The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses

Accepted Manuscript Title: The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses Author: Tinh Huu Nguyen Van Thai Than Hien Thanh Dang Van Quang Nguyen Kim Hue Nguyen Duc Tan Nguyen Jong-Hwa Park In Sik Chung Dae Gwin Jeong Kyu-Tae Chang Tae-kwang Oh Wonyong Kim PII: DOI: Reference:

S0147-9571(15)00058-2 http://dx.doi.org/doi:10.1016/j.cimid.2015.08.001 CIMID 1020

To appear in: Received date: Revised date: Accepted date:

9-12-2014 9-8-2015 18-8-2015

Please cite this article as: Nguyen TH, Than VT, Dang HT, Nguyen VQ, Nguyen KH, Nguyen DT, Park J-H, Chung IS, Jeong DG, Chang K-T, Oh T-k, Kim W, The evolutionary dynamics of highly pathogenic avian influenza H5N1 in southcentral Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses, Comparative Immunology, Microbiology and Infectious Diseases (2015), http://dx.doi.org/10.1016/j.cimid.2015.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights (for review)

Highlights

• First report of HPAI H5N1 genetic diversity in south-central Vietnam. • We isolated 47 H5N1s from both vaccinated and unvaccinated poultry in 2013–2014.

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• All these viruses were sequenced and we performed phylogenetic analysis

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• Three clades of HPAI H5N1: 1.1.2, 2.3.2.1a, and 2.3.2.1c were identified.

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• These clades are closely related to Chinese and Cambodian clade.

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*Manuscript Click here to view linked References

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The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central

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Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses

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Tinh Huu Nguyena,b, Van Thai Thana, Hien Thanh Danga,b, Van Quang Nguyenb, Kim Hue

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Nguyenb, Duc Tan Nguyenb, Jong-Hwa Parkc, In Sik Chungc, Dae Gwin Jeongd, Kyu-Tae

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Changd,e, Tae-kwang Ohd, and Wonyong Kima*

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Department of Microbiology, Chung-Ang University College of Medicine, Seoul, South Korea

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b

Central Vietnam Veterinary Institute, Nha Trang, Vietnam

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c

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University, Yongin 446-701, South Korea

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d

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Biotechnology, Daejeon, South Korea

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e

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Ochang, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea

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Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee

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Viral Infectious Disease Research Center, Korea Research Institute of Bioscience and

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National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology,

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*Corresponding author

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Wonyong Kim, DVM, Ph.D.

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Professor

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Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, South

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Korea.

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Phone: +82 2 820 5685; Fax: +82 2 822 5685; E-mail: [email protected]

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ABSTRACT

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In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is

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the most highly contagious infectious disease that has been affecting domestic poultry in recent

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years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic

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strains. However, few studies have reported viruses circulating in the south-central region of

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Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated

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and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013–2014,

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and their genetic diversity was analyzed. A common sequence motif for HPAI virus was

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identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a)

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or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of

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HPAI H5N1: 1.1.2 (n = 1), 2.3.2.1a (n = 1), and 2.3.2.1c (n = 45). The phylogenetic analysis

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indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus

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clades isolated in 2012–2013 but are less closely related to the clades detected from the Tyva

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Republic, Bulgaria, Mongolia, Japan, and Korea in 2009–2011. Detection of the coexistence of

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virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local

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importance and highlights concerns regarding their spread, both northwards and southwards, as

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well as the potential for reassortment. The obtained data highlight the importance of regular

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identification of viral evolution and the development and use of region-specific vaccines.

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Keywords: Highly pathogenic avian influenza H5N1, Poultry, South-central Vietnam

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1. Introduction

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Influenza A virus belongs to the Orthomyxoviridae family. The viral genome consists of eight

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segments of a single-stranded negative RNA, encoding at least 10 known functional proteins

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(PB1, PB2, PA, HA, NP, NA, M1, M2, NS1, and NS2), and more recently, five little-known

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functional proteins (PB1-F2, PB1-N40, PA-X, PA-N155, and PA-N182) [1-3]. Hemagglutinin

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(HA) and neuraminidase (NA) are surface antigen proteins that play a major role in the host

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humoral immune response against viruses. Based on the presence of HA and NA antigens,

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influenza A viruses are divided into Hx and Ny subtypes, respectively. To date, 18 HA (H1–

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H18) and 11 NA (N1–N11) subtypes have been identified in aquatic fowls and bats. This

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suggests the possibility of genetic assortment between these genes, thereby generating new

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HxNy subtypes [3, 4].

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The highly pathogenic avian influenza (HPAI) virus H5N1 was first identified in 1996 in a

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domestic goose in the Guangdong province of China [5]. Since then, the virus has spread rapidly

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among birds in more than 60 Eurasian and African countries, threatening to spread further into

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the American and Australian continents [6]. The HPAI H5N1 virus has caused severe economic

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repercussions due to millions of poultry deaths and the resultant culling policy. Although the

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evident risk of transmission of these viruses to humans remains low, its high mortality rate in

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humans (causing fatalities in approximately 59% of the HPAI H5N1-infected patients from 15

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countries since 2003) [7] has brought it under the scanner of both animal and public health

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authorities worldwide.

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The rapid changes in HPAI H5N1 virus have generated numerous distinct clades (clades 0–9)

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and subclades. In 2008, at one point, clades 0, 3, 4, 5, 6, 8, and 9 and several subclades from

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clade 2 stopped circulating. By 2014, clades 1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4, and 7 continued to

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evolve and expand worldwide despite control efforts. Viruses from clade 2.3.2 have emerged in

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many Asian countries, including China, Vietnam, Hong Kong, Japan, Korea, Laos, Bangladesh,

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Nepal, Mongolia, and the Tyva Republic, as well as eastern Europe, particularly in Bulgaria and

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Romania [8]. Other circulating virus clades have persisted in specific geographical areas as

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follows: clade 1, mainly in the Mekong River Delta region in 2004 (southern Vietnam and

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Cambodia), evolving into two new clades termed 1.1.1 and 1.1.2 in 2010 [9]; clade 2.1, in

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Indonesia in 2003, evolving into a new clade 2.1.3.2a in 2010; clades 2.2 and 2.2.2, enzootic in

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India and Bangladesh in 2008; clade 2.2.1.1, in Egypt and Israel in 2007, evolving into new

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clade 2.2.1.1a in 2011; clade 2.3.4, in China, Hong Kong, Vietnam, Thailand, and Laos in 2007,

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evolving to six subclades 2.3.4.1 to 2.3.4.6 in 2011; and clade 7 viruses, in China and northern

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Vietnam in 2009 [10].

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In Vietnam, the HPAI H5N1 virus was first detected in 2001, and H5N1 outbreaks in poultry

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have been reported frequently since early 2004 [11, 12]. Most outbreaks in Vietnam were

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monitored by a passive surveillance system, where the nature of poultry farming practices (e.g.,

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backyard or grazing frame methods) were considered to play an important role in the

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maintenance and spread of viruses throughout the country [13]. To control the outbreak of HPAI

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H5N1, the Vietnamese government implemented a mass vaccination program for domestic

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poultry in October 2005. Although no H5N1 outbreaks occurred until October 2006, outbreaks

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of a new subtype of H5N1 have been reported after November 2006 [14]. In addition, HPAI

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H5N1 infections in humans, having a high mortality rate, have been sporadically reported in the

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vicinity of severe poultry outbreaks [15]. Close contact with infected birds has been suggested as

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a potential source of HPAI H5N1 infection in humans, raising the possibility of poultry-to-

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human viral transmission [16].

