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\begin{document}

\title{Phylogeography of the Highly Pathogenic Avian Influenza H5N6 Virus
Determine Transmission from Guangdong Province, China to South Korea and
Japan by Wild Bids in 2016}



\author[1]{Jung-hoon Kwon}%
\affil[1]{Kyungpook National University}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
The Goose/Guangdong lineage highly pathogenic avian influenza (HPAI)
clade 2.3.4.4 viruses have spilled over to wild birds and caused
transboundary epizootics. In 2016, the H5N6 subtype of clade 2.3.4.4
HPAI viruses was introduced to Korea and Japan by wild waterfowls.
However, the detailed geographic location of the origin of this outbreak
was not fully defined yet. In this study, the geographical origin of
this outbreak was estimated using phylogeographic analysis. The results
indicate that the H5N6 viruses of wild birds originated from a virus in
Guangdong province, China. In addition, Guangdong province was an
epicenter for H5N6 viruses, and multiple spillovers of viruses from
poultry into wild birds have been detected. Thus, to predict and prepare
for the next HPAI outbreak in Korea and Japan, viruses in Guangdong
province should be carefully monitored and investigated.%
\end{abstract}%



\sloppy


\emph{Short communication}

\textbf{Title:} Phylogeography of the Highly Pathogenic Avian Influenza
H5N6 Virus Determine Transmission from Guangdong Province, China to
South Korea and Japan by Wild Bids in 2016

\textbf{Running Title} : Phylogeographic Analysis of H5N6 HPAIV

\textbf{Author:} Jung-Hoon Kwon

\textbf{Affiliation:} College of Veterinary Medicine, Kyungpook National
University, Daegu, 41566, Republic of Korea.

\textbf{Corresponding Author}

Jung-Hoon Kwon

College of Veterinary Medicine, Kyungpook National University

Daegu, 41566, Republic of Korea.

E-mail: Junghoon.kwon@knu.ac.kr

Phone: +82.53.950.5968

\textbf{Summary}

The Goose/Guangdong lineage highly pathogenic avian influenza (HPAI)
clade 2.3.4.4 viruses have spilled over to wild birds and caused
transboundary epizootics. In 2016, the H5N6 subtype of clade 2.3.4.4
HPAI viruses was introduced to Korea and Japan by wild waterfowls.
However, the detailed geographic location of the origin of this outbreak
was not fully defined yet. In this study, the geographical origin of
this outbreak was estimated using phylogeographic analysis. The results
indicate that the H5N6 viruses of wild birds originated from a virus in
Guangdong province, China. In addition, Guangdong province was an
epicenter for H5N6 viruses, and multiple spillovers of viruses from
poultry into wild birds have been detected. Thus, to predict and prepare
for the next HPAI outbreak in Korea and Japan, viruses in Guangdong
province should be carefully monitored and investigated.

\textbf{Keywords} : H5N6, highly pathogenic avian influenza virus,
phylogeography, wild birds

\textbf{Introduction}

The Goose/Guangdong (Gs/Gd) lineage highly pathogenic avian influenza
viruses (HPAIVs) have been circulating in poultries in endemic countries
since it was first detected in 1996 (Swayne, 2016; Swayne, Suarez \&
Sims, 2020). The HPAIV spillovers from poultry into wild birds have
caused long-distance transmission beyond national borders (Lee et al.,
2015; Olsen et al., 2006). Transmission of HPAIVs by wild birds has
caused significant economic loss in the poultry industry, because most
non-endemic countries do not use vaccines and instead have applied a
stamping-out policy for the eradication of the virus (Swayne, Pavade,
Hamilton, Vallat, \& Miyagishima, 2011).

In South Korea and Japan, a total of seven HPAIV outbreaks have been
reported: 2003 (H5N1 clade 2.5), 2006--2007 (H5N1 clade 2.2), 2008 (H5N1
clade 2.3.2.), 2010--2011 (H5N1 clade 2.3.2.1), 2014--2016 (H5N8 clade
2.3.4.4), 2016--2017 (H5N6 and H5N8, clade 2.3.4.4) and 2017--2018
(H5N6, clade 2.3.4.4) (Kwon et al., 2018; Kwon et al., 2017; Mo et al.,
2016; Woo et al., 2017). In addition, these viruses disseminated to
Europe, North America, and Africa by migratory wild waterfowls (Lee,
Bertran, Kwon, \& Swayne, 2017; Lee et al., 2015; Olsen et al., 2006).
Previous studies using phylogenetic analysis indicated that these
viruses originated from endemic regions, including China (Kwon et al.,
2017; Lee et al., 2017; Olsen et al., 2006).

