Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
OR I G I N A L A R T I C L E
Emergence and adaptation of H3N2 canine influenza virusfrom avian influenza virus: An overlooked role of dogs ininterspecies transmission
Wanting He1 | Gairu Li1 | Henan Zhu2 | Weifeng Shi3 | Ruyi Wang1 |
Cheng Zhang 1 | Yuhai Bi4,5 | Alexander Lai6 | George F. Gao4,5 | Shuo Su1
1MOE Joint International Research
Laboratory of Animal Health and Food
Safety, Engineering Laboratory of Animal
Immunity of Jiangsu Province, College of
Veterinary Medicine, Nanjing Agricultural
University, Nanjing, China
2MRC-University of Glasgow Centre for
Virus Research, Glasgow, UK
3Institute of Pathogen Biology, Taishan
Medical College, Taian, China
4Chinese Center for Disease Control and
Prevention (China CDC), National Institute
for Viral Disease Control and Prevention,
Beijing, China
5CAS Key Laboratory of Pathogenic
Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of
Sciences, Beijing, China
6College of Natural, Applied, and Health
Sciences, Kentucky State University,
Frankfort, Kentucky, USA
Correspondence
Shuo Su, MOE Joint International Research
Laboratory of Animal Health and Food
Safety, Engineering Laboratory of Animal
Immunity of Jiangsu Province, College of
Veterinary Medicine, Nanjing Agricultural
University, Nanjing, China.
Email: [email protected]
Funding information
The National Key Research and
Development Program of China, Grant/
Award Number: 2017YFD0500101; the
Youth Talent Lift Project of China
Association for Science and Technology; the
Fundamental Research Funds for the Central
Universities, Grant/Award Number:
Y0201600147; the Priority Academic
Program Development of Jiangsu Higher
Education Institutions
Abstract
H3N2 canine influenza virus (CIV) originated from avian species and emerged in
dogs in Asia around 2005 where it became enzootic before reaching the USA in
2015. To investigate the key aspects of the evolution of H3N2 CIV regarding its
emergence and adaptation in the canine host, we conducted an extensive analysis
of all publicly available H3N2 CIV sequences spanning a 10‐year period. We believe
that H3N2 AIVs transferred to canines around 2002–2004. Furthermore, H3N2
CIVs could be divided into seven major clades with strong geographic clustering and
some changed sites evidence of adaptive evolution. Most notably, the dN/dS of
each H3N2 CIVs segment was higher than the correspondent of H3N2 AIVs and
the U content of HA and NA was increasing over time, suggesting the idea that this
avian‐origin virus may be gradually adapting to the host. Our results provide a
framework to elucidate a general mechanism for emergence of novel influenza
viruses.
K E YWORD S
evolution, H3N2 canine influenza virus, virus host-adaptation
Received: 9 July 2018 | Revised: 15 October 2018 | Accepted: 7 November 2018
DOI: 10.1111/tbed.13093
842 | © 2018 Blackwell Verlag GmbH wileyonlinelibrary.com/journal/tbed Transbound Emerg Dis. 2019;66:842–851.
1 | INTRODUCTION
Understanding viral emergence involves unravelling how novel
viruses are transmitted and the characteristics they possess that
allow sufficient replication within the new host and spread leading
to outbreaks/epidemics/pandemics (Geoghegan, Duchêne, & Holmes,
2017). While influenza virus infections are typically species‐specific,they can occasionally cross the species barrier, infect and rapidly
adapt to a new host to establish new variants and lineages. During
the influenza pandemic of 1968, studies showed that companion ani-
mals, such as dogs and cats, were seropositive for human H3N2
influenza A virus (IAV; Kilbourne & Kehoe, 1975). Prior to the epi-
zootic of respiratory disease and subsequent identification of H3N8
canine influenza virus from greyhounds in Florida, 2004, there was
no evidence of canine‐specific influenza virus. Of note, serological
evidence suggested that the interspecies transmission of H3N8
equine influenza to canine species had occurred more than once (Ieh
et al., 2017; Payungporn et al., 2008). H3N2 canine influenza virus
(CIV) emerged in dogs in China or Korea around 2005 and was first
reported in 2008, followed by China and Thailand (Bunpapong et al.,
2014; Li et al., 2010; Song et al., 2008). In February 2015, H3N2
CIV was also reported in Chicago, USA (Voorhees et al., 2017). By
May 2015, H3N2 CIV was found in eastern and south‐eastern USA
(Voorhees et al., 2017), with over 1,000 dogs infected (Sun et al.,
2017). Since that time, H3N2 CIV has established as an enzootic
virus in pet dogs and sheltered dogs throughout South‐East Asia and
in the United States, causing occasional epizootics. Reassortment of
H3N2 CIV with other IAVs has been previously reported (Moon et
al., 2015; Zhu, Hughes, & Murcia, 2015). Epizootics by H3N2 CIV
was resulted from a single cross‐species transmission event with rel-
atively little reassortment taking place during dog‐to‐dog spread
(Song et al., 2012). Thus, CIVs provide a useful and relatively simple
model to understand how IAVs emerge and spread in new hosts and
therefore how IAVs are maintained in nature (Crawford et al., 2005).
Companion animals, such as cats and dogs, have been suggested
to be a source of new avian‐origin AIVs, with the potential to cause
severe respiratory disease in humans. In 2016, a veterinarian was
infected with a low pathogenic avian influenza virus H7N2, which
was also isolated from a cat shelter in New York City (Belser et al.,
2017). Canines carrying avian‐origin H3N2 CIV have also the poten-
tial to transmit the virus to other species in frequent close contact
such as cats (Jeoung et al., 2013; Park et al., 2013). Although no
human infections have yet been reported with H3N2 CIVs, more
and more new H3N2 CIV cases are being reported in the USA and
Eastern Asia and it may be a matter of time before human infections
are reported or passed as unrecognized cases. Thus, detailed geno-
typing and evolution of emerging H3N2 CIV is important for under-
standing and estimating the risk of ongoing infections amongst dogs
and potential adaptation to humans as well as for developing effec-
tive countermeasures.
In this study, we investigated the phylogenetic and evolution
dynamics of H3N2 CIV to accurately identify its origin and geno-
types, as well as underlying cross‐species transmission and spread in
new host populations. Notably, our results fill up some gaps in
understanding what has driven sustained H3N2 CIVs transmission
and the mechanism of emergence of novel influenza viruses in new
hosts.
2 | MATERIALS AND METHODS
2.1 | Sequence information
2.1.1 | Influenza A virus dataset compilation
A total of 658 HA of the H3 subtype avian influenza virus (AIV), 840
NA of the N2 subtype AIV, 67 H3N2 CIV HA and NA, 10 H3N2
feline influenza virus (FIV) HA and NA, 1 H7N2 FIV HA and NA, 82
H3N8 equine influenza virus (EIV) HA and NA (Supporting Informa-
tion Table S1) coding regions were collected from the NCBI Gen-
Bank database (https://www.ncbi.nlm.nih.gov/). H3N2 FIV isolates
were included in the data set for the origin and genotyping analysis.
