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: shuosu@njau.edu.cn

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

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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

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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

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