
Dog 5 3/20/09 Tracheal wash Shelter A
Dog 6 3/18/09 Tracheal wash Shelter A
Dog 7 11/18/08 No further details Shelter B
Dog 8 11/20/08 No further details Shelter B
Dog 9 11/20/08 No further details Shelter B
Dog 21 12/18/09 Swab Shelter B
Dog 22 9/22/09 Lung Shelter A
Dog 23 9/22/09 Lung Shelter A
Long Is/2005 September 2005 Virus (AF) Long Island, NY
NewYork/Jan2006 1/16/06 Virus (AF) New York
A/2006 8/16/06 Virus (TC) Shelter A
NewYork/Dec2006 12/18/06 Virus (TC) New York
B/2006 8/16/06 Virus (AF) Shelter B
Colo/2006 January 2006 Virus (AF) Colorado
Staten Is/2007 6/9/07 Virus (TC) Staten Island, NY
Colo/2008 January 2008 Virus (AF) Colorado
Phil/2008 November 2008 No further details Philadelphia
Vir/2009 August 2009 Virus (AF) Virginia
a
Unless otherwise noted, sampling dates are shown as month/day/year.
b
AF, allantoic fluid; Tc, tissue culture.
TABLE 2. Locations of H3N8-specific primers used for RT-PCR
Gene
segment
Position ina
:
Fragment 1 Fragment 2
Forward
primer
Reverse
primer
Forward
primer
Reverse
primer
PB2 1 1250 1150 2342
PB1 1 1253 1148 2341
PA 1 1252 1150 2233
HA 1 1008 927 1773
NP 1 1567
NA 1 1465
M 1 1027
NS 1 892
a
The numbering shown is from A/equine/Miami/1963 (GenBank accession no.
CY028836 to CY028843).
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Cloning and analysis of single sequences. PCR products were cloned into the
pCR4Blunt-TOPO vector in the Zero Blunt TOPO PCR cloning kit (Invitrogen)
and electrotransformed into supplied TOP10 electrocompetent Escherichia coli
cells. Plasmid DNAs prepared by the Miniprep method (Promega, Madison, WI)
were sequenced with the T3 and T7 plasmid primers. Forward and reverse
sequences were assembled with SeqMan (DNASTAR, Madison, WI) and visu-
ally checked. Primer regions were trimmed.
Evolutionary analysis. All EIV sequences (between 77 and 205 per gene
segment) and CIV sequences (between 4 and 10 per gene segment) available up
to 1 March 2010 were downloaded from the Influenza Virus Resource, National
Center for Biotechnology Information (NCBI). A multiple alignment of the
sequences for each gene region was done in ClustalX v1.83.1 (6, 27). The output
alignment was visually checked and edited in Se-Al v2.0a11 (kindly provided by
Andrew Rambaut, University of Edinburgh). Maximum likelihood (ML) trees
were then inferred in PAUP* (48), using substitution model parameters as
determined by Modeltest3.7 (40). Specifically, for the trees containing both EIV
and CIV sequences, these were the K81ufIG, TIMIG, and TVMI
substitution models for the HA1, M, and NS segments, respectively. For the trees
with the shelter CIV sequences, the models used were TIM, HKYI, and TVM
for HA1, M, and NS, respectively. In all cases, a heuristic search was used, with
10 replicates, random sequence addition, and tree bisection-reconnection
branch-swapping. Bootstrap analysis was also performed in PAUP* with 1,000
replicates of neighbor-joining trees under the ML substitution model.
We used the Bayesian Markov Chain Monte Carlo (MCMC) approach avail-
able in the BEAST package (11) to estimate both rates of nucleotide substitution
and the time to the most recent common ancestor (TMRCA) of the HA1, M,
NA, and NS gene segments from CIV and EIV using information on the month
and year of sampling. The sequences in question were very closely related, so
there will be little multiple substitution at single-nucleotide sites. Because of this,
we used the relatively simple HKY85 model of nucleotide substitution in each
case (which accords with the models described above) and assumed a relaxed
log-normal clock and a Bayesian skyline coalescent tree prior (12). The MCMC
chain was run for 200 or 300 million generations with a 10% burn-in. Statistical
uncertainty is provided by values of the 95% highest-probability density (HPD).