Although H3N2 FIV has been isolated in cats, it is essentially H3N2
CIV (Park et al., 2013; Zhu et al., 2015).
2.1.2 | H3N2 CIV datasets for adaption evolutionanalysis
While H3N2 CIV has been known to reassort with IAV strains from
other hosts (Zhu et al., 2015), these events have not been shown to
produce dog‐to‐dog transmissible viruses. Therefore, we excluded
segments resulting from reassortment with other non‐H3N2 CIVs,
AIVs, non‐full‐length strains and H3N2 FIV sequences. A total of 43
H3N2 CIVs full genomes remained. The length of each segment after
alignment was: HA 1,698 nucleotides (nt), MP 1,044 nt, NA 1,416
nt, NP 1,494 nt, NS 1,051 nt, PA 2,148 nt, PB1 2,271 nt and PB2
2,277 nt. Concatenation of the eight H3N2 CIV segments yield a
sequence of 13,390 nt.
2.2 | Alignment and model selection
Multiple sequence alignments (MSA) were generated and assembled
using MUSCLE (version 3.8.31; Edgar, 2004), followed by manually
editing using MEGA (version 7.0; Kumar, Stecher, & Tamura, 2016).
The jModelTest program was used to select the best‐fit nucleotide
substitution model. TempEst (version1.5.1) was used to analyse the
Root‐to‐tip genetic distance against sampling dates analysis in
selected sequences (Rambaut, Lam, Max Carvalho, & Pybus, 2016).
2.3 | Phylogenetic and evolutionary dynamicsanalyses
The maximum‐likelihood (ML) tree for origin analysis, was con-
structed by RAxML (version 8.2.4) using the general time reversible
model, gamma distributed rate heterogeneity (GTR + G) with 1,000
bootstrap replications (Stamatakis, 2014). While, for genotyping anal-
ysis the Hasegawa‐Kishino‐Yano model plus gamma distributed rate
HE ET AL. | 843
heterogeneity (HKY + G) model was used. All trees were rooted
with the earliest isolated strain. The maximum clade credibility
(MCC) trees were reconstructed using best‐fit HKY + G model with
a lognormal relaxed molecular clock using the BEAST (version 1.8.4)
package (Drummond & Rambaut, 2007). A Bayesian skyline coales-
cent model was set as the tree prior. Markov Chain Monte Carlo
(MCMC) sampling was run for 1 × 108 generations, with trees and
posteriors sampled every 1 × 104 steps. Each tree was run twice
independently and combined using LogCombiner. When the effec-
tive sampling size (ESS) was >200, the result was accepted. After a
burn‐in of 10%, the final tree was summarized using TreeAnnotator
(Helfrich, Rieb, Abrami, Lucking, & Mehler, 2018) and replayed in
Figtree (Drummond & Rambaut, 2007). Root‐to‐tip genetic distance
analysis was performed based on ML tree against sampling dates
using TempEst (Rambaut et al., 2016). We also conducted a more
detailed analysis of evolutionary rates using the Bayesian MCMC
method implemented in the BEAST package. The MCC trees of each
segment were built using the same methods as above. The time of
most recent common ancestor (TMRCA) and evolutionary rates were
estimated using the Bayesian MCMC process.
2.4 | Geographical correlation of H3N2 CIV
BaTS (Bayesian Tip‐Significance testing) was used to analyse the
correlation between each H3N2 CIV segment and geographical
structure. Geographic structure was defined according to the
sequence separation of countries (including China, South Korea and
the USA; Parker, Rambaut, & Pybus, 2008). The association index
(AI) and parsimony score (PS) statistic were calculated using the
MCC trees of each segment. P‐value of AI and PS less than 0.05
indicate significant correlation with H3N2 CIVs and geographical
distribution.
2.5 | Amino acid analysis and U content ofinfluenza A viruses
Amino acids changes associated with canine infection of H3N2 CIV
and H3N8 CIV were analysed using MEGA7.0. Consensus sequences
were aligned and mutations were recorded. The positions of the
mutations of each enzootic cluster were confirmed manually. The
number of amino acid changes in each enzootic cluster was counted.
The U content of HA and NA of multiple influenza A virus was cal-
culated using Bioedit (Hall, 1999). Correlation analysis was per-
formed using Graphpad 7.0 (Motulsky, 1999).
2.6 | Selection analysis
ML trees (Figure 2) and MCC trees (Figure 3) were used as the input
reference trees in DATAMONKEY (Delport, Poon, Frost, & Pond,
2010; http://www.datamonkey.org/), which was used to estimate
selection pressures. The SLAC (Single Likelihood Ancestry Counting),
FEL (Fixed Impact Probability), MEME (Evolutionary Mixed Effects
Model) and FUBAR (Fast, Unconstrained Bayesian Approximation)
methods were used to identify codons under positive selection. The
Branch Site REL model was used to determine the selection analysis
along branches (Kosakovsky Pond & Frost, 2005; Murrell et al.,
2012; Murrell et al., 2013; Smith et al., 2015).
We considered p‐values of SLAC, FEL and MEME < 0.1, FUBAR
posterior probability > 0.9 as significant levels and only sites sup-
ported with at least three methods were reported. Additionally, the
non‐synonymous and synonymous substitutions per rate (dN/dS) val-
ues of each segment of H3N2 AIV, H3N2 CIV and H3N2 FIV were
estimated using SLAC.
3 | RESULTS
3.1 | Insights into the origin of HA and NA ofH3N2 CIV
It has been reported that H3N2 CIV originated from H3N2 AIV (Zhu
et al., 2015); however, the precise time of H3N2 CIV divergence
from H3N2 AIV has not been determined. In order to identify the
origin of HA and NA of H3N2 CIV, the H3 HA and the N2 NA
sequences of avian, canine and feline were collected from the Gen-
Bank database. The ML tree (Supporting Information Figure S1) indi-
cated that the HA and NA segments of H3N2 CIV were closely
related to the Eurasian avian influenza lineage. In particular, the clos-
est cluster consisted of the major isolates in South Korea. Then, we
chose the most closely related Eurasian avian influenza lineage and
all of H3N2 CIV and FIV sequences to build a more accurate tree
using the Bayesian MCMC method (Figure 1). We found that all
H3N2 CIV HA (Figure 1a) and NA (Figure 1b) sequences were clo-
sely related to H3N2 AIV (A/duck/Korea/JS53/2004) with a high pos-
terior probability (1.0). The HA segment of H3N2 CIV divergence
time from East Asia AIV was around 1998 (95% HPD 1993–2001);however, the NA H3N2 CIV divergence time was later than HA,
around 2002 (95% HPD 1999–2004). This finding suggested that
H3N2 CIV may have originated from a putative reassortment event
of AIVs. In addition, we found distinct geographical clustering with
CIVs from the same country, probably the result of a founder effect
(Figure 1). In summary, H3N2 CIV likely originated from H3N2 AIVs
from China or South Korea.