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HPAI H5N1 viruses have been classified into different genetic groups in Vietnam. Although

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some H5N1 clades disappeared a short time after their emergence, two major clades, clade 1 and

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clade 2, are still in circulation. Many of their subclades have also been subsequently identified in

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poultry [14, 17-19]. Studies have shown that by 2012, clade 1.1 viruses were mostly circulating

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in southern Vietnam, while viruses isolated from influenza cases in the northern and other

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regions belonged to clade 2.3.2.1 [20, 21]. H5N1 outbreaks have been observed to typically

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originate from and be predominant in the northern regions, rapidly spreading to the southern

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regions of Vietnam [17]. In addition, the predominance and spread of each genotype depends on

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many ecological and socioeconomic factors, e.g., seasons, the migration and movement of wild

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birds and poultry, national poultry import controls, national vaccination strategies, and efforts of

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virological surveillance. [22]. However, the lack of epidemiology and vaccination records in

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many previous H5N1 studies in Vietnam may obscure the true reasons underlying the occurrence

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of H5N1 outbreaks [11, 14, 17, 18, 20, 23].

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Information on H5N1 outbreaks in the central regions of Vietnam is currently limited [17, 21,

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23, 24]. In this study, we have reported on the phylogenetic and molecular analyses of 47 H5N1-

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positive samples collected from outbreaks in both vaccinated and unvaccinated poultry in the

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Khanh Hoa and Phu Yen provinces between January 2013 and February 2014 are reported.

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The present study is the first from this intersecting area between the North, with its widely

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circulating clade 2.3.2.1c viruses, which have supplanted the clade 1.1 viruses, and the South,

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with clade 1.1.1/1.1.2 viruses derived from the highly virulent Cambodian clade 1.1 viruses. The

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potential for the combination of genetic drift and genetic reassortment of HA and NA genes to

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generate more virulent and transmissible pandemic strains has been frequently demonstrated [3].

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These data will provide comprehensive and updated information on the genetic diversity of

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HPAI H5N1 viruses circulating in the central regions of Vietnam.

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2. Materials and methods

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2.1. Sample collection

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The Central Vietnam Veterinary Institute (CVVI), in collaboration with the Provincial Animal

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Health Department laboratory, collected all samples in a passive surveillance program. Here,

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H5N1 virus detection relied on suspected cases from poultry farms being reported to the

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authorities. In general, two sick or dead birds from each farm were collected and transported to

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the CVVI laboratory, where tissue samples from the brain, lung, spleen, bronchus, and intestine

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were collected and stored at −80°C for further examination. Accurate poultry farm information

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was supplied by the owners.

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2.2. RNA extraction and real-time RT-PCR

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The pooled-tissue samples of birds from each farm were homogenized in phosphate-buffered

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saline (PBS; pH 7.4) and clarified by centrifugation at 400 × g for 10 min. The viral RNA was

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extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA), according to the

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manufacturer’s instructions. Extracted RNA was resuspended in RNase-free water and stored at

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−80°C until RT-PCR analysis was performed. Real-time RT-PCR was performed per the WHO

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recommendation [25]. Briefly, the QuantiTect Probe RT-PCR Kit (Qiagen, Valencia, CA, USA)

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was used with 12.5 µl of master mix, 1.5 µl each of forward and reverse primers (10 µM), 0.5 µl

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of probe (5 µM), 0.25 µl of QuantiTect RT Mix, 3.75 µl of RNase-free water, and 5.0 µl RNA

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template in a 25-µl total volume. Using the ABI 7500 (Life Technologies, Grand Island, NY,

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USA) real-time thermocycler, reverse transcription was carried out for 30 min at 50°C followed

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by polymerase activation for 15 min at 95°C. Denaturation for 15 s at 94°C and annealing-

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extension for 1 min at 56°C were performed for 45 cycles to evaluate cycle threshold (Ct) values.

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2.3. Reverse transcription-polymerase chain reaction

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The SuperScript III First-Strand Synthesis SuperMix kit (Invitrogen, Carlsbad, CA, USA) was

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used to prepare cDNA from the extracted RNA with Uni12 primer (5′-AGCRAAAGCAGG-3′).

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The reaction was carried out at 42°C for 60 min, followed by 72°C for 10 min. Full-length HA,

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NA, and M genes were amplified as described previously [26-28]. Each PCR product was

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separated on a 1.2% SeaKem LE agarose gel (FMC Bioproducts, Rockland, ME, USA) and

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stained with ethidium bromide. The gels were viewed on a Gel Doc XR image-analysis system

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(Bio-Rad, Hercules, CA, USA).

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2.4. Nucleotide sequencing and sequence analysis

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The amplified PCR products were purified using the QIAquick Gel Extraction kit (Qiagen). RT-

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PCR primers were used for the direct sequencing of the HA, NA, and M genes by using a

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BigDye Terminator Cycle Sequencing Kit and an automatic DNA sequencer (Model 3730;

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Applied Biosystems, Foster City, CA, USA). Walking primers were designed to obtain the

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sequences of the 5′- and 3′-ends of the genes (Table 1). The resultant nucleotide and deduced

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amino acid sequences were aligned using the ClustalX 2.1 program [29] and Lasergene software

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(DNASTAR; Madison, WI, USA) by using the parameters set against the corresponding H5N1

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viral sequences from the NCBI GenBank. The nucleotide sequences obtained in this study were

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deposited in NCBI GenBank under the accession numbers KM821606–KM821746.

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2.5. Phylogenetic analysis

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The nucleotide sequences of the HA, NA, and M genes from the 47 HPAI H5N1 samples

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examined in this study were compared against representative HA, NA, and M gene sequences

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from the available HPAI H5N1 sequences in the GenBank database. Phylogenetic trees were

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constructed using the neighbor-joining algorithms in the PHYLIP suite and Kimura two-

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parameter model using MEGA 6.06 software [30-32]. Evolutionary distances for the neighbor-

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joining analyses were based on the model described by Jukes and Cantor [33]. Tree topology was

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evaluated using the bootstrap resampling method, using 1000 replicates of the neighbor-joining

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dataset, with the SEQBOOT and CONSENSE programs from the PHYLIP suite [30].

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3. Results

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3.1. H5N1 outbreak determination

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Between January 2013 and February 2014, 47 H5N1 outbreaks were reported from chicken and

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duck farms located in the Khanh Hoa and Phu Yen provinces of south-central Vietnam. In 15

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chicken farms, poultry was raised in the backyard or open system, with netting to prevent the

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entrance of passerine birds. In 32 duck farms, ducks were bred in the grazing duck raising system,

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from where they moved outdoors to rice fields without bio-security measures. The 47 H5N1

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virus strains were rapidly identified by real-time RT-PCR using specific primer sets to detect H5

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and N1 genes [25]. The real time RT-PCR cycle threshold values varied, ranging from 9.03 to

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21.61 and from 10.69 to 22.07 for H5 and N1 genes, respectively (Table 2).

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3.2. Phylogenetic analysis

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Phylogenetic trees of the HA, NA, and M genes were constructed to examine the H5N1 genetic

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groups using the sequences obtained in this study and the available sequences in GenBank.

