DR.

RYOTA TSUNEKUNI (Orcid ID : 0000-0002-6204-1233)

MS. MOMOKO NAKAYAMA (Orcid ID : 0000-0002-9284-8425)

DR. NOBUHIRO TAKEMAE (Orcid ID : 0000-0001-5152-8938)

Article type : Rapid Communication

Isolation of highly pathogenic H5N6 avian influenza virus in Southern Vietnam with

genetic similarity to those infecting humans in China

Running title: Highly pathogenic H5N6 avian influenza virus in Southern Vietnam

Ryota Tsunekunia,b,*, Kasumi Sudoc,*, Phuong Thanh Nguyend, Bach Duc Luud, Thai Duy

Phuongd, Tran Minh Tand, Nguyen Tunge, Junki Minea,b, Momoko Nakayamaa,b, Taichiro

Tanikawaa,b, Kirill Sharshovf, Nobuhiro Takemaea,b, Takehiko Saitoa,b,g,#

This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences
between this version and the Version of Record. Please cite this article as doi: 10.1111/tbed.13294

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aDivision of Transboundary Animal Disease, National Institute of Animal Health,

NARO, Tsukuba, Japan

bThailand–Japan Zoonotic Diseases Collaboration Center, Bangkok, Thailand

cNational Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries,

Japan

dRegional Animal Health Office No. 6, Department of Animal Health, Vietnam

eDivision of International Cooperation and Communications, Department of Animal

Health, Hanoi, Vietnam.

fFederal Research Center of Fundamental and Translational Medicine, Novosibirsk,

Russia

gUnited Graduate School of Veterinary Sciences, Gifu University, Japan

*These authors contributed equally.

#Corresponding author: Takehiko Saito, DVM, Ph.D.

Phone & Fax: +81-29-838-7738

taksaito@affrc.go.jp,tune@affrc.go.jp

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Summary

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have

been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is

also a public concern due to sporadic human infections being reported in China. In the

current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long

An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in

Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment

Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were

most closely related (99.6–99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which

was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4–99.9%

homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in

China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was

directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and

clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to

2018. This indicated that AI470 was introduced into Vietnam from China. In addition,

molecular characteristics related to mammalian adaptation among the recent human

H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are

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cause for concern since H5N6 HPAIV strains that possess a risk of human infection have

crossed the Chinese border.

Keywords: Highly pathogenic avian influenza virus, human infection, Vietnam, G1.1,

South China

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1 | INTRODUCTION

Highly pathogenic avian influenza viruses (HPAIVs) of the Goose/Guangdong

(Gs/Gd) lineage have become diversified since their original emergence in 1996 (Xu,

Subbarao, Cox, & Guo, 1999). Gs/Gd HPAIVs had been exclusively of the H5N1 subtype

until an H5N5 HPAIV Gs/Gd strain appeared in China in 2008–2009 due to genetic

reassortment (Gu et al., 2011). Since then, the so-called H5Nx HPAIVs have expanded

worldwide and have been responsible for poultry outbreaks on the Eurasian, North

American, and African continents (World Organisation for Animal Health, 2018a). The

clade 2.3.4.4 has been a major branch of the H5Nx HPAIVs of the Gs/Gd lineage and

classified into four sub-lineages, groups A, B, C, and D (D. H. Lee et al., 2016; Lee,

Bertran, Kwon, & Swayne, 2017). The clade 2.3.4.4 group A H5 HPAIVs were first

identified in China in 2013 as H5N8 (Fan et al., 2014) and were subsequently responsible

for outbreaks in South Korea and Japan in 2014 (Kanehira et al., 2015; Lee et al., 2014).

This sub-lineage disseminated to Eastern Siberia, Eurasia, and North America through

bird migration (Global Consortium for H5N8 and Related Influenza Viruses, 2016; Lee et

al., 2015; M. S. Lee et al., 2016; Marchenko et al., 2015; Tanikawa et al., 2016). The clade

2.3.4.4 group B H5 HPAIVs were identified in China and Korea in 2013–2014 (Fan et al.,

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2014; Lee et al., 2015) and widely isolated in Eurasian countries in 2016–2017 (Lee,

Sharshov, et al., 2017; Pohlmann et al., 2017). While group A and group B HPAIVs have

expanded worldwide, group C H5N6 HPAIVs have been mainly isolated from 2013 to

2017 in Asian countries such as China, Japan, Korea, Laos, and Vietnam (Hiono et al.,

2017; Mok et al., 2015; Okamatsu et al., 2017; Takemae et al., 2017; Wong et al., 2015).