To avoid being biased by the large number of intrahost CIV sequences available
for the HA1, M, and NS gene regions, we also used an alignment of only the
consensus sequences from each dog to estimate the rates of nucleotide substi-
tution in these cases.
To determine the strength of geographical structure in these data, and spe-
cifically whether sequences were clustered according to (i) host dog, (ii) dog
shelter, and (iii) sampling date, we employed a Bayesian MCMC approach that
accounts for phylogenetic uncertainty by considering a large set of plausible trees
(36). Hence, using the Bayesian Tip-Associated Analysis (BaTS) program, we
computed the parsimony score (PS) and association index (AI) statistics of
phylogeny-trait association, making use of the posterior sample of trees previ-
ously output from the BEAST analysis and employing 1,000 randomizations.
Finally, to determine the nature of the selection pressures acting on CIV, we
estimated the relative numbers of nonsynonymous (dN) and synonymous (dS)
nucleotide substitutions per site (dN/dS) in the protein-coding gene regions of
HA1, M, NS, and NA, using HyPhy (39). For each gene segment, a neighbor-
joining tree was used and the single likelihood ancestor counting (SLAC), two-
rate fixed-effects likelihood (FEL), and random-effects likelihood (REL) meth-
ods were all employed. In addition, we used the TestBranchDNDS method in
HyPhy to look for differences in the dN/dS ratio on the EIV-to-CIV main
internal branches compared to the rest of the phylogeny. Finally, we used the
two-ratio model implemented in the CODEML program from the PAML pack-
age (54) to determine the dN/dS ratio on external branches and internal branches
of an ML tree constructed with only a single representative sequence from each
dog. If those mutations fixed on internal branches were primarily due to positive
selection, we would expect a higher dN/dS ratio on internal branches than on
external branches of the phylogeny. Conversely, an elevated dN/dS ratio on
external branches is suggestive of the presence of transient deleterious mutations
that have yet to be removed by purifying selection.
Nucleotide sequence accession numbers. New sequences generated here have
been submitted to GenBank and assigned accession numbers HQ237502 to
HQ238128.
RESULTS
Evolution of CIV in dogs since 2003. We examined the
evolution of CIV since its initial recognition in Florida, with a
particular focus on those viruses circulating in the northeastern
United States, including those transmitted within two large
shelters in New York City during 2008 and 2009. We examined
sequences from all gene segments, but since there were many
more sequences for the HA1, NA, M, and NS segments, we
focused our studies of the dynamic aspects of the viral evolu-
tion on these segments. Our phylogenetic analysis confirmed
that the CIV lineage was derived from a single EIV strain
circulating around 2000 (Fig. 1). More notably, our analysis of
multiple gene segments from viruses collected at different
times and places since it emerged suggested a marked degree
of sequence variation among the CIV lineages (Fig. 1 and 2;
see Fig. S1 in the supplemental material). One lineage con-
tained the viruses collected from New York City between 2006
and 2009, and as expected for a rapidly evolving RNA virus
such as influenza virus, additional mutations were observed in
the later samples compared to those collected in earlier years
(Fig. 1 and see Table 4). Samples taken from dogs in Virginia
in 2009 and Philadelphia in 2008 were related to the other
viruses from the northeast United States, although they were
sufficiently distinct to indicate that the virus was not derived
directly from the New York virus population (Fig. 1).
Viruses from dogs in Colorado in 2006 and 2008 were dis-
tinct from those seen in New York City, and in many segments
were very similar to the viruses from the original Florida out-
breaks, suggesting that they represent a separate lineage (Fig.
1). Our analysis of rates of nucleotide substitution and times to
common ancestry of the HA1, NS, and M segment sequences
(see below) suggests that the Colorado lineage emerged be-
tween December 2004 and December 2005 (range of 95%
HPD values), likely during the original spread of CIV with
racing greyhounds.
Finally, it is important to note that there appeared to be
little variation deriving from the virus isolation in eggs, as
comparison of two New York 2006 egg isolation virus M se-
quences with two New York 2006 tissue culture-derived virus
M sequences showed there is only one mutation, in one of the
latter sequences, not seen in earlier canine or equine reference
sequences.
Two canine sequences isolated from a dog in Sydney, Aus-
tralia, during the 2007 EIV outbreak (24) are included in the
HA1 and M phylogenies (Fig. 1) and NA phylogeny (see Fig.