However, the detailed geographical origin and transmission of these
outbreaks had not fully been determined, because only a few nucleotide
sequences were available in the database. For instance, only nine
hemagglutinin sequences of H5N8 viruses were available in GenBank
(https://www.ncbi.nlm.nih.gov/genbank/) before 2104, when it was first
detected in Korea (Lee et al., 2014). Recently, however, the
implementation of next-generation sequencing systems has allowed for
high-throughput sequence analyses of avian influenza viruses, so there
are a considerable number of sequences available now (Lee, 2020). In
particular, clade 2.3.4.4 H5N6 HPAIVs has been a dominant virus in wide
geographic regions in China since 2014, and numerous nucleotide
sequences of these viruses have been analyzed (Bi et al., 2016).

Using these sequencing data, in this study, we inferred the geographical
origin of the H5N6 outbreak in South Korea and Japan and estimated the
time for H5N6 virus spillover from poultry into wild birds for this
outbreak.

\textbf{Materials and Methods}

All available hemagglutinin (HA) gene sequences of H5N6 viruses
belonging to clade 2.3.4.4 subgroup C (n = 1388) were downloaded from
the Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu
database (https://www.gisaid.org/) on August 13, 2020. After excluding
the sequences that did not have an exact collection date or region, we
reduced the data set based on the sequence identity at a 99.9\% level
using the program cd-hit (Li \& Godzik, 2006). The reduced data set
consisted of 679 HA sequences, and the sequences were aligned by the
Multiple Alignment using Fast Fourier Transform (MAFFT) method (Katoh,
Misawa, Kuma, \& Miyata, 2002) and were manually trimmed to equal
lengths (1674 bp) using Geneious Prime (https://www.geneious.com/).
After phylogenetic analysis using FastTree (Price, Dehal, \& Arkin,
2009) (Figure S1), the sequences of a subclade related to the South
Korea and Japan outbreak showed \textgreater{}0.9 FastTree support value
was extracted (n = 241), which was used for the final data set. The taxa
were coded into nine discrete groups: Fujian, Guangdong, Hunan, Japan,
Jiangxi, Korea, Vietnam, Wild bird, and Yunnan. The Wild bird
classification included all wild bird isolates regardless of location.

The phylogeography in discrete space was estimated using BEAST v.1.10.4
(http://beast.bio.ed.ac.uk) with an uncorrelated lognormal relaxed
molecular clock. The HKY + G nucleotide substitution model (Hasegawa,
Kishino, \& Yano, 1985) was used along with a Gaussian Markov random
field (GMRF) Bayesian Skyride coalescent tree (Minin, Bloomquist, \&
Suchard, 2008). The ancestral locations were reconstructed, and the
asymmetric viral exchanges between regions were estimated using the
non-reversible continuous-time Markov chain (CTMC) model. We applied a
Bayesian stochastic search variable selection (BSSVS) procedure to
identify the best-supported transitions between discrete states using
Bayes factors test, which is a feature of SPREAD software v1.0.7. We
identified a transition as significant when the posterior probability
was \textgreater{}0.5 and the Bayesian factor (BF) was \textgreater{}6.

The Markov chain Monte Carlo (MCMC) method was run in parallel for four
chains, each with 100 million steps, and the parameters and trees were
sampled every 10,000 steps. After burn-in first 10\% of each run,
samples across the chains combined, yielding a total of 36,000 parameter
states and posterior trees. The parameters were analyzed with TRACER
v1.5 (http://tree.bio.ed.ac.uk/software/tracer/), which showed that all
parameters had an effective sample size greater than 200. A maximum
clade credibility (MCC) tree was generated using TreeAnnotator in BEAST
and visualized using FigTree 1.4.2
(http://tree.bio.ed.ac.uk/software/figtree/). We also estimated the rate
and number of transitions between hosts (Markov jumps) using stochastic
mapping techniques implemented in the BEAST package (Minin \& Suchard,
2008)

\textbf{Results}

The phylogeographic analysis was conducted against viruses belonging to
a subgroup of clade 2.3.4.4C viruses, including those of the South Korea
and Japan outbreak (n = 241; Figure S1). The emergence time of this
subgroup was estimated to be February 2014 (95\% Bayesian credibility
intervals, BCI: October 2013 to March 2014), and the geographic location
of the ancestor of this subgroup was estimated to be Guangdong province
with 97.87\% posterior probability (Figure 1 and Figure S2).