Group D H5N6 HPAIVs have been enzootic in China and Vietnam (Chu et al., 2016; Lee

et al., 2015; Qi, Cui, Yu, Ge, & Tang, 2014).

In Vietnam, the first wave of massive outbreaks started on December 23, 2003,

by H5N1 HPAIV of the clade 1 Gs/Gd lineage (Nguyen, 2005) with most poultry

outbreaks during this period being caused by clade 1 viruses (Wan et al., 2008).

Subsequently, clade 2.3.4 viruses gradually replaced the clade 1 viruses in parts of

Northern and Central Vietnam between 2005 and 2010 with the descendants of the clade

1 viruses, so-called clade 1.1, being followed by clade 1.1.2 viruses, which have continued

to predominate in Southern Vietnam (Nguyen et al., 2012; Wan et al., 2008). Three

genetically distinct subgroups of clade 2.3.2.1 (2.3.2.1a, 2.3.2.1b, and 2.3.2.1c) replaced

clade 2.3.4 viruses in parts of Northern and Central Vietnam between 2010 and 2012

(Creanga et al., 2013). Clade 2.3.2.1c viruses have spread to parts of Southern Vietnam

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after 2012. Clade 2.3.4.4 (H5N6) has become enzootic in parts of Northern and Central

Vietnam since 2014.

Since the first reported case of human infection with H5N1 that occurred in

1997 in Hong Kong, more than 900 cases in 16 countries have been reported by 2015

with half of them being fatal (Lai et al., 2016). Subsequently, as of March 6, 2019, the

incidence of human cases of H5N1 infection has been decreasing with none being

reported since a case in Indonesia in September 2017. In Vietnam, 127 confirmed

human cases of H5N1 virus infections were recorded between 2003 and 2014 with 64 of

them being fatal (https://apps.who.int/iris/handle/10665/279855). However, there have

been no reports of human cases of H5N1 or H5N6 HPAIV infections since reports in

Vietnam in 2015. Recently, the H5N6 HPAIV clade 2.3.4.4 groups C and D have been

responsible for 23 human infections, leading to seven deaths in China since 2014

(https://apps.who.int/iris/bitstream/handle/10665/279855/AI-20190201.pdf?sequence=4

&isAllowed=y).

In the current study, we isolated an H5N6 HPAIV strain from specimens

collected during an HPAI outbreak on a Muscovy duck farm in Long An Province in

Southern Vietnam in July 2018. The entire genome of the isolate was sequenced for
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detailed molecular analyses, including a comparison of the new isolate with recent

human HPAIV isolates from China.

2 | MATERIALS AND METHODS

2.1 | Virus isolation

On July 25, 2018, six Muscovy ducks on a farm in Long An Province, Vietnam,

died suddenly after exhibiting clinical signs of HPAI. The farm included 1,202 Muscovy

ducks, which had not been vaccinated against HPAIV and had been scavenging in rice

fields during the days. A spleen tissue sample was collected from a dead Muscovy duck

on an affected farm, and the tissue homogenate was inoculated into 10-day-old

embryonated chicken eggs in a BSL-3 facility at the National Institute of Animal Health,

NARO, Tsukuba, Japan. Allantoic fluid recovered after incubation for 24 h at 37 ℃

showed hemagglutination activity with 0.55% chicken red blood cells.

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2.2 | Sequencing

RNA was purified from the allantoic fluid with the RNeasy Micro kit (Qiagen,

Valencia, CA, USA). The sequence library was prepared using an NEBNext Ultra RNA

Library Prep Kit for Illumina (NEB, Valencia, CA, USA) and subjected to whole-genome

sequence analysis using a MiSeq system (Illumina, San Diego, CA, USA) with MiSeq

Reagent Kit v2 as described previously (Tanikawa et al., 2019). The sequence data were

analyzed using FluGAS software v0.9.0 (World Fusion, Tokyo, Japan) to create

consensus sequences of each genomic segment (GISAID accession number

EPI1363700-7).