S1 in the supplemental material). These Sydney CIV se-
quences are identical to an EIV isolated from a horse in the
same stable as the dog, and there have been no reports of
interdog CIV transmission in Australia (24). As a result, the
Sydney CIV sequences are considered equine viruses isolated
from dogs and were not included in further CIV analyses in
this study.
Rates of molecular evolution. To explore the evolutionary
dynamics of CIV in its new host species, we estimated rates of
nucleotide substitution for the HA, NA, M, and NS gene seg-
ments and compared those to the rates estimated from publicly
available EIV isolates (Table 3). The highest EIV substitution
rates were observed in the HA1 and NA gene segments (mean
rates of 1.5  103
substitutions/site/year; 95% HPD  1.1 
103
to 1.8  103
), while rates estimated for other gene
segments were between 0.7  103
and 0.8  103
substitu-
tions/site/year (combined 95% HPD  0.6  103
to 1.1 
103
). These rates are broadly similar to the 0.5  103
substitutions/site/year documented for M and NS in a previous
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FIG. 1. Phylogenetic trees of EIV (from 1963 to the present), CIV, and representative shelter sequences. CIV sequences are shown in blue;
their detailed phylogenetic relationships are shown in the insets. Virus samples in black are equine sequences, and those in pink are canine
sequences. Sequences of viruses from the dogs in the New York shelters are color coded and indicate the consensus sequence of the virus from
an individual dog. All horizontal branches are drawn to a scale of nucleotide substitutions per site, and bootstrap values of 70% are shown.
(A) HA1; (B) M; (C) NS.
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EIV study (29), although a rather different methodology has
been employed here. The highest CIV substitution rates were
observed in the NA (6.2  103
substitutions/site/year; 95%
HPD  3.8  103
to 9.0  103
) and M (4.0  103
substi-
tutions/site/year; 95% HPD  2.9  103
to 5.2  103
) gene
segments and hence were higher than those observed in EIV.
As expected, the HA1, M, and NS substitution rates obtained
using only the consensus sequences were lower than those
calculated with all the sequence data included (Table 3), sug-
gesting that the intrahost data sets contain an abundance of
deleterious mutational polymorphisms. Indeed, it is important
to note that the CIV sequences were sampled over a far shorter
time frame than the EIV sequences, which will generally result
in an inflation of substitution rates due to the presence of
transient deleterious mutations yet to be purged by purifying
selection, as has also been suggested for the swine-origin
H1N1/09 influenza virus (21, 45).
Our analysis of selection pressures in CIV using dN/dS re-
vealed no evidence for positive selection in the HA1, HA2, M1,
NS1, NA, or NP gene regions, with global dN/dS ratios ranging
from 0.27 to 0.66. Similarly, we found no significant difference
in the dN/dS ratios on main EIV-to-CIV trunk branches com-
pared to the rest of the phylogeny. Finally, in all segments
analyzed, the external branches of the ML trees were charac-
terized by higher dN/dS ratios than the internal branches (dif-
ferences in dN/dS ratios for external and internal branches
ranged from 0.06 to 0.51), strongly suggesting the presence of
transient deleterious mutations in this viral population.
Microevolution of CIV in dog shelters. CIV transmission is
sustained by the high numbers of susceptible dogs entering
animal shelters and kennels, where the animals are housed in
close proximity. We examined the viruses circulating in 2006,
2008, and 2009 in two large shelters (denoted A and B) located
in New York City. Phylogenies of multiple cloned CIV se-
quences from individual dogs in the shelters are shown for
three gene regions: HA1, M, and NS (Fig. 2). These trees
showed that all of the 2008/2009 viruses in the two shelters
derive from a single common ancestor that likely existed be-
tween August 2007 and September 2008 (range of 95% HPD
values). The 2006 isolates from each of these shelters (A/2006
and B/2006) were interspersed with the earlier northeast virus
samples and do not appear to be the direct ancestors of the
2008 and 2009 viruses. The patterns obtained suggest either
that the viruses had evolved in situ or had been replaced after
2006 by a different viral lineage, or possibly the viruses in these
shelters were seeding other areas. For the viruses collected
during November 2008 for shelter B and March 2009 for shel-
ter A, there was a separation of sequences, suggesting that
each shelter was maintaining a distinct viral population. In-
deed, an analysis of the extent of phylogeographic structure
revealed a highly significant geographical clustering by shelter
(P  0.001 for both the PS and AI tests), indicative of relatively
little viral gene flow even among shelters that were only 8 km
apart. However, some limited mixing of the viruses between
the two shelters during 2008 and 2009 was observed. For ex-
ample, two M sequences from a shelter A dog grouped within
the shelter B sequences (Fig. 2B). Additionally, HA1 and M
sequences from a dog sampled in December 2009 from shelter
B were located within the shelter A clade (Fig. 2).
