Genetic characterization of highly pathogenic H5 ...

Infection, Genetics and Evolution 38 (2016) 96–100

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Genetic characterization of highly pathogenic H5 influenza viruses from poultry in Taiwan, 2015 Pei-Yu Huang a,b, Chang-Chun David Lee c, Chun-Hung Yip b,d, Chung-Lam Cheung b, Guangchuang Yu b, Tommy Tsan-Yuk Lam b, David K. Smith b, Huachen Zhu a,b,d,⁎, Yi Guan a,b,d,⁎ a

Joint Influenza Research Centre, Shantou University Medical College, Shantou, Guangdong, China Centre of Influenza Research, School of Public Health, The University of Hong Kong, Hong Kong Special Administrative Region Genomics Research Center, Academia Sinica, Taipei, Taiwan d State Key Laboratory of Emerging Infectious Diseases (Shenzhen Base), Shenzhen Third People's Hospital, Shenzhen, China b c

a r t i c l e

i n f o

Article history: Received 24 September 2015 Received in revised form 8 December 2015 Accepted 9 December 2015 Available online 10 December 2015 Keywords: H5N8 Reassortment Genotype Migratory bird Clade 2.3.4.4 Eurasian gene pool

a b s t r a c t Phylogenetic analysis of the highly pathogenic avian influenza (HPAI) H5 viruses causing recent outbreaks in Taiwan showed that they belonged to the Asian HPAI H5 lineage, clade 2.3.4.4 viruses, and were apparently introduced by migratory birds. These viruses reassorted with Eurasian influenza gene pool viruses and formed five genotypic variants. As Taiwan has a similar influenza ecosystem to southern China, the HPAI H5 lineage could become established and enzootic in the island. © 2015 Elsevier B.V. All rights reserved.

The enzootic state of the Asian highly pathogenic avian influenza (HPAI) H5N1 viruses in poultry in China has led to repeated disseminations to other countries (WHO/OIE/FAO, 2014). The most well known case is the clade 2.2 (Qinghai-like) virus, which spread apparently by migratory birds across Asia and into Europe and Africa (WHO/OIE/ FAO, 2014). However, the Asian HPAI H5N1 lineage had not crossed the Bering Strait to North America, nor spread to Taiwan, until the emergence of A/Duck/Korea/Buan2/2014 (Buan2)-like H5N8 variants

Abbreviations: KU1, Crane/Kagoshima/KU1/2014; KU13, Crane/Kagoshima/KU13/ 2014; HA, hemagglutinin; NA, neuraminidase; Tonghai/5, A/chicken/Tonghai/5/2014 (H5N1); GD01, A/duck/Guangdong/GD01/2014 (H5N6); Buan2, A/duck/Korea/Buan2/ 2014 (H5N8); Gochang1, A/duck/Korea/Gochang1/2014 (H5N8); Sichuan/26221, A/Sichuan/26221/2014 (H5N6); H5Nx-like, Buan2-like H5N8 variants of clade 2.3.4.4 lineage; GP-EA, Eurasian gene pool; GP, gene pool; EA, Eurasian; NorAm, North American; Gs, goose; Dk, duck; Ck, chicken; Tk, turkey; AgwTeal, American green-winged teal; GFc, gyrfalcon; EWg, Eurasian wigeon; Wg, wigeon; MDk, mallard duck; BTeal, Baikal teal; WDk, wild duck; Qa, quail; RbPc, rosy-billed pochard; AcB, aquatic bird; Shv, shoveler; NPt, northern pintail; NSh, northern shoveler; CDk, call duck; WB, wild bird; Gw, gadwall; BWTeal, blue-winged teal; RDv, rock dove; BhGu, black-headed gull; WGs, wild goose; Av, avian; RTs, ruddy turnstone; RsDk, ruddy shelduck; MSwan, mute swan; Env, environment; COA, Council of Agriculture; TW, Taiwan; OIE, World Organization for Animal Health; WHO, World Health Organization; FAO, Food and Agricultural Organization. ⁎ Corresponding authors at: 5th Floor, Lab Block, 21 Sassoon Road, Pokfulam, Hong Kong Special Administrative Region. E-mail addresses: [email protected] (H. Zhu), [email protected] (Y. Guan).

http://dx.doi.org/10.1016/j.meegid.2015.12.006 1567-1348/© 2015 Elsevier B.V. All rights reserved.