3.2 | H3N2 CIV genotyping
A total of 77 H3N2 HA and 77 NA complete coding sequences of
CIV and FIV were used for genotype analysis (Figure 2). Both the
HA and NA ML trees were rooted to A/canine/Guangdong/1/2006,
the earliest CIV sequence reported. According to the structure of
the ML tree of HA and NA, H3N2 CIV could be divided into seven
major clades (Figure 2 and Supporting Information Figure S2). The
early origin sequences could be divided into two clades: clade I con-
sists of early viruses isolated during 2006–2007 from Guangdong,
China, and clade II consists of virual isolates exclusively from South
Korea since 2007. Moreover, the China isolates could be divided
into three major clades: clade III including isolates exclusively from
844 | HE ET AL.
the Jiangsu Provinces from 2009 to 2010 and clade IV including iso-
lates from the Guangdong Provinces and the North‐eastern Pro-
vinces of China. Additionally, clade VII consisted of Beijing isolates.
Regarding the Korea/USA isolates, clade V consisted of strains circu-
lating in South Korea from 2010 to 2014. Clade VI contained viruses
from the South Korea strain, the ancestor of the USA H3N2 CIV iso-
lates, from 2013 to 2014 and all USA isolates. As shown in Fig-
ure 2b, the phylogenetic tree of H3N2 NA showed a similar
structure to that of the HA tree and could be divided into six major
clades. Of note, all the HA sequences from isolates from Beijing that
clustered in clade VII in the HA tree grouped in clade III in the NA
tree. According to the structure of the trees of Figures 1 and 2 and
inspection of all available H3N2 CIV sequences isolated from cats,
we found that all feline isolates clustered with canine isolates, indi-
cating H3N2 CIV could infect not only canine species, but also
feline, and it may already circulating in feline. Additionally, all the
sequences in the same clade strictly clustered according to geo-
graphical location.
3.3 | Evolutionary dynamics of H3N2 CIV
A total of 43 full length CIV genomes were used to reconstruct a
ML tree that was used for regression analysis with root‐to‐tip dis-
tance (Supporting Information Figure S3). The estimated R2 was
0.9329, indicating a significant linear regression of nucleotide diver-
gence over time. Given this strong clock‐like evolution signal, the
Bayesian MCMC method was used to estimate the evolutionary rate
and the TMRCA of each gene segment. Tree structures remained
generally consistent across all genomic segments, with Korean/USA
and Chinese clusters bifurcating before 2008 (Figure 3 and Support-
ing Information Figure S3). Interestingly, despite the limited number
of sequences available (Figure 3), the 43 H3N2 CIVs still formed
three stable enzootic clusters according to countries in all eight seg-
ments. Of note, the p‐value of AI and PS of each segment were less
than 0.05 (Supporting Information Table S2). This is consistent with
the structure of phylogenetic trees and indicates that the phylogeny‐geography association was significant.
For these reasons, we re‐grouped them in three clusters for fur-
ther analysis. The TMRCA of H3N2 CIV segments and enzootic clus-
ters were estimated (Table 1). The TMRCA of HA was 2002 (95%
highest probability density (HPD) 1999–2004), for MP, NA and PA
were 2004 (95% HPD 2004–2006), for NP was 2000 (95% HPD
1996–2003), for NS was 2001 (95% HPD 1997–2005), for PB1 was
2003 (95% HPD 1999–2005) and for PB2 was 2004 (95% HPD
2001–2005). All of them earlier than previously reported (Zhu et al.,
2015), due to the increase in the number of sequences. The result of
the skyline plot indicated that the effective population size of HA
increased between 2008 and 2009. When mapping this time to the
phylogenetic tree, we found that there were more subclades formed
around this time indicating that HA divergence occurred during this
period. We then compared the evolutionary rates of the different
enzootic clusters. Overall, the virus evolved at a mean rate of
F IGURE 1 MCC trees of the HA (a) and NA (b) segments. Trees were reconstructed using the HKY + G nucleotide substitution model andlognormal relaxed clock in BEAST program. The skyline coalescent model length chain was set with 1 × 108 generations and resampled every1 × 104 steps. Rectangles are coloured according to different characters. The first column represents different countries. The second columnrepresents different hosts. The black star indicates posterior probability > 0.7 [Colour figure can be viewed at wileyonlinelibrary.com]
HE ET AL. | 845
1.5 × 10−3 (95% HPD 1.2–1.8 × 10−3 subs site−1 year−1) over the
10 years since it was first reported in dogs. The evolutionary rate of
the China enzootic cluster was 1.7 × 10−3 (95% HPD 0.3–3.7 × 10−3 subs site−1 year−1), the Korea/USA enzootic cluster was
1.8 × 10−3 (95% HPD 0.5–3.5 × 10−3 subs site−1 year−1) and the
USA was 1.6 × 10−3 (95% HPD 0.2–3.8 × 10−3 subs site−1 year−1;
Figure 4a). In addition, we found that the evolutionary rates of each
H3N2 CIV segment were at the lower end of the 95% HPD intervals
for what has been shown in various IAVs circulating in human (West-
geest et al., 2014) but higher than H3N8 EIV, except for HA (Murcia,
Wood, & Holmes, 2011; Figure 4b).
3.4 | Host adaptation of H3N2 CIV
In order to investigate the changes of H3N2 CIV in the population,
we analysed the change in these amino acid sites among geographic
enzootic clusters. We found a number of amino acid changes relative
to the early origin enzootic cluster sequences compared to the China
and the Korea/US enzootic cluster sequences (Supporting Informa-
tion Table S3). In HA, position 146 (based on H3 numbering), near
by the receptor binding site (Ieh et al., 2017), changed from G to S
in the USA enzootic cluster. Site 218 changed in the Korea/USA
enzootic cluster from G to E. Additionally, NA amino acid positions
155 and 222 (near the enzymatic active site) were different among
the three enzootic clusters: I155T mutation in the Korea/USA enzoo-
tic cluster and R222Q in the China enzootic cluster. Additionally,
compared to H3N8 CIV, some H3N2 CIV amino acid site changes
coincided with the corresponding sites of H3N8 CIV, including
V200I on PB1, A208T and T387I on PA, among others.
In order to further investigate the adaptability of H3N2 CIV to
dogs, we used the following methods: selection analysis and base
composition. We used the HA and NA ML trees (Figure 2 and Sup-
porting Information Figure S2) for selection analysis. Interestingly,
we found four sites under positive selection in HA including: 4, 218,
436 and 453 (Table 2). Of note, we found the positive selected site
in position 218 can affect protein structure and function. Indeed, it
might affect the pH of membrane fusion and receptor binding
(Keleta, Ibricevic, Bovin, Brody, & Brown, 2008). This indicates that
this site is an adaptive evolutionary site in dogs. However, we only
found site 222 to be under positive selection in NA (Figure 2). In
addition, the mean dN/dS was calculated using SLAC (Figure 5).