All the poultry isolates of South Korea and Japan were clustered with
wild bird isolates. The ancestral reconstruction and the Bayes factor
analysis indicated that all HPAIVs of the South Korea and Japan outbreak
originated from H5N6 HPAIVs of wild birds (Figure 1, Figure S2, and
Table 1). At least thirteen independent introduction events from wild
birds into poultries in South Korea and Japan were detected in this
study (Figure 1, Table 1). Four Vietnam isolates were also clustered
with the South Korean, Japanese, and wild bird viruses, indicating at
least two independent introduction events from wild birds to Vietnam
(Figure 1 and Figure S2). The transmission from poultry to wild birds or
between poultries in different countries was not detected.

The wild bird HPAI isolates in South Korea, Japan, and Shanghai were
clustered with Korean, Japanese, and Vietnamese viruses. HPAIV
infections in wild birds were also detected from Hong Kong, Guangdong,
Hunan, and Vietnam, but these were phylogenetically distinct from the
South Korea and Japan isolates (Figure 1 and Figure S2). However, in
this phylogeographic analysis, the ancestral locations of all the wild
bird isolates were traced to Guangdong province, showing
\textgreater{}0.9 posterior probability (Figure 1 and Figure S2). The
Markov jump count from Guangdong to the wild birds was estimated with a
mean of 6.89 (95\% BCI: 6--8) in this study (Table 1).

The time period of the most recent common ancestor (tMRCA) of the South
Korea and Japan outbreak and related wild bird viruses (Figure 1) was
estimated to be March 2016 (95\% BCI: January to April 2016). The tMRCA
of the Guangdong viruses and wild bird viruses that caused the South
Korea and Japan outbreak was estimated to be October 2015 (95\% BCI:
September to November 2015), which indicates that the spillover of HPAIV
from poultries to wild birds occurred during this period. The first
real-world detection of wild bird infection with these viruses was
October 28, 2016 (J. H. Kwon et al., 2017), which shows a one year
interval from the estimated spillover time.

\textbf{Discussion}

Despite repeated transboundary transmission of HPAIVs, the detailed
geographic origins and transmission routes of the evolution and
epidemiology of the viruses have not fully been determined yet. The
migration of wild birds, which is a major contributor to long-distance
HPAIV transmission, is highly complex, unpredictable, and impacted by
climate change (Fusaro et al., 2019). In this study, we estimated the
origin of the 2016--2017 South Korea and Japan H5N6 HPAI outbreak using
Bayesian phylogenetic analysis.

Guangdong province, China was estimated to be the site for wild bird
infection of H5N6 HPAIV by poultry in this study. Guangdong province is
known as one of the major regions for duck and geese production in
China, and a high incidence of H5N6 HPAIV infection was detected in a
previous study (Bi et al., 2016). In addition, a previous
phylogeographic study showed that Guangdong province and southern China
were the epicenters for H5N6 HPAIV in China (Zhang et al., 2020). These
results of the previous studies support the finding in this study that
wild bird H5N6 viruses originated from Guangdong province, China.

The tMRCA analysis result indicated that H5N6 HPAIV spilled over to wild
birds around October 2015, and then circulated in the wild bird
population for approximately one year. However, the virus was first
detected in October 2016. A previous experimental infection study
suggests possible subclinical infection and replication of H5N6 HPAIV in
Mandarin ducks (\emph{Aix galericulata} ) (Son et al., 2018). In
addition, simultaneous detection of multiple reassortants of H5N6 HPAIVs
in South Korea and Japan also supports the possibility of long-term
silent circulation of H5N6 HPAIVs in wild birds (Lee et al., 2018;
Takemae et al., 2017). The results suggest that spring migration of wild
birds in 2016 caused the dissemination of viruses to other population of
wild birds, and fall migration in 2016 introduced the virus into South
Korea, Japan, and Shanghai.