2.3 | Sequence analysis

The sequence similarity of each segment was determined using Nucleotide

Basic Local Alignment Search Tool (BLAST) search of Global Initiative on Sharing All

Influenza Data (GISAID). For phylogenetic analysis, all AIV sequences registered to
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GISAID as of March 8, 2019 were downloaded and aligned using MAFFT (Katoh et al,

2013). Non-coding regions of each segment were trimmed and sequences that included

ambiguous and mixed bases were removed. Segment sets in the maximum likelihood

(ML) tree were screened as having 0.995 (H5) or 0.970 (PB2, PB1, PA, NP, N6, M and

NS) sequence similarity by CD-HIT and added to the sequences of the top 250 blast hits

for AI470 on GISAID. The number of analyzed sequences of each segment included 1399

for PB2, 1414 for PB1, 1342 for PA, 701 for H5, 1235 for NP, 348 for N6, 1008 for M, and

1104 for NS. Phylogenetic trees of the eight gene segments of HPAIVs were constructed

with MEGA7.0.26 software using the ML method. Bootstrap analysis was performed

with 200 replicates. For tanglegrams, all segment sets of strains belonging to clade

2.3.4.4 in the ML tree of H5 genes were downloaded from GISAID. The sequence sets

were added the sequences of each top 250 blast hits for AI470 and human-origin H5N6

HPAIVs on GISAID with the duplicate strains being removed. Tanglegrams were

generated based on the phylogenetic trees using the Dendroscope 3 program. A Bayesian

maximum clade credibility (MCC) tree of the hemagglutinin (HA) gene was constructed

from the sequences of the top 100 blast hits for AI470 on GISAID using BEAST 1.8.4

software. A world map was generated using SPREAD software to perform discrete

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phylogeographical analyses, and the results were visualized using Google Earth

(https://www.google.co.jp/intl/ja/earth/).

3 | RESULTS AND DISCUSSION

The new isolate was determined to be an H5N6 subtype and was designated as

A/ Muscovy duck/Long An/AI470/2018 (H5N6; AI470). The amino acid sequence of the

HA protein cleavage site of AI470 was RERRRKRGLF, which indicated it was an HPAIV

according to the molecular criteria of the World Organisation for Animal Health (OIE)

Terrestrial Animal Health Code (World Organisation for Animal Health, 2018b). A

BLAST search revealed that the new isolate had the highest HA gene homology with

A/common gull/Saratov/1676/2018 (H5N6), which was collected on October 1, 2018,

followed by A/Guangxi/32797/2018 (H5N6), which was isolated on October 25, 2018, from

specimens of a patient. The HA gene homology of AI470 compared to that of other H5N6

HPAIVs from human patients is shown in Table S1. The other seven genomic segments

of AI470 demonstrated similar results as those for the HA segment.

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 To clarify the origin of AI470, phylogenetic trees including AI470 were

constructed using the eight genomic segments. The phylogenetic trees revealed that the

HA segment of AI470 was derived from H5N6 HPAIV clade 2.3.4.4 group C in South

China in 2015–2018 (Figure 1). The other segments, except polymerase basic protein 2

(PB2), were shown by phylogenic analyses to have origins similar to that of HA (Figure 2

and Figures S1–S7). In contrast, the PB2 segment of AI470 was derived from H5N6

HPAIV strains in China in 2014 to 2017, which initially originated from an H6 avian

influenza virus that circulated among wild birds. Intriguingly, five HPAIVs of human

origin in South China from 2017 to 2018 clustered with AI470 in the HA tree, with four of

them also demonstrating results similar to those of the other seven segments. Analysis

based on the genetic nomenclature defined by Bi et al. demonstrated that AI470 and the

four HPAIVs of human origin in South China from 2017 to 2018 were characterized to be

of the G1.1 genotype (Bi et al., 2016). The transmission of four HPAIVs of human origin

to humans suggests that other H5N6 HPAIV strains in this cluster may possess the

potential to infect humans, including AI470.