Interestingly, the mean dN/dS values of H3N2 CIVs were higher than
that of H3N2 AIVs, indicating that CIV accumulated more non‐synonymous substitutions after entering canines. The mean dN/dS of
the major coding regions of the larger genomic segments in CIVs
ranged from 0.17 to 0.35 except for NS that showed more variability
probably due to its short sequence length. In contrast, the dN/dS of
avian H3N2 ranged from 0.03 to 0.12 (Figure 5). The elevated mean
dN/dS in CIV is reflected by numerous lineage‐defining amino acid
changes that appeared during its circulation in Asia and spread in
the USA. To investigate the host suitability of H3N2 CIV, we also
calculated the U content of multiple IAVs. We found that the U con-
tent of AIV was within a certain range (HA: 31.27%–33.51%, NA:
29.35%–32.19%). The U content of H3N8 EIV was calculated for
reference. Furthermore, the mean U content of H3N2 CIV HA and
NA were 33.21 and 30.82, respectively. It is worth noting that the U
content of both H3N2 CIV and H3N8 EIV showed an increasing
trend over time (p < 0.01). This means that H3N2 CIV is gradually
F IGURE 2 ML trees of H3N2 CIV and H3N2 FIV HA (a) and NA (b) segments. Trees were reconstructed using the HKY + G nucleotidesubstitution model and 1,000 bootstraps using RAxML. The horizontal lines in different colours represent the different countries. The rectanglerepresents the H3N2 CIV genotyping. The black star indicates bootstrap values > 70 [Colour figure can be viewed at wileyonlinelibrary.com]
846 | HE ET AL.
adapting to canines. The U content of H3N2 FIV was also calculated.
In contrast, although the U content of H3N2 FIV also increased, the
calculated p‐value > 0.05 and deemed not significant, this may be
due to the small number of sequence data available.
4 | DISCUSSION
Cross‐species transmission is common for influenza virus, and it is
now generally accepted that all mammalian influenza virus originated
from AIV, as waterfowl are natural hosts for influenza viruses
(Sturmramirez et al., 2004; Sturmramirez et al., 2005; Yoon, Webby,
& Webster, 1992). Avian to mammal transmission and mammalian
adaptation of AIV are of great concern to the public health. Most
previous studies focused on interspecies transmission of AIV to
humans (Herfst et al., 2012; Su et al., 2017) and have shown that
altered receptor binding (Connor, Kawaoka, Webster, & Paulson,
1994; Glaser et al., 2005; Matrosovich et al., 2000; Shinya et al.,
2006), reassortment with circulating strains (Castrucci et al., 1993;
Smirnov et al., 2000; Webster, Sharp, & Claas, 1995) and adaptation
of the viral polymerase gene (Li et al., 2009; Mänz, Schwemmle, &
Brunotte, 2013; Min et al., 2013; Yasuha et al., 2016) are possible
mechanisms that allow species jumps. Moreover, if dogs and cats
serve as potential mixing vessels for avian and mammalian IAVs by
their susceptibility to these viruses, they pose a significant public
health concern by their close proximity to humans and by their sheer
number. However, there remains a huge knowledge gap to address
the mechanisms by which AIV emerge and adapt. Recently an epi-
zootic and transmission of feline H7N2 influenza virus to a human in
a New York animal shelter during 2016 (Hatta et al., 2018; Marino-
vapetkova et al., 2017) showed that the close contact between
humans and cats resulted in virus transmission. Therefore, the emer-
gence and spread of novel avian‐origin H3N2 CIVs in companion
animals represents a potentially neglected threat to public health
due to the high level of exposure of humans that may potentially
allow these novel H3N2 CIVs to adapt to infect humans.
Here, we confirmed that H3N2 CIV emerged in dogs by a single
transmission event from an avian host that leads to a widespread
epidemic in dogs in China and Korea followed by introduction into
the USA. We estimated that H3N2 CIV transferred to canines during
the period between 2002 and 2004, probably by a putative reassort-
ment event of AIVs in East Asia, differently from what previously
2002.5 2005 2007.5 2010 2012.5 2015 2017.5
0.11
0.91
0.73
1
0.19
1
0.14
0.8
11
0.92
0.17
1
1
1
0.96
0.37
10.41
0.43
0.111
1
0.24
1
0.28
0.61
0.98
0.45
0.20.08
0.29
0.48
0.97
0.88
1
0.31
0.35
0.52
0.261
1997.5 2000 2002.5 2005 2007.5 2010 2012.5 2015 2017.5
0.98
0.23
1
0.92
0.29
0.84
0.91
1
0.41
0.55
1
1
1
1
1
0.44
0.23
0.38
1
0.730.5
0.87
1
0.99
0.99
0.33
1
1
10.81
1
1
1
0.32
0.99
1
0.36
0.27
1
1
0.94
2000 2002.5 2005 2007.5 2010 2012.5 2015 2017.5
1
1
0.06
0.63
0.06
0.59
0.2
1
0
0
1
0.01
1
0.01
0.84
1
0.55
1
1
0.05
0.33
0.01
1
0.09
0.49
1
0.42
0.2
0.17
0.74
1
1
0.75
0.22
0.95
0.38
1
0.06
0.19
0.42
0.16
2002.5 2005 2007.5 2010 2012.5 2015 2017.5
0.58
0.86
0.99
1
1
0.89
1
1
0.48
1
1
1
1
0.37
0.54
1
1
0.97
0.38
1
1
0.62
1
0.99
0.93
0.83
1
0.38
1
0.91
1
0.36
0.94
0.44
1
1
0.9
0.9
0.38
1
1
2002.5 2005 2007.5 2010 2012.5 2015 2017.5
10.87
0.38
0.33
0.48
1
1
1
0.330.84
0.59
1
1
0.75
1
1
0.241
1
0.53
1
0.47
0.45
0.740.98
0.29
1
0.19
0.12
0.4
0.93
1
1
1
1
1
0.97
0.57
0.68
1
1
2002.5 2005 2007.5 2010 2012.5 2015 2017.5
0.95
1
1
1
1
0.9
0.63
0.9
0.921
0.73
1
1
1
1
1
0.48
0.7
1
0.4
0.65
1
0.99
1
1
0.91
1
0.34
1
0.36
1
0.77
1
0.96
0.53
1
0.97
0.82
1
0.8
1
2002.5 2005 2007.5 2010 2012.5 2015 2017.5
1
1
0.01
0.09
0.98
0.77
1
1
1
0.16
0.32
0.73
0.28
1
0.84
0.99
0.6
1
0.56
0.09
1
0.1
0.45
0.83
0.27
0.9
0.6
0.98
1
0.130.39
0.02
1
0.67
0.09
0.16
0.78
0.56
0.36
0.01
0.36
Time2005 2007.5 2010 2012.5 2015
1.E0
1.E1
1.E2
2002.5 2005 2007.5 2010 2012.5 2015 2017.5
A/canine/Korea/KRIBB01/2011