of clade 2.3.4.4 viruses in late 2013 (Lee et al., 2015; Lee et al., 2014a and OIE, 2015). Clade 2.3.4.4 H5 viruses have evolved into subgroups (Fig. 1; Hu et al., 2015) and some have dispersed following bird migration pathways to Europe and North America (Hill et al., 2015; Lee et al., 2015 and Verhagen et al., 2015). In early 2015, outbreaks of highly pathogenic H5N2, H5N3 and H5N8 viruses were detected, mainly in the west of Taiwan, affecting more than 800 widespread poultry farms and causing a loss of 3.2 million birds within half a year. These losses were 1.3 million geese (58.6% of the population of 2.2 million birds held in 1011 farms), 1.8 million chickens (1.9% of the 94 million birds held in 5716 farms), 0.1 million domestic ducks (1.1% of 8.7 million birds from 2790 farms) and 0.03 million domestic turkeys (26.8% of 0.11 million birds held in 92 farms) (OIE, 2015 & COA, Taiwan, 2014). To investigate the origin and development of these H5 HPAI viruses, we conducted a survey for influenza viruses in January 2015 at two goose farms in southwestern Taiwan. Paired cloacal and oropharyngeal swabs were collected on one farm from 14 geese with suspected HPAI infections due to their showing signs of hemorrhages in the eyes and beaks, paralysis or torticollis, and watery diarrhea. Oropharyngeal swabs were taken from another six birds in a moribund state. Fecal materials (n = 17) were sampled at a neighboring goose farm where all birds seemed healthy. Each sample was placed into a transport medium with antibiotics and RNA was extracted from the samples using the QIAamp Viral RNA Minikit

P.-Y. Huang et al. / Infection, Genetics and Evolution 38 (2016) 96–100

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Fig. 1. Maximum likelihood phylogeny of the H5 hemagglutinin gene. The inset shows the hemagglutinin (HA) phylogeny of clade 2.3.4.4 H5 viruses, generated from sequences obtained from this study and public databases accessed on 22 July 2015. Five sub-lineages were identified and labeled (bottom to top A to E and colored as purple, pink, orange, green and blue, respectively). Example viruses of each sublineage are shown in parentheses. Within the Buan2-like sublineage, the groups identified in Japan and Taiwan and in North America are colored red and green, respectively. Together with ten Mexican-like H5N2 viruses, HA genes of 42 selected Buan2-like viruses were phylogenetically analyzed. Sequences reported during the highly pathogenic avian influenza (HPAI) H5 outbreaks in Taiwan in 2015 are labeled in blue (this study) and green (Council of Agriculture, Taiwan). Subtypes are shown in parentheses. Bootstrap support values (percentages) from 1000 pseudo-replicates are shown for selected lineages. Numbers (n) of sequences identical at the nucleotide level to the examples given are indicated. Lineage origins of genotypes are shown as eight blocks to the right of the tree (left to right HA, NA, PB2, PB1, PA, NP, M and NS) colored as given by the lineage legend. The five genotypes of Taiwanese H5 viruses are indicated by roman numerals. Branch scale represents 0.01 substitutions per site. The figure was prepared using ggtree (Yu G and Lam TTY [http://www.bioconductor.org/packages/ggtree]). See Abbreviations.

(Qiagen). Diagnostic real-time RT-PCR of the matrix gene was conducted to detect the presence of influenza A virus as previously described (Zhu et al., 2010). Only swabs from the farm with geese showing clinical symptoms (all, n = 34) were positive for influenza viruses. Full genomic sequences of 33 H5N8 viruses and partial sequences of one H5N8 isolate were obtained using a next generation sequencing protocol. All gene segments from each positive sample were amplified using the universal oligomer sets (Hoffmann et al., 2001) and purified with the QIAquick PCR Purification Kit (Qiagen). Equimolar volumes of these PCR products were mixed and a library was prepared using the Nextera® XT DNA Library Preparation Kit (Illumina) for each sample. The concentration of the library was verified with quantitative real-time PCR and samples were adjusted to equal concentrations. The library was hybridized with oligomers on a flow cell and DNA clusters were generated. Dual indexed paired-end sequencing was performed on the MiSeq platform (Illumina) following the manufacturer's protocol. Contigs were assembled using GS De Novo assembler v2.9 from reads and identified as influenza A sequences by BLAST against GenBank (Lam

et al., 2015). The sequences obtained have been deposited in GenBank (accession numbers KT388441–KT388712). Phylogenetic analysis, as described previously (Lee et al., 2014b), was performed using these full genome sequences and closely related sequences from GenBank, GISAID and the publicly available government website (http://ai.gov.tw/index.php?id=720), which gave the sequences of the 16 H5 viruses isolated by the Council of Agriculture (COA), Taiwan during the recent outbreaks. While all influenza viruses detected in this study were H5N8 subtype viruses from geese, the 16 viruses reported by COA, Taiwan were four H5N8 (goose 2, duck 1, chicken 1), nine H5N2 (goose 1, duck 1, chicken 7) and three H5N3 (goose, duck and chicken, 1 each) viruses. Six of these H5N2 chicken isolates belonged to the Mexican/95-like H5N2 viruses circulating in Taiwan since 2003, which obtained their internal genes from the enzootic chicken H6N1 lineage (Lee et al., 2014b). In the phylogeny of the hemagglutinin (HA) gene, all H5 viruses from Taiwan, except the Mexican/95-like H5N2 viruses, formed a monophyletic cluster in the Buan2-like lineage with two Japanese