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Phylogenetic analysis indicated that the HA genes from the 47 H5N1 strains were clustered into

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clade 1.1.2 (n = 1) and clade 2.3.2.1 (n = 46) (Fig. 1). The majority of the strains were clustered

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into clade 2.3.2.1c (n = 45); only one strain fell into clade 2.3.2.1a, together with the strains

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isolated from China and Vietnam in 2011–2012. The strains in clade 2.3.2.1c obtained in this

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study were closely related to the strains from China, Taiwan, and Indonesia isolated in 2012–

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2013 but more distant from strains previously isolated in 2009–2011 in China, Mongolia, the

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Tyva Republic, Bulgaria, Japan, and Korea. The nucleotide sequence identity among the HA

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genes (clade 2.3.2.1c) in this study was 97.8–100%. These strains shared nucleotide sequence

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identity with the A/Hong Kong/6841/2010 prototype strain at 96.9–97.9%. The CVVI-18/2013

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strain (clade 2.3.2.1a) shared the highest nucleotide identity (98.1–98.9%) with strains isolated in

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China (Hubei/1/2010) and Vietnam (OIE-1287/2012). The CVVI-24/2013 strain (clade 1.1.2)

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shared 99.1–99.4% nucleotide sequence identity with strains isolated in Vietnam (OIE-

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0043/2012) and Cambodia (X0815301/2013). The phylogenic tree of the NA genes isolated in

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this study displayed three distinct groups: Hong Kong/1161-like, Hubei-like, and HK/821-like

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(Fig. 2). Most of the NA genes belonged to the Hong Kong/1161-like group, together with

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strains isolated from Hong Kong in 2010 (Hong Kong/1161), from China in 2011–2013

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(Zhejiang/224, Jiangsu/01), and from Vietnam in 2012 (QB1207). One strain belonged to the

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Hubei-like group (CVVI-18), and one strain belonged to the HK/821-like group (CVVI-24),

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along with strains isolated in Hong Kong, Vietnam, Thailand, China, and Cambodia. The

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nucleotide sequence identity of the NA strains obtained in this study was 97.9–100% with the

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strains isolated in China (Hubei/1/2010), Hong Kong (Hong Kong/1161/2010), Vietnam

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(QB1207/2012, OIE-1287/2012), and Cambodia (X0815301/2013). On the basis of the M gene

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sequence identity, the Vietnamese strains obtained in this study were separated into two groups,

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Zhejiang/224-like and Hong Kong/1161-like, together with Hong Kong/1161, Hubei/1,

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Zhejiang/224, Jiangsu/01, QB1207, OIE-2211, OIE-2533, OIE-1287, and X0815301 (Fig. 3).

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3.3. Genetic analysis

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The amino acid sequences of the HA, NA, and M genes of the H5N1 study strains were deduced

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by comparison with the H5N1 ancestor strain A/Goose/Guangdong/1/1996 (clade 0) and the

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H5N1 strains recently isolated in Vietnam: A/Muscovy duck/Vietnam/OIE-0043/2012 (clade

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1.1.2), A/duck/Vietnam/QB1207/2012 (clade 2.3.2.1c), and A/duck/Vietnam/LBM140/2012

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(clade 2.3.2.1a). In the study strains, the cleavage site of the HA sequence at position 323–332

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contains multiple basic amino acids of the HPAI H5N1 viruses. This site possesses the

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RERRRKR/G sequences from clade 2.3.2.1c and 2.3.2.1a, and the REGRRKKR/G sequence

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from clade 1.1.2 (Table 3). Amino acid residues at the receptor-binding, antigenic, and

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glycosylation sites that have been reported to play an important role in neutralization and

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antibody

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A/Goose/Guangdong/1/1996, the present study sequences showed nine accumulated amino acid

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changes at positions 86A-V, 140R-N,Q, 156A-T, 189K-R, and 277K-R (antigenic site), and 123S-P, 129S-

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positions 141 and 263 (antigenic site) and 222 and 224 (receptor-binding site). The N-linked

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glycosylation site at position

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2.3.2.1a and 2.3.2.1c. However, this position was lost in the CVVI-24 strain belonging to the HA

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clade 1.1.2, as well as the A/Goose/Guangdong/1/1996 prototype strain. On the other hand, the

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binding

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, 175L-M, and 223S-R (receptor-binding site). Four amino acid substitutions were detected at

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NSS142 was found in all strains belonging to the HA subclades

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NNT156 was found in the CVVI-24 strain but lost in the other

glycosylation site at position

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strains belonging to clade 0 and subclades 2.3.2.1a and 2.3.2.1c. Genetic analysis of the NA

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genes identified in previous studies indicated an approximately 20-amino acid deletion in the NA

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stalk, a sequence known to be related to the increase in viral pathogenicity [34-36]. All the 47

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study strains showed a deletion of these 20 amino acids at the NA stalk region, when compared

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to the OIE0043, LBM140, and QB1207 reference strains (Table 4). In addition, the NA region of

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the CVVI-24 strain contains an amino acid substitution at position 253Y- might be involved in

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the increase in binding affinity of the virus towards oseltamivir [37] (Table 4). In the M2 protein,

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five amino acids involved in amantadine resistance, at positions 26, 27, 30, 31, and 34, were

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analyzed. When compared to the A/Goose/Guangdong/1/1996 prototype strain, 25 strains

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displayed an amino acid substitution at position 27V-I, together with the QB1207 strain isolated

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from a domestic duck in the Quang Binh province in 2012.

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4. Discussion

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There is an increasing emphasis on the importance of H5N1 diagnosis in poultry because of the

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dramatic economic losses suffered by the national poultry industry from the deaths or culling of

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several hundred million birds [6]. The viruses also pose a potentially great public health risk, as

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indicated by the several hundred cases of human infections, suggesting the possibility of

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transmission to the human population and its pandemic potential [15]. The present study reports

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the detection and genetic characterization of HPAI H5N1 viruses in the Khanh Hoa and Phu Yen

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provinces located in south-central Vietnam. In total, 47 HPAI H5N1 outbreaks were detected in

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32 domestic duck farms and 15 chicken farms in these two provinces between January 2013 and

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February 2014.

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Surveillance studies indicate that ducks and other wild waterfowl play an important role in

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the spread and maintenance of HPAI H5N1 viruses in the environment [38]. In Vietnam,

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according to traditional village livestock rearing practices, free-range ducks are kept in breeding

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houses during their first month of life, subsequently being released into paddy fields. In these

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open systems, healthy ducks may be exposed to the HPAI H5N1 virus, especially in the shared

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scavenging area occupied by other duck farms. In this study, the HPAI H5N1 outbreaks mostly

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occurred in unvaccinated duck and chicken farms that were raised in the grazing and backyard

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systems, respectively. These results suggest that ducks and chickens should be vaccinated and

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protected by biosecurity measures before they are moved to possible high-risk areas of HPAI

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H5N1 infections.

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Phylogenetic analysis of the HA genes indicated six broad types of HA clades circulating in

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Vietnam since 2007: 1.1, 2.3.2.1, 2.3.4.1, 2.3.4.2, 2.3.4.3, and 7.1 [18]. Clade 1 viruses have

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been detected in the northern and central regions of Vietnam between 2003 and 2007, being

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subsequently replaced by clade 2.3.4 viruses after 2005 [17]. Clades 1.1.1 and 1.1.2 have

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emerged as a result of evolution in clade 1, and clade 1.1.2 viruses have been persistently

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circulating in the south of Vietnam [23, 39]. Viruses from clade 2.3.2.1, containing three large

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subclades, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c, have been detected in many countries, including

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China, Southeast Asia, Japan, Korea, Mongolia, Russia, and Europe. In Vietnam, viruses from

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clade 2.3.2.1 were introduced in 2009 and have since rapidly spread out to the northern and

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central parts of the country [21]. In this study, 46 clade 2.3.2.1 viruses and one clade 1 virus

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were identified. The majority of the strains belonged to subclade 2.3.2.1(a,c) and may have

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evolved from the Chinese viruses isolated in 2012–2013. At the phylogenetic level, they are

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distinct from viruses in subclades isolated in 2009–2011 from China, Lao, Mongolia, the Tyva

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Republic, Bulgaria, Japan, and Korea. The remaining strain belonging to clade 1.1.2 may have

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evolved from the Cambodia virus clade. HPAI H5N1 epi-zones in Asia have been delineated by