A/canine/Indiana/003018/2016
A/canine/Texas/343907/2015
A/canine/South_Korea/0173915/2015
A/canine/Jiangsu/03/2010
A/canine/Korea/0589318/2015
A/canine/Jiangsu/01/2009
A/canine/Florida/269770/2015
A/canine/Guangdong/1/2006
A/canine/Jiangsu/05/2010
A/canine/Korea/CY009/2010
A/canine/Heilongjiang/L1/2013A/canine/Liaoning/H6/2012
A/canine/Korea/DG1/2014
A/canine/Guangdong/23/2012
A/canine/Indiana/96198/2015
A/canine/Illinois/12191/2015
A/canine/Zhejiang/1/2010
A/canine/Guangdong/1/2007
A/canine/Korea/01/2007
A/canine/Illinois/077753/2016
A/canine/Guangdong/2/2006
A/canine/Guangdong/12/2012
A/canine/Illinois/328292/2015
A/canine/Georgia/104940/2015
A/canine/Korea/S1/2012
A/canine/NorthCarolina/109904/2015
A/canine/Liaoning/27/2012
A/canine/Guangdong/3/2011
A/canine/Korea/CY053/2014
A/canine/Jiangsu/06/2010
A/canine/China/JLM2/2015
A/canine/Jiangsu/02/2010
A/canine/Wisconsin/19137/2016
A/canine/Guangdong/2/2011
A/canine/Illinois/283066/2015
A/canine/Korea/GCVP01/2007
A/canine/China/JLM1/2015
A/canine/Jiangsu/04/2010
A/canine/Korea/BD-1/2013
A/canine/Guangdong/05/2011
A/canine/Georgia/95391/2015A/canine/Illinois/1619144/2015
0.1
1
0.34
0.23
1
0.99
0.41
1
0.85
0.51
0.52
0.87
1
0.42
1
0.78
1
1
1
1
1
0.99
0.2
0.65
0.9
0.82
1
10.85
0.17
1
0.99
0.96
0.69
1
1
0.22
0.821
0.61
0.47
Origin
China
Korea/USAOrigin
China
Korea/USAHA
MP
NP
PA
PB2
PB1
NS
NA
F IGURE 3 MCC trees of each H3N2 CIV segment. The different coloured lines indicate different enzootic clusters. Blue line is the originenzootic cluster, red line is the China enzootic cluster, and orange line is the Korea/USA enzootic cluster. The graph above the HA MCC tree isthe relative genetic divergence over time of the HA segment. The black line is the mean value of genetic diversity and the blue line is the 95%HPD. The red vertical bar in both panels indicates the period of divergence of major H3N2 lineages in canine [Colour figure can be viewed atwileyonlinelibrary.com]
HE ET AL. | 847
reported (Zhu et al., 2015). Moreover, we propose for the first time
the classification of H3N2 CIV into seven major clades. Notably, we
observed that the distribution of H3N2 CIV sequences in the same
clade was geographically aggregated revealing important epidemio-
logical features of H3N2 CIV. In Korea and the USA, H3N2 CIVs
transmission tends to occur in pet shelters in large cities (Ieh et al.,
2017). Similarly, transmission in China seems to be supported by
large interconnected networks of veterinary hospitals, farmed dogs
or pet markets (Murcia et al., 2011). This association between
sequences and geographic location is further supported by BaTS
algorithm analysis. This characteristic is probably a result of a foun-
der effect. Given that H3N2 CIV generally emerges in high‐densitydog populations like veterinary hospitals, pet markets, dog farms and
animal shelters, we speculate that H3N2 CIV may be more likely to
erupt again in areas where it has already appeared. Therefore, we
recommend dogs in these areas (Supporting Information Figure S4)
to be vaccinated along with ventilation isolation and strengthening
quarantine before transportation.
We also analysed host adaptability of CIV after transfer from
avians to dogs and subsequent spread. The interspecies transfer of
AIV to canine species and its establishment as enzootic H3N2 CIV in
China and in Korea occurred over a 10‐year period, followed by
spread to the USA. The analysis of virus population dynamic indi-
cated that the genetic diversity of HA increased quickly and the vari-
ants were generated between 2008 and 2009 in canines. In general,
the evolutionary rates of each segment were similar to each other.
However, the evolutionary rate of NA was higher than others, which
may indicate that the adaptation of H3N2 CIV to canines is more
reflected in changes in the NA segment. In addition, we also anal-
ysed the evolutionary rates and TMRCA of each segment according
to enzootic clusters and after entering the USA. The consistence and
the relatively low rates of genomic evolution (among IAVs) suggest
that the evolutionary of H3N2 CIV is mainly shaped by the underly-
ing mutation rate.
H3N2 CIV has been known to reassort with IAV strains from
other hosts; however, it seems that reassortment was not involved
in the emergence of the dog‐to‐dog transmissible virus (Ieh et al.,
2017; Zhu et al., 2015). Therefore, H3N2 CIV can be used as a
model for studying IAV cross species transmission (Supporting Infor-
mation Figure S5). Adaptive evolution can be characterized by
TABLE 1 The TMRCA of each segment of H3N2 CIV
TMRCA Mean 95% HPD
Previously reportedmean TMRCA(Zhu et al., 2015)
HA 2002 1999 2004 2004
China enzootic cluster 2007 2006 2008
Korea/USA enzootic
cluster
2008 2007 2009
MP 2004 2002 2006 2005
China enzootic cluster 2008 2007 2009
Korea/USA enzootic
cluster
2010 2009 2010
NA 2004 2002 2006 2005
China enzootic cluster 2008 2007 2009
Korea/USA enzootic
cluster
2008 2007 2009
NP 2000 1996 2003 2003
China enzootic cluster 2008 2007 2009
Korea/USA enzootic
cluster
2008 2007 2009
NS 2001 1997 2005 2004
China enzootic cluster 2008 2006 2009
Korea/USA enzootic
cluster
2008 2006 2009
PA 2004 2002 2006 2004
China enzootic cluster 2008 2007 2009
Korea/USA enzootic
cluster
2009 2008 2010
PB1 2003 1999 2005 2003
China enzootic cluster 2008 2007 2009
Korea/USA enzootic
cluster
2009 2007 2010
PB2 2004 2001 2005 2004
China enzootic cluster 2008 2007 2009
Korea/USA enzootic
cluster
2008 2007 2009
Note. The time of most recent common ancestor (TMRCA).
