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Infection, Genetics and Evolution 44 (2016) 530–540

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Research paper

Prevalence and diversity of H9N2 avian influenza in chickens of Northern


Vietnam, 2014
Duong Mai Thuy a, Thomas P. Peacock b,c, Vu Thi Ngoc Bich d, Thomas Fabrizio e, Dang Nguyen Hoang f,
Nguyen Dang Tho g, Nguyen Thi Diep f, Minh Nguyen d, Le Nguyen Minh Hoa d, Hau Thi Thu Trang d,
Marc Choisy d,g, Ken Inui h, Scott Newman h, Nguyen vu Trung i, Rogier van Doorn d,j, Thanh Long To a,
Munir Iqbal b, Juliet E. Bryant d,j,⁎
a
National Center for Veterinary Diagnostics, Department of Animal Health, Hanoi, Vietnam
b
Avian Viral Diseases programme, The Pirbright Institute, Woking, UK
c
St Mary's Campus, Imperial College London, London, UK
d
Oxford University Clinical Research Unit and Wellcome Trust Major Overseas Programme, Hanoi, Vietnam
e
St Jude's Center for Excellence in Influenza Research and Surveillance, Memphis, TN, USA
f
Division of Epidemiology, Department of Animal Health, Hanoi, Vietnam
g
MIVEGEC (UM1-UM2-CNRS 5290-IRD 224), Centre de Recherche IRD, Montpellier, France
h
Food and Agriculture Organization of the United Nations, Hanoi, Vietnam
i
National Hospital Tropical Diseases, Hanoi, Vietnam
j
Center for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK

a r t i c l e i n f o a b s t r a c t

Article history: Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant
Received 18 May 2016 losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveil-
Received in revised form 17 June 2016 lance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza
Accepted 19 June 2016
H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in
Available online 20 June 2016
chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs
Keywords:
collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled
Avian influenza swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools)
Vietnam were 5.8% (CI 5.4–6.0). Subtyping was performed on 468 pooled swabs with M gene Ct b 26. No influenza H7 was
H9N2 detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interac-
Poultry tion between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11
Chicken partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to
H5 Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 dis-
Zoonotic
tinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam
rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Viet-
nam are warranted to assess H9 endemicity elsewhere in the country.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction monitoring genetic diversity and ongoing evolution of HPAI H5 subtype


viruses within duck populations (Nguyen et al., 2014). Starting in 2013,
Vietnam has a well-developed system for the active surveillance of additional surveillance activity was initiated in provinces along the Chi-
highly pathogenic avian influenza (HPAI) strains within live bird mar- nese border to promote early detection of H7N9 viruses, and was fo-
kets (LBMs). From 2010 to 2015, surveillance focused primarily on cused largely on collections from chickens and environmental
samples. To date, this second surveillance activity has screened
N33,480 pooled chicken oropharyngeal swabs and environmental sam-
Abbreviations: LPAI, low pathogenicity avian influenza; HPAI, highly pathogenic avian ples, however no A/H7N9 viruses have been identified in Vietnam (FAO,
influenza; FAO, Food and Agriculture Organization of the United Nations; LBM, live bird 2014).
market.
⁎ Corresponding author at: Oxford University Clinical research Unit and Wellcome Trust
Low pathogenicity avian influenza (LPAI) H9N2 is the most preva-
Major Overseas Programme, Hanoi, Vietnam. lent influenza subtype circulating endemically in chickens worldwide.
E-mail address: [email protected] (J.E. Bryant). H9N2 viruses are endemic in poultry across Asia, the Middle East and

http://dx.doi.org/10.1016/j.meegid.2016.06.038
1567-1348/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540 531