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 Amino acid substitutions related to host adaptation and virulence were shared

among AI470 and the four human-origin H5N6 HPAIVs in the same cluster of a G1.1

genotype (Table 1). The substitutions of D101N and T160A in the HA protein (using H3

numbering) increase HA binding to sialic acid α2,6-galactose, which is known as a

human-like receptor (Gao et al., 2018; Su, Yang, Zhang, Jia, & Tien, 2008), and these

substitutions are commonly found in clade 2.3.4.4. The receptor binding site (RBS) of HA

has conserved secondary elements (130-loop, 190-helix, and 220-loop), and substitutions

proximal to the RBS can affect receptor binding specificity (Tharakaraman et al., 2013;

Yang et al., 2007). Remarkably, a deletion of HA D130 created a putative glycosylation

site at amino acid 129 of AI470. This new glycosylation site adjoins the 130-loop of the

RBS of HA and may affect receptor specificity. In addition, this putative glycosylation

site is characteristic of most H5N6 viruses of human origin and may play a role in human

adaptation. Some amino acid substitutions that are considered to increase virulence in

mammalian hosts such as mice were shared by AI470, recent H5N6 HPAIVs from

patients, and closely related poultry isolates. Those substitutions include a deletion in

the neuraminidase (NA) stalk region (position 59–69), N30D and T215A substitutions in

the matrix protein 1 (M1), and a S42 substitution and amino acid deletion (position

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80–84) in nonstructural protein 1 (NS1) (Fan et al., 2009; Jiao et al., 2008; Long, Peng,

Liu, Wu, & Liu, 2008; Matsuoka et al., 2009). AI470 also shared a G309D substitution in

PB2, which is known to enhance polymerase activity in mammalian cells (Hatta, Gao,

Halfmann, & Kawaoka, 2001; Subbarao, London, & Murphy, 1993; Yamaji et al., 2015).

AI470 did not possess PB2 substitutions E627K and D701N, but some of the human

isolates did have the E627K substitution. No substitutions related to oseltamivir or

amantadine resistance were present in NA or matrix 2 (M2) protein of AI470 or five of

the H5N6 HPAIVs of human origin.

Our genetic analyses revealed that the H5N6 HPAIV AI470 was closely related

to H5N6 HPAIV strains isolated from patients in China and that it was introduced into

Southern Vietnam (Figure 3). Vietnam has been at risk of HPAIV invasion because of its

geographical relation to Southern China, a possible “epicenter” of HPAIVs (Smith et al.,

2006). After crossing the border from China into Vietnam, HPAIVs have usually

disseminated from the North to the South through the poultry trade and other human

activities (Nguyen et al., 2017). It is surprising that there has been no report prior to our

study of an H5N6 HPAIV in Vietnam that is genetically related to AI470. This suggests

that the dissemination route of AI470 may have been different than that described

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above. Because of the genetic similarity of AI470 to A/common gull/Saratov/1676/2018

(H5N6), which was isolated from wild bird in Russia, it is conceivable that AI470 was

transmitted from China to Southern Vietnam by migratory birds. As the details

regarding how A/common gull/Saratov/1676/2018 (H5N6) was transmitted to Russia

have not been determined, this hypothesis should be further scrutinized. Another

potential route of virus transmission may be the illegal transportation of live birds or

poultry products, which was observed in cases of HPAIV being isolated from eagles that

had been illegally smuggled into Belgium (Borm et al., 2005) and from raw poultry

illegally imported from China into Japan by airline passengers (Shibata et al., 2018).

Since the outbreak by AI470, eight outbreaks of H5N6 viruses in Vietnam have been

reported to OIE through 2018 (Table S2). Therefore, AI470-like viruses may have

already been circulating in Vietnam, and the genetic analyses of those viruses are

needed.