F IGURE 4 Evolutionary rates of canine, equine and human IAVs.H3N2 CIV genome and each enzootic cluster evolutionary rates (a),blue is the evolutionary rate of H3N2 CIV, red is the China enzooticcluster, orange is the Korea/USA enzootic cluster and yellow is theUSA enzootic cluster. Evolutionary rates of each segment andenzootic cluster of H3N2 CIV H3N8 EIV and H3N2 hIAV (b). Thebrown is H3N8 EIV and gary is the H3N2 human influenza virus[Colour figure can be viewed at wileyonlinelibrary.com]
848 | HE ET AL.
positive selection. Here we found four sites under positive selection
in HA, including site 218, a common amino acid site associated with
adaptation in mice and canines (Keleta et al., 2008). Additionally,
compared to H3N8 CIV, some H3N2 CIV amino acid changes coin-
cided with the corresponding sites of H3N8 CIV, such as V200I on
PB1, A208T and T387I on PA among others, whether these site
changes contribute to the further adaptation of H3N2 CIV to cani-
nes is still subject to experimental validation. Our amino acid site
analysis and selection analysis show that for more than 10 years,
H3N2 CIV had undergone certain degree of adaptive evolution in
dogs, and that this adaptation level was higher than at the early
stage of the virus epidemic. An increase in U content in IAV gen-
omes correlates with adaptation in mammalian host, as shown by
Figure 5 (Rabadan, Levine, & Robins, 2006; Worobey & Al, 2014).
The mean dN/dS of H3N2 CIV in canines was higher than the avian
counterpart and the U content increased over time indicating an ini-
tial gradual adaptation to dogs. Voorhees et al. (2018) pointed out
that although the reproduction number (R0) of H3N2 CIV range from
1 to 1.5, due to the heterogeneity of transmission, CIV is mostly
infected and faded in shelters in the United States. This decentral-
ized shelter cannot form a continuous communication network.
Therefore, H3N2 may gradually disappear into the dog (Voorhees et
al., 2018). However, considering that H3N2CIV is gradually adapting
to dogs, it is still unknown whether H3N2 CIV will face complete
epidemics extinction. The evidence of host adaptation of the emer-
gence of H3N2 CIV from AIV provides a unique opportunity to
study the mechanism of interspecies transmission and adaptation of
emerging influenza virus. We find that close proximity and some-
what weakened susceptible hosts are still required for sustained cir-
culation of the H3N2 CIV. Given that H3N2 CIV reassortants pose a
risk to public health (Song et al., 2012), an enhanced surveillance for
influenza viruses in canines, particularly in high‐density populations,
such as in shelters, urban areas, and dog shows or competitions. The
application of such knowledge and findings is invaluable not just for
veterinarians but also for public health, as the threat of a more seri-
ous influenza pandemic is always a concern.
ACKNOWLEDGEMENTS
This work was financially supported by the National Key Research
and Development Program of China (2017YFD0500101), the Youth
Talent Lift Project of China Association for Science and Technol-
ogy, the Fundamental Research Funds for the Central Universities
Y0201600147, and the Priority Academic Program Development of
Jiangsu Higher Education Institutions. Prof. Weifeng Shi was sup-
ported by the Taishan Scholars program of Shandong Province
(ts201511056).
CONFLICT OF INTEREST
The authors, taken part in this study, declared no conflict of interest
and nothing to disclose regarding funding.
TABLE 2 Selection analysis of H3N2CIV HA and NA segments
Segment Codon
Test methods
FEL SLAC FUBAR MEME
dN‐dS p‐value dN‐dS p‐value dN‐dS Post.Pro w+ p‐value
HA 4 3.15 0.08 0.48 0.15 1.71 0.94 6.10 0.02
218 3.46 0.06 0.71 0.08 3.31 0.99 4.21 0.06
436 3.17 0.07 0.56 0.14 2.52 0.98 4.60 0.05
453 4.24 0.04 0.65 0.11 3.47 0.99 5.63 0.03
NA 222 47.03 0.04 4.18 0.08 3.30 0.98 4.74 0.04
Notes. Position on HA is based on H3 numbering.
The bold representing the p < 0.1 and posterior p > 0.9, with significant difference.
F IGURE 5 H3N2 CIV host adaptation. Mean dN/dS of each AIV segment and it was estimated according to SLAC algorithm in Datamonkeywebsite (a), U content and regression curve of time of H3N2 AIV (green frame), H3N2 CIV (green line), H3N8 EIV (orange line), H3N2 FIV(blue line) and infected with human H7N2 FIV (blue star). HA segment (b) and NA (c) segments. It was calculated using Bioedit and analysis inGraphpad 7 [Colour figure can be viewed at wileyonlinelibrary.com]
HE ET AL. | 849
ORCID
Shuo Su https://orcid.org/0000-0003-0187-1185
REFERENCES
Belser, J. A., Pulitpenaloza, J. A., Sun, X., Brock, N., Pappas, C., Creager,
H. M., … Maines, T. R. (2017). A novel A(H7N2) influenza virus iso-
lated from a veterinarian caring for cats in a New York City animal
shelter causes mild disease and transmits poorly in the ferret model.
Journal of Virology, 91, e00672–00617. https://doi.org/10.1093/mol
bev/msw054
Bunpapong, N., Nonthabenjawan, N., Chaiwong, S., Tangwangvivat, R.,
Boonyapisitsopa, S., Jairak, W., … Amonsin, A. (2014). Genetic char-
acterization of canine influenza A virus (H3N2) in Thailand. Virus
Genes, 48, 56–63. https://doi.org/10.1007/s11262-013-0978-zCastrucci, M. R., Donatelli, I., Sidoli, L., Barigazzi, G., Kawaoka, Y., & Web-
ster, R. G. (1993). Genetic reassortment between avian and human
influenza A viruses in Italian pigs. Virology, 193, 503–506. https://doi.org/10.1006/viro.1993.1155
Connor, R. J., Kawaoka, Y., Webster, R. G., & Paulson, J. C. (1994).
Receptor specificity in human, avian, and equine H2 and H3 influenza
virus isolates. Virology, 205, 17–23. https://doi.org/10.1006/viro.
1994.1615
Crawford, P. C., Dubovi, E. J., Castleman, W. L., Stephenson, I., Gibbs, E.
P., Chen, L., … Donis, R. O. (2005). Transmission of equine influenza
virus to dogs. Science, 310, 482–485. https://doi.org/10.1126/scie
nce.1117950
Delport, W., Poon, A. F. Y., Frost, S. D. W., & Pond, S. L. K. (2010). Data-
monkey 2010: A suite of phylogenetic analysis tools for evolutionary
biology. Bioinformatics, 26, 2455. https://doi.org/10.1093/bioinforma
tics/btq429
Drummond, A. J., & Rambaut, A. (2007). BEAST: Bayesian evolutionary
analysis by sampling trees. BMC Evolutionary Biology, 7, 214.
https://doi.org/10.1186/1471-2148-7-214
Edgar, R. C. (2004). MUSCLE: Multiple sequence alignment with high
accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797. https://doi.org/10.1093/nar/gkh340
Geoghegan, J. L., Duchêne, S., & Holmes, E. C. (2017). Comparative anal-
ysis estimates the relative frequencies of co‐divergence and cross‐species transmission within viral families. Plos Pathogens, 13,
e1006215. https://doi.org/10.1371/journal.ppat.1006215
Glaser, L., Stevens, J., Zamarin, D., Wilson, I. A., Garcíasastre, A., Tumpey,
T. M., … Palese, P. (2005). A single amino acid substitution in 1918
influenza virus hemagglutinin changes receptor binding specificity.