By comparing sequences of up to 20 clones of three gene
segments from the same dog sample, we also sought to explore
some aspects of dog infection bottleneck size and intrahost
evolution. Contrasting with the variation among samples from
different dogs, the multiple sequences recovered from each
dog were mostly identical in sequence or very closely related,
indicative of a single founding virus within each animal. This
was supported by our BaTS analysis, which showed significant
clustering by host dog (for the PS and AI tests, P  0.001).
However, as the sequences found within each shelter were
FIG. 2. Phylogenetic trees of cloned CIV sequences prepared after direct RT-PCR of virus in swabs of infected dogs sampled from the New
York City shelters, as well as some other isolates. Samples were taken from shelter B in November 2008 (light pink boxes) and December 2009
(dark pink boxes) and from shelter A in March 2009 (light blue boxes) and September 2009 (dark blue boxes). Each colored circle represents a
sequence, and sequences from the same dog are colored the same across all trees. All horizontal branches are drawn to a scale of nucleotide
substitutions per site, and bootstrap values of 70% are shown. (A) HA1; (B) M; (C) NS.
TABLE 3. Rates of nucleotide substitutions per site for CIV and EIV sequences
CIV EIV
Gene segment CIV no.
Mean no. of nta
substitutions/
site/yr (103
)
95% HPD
EIV no.
Mean no. of nta
substitutions/
site/yr (103
)
95% HPD
Lower Upper Lower Upper
HA1 104 2.1 1.1 2.9 205 1.5 1.2 1.7
Consensus 26 1.8 1.1 2.6
M 149 4.0 2.9 5.2 104 0.7 0.6 0.9
Consensus 26 2.7 1.9 3.7
NS 156 3.6 2.8 4.6 96 0.8 0.6 1.1
Consensus 24 3.3 1.2 5.1
NA 36 6.2 3.8 9.0 91 1.5 1.1 1.8
a
nt, nucleotide.
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generally very similar, there may not be sufficient phylogenetic
resolution to determine whether dogs were multiply infected.
Sequence and amino acid changes associated with canine
infection. Our analysis of viruses associated with the dog shel-
ters in New York City along with the database CIV and EIV
sequences also enabled us to identify, on a genome-wide basis,
mutations that are unique or enriched in the CIV sequences
compared to those in EIV (Table 4; see Table S1 in the sup-
plemental material). In each gene segment, between one and
nine nonsynonymous mutations were identified as exclusively
present in CIV isolates. HA has been the particular focus for
studies of genetic variation in influenza virus strains, and sev-
eral changes in HA that have been previously reported as CIV
specific were seen in all our CIV sequences (7, 38). Interest-
ingly, two A/H3N8 viruses recently isolated from swine in
China differ from early European equine A/H3N8 sequences
at six HA sites (50), three of which (N159S, W222L, and
I328T) were also found in the CIV sequences, suggesting that
these mutations may play an important role in the infection of
new host species.
In Table S1 in the supplemental material, we identify all of
the nucleotide substitutions that are CIV specific or that
showed increased occurrence in CIV compared to EIV (some
of which were identified in previous studies [7, 38]). Some
changes found in only a small proportion of the EIV sequences
appeared to become fixed in the CIV isolates, including eight
additional sites in HA, six of which were nonsynonymous.
