Emergence and adaptation of H3N2 canine influenza virus ... · the influenza pandemic of 1968, studies showed that companion ani-mals, such as dogs and cats, were seropositive for
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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
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
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
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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