Journal of Virology, 79, 11533–11536. https://doi.org/10.1128/JVI.
79.17.11533-11536.2005
Hall, T. A. (1999). BioEdit: A user‐friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucleic Acids
Symposium Series, 41, 95–98.Hatta, M., Zhong, G., Gao, Y., Nakajima, N., Fan, S., Chiba, S., … Kiso, M.
(2018). Characterization of a feline influenza A(H7N2) virus. Emerging
Infectious Diseases, 24, 75. https://doi.org/10.3201/eid2401.171240
Helfrich, P., Rieb, E., Abrami, G., Lucking, A., & Mehler, A. (2018).
TreeAnnotator: Versatile Visual Annotation of Hierarchical Text Rela-
tions. Lrec 2018: Edition of the Language Resources and Evaluation
Conference. https://doi.org/10.1128/JVI.03395-14
Herfst, S., Schrauwen, E. J., Linster, M., Chutinimitkul, S., De, W. E., Mun-
ster, V. J., … Smith, D. J. (2012). Airborne transmission of influenza
A/H5N1 virus between ferrets. Science, 336, 1534. https://doi.org/
10.1126/science.1213362
Ieh, V., Glaser, A. L., Toohey-Kurth, K., Newbury, S., Dalziel, B. D.,
Dubovi, E. J., … Brisbane-Cohen, L. (2017). Spread of canine
influenza A(H3N2) virus, United States. Emerging Infectious Diseases,
23, 1950–1957.Jeoung, H. Y., Lim, S. I., Shin, B. H., Lim, J. A., Song, J. Y., Song, D. S., …
An, D. J. (2013). A novel canine influenza H3N2 virus isolated from
cats in an animal shelter. Veterinary Microbiology, 165, 281–286.https://doi.org/10.1016/j.vetmic.2013.03.021
Keleta, L., Ibricevic, A., Bovin, N. V., Brody, S. L., & Brown, E. G. (2008).
Experimental evolution of human influenza virus H3 hemagglutinin in
the mouse lung identifies adaptive regions in HA1 and HA2. Journal
of Virology, 82, 11599. https://doi.org/10.1128/JVI.01393-08
Kilbourne, E. D., & Kehoe, J. M. (1975). Demonstration of antibodies to
both hemagglutinin and neuraminidase antigens of H3N2 influenza A
virus in domestic dogs. Intervirology, 6, 315–318. https://doi.org/10.1159/000149485
Kosakovsky Pond, S. L., & Frost, S. D. (2005). Not so different after all: A
comparison of methods for detecting amino acid sites under selec-
tion. Molecular Biology and Evolution, 22, 1208. https://doi.org/10.
1093/molbev/msi105
Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolu-
tionary genetics analysis version 7.0 for bigger datasets. Molecular
Biology and Evolution, 33, 1870–1874.Li, O. T., Chan, M. C., Leung, C. S., Chan, R. W., Guan, Y., Nicholls, J. M.,
& Poon, L. L. (2009). Full factorial analysis of mammalian and avian
influenza polymerase subunits suggests a role of an efficient poly-
merase for virus adaptation. PLoS ONE, 4, e5658. https://doi.org/10.
1371/journal.pone.0005658
Li, S., Shi, Z., Jiao, P., Zhang, G., Zhong, Z., Tian, W., … Wan, X. F.
(2010). Avian‐origin H3N2 canine influenza A viruses in Southern
China. Infection, Genetics and Evolution, 10, 1286–1288. https://doi.org/10.1016/j.meegid.2010.08.010
Mänz, B., Schwemmle, M., & Brunotte, L. (2013). Adaptation of avian
influenza A virus polymerase in mammals to overcome the host spe-
cies barrier. Journal of Virology, 87, 7200–7209. https://doi.org/10.
1128/JVI.00980-13
Marinovapetkova, A., Laplante, J., Jang, Y., Lynch, B., Zanders, N., Rodri-
guez, M., … Cruz, J. A. D. L. (2017). Avian influenza A(H7N2) virus in
human exposed to sick cats, New York, USA, 2016. Emerging Infec-
tious Diseases, 23, 2046–2049.Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Cas-
trucci, M. R., … Kawaoka, Y. (2000). Early alterations of the receptor‐binding properties of H1, H2, and H3 avian influenza virus hemagglu-
tinins after their introduction into mammals. Journal of Virology, 74,
8502–8512. https://doi.org/10.1128/JVI.74.18.8502-8512.2000Min, J. Y., Santos, C., Fitch, A., Twaddle, A., Toyoda, Y., Depasse, J. V., …
Subbarao, K. (2013). Mammalian adaptation in the PB2 gene of avian
H5N1 influenza virus. Journal of Virology, 87, 10884. https://doi.org/
10.1128/JVI.01016-13
Moon, H., Hong, M., Kim, J. K., Seon, B., Na, W., Park, S. J., … Kim, J. M.
(2015). H3N2 canine influenza virus with the matrix gene from the
pandemic A/H1N1 virus: Infection dynamics in dogs and ferrets. Epi-
demiology & Infection, 143, 772–780. https://doi.org/10.1017/
S0950268814001617
Motulsky, H. J. (1999). Analyzing data with GraphPad prism. San Diego
CA: Graphpad Software Inc.
Murcia, P. R., Wood, J. L., & Holmes, E. C. (2011). Genome‐scale evolu-
tion and phylodynamics of equine H3N8 influenza A virus. Journal of
Virology, 85, 5312–5322. https://doi.org/10.1128/JVI.02619-10Murrell, B., Moola, S., Mabona, A., Weighill, T., Sheward, D., Pond, S. L.
K., & Scheffler, K. (2013). FUBAR: A fast, unconstrained bayesian
approximation for inferring selection. Molecular Biology and Evolution,
30, 1196–1205. https://doi.org/10.1093/molbev/mst030
Murrell, B., Wertheim, J. O., Moola, S., Weighill, T., Scheffler, K., & Pond,
S. L. K. (2012). Detecting individual sites subject to episodic diversify-
ing selection. PLoS Genetics, 8, e1002764. https://doi.org/10.1371/
journal.pgen.1002764
850 | HE ET AL.
Park, S. J., Kang, B. K., Jeoung, H. Y., Moon, H. J., Hong, M., Na, W., …An, D. J. (2013). Complete genome sequence of a canine‐originH3N2 feline influenza virus isolated from domestic cats in South
Korea. Genome Announcements, 1, e0025312. https://doi.org/10.