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determining regions that share closely related viruses and common epidemiological features and

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risk factors, e.g., virus characterization, geographical location, and migrant birds etc. [40]. HPAI

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H5N1 viruses in Vietnam belong to epi-zone 3 (south China, Hong Kong, north Vietnam, and

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Laos) and epi-zone 4 (Cambodia and south Vietnam), with the most recent predominant Chinese

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and Cambodian viruses of clades 2.3.2.1 and 1.1.2 in the north and south of Vietnam,

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respectively [40]. The repeated detection and coexistence of these two predominant virus clades

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in the central regions of Vietnam suggest that these strains might be locally significant. In

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addition, the migration of these virus clades in the central regions alerts to the possible spread of

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these virus clades in both directions, northwards and southwards, via poultry trading or bird

286

migration in the near future [17, 24, 41].

d

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275

Two commercial poultry influenza vaccines, Re-1 and Re-5 reassortant vaccines (Harbin

288

Veterinary Research Institute, China), have been used since 2005 in the nationwide

289

immunization program for poultry for the prevention and control of HPAI H5N1 virus outbreaks

290

in Vietnam. Re-1 and Re-5 vaccines contain reassortant viruses bearing the HA and NA genes of

291

the A/goose/Guangdong/1/1996 strain (clade 0) and A/duck/Anhui/1/2006 strain (clade 2.3.4),

292

respectively [42]. Re-1 was used widely between 2006 and 2010, while Re-5 was mainly used in

293

the southern regions between 2011 and 2012. The H5N1 viruses have been mutating rapidly,

294

possibly because of an antigenic drift and/or positive selection under immunological pressure

295

[43]. The mutations in the HA gene relate to positions at antigenic sites that are involved in

296

antibody binding and neutralizing epitopes. These mutations have resulted in the modification of

297

N-linked glycosylation and escape of viruses from antibody binding. In this study, the recent

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13

Page 14 of 31

emergence of new HPAI H5N1 virus clades 1.1.2, 2.3.2.1a, and 2.3.2.1c occurred in both

299

vaccinated and unvaccinated poultry farms, and the emergence of the 2.3.2.1c virus clade in the

300

northern regions suggests that these vaccines may not have sufficient efficacy against these new

301

HPAI H5N1 virus clades [44]. These data highlight the importance of regular identification of

302

virus evolution and updating of vaccines accordingly, as well as the need for the use of region-

303

specific vaccines.

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Infection of humans by HPAI H5N1 viruses poses a great public health concern. Between

305

2003 and June 2014, approximately 667 laboratory-confirmed human cases of H5N1 virus

306

infection were reported in 15 countries; of these cases, 393 were fatal [7]. Vietnam is among the

307

countries with a high rate of human H5N1 infections, with a reported 127 laboratory-confirmed

308

human cases, including 62 confirmed deaths [45]. In addition, Vietnam and Canada have

309

reported human cases infected with the new H5N1 viruses, belonging to the 1.1.2 and 2.3.2.1c

310

clades [39, 46].

te

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Drug resistance and monitoring of the emergence of drug resistance play an important role in

312

choosing the appropriate treatment method for H5N1 patients. In this study, no predicted amino

313

acid sequence analysis was performed to determine amino acid substitutions known to be related

314

to oseltamivir resistance. However, the M2 genes of the 25 strains in this study displayed an

315

amino acid substitution at position 27V-I, which may cause amantadine resistance [47].

Ac ce p

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316

In conclusion, the present findings provide important information on the genetic

317

characterization of the HPAI H5N1 viruses in the Khanh Hoa and Phu Yen provinces in the

318

south-central region of Vietnam. The results highlight the genetic variation between the multiple

319

HPAI H5N1 viral clades identified, underscoring the importance of regular identification of viral

320

evolution and the development and use of region-specific vaccines. In addition, the introduction

14

Page 15 of 31

321

of new HPAI H5N1 virus clades in these regions suggests that stringent disease prevention and

322

control methods must be applied in order to prevent the threat to poultry and wild birds, as well

323

as humans and animals.

ip t

324

Acknowledgments:

326

This research was supported by a grant from KRIBB Research Initiative Program.

cr

325

Conflict of interest

329

No competing interests exist.

an

328

us

327

330

References

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[1] Shaw M, Palese P. Orthomyxoviridae. In: Knipe DM, Howley P, editors. Field virology. 6th

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ed. Vol. 1. Philadelphia: Lippincott Williams & Wilkins. 2013:1151-85.

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[2] Webster RG, Bean WJ, Gorman OT et al. Evolution and ecology of influenza A viruses.

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Microbiol Rev 1992;56(1):152-79.

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[3] Sonnberg S, Webby RJ, Webster RG. Natural history of highly pathogenic avian influenza

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[4] Tong S, Zhu X, Li Y et al. New world bats harbor diverse influenza A viruses. PLoS Pathog

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all influenza A viruses. Arch Virol 2001;146:2275-89.

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[27] Lee MS, Chang PC, Shien JH et al. Identification and subtyping of avian influenza viruses

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by reverse transcription-PCR. J Virol Methods 2001;97:13-22.

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transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of

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avian influenza viruses and comparison to standard serological subtyping tests. J Clin Microbiol

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[32] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing

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Protein Metabolism, pp. 21-132, Academic Press, New York. 1969.

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[34] Colman PM, Hoyne PA, Lawrence MC. Sequence and structure alignment of

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paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase. J Virol

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[35] Zhou H, Yu Z, Hu Y et al. The special neuraminidase stalk-motif responsible for increased

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virulence and pathogenesis of H5N1 influenza A virus. PLoS One 2009;4:e6277.

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[36] Li Y, Chen S, Zhang X et al. A 20-amino-acid deletion in the neuraminidase stalk and a

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five-amino-acid deletion in the NS1 protein both contribute to the pathogenicity of H5N1 avian

419

influenza viruses in mallard ducks. PLoS One 2014;9:e95539.

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[37] Collins PJ, Haire LF, Lin YP et al. Crystal structures of oseltamivir-resistant influenza virus

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neuraminidase mutants. Nature 2008;453:1258-61.

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[38] Hulse-Post DJ, Sturm-Ramirez KM, Humberd J et al. Role of domestic ducks in the

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propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia. Proc

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Natl Acad Sci U S A 2005;102:10682-7.

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[39] WHO. Candidate vaccine viruses and potency testing reagents for influenza A (H5N1).

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http://www.who.int/influenza/vaccines/virus/201402_h5h7h9h10_vaccinevirusupdate.pdf?ua=1.

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Accessed 12 July 2014.

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[40] Inui K. Epi-zone approach for HPAI H5N1 surveillance and control [Internet]. Available

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from:

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/2-1_Dr_Inui.pdf. 2014.

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[41] Kilpatrick AM, Chmura AA, Gibbons DW et al. Predicting the global spread of H5N1 avian

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influenza. Proc Natl Acad Sci U S A 2006;103:19368-73.

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[42] Chen H. Avian influenza vaccination: the experience in China. Rev Sci Tech 2009;28:267-

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74.

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[43] Bui VN, Ogawa H, Trinh DQ et al. Genetic characterization of an H5N1 avian influenza

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virus from a vaccinated duck flock in Vietnam. Virus Genes 2014;49:278-85.

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[44] Gu M, Zhao G, Zhao K et al. Novel variants of clade 2.3.4 highly pathogenic avian

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influenza A (H5N1) viruses, China. Emerg Infect Dis 2013;19:2021-4.

440

[45] WHO. Cumulative number of confirmed human cases for avian influenza A (H5N1)

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reported

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http://www.who.int/influenza/human_animal_interface/EN_GIP_20140124CumulativeNumberH

443

5N1cases.pdf. Assessed 24 January 2014.