A470 and A470-like strains of influenza may be threats to public health since

the genetic features of A470 resemble those of human isolates. HA proteins of the viruses

belonging to clade 2.3.4.4 have the ability to bind to both avian-like (α-2,3) and
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mammalian-like (α-2,6) receptors (Gao et al., 2018). However, in addition to this

characteristic, other mutations in the virus genome are necessary for avian HPAIVs to

replicate efficiently in humans. For instance, the E627K substitution in PB2 is an

important change that is required for efficient replication in humans (Moncorge, Mura, &

Barclay, 2010). It has been reported that when an H5N1 HPAIV with E627 in PB2 is

inoculated into mice, the E627K substitution becomes predominant by 6 days post

infection (dpi) (Min et al., 2013). Therefore, while AI470-like strains in poultry may not

possess the E627K substitution in PB2, it may be possible for them to acquire this

change once they infect humans.

H5N6 HPAIVs related to human isolates have been previously isolated in

Vietnam. For instance, the H5N6 HPAIV A/Muscovy duck/Quang Ninh/4c111/2013 of the

G2 genotype was isolated in April 2013 in Vietnam and has the same origin as

A/Sichuan/26221/2014 (H5N6; Sichuan), which was isolated in April 2014 from a patient

in South China that had an H5N6 HPAIV infection (Figure 2 and Figure S8) (Pan et al.,

2016). Subsequently, Sichuan-like H5N6 HPAIVs have circulated in Vietnam, at least

through January 2016 (Nguyen et al., 2017). H5N6 HPAIV A/Muscovy duck/Quang

Ninh/4c111/2013 and A/Sichuan/26221/2014 share some amino acid substitutions,

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including D101N and T160A in HA and G309D in PB2, as well as the deletion of amino

acids 80–84 in NS1. These changes are related to the adaptation of the Sichuan strain for

replication in mammalian cells (Table S3). A D701N substitution in PB2 was unique to

the Sichuan strain, while deletions of HA amino acid E130 and amino acids 59–69 in the

NA stalk region were not detected in either strain (Long et al., 2008; Matsuoka et al.,

2009). The putative glycosylation site at HA amino acid 129 caused by the deletion of HA

E130 was found in 19 of 27 human H5N6 HPAIVs evaluated (Table S3) and in 4 of 19

strains that belonged to the G1.1 genotype and have infected humans for at least one

year. In contrast, 8 of 27 virus strains without the glycosylation site at amino acid 129

were scattered among six different genotypes and have been known to only sporadically

infect humans. These results suggested that the additional glycosylation site at amino

acid 129 of HA may be related to the adaptation of HPAIVs for replication in humans;

however, further investigation is needed. Regarding these molecular features, AI470-like

H5N6 HPAIVs may possess a greater potential for human infection than Sichuan-like

H5N6 HPAIVs. In fact, human infection by Sichuan H5N6 HPAIVs has not been

reported since 2014, and human infection by Sichuan-like H5N6 HPAIVs has never been

reported.

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 In the current study, we determined that AI470 was genetically closely related

to the H5N6 HPAIVs that infect humans in China, suggesting that AI470 may be a risk

for human infection, at least based on genetic analyses. Genome analyses of HPAIVs

may be useful for evaluating the potential of human infection by HPAIVs based on

known genetic signatures. However, genetic analyses alone may be insufficient for the

evaluation since such signatures may act in combination with other unknown changes

that may be involved in the potential for human infection. Therefore, further studies,

such as receptor binding assays and infection and transmissibility studies in ferrets, are

needed to properly evaluate the potential of AI470 and AI470-like virus for human

infection. It is also important to monitor AI470-like viruses to determine if they have

established in Vietnam in order to provide a proper risk assessment to the public.

Acknowledgments

We gratefully acknowledge the investigators that produced and submitted the

sequences that were used in the present study to GISAID. The current research was

supported by the Japan Initiative for Global Research Network on Infectious Diseases

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(J-GRID) from the Ministry of Education, Culture, Sports, Science & Technology in

Japan and the Japan Agency for Medical Research and Development (AMED; grant

number 18fm0108008) and by a joint grant from a pilot program for international

collaborative research between the Agriculture, Forestry and Fisheries Research Council

(AFFRC), the Ministry of Agriculture, Forestry and Fisheries (MA) of Japan and the

Russian Science Foundation (grant number 17-44-07001).

Conflict of Interest statement

The authors declare no competing financial interests.

Ethics

Ethical approval was not required for this study.