1128/genomeA.00253-12
Parker, J., Rambaut, A., & Pybus, O. G. (2008). Correlating viral pheno-
types with phylogeny: Accounting for phylogenetic uncertainty. Infec-
tion, Genetics and Evolution, 8, 239–246. https://doi.org/10.1016/j.
meegid.2007.08.001
Payungporn, S., Crawford, P. C., Kouo, T. S., Chen, L. M., Pompey, J.,
Castleman, W. L., … Donis, R. O. (2008). Influenza A virus (H3N8) in
dogs with respiratory disease, Florida. Emerging Infectious Diseases,
14, 902–908. https://doi.org/10.3201/eid1406.071270Rabadan, R., Levine, A. J., & Robins, H. (2006). Comparison of avian and
human influenza A viruses reveals a mutational bias on the viral gen-
omes. Journal of Virology, 80, 11887–11891. https://doi.org/10.1128/JVI.01414-06
Rambaut, A., Lam, T. T., Max Carvalho, L., & Pybus, O. G. (2016). Explor-
ing the temporal structure of heterochronous sequences using Tem-
pEst (formerly Path‐O‐Gen). Virus Evolution, 2, vew007.
Shinya, K., Ebina, M., Yamada, S., Ono, M., Kasai, N., Kawaoka, Y. (2006).
Avian flu: Influenza virus receptors in the human airway.. Nature,
440, 435–436. https://doi.org/10.1038/440435aSmirnov, Y. A., Lipatov, A. S., Beek, R. V., Gitelman, A. K., Osterhaus, A. D. M.
E., & Claas, E. C. J. (2000). Characterization of adaptation of an avian
influenza a (h5n2) virus to a mammalian host. Acta Virologica, 44, 1–8.Smith, M. D., Wertheim, J. O., Weaver, S., Murrell, B., Scheffler, K., &
Pond, S. L. K. (2015). Less is more: An adaptive branch‐site random
effects model for efficient detection of episodic diversifying selec-
tion. Molecular Biology and Evolution, 32, 1342–1353. https://doi.org/10.1093/molbev/msv022
Song, D., Kang, B., Lee, C., Jung, K., Ha, G., Kang, D., … Oh, J. (2008).
Transmission of avian influenza virus (H3N2) to dogs. Emerging Infec-
tious Diseases, 14, 741–746. https://doi.org/10.3201/eid1405.071471Song, D., Moon, H. J., An, D. J., Jeoung, H. Y., Kim, H., Yeom, M. J., …
Park, B. K. (2012). A novel reassortant canine H3N1 influenza virus
between pandemic H1N1 and canine H3N2 influenza viruses in
Korea. Journal of General Virology, 93, 551. https://doi.org/10.1099/
vir.0.037739-0
Stamatakis, A. (2014). RAxML version 8: A tool for phylogenetic analysis
and post‐analysis of large phylogenies. Bioinformatics, 30, 1312–1313. https://doi.org/10.1093/bioinformatics/btu033
Sturmramirez, K. M., Ellis, T., Bousfield, B., Bissett, L., Dyrting, K., Rehg,
J. E., … Webster, R. G. (2004). Reemerging H5N1 influenza viruses in
Hong Kong in 2002 are highly pathogenic to ducks. Journal of Virol-
ogy, 78, 4892. https://doi.org/10.1128/JVI.78.9.4892-4901.2004
Sturmramirez, K. M., Hulsepost, D. J., Govorkova, E. A., Humberd, J., Sei-
ler, P., Puthavathana, P., … Long, H. T. (2005). Are ducks contributing
to the endemicity of highly pathogenic H5N1 influenza virus in Asia?
Journal of Virology, 79, 11269–11279. https://doi.org/10.1128/JVI.
79.17.11269-11279.2005
Su, S., Gu, M., Liu, D., Cui, J., Gao, G. F., Zhou, J., & Liu, X. (2017). Epi-
demiology, evolution, and pathogenesis of H7N9 influenza viruses in
five epidemic waves since 2013 in China. Trends in Microbiology, 25,
713. https://doi.org/10.1016/j.tim.2017.06.008
Sun, H., Blackmon, S., Yang, G., Waters, K., Li, T., Tangwangvivat, R., …Wan, X. F. (2017). Zoonotic risk, pathogenesis, and transmission of
avian‐origin H3N2 canine influenza virus. Journal of Virology, 91,
e00637–00617.Voorhees, I. E. H., Dalziel, B. D., Glaser, A., Dubovi, E. J., Murcia, P. R.,
Newbury, S., … Parrish, C. R. (2018). Multiple incursions and recur-
rent epidemic fade‐out of H3N2 canine influenza A virus in the Uni-
ted States. Journal of Virology, 92, e00323–00318.Voorhees, I. E. H., Glaser, A. L., Toohey-Kurth, K., Newbury, S., Dalziel, B.
D., Dubovi, E. J., … Parrish, C. R. (2017). Spread of canine influenza
A(H3N2) virus, United States. Emerging Infectious Diseases, 23, 1950–1957. https://doi.org/10.3201/eid2312.170246
Webster, R. G., Sharp, G. B., & Claas, E. C. J. (1995). Interspecies trans-
mission of influenza viruses. American Journal of Respiratory and Criti-
cal Care Medicine, 152, 25–30. https://doi.org/10.1164/ajrccm/152.
4_Pt_2.S25
Westgeest, K. B., Russell, C. A., Lin, X., Spronken, M. I., Bestebroer, T.
M., Bahl, J., … de Jong, J. C. (2014). Genomewide analysis of reas-
sortment and evolution of human influenza A(H3N2) viruses circulat-
ing between 1968 and 2011. Journal of Virology, 88, 2844. https://d
oi.org/10.1128/JVI.02163-13
Worobey, M., & Al, E. (2014). A synchronized global sweep of the inter-
nal genes of modern avian influenza virus. Nature, 508, 254.
https://doi.org/10.1038/nature13016
Yasuha, A., Norihito, K., Tomo, D., Ibrahim, M. S., El-Gendy, E. M., Tat-
suya, T., … Takaaki, N. (2016). Novel polymerase gene mutations for
human adaptation in clinical isolates of avian H5N1 influenza viruses.
PLoS Pathogens, 12, e1005583.
Yoon, S. W., Webby, R. J., & Webster, R. G. (1992). Evolution and ecol-
ogy of influenza A viruses. Current Topics in Microbiology and
Immunology, 56, 152–179.Zhu, H., Hughes, J., & Murcia, P. R. (2015). Origins and evolutionary
dynamics of H3N2 canine influenza virus. Journal of Virology, 89,
5406–5418.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
How to cite this article: He W, Li G, Zhu H, et al. Emergence
and adaptation of H3N2 canine influenza virus from avian
influenza virus: An overlooked role of dogs in interspecies
transmission. Transbound Emerg Dis. 2019;66:842–851.https://doi.org/10.1111/tbed.13093
HE ET AL. | 851