444

[46] Pabbaraju K, Tellier R, Wong S et al. Full-genome analysis of avian influenza A(H5N1)

445

virus from a human, North America, 2013. Emerg Infect Dis 2014;20:887-91.

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[47] Cheung CL, Rayner JM, Smith GJ et al. Distribution of amantadine-resistant H5N1 avian

447

influenza variants in Asia. J Infect Dis 2006;193:1626-9.

M

an

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ip t

430

WHO,

2003-2014.

Ac ce p

te

d

to

20

Page 21 of 31

Table 1. Primers for amplifying and sequencing the HA, NA, and M genes used in this study

ip t

CCTTCTCCACTATGTANGACCATTC

[25]

FAM-AGCCAYCCAGCTACRCTACA-MGB

FAM-AGCCATCCCGCAACACTACA-MGB

N1-For-474-502-v2

TAYAACTCAAGGTTTGAGTCTGTYGCTTG

N1-Rev-603-631-v2

ATGTTRTTCCTCCAACTCTTGATRGTGTC

N1-Probe-501-525-v3

FAM-TCAGCRAGTGCYTGCCATGATGGCA-MGB

Bm-HA-1

TATTCGTCTCAGGGAGCAAAAGCAGGGG

Bm-NS-890R

ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT

H5-699R

CTYTGRTTYAGTGTTGATGT

[27]

H5-317F

CCAATCCAGCCAATGACCTC

This study

H5-918F

CCARTRGGKGCKATAAAYTC

H5-1178R

GTCTGCAGC RTAYCCACTYC

Bm-NA-1

TATTGGTCTCAGGGAGCAAAAGCAGGAGT

Bm-NA-1413R

ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTT

N1-660F

GACACTATCAAGAGTTGGAGG

N1-961R

an

[25]

[26]

[28]

[26]

This study

GAGCCATGCCAATTATCCCTG TATTCGTCTCAGGGAGCAAAAGCAGGTAG

Ac ce p

Bm-M-1

cr

H5-Probe-239-RVb

NA

M

CGATCTAGAYGGGGTGAARCCTC

M

HA

H5HA-205-227v2For H5HA-326-302v2Rev H5-Probe-239-RVa

References

d

N1

Primer sequences (5ʹ-3ʹ)

te

H5

Primers/Probes

us

Gene segment

[26]

Bm-M-1027R

ATATCGTCTCGTATTAGTAGAAACAAGGTAGTTTTT

M-370F

CGCACTCAGTTACTCAACCG

M-771R

TGCATCTGCACTCCCATTCG

This study

21

Page 22 of 31

Table 2. Outbreak information and diagnostic data in the central region of Vietnam between January 2013 and February 2014

Kind of bird

Total number

Number of deaths

Age (months)

Real-time Ct value H5

Real-time Ct value N1

Vaccinated status

Sample ID

Unvaccinated

CVVI-01

16.47

Unvaccinated

CVVI-02

19.48

Unvaccinated

CVVI-03

duck

6000

220

2

12.63

January 2, 2013

chicken

1200

300

3

12.8

January 5, 2013

duck

2200

110

2

17.03

January 5, 2013

duck

1500

100

2,5

12.13

15.38

Unvaccinated

CVVI-04

January 7, 2013

duck

2000

300

1,5

12.86

16.6

Unvaccinated

CVVI-05

January 10, 2013

duck

1200

190

2

12.24

15.6

Unvaccinated

CVVI-06

January 14, 2013

duck

3000

200

2

13.39

16.74

Unvaccinated

CVVI-07

January 15, 2013

duck

3700

150

1,5

12.72

16.82

Unvaccinated

CVVI-08

January 15, 2013

chicken

1000

200

4

18.35

20.93

Unvaccinated

CVVI-09

January 18, 2013

duck

1500

200

2

15.5

19.45

Unvaccinated

CVVI-10

February 5, 2013

chicken

150

90

3

16.28

19

Unvaccinated

CVVI-11

February 18, 2013

chicken

500

95

2

13.43

17.14

Unvaccinated

CVVI-12

February 19, 2013

chicken

300

74

2

15.84

19.1

Unvaccinated

CVVI-13

February 20, 2013

duck

1300

375

1

15.66

13.8

Unvaccinated

CVVI-14

March 1, 2013

duck

1000

50

1

12.36

16.35

Unvaccinated

CVVI-15

March 4, 2013

duck

4500

100

1,5

17.26

16.6

Unvaccinated

CVVI-16

March 4, 2013

duck

2000

300

1,5

14.18

17.09

Unvaccinated

CVVI-17

March 6, 2013

chicken

200

15

1

17.16

22.07

Unvaccinated

CVVI-18

March 7, 2013

duck

1600

90

2

12.43

15.35

Unvaccinated

CVVI-19

March 14, 2013

duck

2000

200

2

13.31

14.08

Unvaccinated

CVVI-20

March 20, 2013

duck

1660

400

2

15.82

16.07

Unvaccinated

CVVI-21

March 22, 2013

duck

1200

15

1,5

12.36

12.36

Unvaccinated

CVVI-22

March 30, 2013

duck

630

150

1,5

12.11

12.16

Unvaccinated

CVVI-23

April 1, 2013

duck

800

200

1,5

14.33

16.05

Unvaccinated

CVVI-24

February 3, 2014

duck

1000

80

8

12.97

16.63

Vaccinated

CVVI-25

February 3, 2014

duck

2500

320

8

14.38

15.72

Vaccinated

CVVI-26

February 4, 2014

chicken

400

50

3

16.54

20.12

Unvaccinated

CVVI-27

February 5, 2014

chicken

1350

550

2

14.62

21.07

Unvaccinated

CVVI-28

us

an

M

d

Ac ce p

15.28

cr

January 2, 2013

te

Khanh Hoa province

ip t

Date received

22

Page 23 of 31

duck

2200

300

1

12.22

15.23

Unvaccinated

CVVI-29

February 6, 2014

duck

700

500

2

12.15

13.38

Unvaccinated

CVVI-30

February 7, 2014

chicken

350

220

2

12.11

15.88

Unvaccinated

CVVI-31

February 8, 2014

duck

1500

100

2

9.08

12.23

Unvaccinated

CVVI-32

February 10, 2014

duck

500

20

1,5

12.03

14.3

Unvaccinated

CVVI-33

February 10, 2014

duck

2000

600

2

14.52

16.7

February 11, 2014

duck

2000

200

1.5

12.16

13.83

February 14, 2014

duck

1500

200

1,5

9.09

12.19

February 16, 2014

duck

4000

200

1

9.06

February 16, 2014

duck

350

60

1

9.03

February 16, 2014

chicken

500

100

10

February 18, 2014

chicken

400

100

February 19, 2014

chicken

1060

February 21, 2014

chicken

February 21, 2014

ip t

February 5, 2014

CVVI-34

Unvaccinated

CVVI-35

Unvaccinated

CVVI-36

12.19

Unvaccinated

CVVI-37

12.11

Unvaccinated

CVVI-38

13.13

15.66

Vaccinated

CVVI-39

3

11.99

12.13

Unvaccinated

CVVI-40

200

1,5

12.69

10.69

Unvaccinated

CVVI-41

300

100

2

12.77

16.43

Unvaccinated

CVVI-42

chicken

900

100

3

13.77

18

Unvaccinated

CVVI-43

February 26, 2014

chicken

330

80

2

13.02

17.64

Unvaccinated

CVVI-44

February 26, 2014

duck

1500

300

1

10.52

15.04

Unvaccinated

CVVI-45

February 26, 2014

duck

5000

300

1,5

12.28

16.75

Unvaccinated

CVVI-46

duck

1000

2

21.61

19.89

Unvaccinated

CVVI-47

us

an

M

d 650

Ac ce p

February 13, 2014

te

Phu Yen province

cr

Unvaccinated

23

Page 24 of 31

ip t

HA clade

Cleavage site

Glycosylation site

Antigen site

us

Strain

cr

Table 3. Comparison of the deduced amino acid sequences of the HA genes of the study strains and available reference strains Receptor-biding site