The Bayesian phylogenetic inference and ancestral reconstruction methods
used in this study can be affected by sampling bias (Kwon et al., 2020).
To minimize the effect of sampling bias, the sequences were equally
subsampled by sequence identity using all available sequences in the
database. However, each province in China has a different surveillance
system and sampling frequency. Thus, the potential role of un-sampled or
less-sampled viruses cannot be completely excluded. Nevertheless, this
study presents the most reliable transmission route using all available
data, and the results strongly indicate the significant role of
poultries in Guangdong province, China, for the H5N6 outbreak in wild
birds. Continuous, constant, and regular surveillance and a nucleotide
sequencing system will improve the quality of epidemiological analysis
of HPAIV, including the molecular epidemiology.

Clade 2.3.4.4 H5N6 HPAIVs are causing continuous outbreaks and emergence
of novel reassortants in endemic countries. This study recommends
careful monitoring and control of HPAIVs in Guangdong province to
prevent wild bird infection and transboundary transmission of HPAIVs.
Although the emergence and spread of novel viruses are unpredictable,
the accumulation of epidemiologic data, such as that reported in this
study, will allow prediction and preparation that can be taken to
minimize the economic loss caused by epidemics in the event of future
HPAIV outbreaks.

\textbf{Acknowledgments}

We gratefully acknowledge the authors and submitting laboratories of the
sequences from GISAID's EpiFlu (https://www.gisaid.org/) database. The
GISAID acknowledgement tables for laboratory contributions are shown in
Table S1.

This research was supported by the National Research Foundation of Korea
(NRF) grant funded by the Korean government (MSIT) (No.
2020R1G1A1101032).

\textbf{Conflict of Interest:} None

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\textbf{Table 1.} Well-supported viral transitions of clade 2.3.4.4C
H5N6 HPAIVs among geographic regions and wild birds.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
From & to & Mean actual migration rate \textsuperscript{a} (95\% BCI
\textsuperscript{b}) & Mean number of Markov jump (95\% BCI) & BF &
Posterior probability\tabularnewline
\midrule
\endhead
Guangdong & Fujian & 1.02 (0.15, 2.20) & 3.16 (3, 4) & \textgreater{}100
& 99.67\%\tabularnewline
Guangdong & Hunan & 2.37 (0.75, 4.37) & 8.77 (7, 11) & \textgreater{}100
& 100.00\%\tabularnewline
Guangdong & Jiangxi & 0.31 (0.00, 1.08) & 0.65 (0, 1) & 12.60 &
63.36\%\tabularnewline
Guangdong & Wild\_Bird & 1.89 (0.53, 3.58) & 6.89 (6, 8) &
\textgreater{}100 & 100.00\%\tabularnewline
Guangdong & Yunnan & 0.43 (0.00, 1.42) & 1.13 (0, 3) & 12.30 &
62.82\%\tabularnewline
Hunan & Guangdong & 0.85 (0.00, 2.14) & 1.32 (0, 3) & 89.57 &
92.48\%\tabularnewline
Wild\_Bird & Japan & 2.67 (0.89, 4.72) & 11.02 (10, 12) &
\textgreater{}100 & 100.00\%\tabularnewline
Wild\_Bird & Korea & 0.89 (0.12, 1.95) & 3.00 (3, 3) & \textgreater{}100
& 99.52\%\tabularnewline
Wild\_Bird & Vietnam & 1.04 (0.00, 2.16) & 3.64 (1, 4) &
\textgreater{}100 & 98.10\%\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a} Actual transition rates were calculated as rate *
indicator.

\textsuperscript{b} BCI, Bayesian credibility intervals.

\textbf{Figure Legends}

\textbf{Figure 1.} Bayesian phylogenetic MCC tree of the HA gene of a
subgroup of clade 2.3.4.4C H5 HPAIVs. Branches are colored according to
discrete traits of geographic regions or wild bird isolates, and the
thickness of branches indicates posterior probabilities of the ancestral
host type. The scale axis indicates the branch lengths by year.

\textbf{Supporting Information}

\textbf{Figure S1.} FastTree Phylogenetic tree of HA gene of clade
2.3.4.4 H5N6 HPAIVs. Node labels indicate FastTree support values. The
scale bar indicates the number of substitutions per site. The sequences
of the highlighted subclade were used for the final dataset.

\textbf{Figure S2.} Bayesian phylogenetic MCC tree of the HA gene of a
subgroup of clade 2.3.4.4C H5 HPAIVs with full taxa names and node bars.
Horizontal node bars indicate 95\% Bayesian credibility intervals for
the divergence in time estimates.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
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