Furthermore, each gene segment also had between 3 and 15
synonymous substitutions that appeared to be fixed in the CIV
sequences, with the exception of NS, in which only nonsynony-
mous CIV-fixed substitutions were seen (see Table S1 in the
supplemental material). Several CIV-specific or CIV-domi-
nant sites may influence viral function. HA1 sites 75 and 216
possibly occur at antigenic sites E and D, respectively (52). HA
sites 222 (Trp to Leu) and 223 (Val to Ile) are adjacent to HA
structures that influence binding to modified sialic acids or
TABLE 4. Amino acids in the different gene regions showing differences in each gene segment found only in CIV and
not in any EIV sequence examined
Gene
segment
Amino acid site
(mature)
Amino acid found in:
Human H3N2
New York
Equine isolates Canine isolates Canine
sequences
(shelter)
Miami 1963
Florida
lineage
2002­2005 2006­2009
HA 29a
I I I I/M M M
54a
S N N K K K
75 (site E) Q H H H H Q
92a
K S S S/N N N
118a
L L L L/V V V
216 (site D) N N N N N/H H
222a
R W W/L L L L
261a
R R K K/N N N
262 S T T T T/P P
328a
(cleavage site) T I I T T T
483a
N N N T T T
NA 62 n/ab
I I I L L
250 n/a Q K K N N
NS 21 Q R R R R/Q Q
193 R R R R R K
214 L F F F F L
M 138 V V V V V/I I
NP 27 A A A A n/a T
375a
G D D D/N n/a N
PA 327 E E E E/K n/a K
444 N N N N N/D D
675a
N N N D N/D D
PB1 200 V V V V/I I I
338 S S S S S/N N
529 V V V V V/I I
591 V V V V V/I I
687 Q Q Q Q Q/H H
754 R R R R R/K K
PB2 389 R R R R n/a K
559 A I I I n/a N
a
These sites have been identified previously (7, 38).
b
n/a, not applicable.
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those in different (2,3 or 2,6) linkages (44). The M1 substi-
tution V15I results in a high-pathogenicity H5N1 influenza
virus in mice (23). A truncated M1 protein due to a termina-
tion codon at site 195 was seen in the consensus sequences
B/2006 and NewYork/Dec2006, which were sampled from dogs
4 months apart. The presence of this clearly deleterious mu-
tation in the consensus sequences suggests the transmission,
for at least 4 months, of a defective virus possibly through
complementation, as has previously been suggested for dengue
virus (1). None of the other CIV sequences had the site 220
stop codon mutation seen in 17 equine sequences and the 2003
and 2004 Florida canine NS sequences.
DISCUSSION
The emergence of CIV provides a unique opportunity to
examine the evolution of an influenza virus before and after it
transferred to a different mammalian host, in which it created
a new self-sustaining epidemic of infections and disease. By
examining the evolutionary history of CIV in the United States
since its emergence from EIV, as well as the smaller-scale
"microevolution" of CIV in two confined areas where the virus
has been circulating continuously for 3 years or more, we can
begin to describe the evolutionary forces that occur during the
circulation of this influenza virus in its new canine host com-
pared to that seen in the donor equine host.
High rates of viral transmission in dog shelters. A feature of
influenza viruses and other pathogens spread by respiratory
routes (such as severe acute respiratory syndrome [SARS]
coronavirus and measles) is that even low-efficiency viruses are
able to spread in dense populations with large numbers of
incoming susceptible hosts (3, 8, 30). This appears to be the
situation for the canine shelter populations that we studied
here, which had sufficient seronegative dogs entering into rel-
atively confined areas to allow continuous endemic CIV trans-
mission. Observations of clinical signs and analysis of viral
sequences indicated that CIV entered the shelters around
2005. We show here that by late 2008 and early 2009, the virus
circulating in both shelters was derived from a single common
ancestor, and there appeared to be only limited mixing of
viruses between the shelters over a period of 2 to 3 years.
Because they likely allow continuing circulation of even inef-
ficiently transmitting viruses, dog shelters are important in
facilitating virus persistence, and they also may represent im-
portant source populations for CIV evolution, generating viral
lineages with a range of phenotypic properties.
Evolution of CIV in dogs over a 9-year period. Overall, CIV
in the United States has experienced rapid evolutionary
change, with substitution rates of 1.8  103
to 6.2  103
substitutions/site/year in the HA1, NA, M, and NS gene seg-
ments (for which we had sufficient samples to undertake an
analysis). These rates overlap with those seen in human
A/H3N2 (3.8  103
to 5.7  103
substitutions/site/year) and
A/H1N1 (5  103
substitutions/site/year) viruses (14, 41, 42),
although the short time scale of sampling of CIV means that
the rates determined for that virus have likely been artificially
inflated due to the presence of transient deleterious mutations.