323–332

10

11

23

140

154

165

286

484

543

86

140

141

156

189

263

277

123

129

175

222

223

224

+

A

R

S

A

K

A

K

S

S

L

Q

S

G

+

V

Q

S

T

K

T

K

P

L

M

Q

S

G

0

RERRRKKR/G

+

+

+

-

-

+

+

+

OIE0043

1.1.2

REERRKKR/G

+

+

+

-

+

+

+

+

LBM140

2.3.2.1a

RERRRK--R/G

+

+

+

+

-

+

+

+

+

A

N

S

A

K

T

R

S

L

L

Q

S

G

QB1207

2.3.2.1c

RERRRK--R/G

+

+

+

+

-

+

+

+

+

A

N

S

A

R

T

R

S

L

L

Q

S

G

CVVI-24 (n* = 1)

1.1.2

REGRRKKR/G

+

+

+

-

+

+

+

+

+

V

Q

S

T

K

T

K

P

L

M

Q

S

G

CVVI-18 (n* = 1)

2.3.2.1a

RERRRK--R/G

+

+

+

+

-

+

+

+

+

A

N

S

A

K

T

R

S

L

M

Q

R

G

CVVI-01 (n* = 38)

2.3.2.1c

RERRRK--R/G

+

+

CVVI-06 (n* = 2)

2.3.2.1c

RERRRK--R/G

+

+

CVVI-22 (n* = 1)

2.3.2.1c

RERRRK--R/G

+

+

CVVI-31 (n* = 2)

2.3.2.1c

RERRRK--R/G

+

+

+

CVVI-41 (n* = 1)

2.3.2.1c

RERRRK--R/G

+

+

CVVI-47 (n* = 1)

2.3.2.1c

RERRRK--R/G

+

+

ed

M an

GD/1/96

+

-

+

+

+

+

A

N

S

A

R

T

R

S

L

L

Q

S

G

+

+

-

+

+

+

+

A

N

S

A

R

T

R

P

L

L

Q

S

G

+

+

-

+

+

+

+

A

N

S

A

R

T

R

S

M

L

Q

S

G

+

-

+

+

+

+

A

N

S

A

K

T

R

S

L

L

Q

S

G

+

+

-

+

+

+

+

A

N

S

A

R

T

K

S

L

L

Q

S

G

+

+

-

+

+

+

+

A

N

S

A

R

T

R

S

L

L

Q

R

G

ce pt

+

Ac

GD/1/96: A/Goose/Guangdong/1/96, OIE0043: A/Muscovy duck/Vietnam/OIE-0043/2012, QB1207: A/duck/Vietnam/QB1207/2012, LBM140: A/duck/Vietnam/LBM140/2012, CVVI-01: A/duck/Khanhhoa/CVVI-01/2013, CVVI-06: A/duck/Khanhhoa/CVVI06/2013, CVVI-18: A/chicken/Khanhhoa/CVVI-18/2013, CVVI-22: A/duck/Khanhhoa/CVVI-22/2013, CVVI-24: A/duck/Khanhhoa/CVVI-24/2013, CVVI-31: A/duck/Khanhhoa/CVVI-31/2014, CVVI-41: A/duck/Khanhhoa/CVVI-41/2014, CVVI47: A/duck/Phuyen/CVVI-47/2014. n*: Number of samples with similar sequences.

24

Page 25 of 31

Table 4. Comparison of the antiviral resistance markers in the NA and M genes in the study strains and available reference strains. HA clade

NA amino acid position

NA stalk region

M2 amino acid position

117

119

150

223

247

253

275

295

26

27

30

31

34

L

V

A

S

G

ip t

Strain

0

No deletion

I

E

K

I

S

Y

H

N

OIE0043

1.1.2

20 aa deletion

I

E

K

I

S

H

H

N

I

V

A

N

G

LBM140

2.3.2.1a

20 aa deletion

I

E

K

I

S

Y

H

N

L

V

A

S

G

2.3.2.1c

20 aa deletion

I

E

K

I

S

Y

H

N

L

I

A

S

G

CVVI-24 (n = 1)

1.1.2

20 aa deletion

I

E

K

I

S

H

CVVI-01 (n* = 25)

2.3.2.1c

20 aa deletion

I

E

K

I

S

Y

2.3.2.1a,c

20 aa deletion

I

E

K

I

S

*

*

CVVI-06 (n = 21)

H

N

L

V

A

S

G

H

N

L

I

A

S

G

L

V

A

S

G

us

QB1207

cr

GD/1/96

Y

H

N

Ac ce p

te

d

M

an

GD/1/96: A/Goose/Guangdong/1/96, OIE0043: A/Muscovy duck/Vietnam/OIE-0043/2012, QB1207: A/duck/Vietnam/QB1207/2012, LBM140: A/duck/Vietnam/LBM140/2012, CVVI-01: A/duck/Khanhhoa/CVVI-01/2013, CVVI-06: A/duck/Khanhhoa/CVVI-06/2013, CVVI-24: A/duck/Khanhhoa/CVVI-24/2013. n*: Number of samples with similar sequences.

25

Page 26 of 31

Figure Legends

Fig. 1. Neighbor-joining phylogenetic tree analyses of the HA genes of the HPAI H5N1 study

449

strains and the available reference strains obtained from the GenBank database. Phylogenetic

450

trees were constructed using the neighbor-joining algorithms in the PHYLIP suite and Kimura

451

two-parameter model using MEGA 6.06 software. Tree topology was evaluated using the

452

bootstrap resampling method, using 1000 replicates of the neighbor-joining dataset. The HPAI

453

H5N1 virus clade nomenclatures were determined based on the criteria and the reference strains,

454

as described by the World Health Organization, World Organization for Animal Health, and

455

Food and Agricultural Organization. The Vietnamese strains obtained in the current study are

456

marked in bold.

d

M

an

us

cr

ip t

448

te

Fig. 2. Neighbor-joining phylogenetic tree analyses of the NA genes of the HPAI H5N1 study

Ac ce p

strains and the available reference strains taken from the GenBank database. Phylogenetic trees were constructed using the neighbor-joining algorithms in the PHYLIP suite and Kimura twoparameter model using MEGA 6.06 software. Tree topology was evaluated using the bootstrap resampling method, using 1000 replicates of the neighbor-joining dataset. The HPAI H5N1 virus clade nomenclatures were determined based on the criteria and the reference strains, as described by the World Health Organization, World Organization for Animal Health, and Food and Agricultural Organization. The Vietnamese strains obtained in the current study are marked in bold.

26

Page 27 of 31

Fig. 3. Neighbor-joining phylogenetic tree analyses of the M genes of the HPAI H5N1 study strains and the available reference strains taken from the GenBank database. Phylogenetic trees were constructed using the neighbor-joining algorithms in the PHYLIP suite and Kimura two-

ip t

parameter model using MEGA 6.06 software. Tree topology was evaluated using the bootstrap resampling method, using 1000 replicates of the neighbor-joining dataset. The HPAI H5N1 virus

cr

clade nomenclatures were determined based on the criteria and the reference strains, as described

us

by the World Health Organization, World Organization for Animal Health, and Food and Agricultural Organization. The Vietnamese strains obtained in the current study are marked in

Ac ce p

te

d

M

an

bold.