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H5N8 viruses, Crane/Kagoshima/KU1/2014 (KU1) and Crane/Kagoshima/KU13/2014 (KU13). The HA genes of two Taiwan H5N3 isolates are identical to KU13 (Fig. 1). These Taiwanese and Japanese viruses share an immediate common ancestor with the Buan2-like H5 2.3.4.4 viruses recently introduced into North America (Hill et al., 2015; Lee et al., 2015 and Ozawa et al., 2015). In the neuraminidase (NA) gene phylogenies of Taiwan H5 Buan2-like viruses, all N8 genes cluster with KU1 and KU13 (Fig. 2a), while the N2 (Fig. 2b) and N3 (data not shown) genes were derived from Eurasian gene pool (GP-EA) viruses, most likely from migratory or local domestic birds. The M genes of all Taiwan H5 Buan2-like viruses (H5N2, N3 and N8) and the PA genes of the Taiwan H5N8 and H5N3 viruses formed monophyletic clusters with the majority of Buan2-like viruses, which obtained these genes from the Asian HPAI H5N1 lineage (Fig. 3c, e). For other internal genes and the PA gene of H5N2 viruses, the Taiwan H5 Buan2like viruses formed different clusters within the GP-EA lineage (Fig. 3a– d, f), indicating various reassortant origins. Five distinctive genotypic groups (Genotypes I–V) were identified among the Taiwan H5 Buan2like viruses (Figs. 1-3). Genotype I viruses were of pure Buan2-like H5N8 lineage common to most viruses of this lineage (Figs. 1-3). The Taiwanese viruses in genotype groups II to V were reassortants of viruses from the GP-EA and the Buan2-like lineages (Figs. 1–3), similar to the Buan2-like H5 viruses in North America, which incorporated gene segments from the American gene pool viruses (Pasick et al., 2015). Genotype II viruses were H5N2 viruses with all internal genes except M derived from GPEA viruses (Fig. 3). Genotype III and IV viruses (H5N8 and H5N3, respectively) shared common ancestors in PB2, PB1 and NP with genes from the GP-EA lineage (Fig. 3a, b, d), but had different origins for their NS genes (Fig. 3f). Genotype V viruses contained GP-EA NP and PB1 genes (Fig. 3b, d), with their other genes from the 2.3.4.4 Buan2-like lineage (Fig. 3). Two genotypes (I and V) of H5N8 viruses were identified at the farm with the outbreak surveyed in this work. Three geese had Genotype V viruses isolated from their oropharynx and Genotype I viruses from their cloacae, while one bird had the reverse situation.

Migratory birds arrive in Taiwan from Northeast Asia during winter and constitute 60% of the wild birds, mainly aggregating on the west coast, where most poultry farms are located (www.bird.org.tw/ index.php/works/lists & COA, Taiwan, 2014). Even though the Asian HPAI H5 virus has been enzootic in Mainland China for almost two decades, it was previously only detected in Taiwan, 130 km away across the Taiwan Strait, from smuggled birds (Lee et al., 2007). The lower pathogenicity of Buan2-like H5 viruses in wild birds (Kang et al., 2015) may facilitate their long-distance dissemination, as both clade 2.2 and 2.3.2.1 viruses had caused outbreaks in Korea and Japan (Lee et al., 2008; Uchida et al., 2008) but were never detected in Taiwan. Reassortment of Buan2-like H5 viruses with gene pool viruses has only been observed in North America and Taiwan. This might be caused by the diversity of influenza viruses in these two ecosystems, together with their abundance of wild birds and their particular intensive poultry farming practices in their local areas. The geographical source of only seven of the sixteen viruses from the Council of Agriculture (COA), Taiwan was available (Fig. S1). However, with the viruses obtained in this study, their locations cover the range of areas affected during the outbreaks in early 2015 in Taiwan (Fig. S1). Regional co-circulation of Mexican-like and Buan2-like H5 viruses, with various NA subtypes and genotypes, was identified in southwestern Taiwan. While more extensive sampling might reveal more genotypes and NA subtypes of highly pathogenic H5 avian influenza viruses, co-circulation of diverse lineages, subtypes and genotypes, including reassortants between Buan2-like H5 viruses and gene pool viruses, has already been observed. This emphasizes the importance of and need for systematic surveillance of avian influenza viruses in poultry and migratory birds to provide warning of novel reassortants and viruses that might lead to outbreaks. Separating poultry types, especially domestic ducks and geese from chickens, and increasing biosecurity should be mandatory to prevent further emergence of influenza viruses. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2015.12.006.

Fig. 2. Maximum likelihood phylogenies of the neuraminidase genes. a, N8 (n = 46) and b, N2 (n = 48). Scale bar represents 0.02 substitutions per site. All other annotations and abbreviations are the same as Fig. 1.


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Fig. 3. Maximum-likelihood phylogenies of internal genes. a, PB2 (n = 73), b, PB1 (n = 70), c, PA (n = 69), d, NP (n = 71), e, M (n = 67) and f, NS (n = 71). All annotations and abbreviations are the same as Fig. 1.

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