Indeed, despite the emergence of mutations that are found
only in the CIV isolates, there was no evidence of strong
positive selection acting on these viruses, although this may be
due to inherent limitations in detecting adaptation on single
nucleotide sites on individual lineages. We have previously
shown that CIV can experience measurable sequence evolu-
tion even within single dogs, including some tentative evidence
for immune selection in the viral HA in partially immune dogs
(20). In the present study, we also saw a concentration of
changes in the internal gene segments, such as M, NS, and NP,
in addition to the genes that encode proteins involved in re-
ceptor or antibody recognition (HA and NA). In the case of
HA, the relative lack of selection at antigenic sites over the 10
years since its emergence may be due to the specific circum-
stances of the transmission of the CIV, with continual intro-
duction of naïve animals into the shelters with little or no
reintroduction of immune animals, so that no evasion of the
host immune response is required.
Evolution of influenza virus in its new host and potential
canine adaptation. One of the most important questions in the
study of disease emergence is the degree to which pre- or
posttransfer adaptation is required for the successful establish-
ment in the new host species. As the emergence of CIV is well
characterized and the canine viruses clearly derive from the
transfer of a single EIV ancestor, it is now possible to directly
examine the evolution of that virus over 10 years of circulation
in dogs.
The host barrier presented by dogs to the recent A/H3N8
EIV viruses may be relatively low, as a number of independent
transfers of the virus into dogs have been reported, including
the current outbreak in the United States and a less extensive
outbreak involving foxhounds in the United Kingdom (9). As
such, it is unclear whether significant amounts of adaptative
evolution are required or would be expected in EIV when it is
transferred to dogs. A number of nucleotide substitutions were
shown to uniquely characterize CIV, and the CIV sequences in
the northeastern United States showed a more rapid rate of
substitution than that reported for EIV over a period of almost
50 years. However, we were unable to conclusively show that
any of the substitutions we document in CIV have been subject
to positive selection, using the bioinformatics methods cur-
rently available. Indeed, any nucleotide changes strongly re-
quired for CIV transmission in dogs likely occurred early in the
evolutionary history of the virus, prior to the first isolates being
collected in 2003, and so would have been fixed in the popu-
lation before the sampling took place. However, even if some
canine adaptation has occurred, the virus appears to be insuf-
ficiently transmissible to be able to spread into the general
population of household dogs. High-density dog populations,
such as those that characterize dog shelters, may therefore be
of critical importance for the maintenance of this host-trans-
ferred virus even after a period of 10 years. This raises the
question of whether a further increase in transmissibility would
allow CIV to circulate widely in household dogs in New York
City or in other regions, potentially exposing large numbers of
people to the virus. The current apparent low transmissibility
also suggests that it may be possible to control CIV by using
currently available methods such as quarantine, vaccination, or
antiviral drugs.
Host range switching in influenza viruses often involves
changes of many different viral genome segments, and a num-
ber of signature changes have been identified in the genomes
of such host-transferred viruses (reviewed in references 5 and
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32­34). However, despite the finding of CIV-associated
changes in most gene segments (Table 4; see Table S1 in the
supplemental material), only a few were in sites that have been
identified as influencing host ranges of other influenza viruses.
Alteration in sialic acid specificity of the HA has been associ-
ated with alterations within, or close to, the sialic acid binding
pocket, including residues 222, 226, and 228 (5, 15, 44). Horses
and most breeds of domestic dogs differ in the sialic acids
expressed, since horses express the sialic acid N-glycolyl neur-
aminic acid, while dogs primarily express the N-acetyl neur-
aminic acid. Those sialic acids in the respiratory tissues of
horses and dogs may also differ in the linkages of the terminal
sialic acids and their acetylation (18, 19, 43, 46, 47, 53, 55). The
differences in residues 222 and 223 from primarily Trp and Val
to Leu and Ile, respectively, are likely associated with changes
in the binding of sialic acids. However, it is clear that the roles
of these or other changes in host adaptation of CIV to dogs
need to be identified by experimental testing.
ACKNOWLEDGMENTS
We thank Wendy S. Weichert, Virginia Scarpino, and Changhao
(Bobby) Yu for technical support and Elodie Ghedin (University of
Pittsburgh), Pablo Murcia, and Karin Hoelzer for assistance with
methods.
This work was supported by NIH grant GM080533-03 to E.C.H. and
C.R.P.
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