27

Page 28 of 31

Fig. 1.

Clade 2.3.2.1c

Ac ce p

te

d

M

an

us

cr

ip t

A/chicken/Khanhhoa/CVVI-11/2013 A/duck/Khanhhoa/CVVI-22/2013 A/chicken/Khanhhoa/CVVI-12/2013 A/duck/Khanhhoa/CVVI-09/2013 A/duck/Khanhhoa/CVVI-07/2013 A/duck/Khanhhoa/CVVI-19/2013 A/duck/Khanhhoa/CVVI-20/2013 A/chicken/Khanhhoa/CVVI-13/2013 A/duck/Khanhhoa/CVVI-08/2013 A/duck/Khanhhoa/CVVI-03/2013 A/duck/Khanhhoa/CVVI-16/2013 A/duck/Khanhhoa/CVVI-17/2013 A/duck/Khanhhoa/CVVI-10/2013 A/chicken/Khanhhoa/CVVI-02/201 A/muscovy duck/Vietnam/LBM344/2013 A/duck/Khanhhoa/CVVI-23/2013 A/duck/Khanhhoa/CVVI-33/2014 A/duck/Khanhhoa/CVVI-15/2013 A/duck/Khanhhoa/CVVI-21/2013 A/duck/Quanh Ninh/21/2013 A/duck/Vietnam/QB1207/2012 A/duck/Khanhhoa/CVVI-14/2013 A/duck/Vietnam/OIE-2211/2012 A/duck/Khanhhoa/CVVI-01/2013 A/duck/Khanhhoa/CVVI-05/2013 A/duck/Khanhhoa/CVVI-04/2013 A/chicken/Khanhhoa/CVVI-28/2014 A/duck/Khanhhoa/CVVI-26/2014 A/duck/Khanhhoa/CVVI-42/2014 A/duck/Khanhhoa/CVVI-34/2014 A/duck/Khanhhoa/CVVI-44/2014 A/chicken/Khanhhoa/CVVI-39/2014 A/duck/Phuyen/CVVI-47/2014 A/duck/Khanhhoa/CVVI-25/2014 A/duck/Khanhhoa/CVVI-46/2014 A/duck/Khanhhoa/CVVI-30/2014 A/duck/Khanhhoa/CVVI-40/2014 A/duck/Khanhhoa/CVVI-31/2014 A/duck/Khanhhoa/CVVI-35/2014 A/duck/Khanhhoa/CVVI-45/2014 A/chicken/Khanhhoa/CVVI-41/2014 A/duck/Khanhhoa/CVVI-27/2014 A/duck/Khanhhoa/CVVI-38/2014 A/duck/Khanhhoa/CVVI-37/2014 A/duck/Khanhhoa/CVVI-29/2014 0.01 A/duck/Khanhhoa/CVVI-43/2014 A/duck/Khanhhoa/CVVI-06/2013 A/duck/Khanhhoa/CVVI-36/2014 0.01 A/duck/Khanhhoa/CVVI-32/2014 A/tiger/Jiangsu/01/2013 A/duck/Sleman/BBVW-1463- 10/2012 A/duck/Tegal/BBVW-1727-11/2012 A/Japanese white-eye/Taoyuan/Q454/2012 A/hill myna/Heilongjiang/0704/2012 A/Hong Kong/6841/2010 A/chicken/Bangladesh/15079/2012 A/duck/Lao/463/2010 A/environment/Chang Sha/2/2009 A/chicken/Nepal/5-1cl/2010 A/great cormorant/Qinghai/1/2009 A/bean goose/Tyva/10/2009 A/bar-headed goose/Mongolia/X53/2009 A/common buzzard/Bulgaria/38WB/2010 0.01 A/black swan/Yamaguchi/1/2011 A/chicken/Chiba/2/2011 A/mandarin duck/Korea/Q2/2011 A/chicken/Khanhhoa/CVVI-18/2013 A/duck/Vietnam/LBM140/2012 0.01 A/duck/Vietnam/OIE-1287/2012 0.02 A/dk/Quangngai/1037/2011 A/environment/Hunan/3/2011 A/duck/Vietnam/QT801/2011 0.01 A/duck/Vietnam/QT802/2011 A/Hubei/1/2010 0.01 A/duck/Vietnam/27386/2009 A/duck/Vietnam/27373/2009 0.01 A/duck/Zhejiang/2242/2011 0.02 A/duck/Zhejiang/224/2011 A/Hong Kong/1161/2010 0.03 A/duck/Hue/V6/2010 A/duck/Vietnam/NA114/2007 A/Muscovy duck/Vietnam/NCVD-46/2007 A/Vietnam/HN31432M/2008

0.01

0.01

0.02

0.01

A/Dk/HK/821/02 A/chicken/Vietnam/53/2004 A/chicken/Vietnam/52/2004 A/Vietnam/CL26/2004 A/Vietnam/CL36/2004 A/chicken/Vietnam/133/2004 A/pigeon/Thailand/1B/2004 A/duck/Vietnam/15/2003 A/duck/Vietnam/NCVD-16/2007 A/Muscovy duck/Ca Mau/1181/2006 0.02 A/chicken/Vietnam/3/2010 0.02 A/chicken/Vietnam/5/2010 A/duck/Vietnam/OIE-0062/2012 A/Cambodia/V0417301/2011 0.02 A/chicken/Soc Trang/1/2012 A/chicken/Soc Trang/5/2012 A/duck/Vietnam/OIE-3038/2012 A/muscovy duck/Vietnam/OIE-0043/2012 A/chicken/Cambodia/X0815301/2013 0.02 A/duck/Khanhhoa/CVVI-24/2013 0.03 0.02 A/chicken/Vietnam/NCVD-016/2008 A/chicken/Vietnam/NCVD-swab19/2008 A/goose/Guangdong/1/1996