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Table 1 Amino acid substitutions in AI470 and other H5N6 highly pathogenic avian influenza viruses (HPAIVs) of avian and human origin.

HA† NA PB2 M1 M2 NS1

Host Strain

D101N E130 130 Loop T160A 190 Helix 220 Loop 59-69 Deletion H275K G309D E627K D701N N30D T215A S31N P42S 80-84 Deletion 92E

Avian A/Muscovy duck/Long An/AI470/2018 N del‡ SSGVSA A EQADLYKN SQVNGQRG Yes H D E D D A S S Yes E

A/common gull/Saratov/1676/2018 N del SSGVSA A EQADLYKN SQVNGQRG Yes H D E D D A S S Yes E

A/chicken/Guangdong/G1012/2018 N del SSGVSA A EQADLYKN SQVNGQRG N.A§ N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A

A/duck/Guangdong/G1378/2018 N del SSGVSA A EQTDLYKN SQVNGQRG N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A

A/goose/Guangdong/QY01/2016 N del SSGVSA A EQTNLYKN SQVNGQRG Yes H D E D D A S S Yes E

A/chicken/Ganzhou/GZ21/2015 N del SSGVSA A EQTNLYKN SQVNGQRG Yes H D E D D A S S No D

A/Pavo cristatus/Jiangxi/JA1/2016 N del SSGVSA A EQTNLYKN SQVNGQRG Yes H D E D D A S S Yes E

A/Chicken/Huizhou/16274/2016 N E SSGVSA A EQTNLYKN SQVNGQRG Yes H D E D D A S S Yes E

Human A/Guangxi/32797/2018 N del SSGVSA A EQADLYKN SQVNGQHG Yes H D K D D A S S Yes E

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 A/Guangxi/31906/2018 N del SSGVSA A EQADLYKN SQVNGQRG Yes H D K D D A S S Yes E

A/Guangdong/18SF020/2018 N del SSGVSA A EQTDLYKN SQVNGQRG Yes H D X D D A S S Yes E

A/Guangxi/13486/2017 N del SSGVSA A EQTNLYKN SQVNGQRG Yes H D K D D A S S Yes E

†
H3 numbering was used.

‡
Deletion at this position creates a putative N-glycosylation site at N129

§
Sequences were not available.

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Figure legends

Figure 1 Phylogenetic trees of hemagglutinin (HA) genes of clade 2.3.4.4 highly

pathogenic avian influenza viruses (HPAIVs). The trees were constructed with

MEGA7.0.26 software using the maximum likelihood (ML) method. Bootstrap

analysis was performed with 200 replicates. The yellow square of the left tree is

enlarged in the right tree. AI470 is indicated in red, human isolates are indicated in

blue, and human isolates of the G1.1 genotype are indicated by the blue circles.

Figure 2 Tanglegrams based on the eight genomic segments of highly pathogenic

avian influenza viruses (HPAIVs). Phylogenetic trees were constructed using the

maximum likelihood (ML) method and then used to generate the tanglegrams using

the Dendroscope 3 program. The groups of H5N6 isolates in this study were

classified according to the tanglegrams. AI470, human H5N6 isolates, and HPAIVs

isolated in Vietnam that are adjacent to each other in the trees are connected by a

line. Colors for the different taxa and lines are as follows: AI470 and its related

H5N6 human isolates, red; H5N6 HPAIVs isolated in Vietnam, green; G1 genotype,

light purple; G1.1 genotype, orange; G1.1b genotype, yellow-green; G1.2 genotype,

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dark purple; G2 genotype, brown. The colors of the different taxa in the trees are as

follows: H5 HPAIVs, black; H6N6 avian influenza viruses (AIVs), blue; H9N2 or

H7N9 AIVs, yellow-green; Other AIVs, blue-green.

Figure 3 Visualization of putative influenza virus transmission to Southern

Vietnam. The connection of areas was based on the Bayesian maximum clade

credibility (MCC) tree of the hemagglutinin (HA) gene constructed using BEAST

1.8.4 software. The world map was generated using SPREAD software to perform

discrete phylogeographical analyses, and the results were visualized using Google Earth

(https://www.google.co.jp/intl/ja/earth/).

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This article is protected by copyright. All rights reserved.
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