Clade 2.3.2.1a

Clade 2.3.2.1b

Clade 2.3.4

Clade 1

Clade 1.1 Clade 1.1.1

Clade 1.1.2

Clade 7 Clade 0

28

Page 29 of 31

us

cr

A/duck/Khanhhoa/CVVI-05/2013 A/duck/Khanhhoa/CVVI-46/2014 A/duck/Khanhhoa/CVVI-04/2013 A/duck/Khanhhoa/CVVI-01/2013 A/duck/Khanhhoa/CVVI-16/2013 A/duck/Khanhhoa/CVVI-17/2013 A/duck/Khanhhoa/CVVI-19/2013 A/duck/Khanhhoa/CVVI-22/2013 A/duck/Khanhhoa/CVVI-06/2013 A/duck/Khanhhoa/CVVI-36/2014 A/duck/Khanhhoa/CVVI-14/2013 A/duck/Khanhhoa/CVVI-10/2013 A/duck/Khanhhoa/CVVI-03/2013 A/duck/Khanhhoa/CVVI-20/2013 A/duck/Khanhhoa/CVVI-08/2013 A/chicken/Khanhhoa/CVVI-02/2013 A/duck/Khanhhoa/CVVI-07/2013 A/duck/Khanhhoa/CVVI-09/2013 A/chicken/Khanhhoa/CVVI-12/2013 A/chicken/Khanhhoa/CVVI-13/2013 A/duck/Khanhhoa/CVVI-33/2014 A/duck/Khanhhoa/CVVI-15/2013 A/duck/Khanhhoa/CVVI-21/2013 A/chicken/Khanhhoa/CVVI-11/2013 A/duck/Vietnam/QB1207/2012 A/tiger/Jiangsu/01/2013 A/duck/Quang Ninh/21/2013 A/duck/Khanhhoa/CVVI-32/2014 A/duck/Khanhhoa/CVVI-31/2014 A/duck/Phuyen/CVVI-47/2014 A/duck/Khanhhoa/CVVI-38/2014 A/duck/Khanhhoa/CVVI-25/2014 A/duck/Khanhhoa/CVVI-27/2014 A/duck/Khanhhoa/CVVI-23/2013 A/duck/Khanhhoa/CVVI-30/2014 0.01 A/duck/Khanhhoa/CVVI-40/2014 A/duck/Khanhhoa/CVVI-42/2014 A/duck/Khanhhoa/CVVI-26/2014 A/duck/Khanhhoa/CVVI-29/2014 A/duck/Khanhhoa/CVVI-43/2014 A/duck/Khanhhoa/CVVI-35/2014 A/chicken/Khanhhoa/CVVI-39/201 A/chicken/Khanhhoa/CVVI-41/201 A/duck/Khanhhoa/CVVI-37/2014 A/duck/Khanhhoa/CVVI-45/2014 0.01 A/duck/Khanhhoa/CVVI-34/2014 A/duck/Khanhhoa/CVVI-44/2014 A/chicken/Khanhhoa/CVVI-28/2014 A/duck/Zhejiang/224/2011 A/Hong Kong/1161/2010 A/duck/Vietnam/QT801/2011 A/environment/Hunan/3/2011 A/duck/Vietnam/LBM14/2011 A/duck/Hunan/S4030/2011 A/duck/Vietnam/LBM57/2011 A/Hubei/1/2010 A/chicken/Khanhhoa/CVVI-18/2013 A/duck/Vietnam/LBM139/2012 A/duck/Vietnam/LBM140/2012 A/duck/Vietnam/LBM133/2012 A/duck/Vietnam/LBM132/2012 A/Muscovy duck/Vietnam/LBM113/2012 A/duck/Vietnam/OIE-1287/2012

ip t

Fig. 2.

Ac ce p

te

0.02

d

M

an

Hongkong/1161-like

0.01

0.01

A/duck/Korea/NSQ263/2008 A/whooper swan/Hokkaido/1/2008 A/whooper swan/Akita/1/2008 A/chicken/Primorje/1/2008 A/whooper swan/Aomori/1/2008 0.02 A/duck/Hunan/3/2007 0.01 A/HongKong/6841/2010 Achicken/Hubei/QE8/2009 A/duck/Lao/471/2010 A/whooper swan/Mongolia/6/2009 A/grebe/Tyva/3/2009 A/ruddy shelduck/Mongolia/911T 2/2009 A/brown-headed gull/Qinghai/9/2009 0.01 A/Muscovy duck/Vietnam/NCVD-46/2007 A/duck/Hue/V6/2010 A/Vietnam/HN31432M/2008 0.01 A/Dk/HK/821/02 A/pigeon/Thailand/1B/2004 A/duck/Vietnam/15/2003 A/chicken/Vietnam/133/2004 A/Vietnam/CL36/2004 A/Vietnam/CL26/2004 A/chicken/Vietnam/53/2004 A/chicken/Vietnam/52/2004 A/Muscovy duck/CaMau/1181/2006 A/duck/Vietnam/OIE-0062/2012 A/Cambodia/V0417301/2011 A/duck/Vietnam/OIE-3038/2012 A/muscovyduck/Vietnam/OIE-0043/2012 A/chicken/Cambodia/X0815301/2013 A/duck/Khanhhoa/CVVI-24/2013 A/goose/Guangdong/1/1996

Hubei-like

Hongkong/6841-like

Vietnam/31432-like

HK/821-like

0.01

29

Page 30 of 31

cr Zhejiang/224-like

0.007

an

us

A/duck/Khanhhoa/CVVI-08/2013 A/duck/Khanhhoa/CVVI-33/2014 A/duck/Khanhhoa/CVVI-03/2013 A/duck/Khanhhoa/CVVI-23/2013 A/duck/Khanhhoa/CVVI-21/2013 A/chicken/Khanhhoa/CVVI-02/2013 A/duck/Khanhhoa/CVVI-19/2013 A/chicken/Khanhhoa/CVVI-12/2013 A/duck/Khanhhoa/CVVI-20/2013 A/duck/Khanhhoa/CVVI-10/2013 A/duck/Khanhhoa/CVVI-15/2013 A/duck/Khanhhoa/CVVI-07/2013 A/duck/Khanhhoa/CVVI-22/2013 A/duck/Khanhhoa/CVVI-09/2013 A/chicken/Khanhhoa/CVVI-11/2013 A/duck/Khanhhoa/CVVI-17/2013 A/chicken/Khanhhoa/CVVI-13/201 A/tiger/Jiangsu/01/2013 A/duck/Khanhhoa/CVVI-01/2013 A/duck/Khanhhoa/CVVI-05/2013 A/duck/Khanhhoa/CVVI-04/2013 A/duck/Khanhhoa/CVVI-14/2013 0.004 A/duck/Khanhhoa/CVVI-16/2013 A/duck/Khanhhoa/CVVI-06/2013 A/duck/Khanhhoa/CVVI-36/2014 A/duck/Vietnam/QB1207/2012 A/duck/Vietnam/OIE-2211/2012 A/duck/Khanhhoa/CVVI-32/2014 0.007 A/duck/QuangNinh/21/2013 A/duck/Zhejiang/224/2011

ip t

Fig. 3.

Ac ce p

te

d

M

A/ Hong Kong/1161/2010 A/Hubei/1/2010 A/duck/Vietnam/OIE-2533/2011 A/chicken/Khanhhoa/CVVI-28/2014 A/duck/Vietnam/LBM140/2012 A/chicken/Khanhhoa/CVVI-18/2013 A/duck/Vietnam/OIE-1287/2012 A/chicken/Cambodia/X0815301/2013 A/duck/Khanhhoa/CVVI-44/2014 A/duck/Phuyen/CVVI-47/2014 A/duck/Khanhhoa/CVVI-42/2014 0.003 A/duck/Khanhhoa/CVVI-24/2013 A/chicken/Khanhhoa/CVVI-39/2014 A/duck/Khanhhoa/CVVI-26/2014 A/duck/Khanhhoa/CVVI-46/2014 Hongkong/1161-like A/duck/Khanhhoa/CVVI-45/2014 A/duck/Khanhhoa/CVVI-25/2014 A/duck/Khanhhoa/CVVI-30/2014 A/duck/Khanhhoa/CVVI-38/2014 A/chicken/Khanhhoa/CVVI-41/2014 A/duck/Khanhhoa/CVVI-43/2014 A/duck/Khanhhoa/CVVI-27/2014 A/duck/Khanhhoa/CVVI-35/2014 A/duck/Khanhhoa/CVVI-40/2014 A/duck/Khanhhoa/CVVI-29/2014 A/duck/Khanhhoa/CVVI-31/2014 A/duck/Khanhhoa/CVVI-37/2014 A/duck/Khanhhoa/CVVI-34/2014 0.007 A/muscovy duck/Vietnam/OIE-0043/2012 0.007 A/Cambodia/V0417301/2011 0.012 A/duck/Vietnam/OIE-0062/2012 0.006 A/duck/Vietnam/15/2003 A/pigeon/Thailand/1B/2004 0.005 A/chicken/Vietnam/53/2004 A/chicken/Vietnam/52/2004 A/Vietnam/CL36/2004 A/Vietnam/CL26/2004 A/chicken/Vietnam/133/2004 A/duck/Lao/471/2010 0.011 A/duck/Hunan/S4150/2011 A/Hong Kong/6841/2010 A/duck/Shandong/2/2011 A/Vietnam/HN31432M/2008 A/goose/Guangdong/1/1996

0.006

0.005

30

Page 31 of 31