Northern Africa (Fusaro et al., 2011), and despite their status as LPAI, inoculated embryonated chicken eggs were clarified by centrifugation
poultry outbreaks of H9N2 are associated with significant economic for 10 min at 4 °C, and presence of virus was determined by hemagglu-
losses, largely due to reduced egg production, reduced feed conversion tination assay as previously described (WHO, 2011).
efficiencies, and highly lethal bacterial or viral co-infections (Li et al.,
2005; Parvin et al., 2014). Epidemiological evidence indicates that the 2.2. Next generation sequencing (NGS)
chicken adapted H9N2 genotype 57 (G57), also known as genotype S,
contains an internal gene cassette (polymerase genes, nucleoprotein, We used NGS protocols for influenza sequencing adopted from St
matrix and non-structural genes) that confers increased host range Jude's Center for Excellence in influenza Research & Surveillance
and zoonotic transmission potential to non-H9N2 influenza strains, (SJCEIRS). Briefly, fresh nucleic acid extracts were prepared and sam-
with recent examples including the zoonotic H7N9 and H10N8 out- ples were pre-amplified to enrich for influenza genomes using
breaks in China (H. Chen et al., 2014; Gu et al., 2014; Pu et al., 2014). Ad- MBTUni12/13 primers as previously described (Zaraket et al., 2015;
ditionally H9N2 viruses pose a zoonotic risk in their own right, having Zhou et al., 2009). DNA libraries were prepared using Nextera XT
caused sporadic human infections in China, Hong Kong, Bangladesh DNA Library Prep Kits (Illumina) with 96 dual-index barcodes accord-
and Egypt (ICDDRB, 2011; Butt et al., 2005; Huang et al., 2015a, ing to manufacturer's instructions. Pooled libraries were sequenced on
2015b; WHO, 2015a, 2015b). It is suggested that the prevalence and a MiSeq Illumina, using the v2 reagents and 300 cycles. Sequence
variation of H9N2 influenza virus in farmed poultry could provide an reads were filtered for quality scores, and the high quality reads aligned
important early warning of the emergence of novel reassortants with to a custom reference library (kindly provided by SJCEIRS) and by
pandemic potential (Liu et al., 2014a, 2014b). Despite increasingly de novo assembly. Genomic sequences and variant frequencies
widespread use of H9 vaccines in neighboring China (Pu et al., 2014; were generated in CLC genomics workbench v. 8.5.1 (http://www.
Wei et al., 2016; P. Zhang et al., 2008), the poultry sector in Vietnam clcbio.com/).
has not yet adopted any vaccination against H9N2 viruses, largely be-
cause poultry production remains dominated by smallholder farms, 2.3. Phylogenetic and genotypic analysis
with little vertical integration and relatively low investment in veteri-
nary vaccines. The only licensed avian influenza vaccines for poultry in Data used for phylogenetic analyses comprised the novel whole
Vietnam are inactivated virus vaccines against the H5 subtype genome sequences generated by this study, as well as additional public-
(Nguyen et al., 2014). ly available sequences of closely related viruses identified by Blastn
Research and surveillance activities in Vietnam from 2003 to analysis of NCBI and GISAID databases. Full datasets for each gene
2013 previously identified a number of LPAI subtypes circulating in segment were aligned using the MUSCLE algorithm in Mega software
duck populations (Hotta et al., 2012; Jadhao et al., 2009; Kim et al., version 6.06 (www.megasoftware.net). Alignments were trimmed to
2013; D. C. Nguyen et al., 2005; Nomura et al., 2012; Masatoshi yield the maximal sequence coverage per segment as follows: 260-
Okamatsu et al., 2013; Takakuwa et al., 2013), including all 3 major 2289 for PB2; 78-2242 for PB1; 232-1981 for PA; 160-1599 for HA;
antigenically divergent lineages of LPAI H9N2 (i.e. the Chinese 49-1507 for NP; 24-1386 for NA; 26-996 for MP; and 56-859 for NS.
BJ94/Y280-like lineage, the Middle Eastern G1-like lineage and the Phylogenetic trees were created using the neighbor-joining algorithm
predominantly Korean Y439-like lineage) (Jadhao et al., 2009; Kim and the Tajima-Nei model, with 1000 bootstrap replications. Bootstrap
et al., 2013; Nomura et al., 2012). In contrast, LPAI transmission values of b 70 were excluded from trees. Genotyping was performed
within chickens has not been well studied. Here we determine the using 2% nucleotide difference cutoff to indicate genetically divergent
prevalence and diversity of LPAI in chickens from northern Vietnam, segments.
and evaluate the feasibility of direct sequencing of AIV from pooled
surveillance swabs.
2.4. Statistical analysis

2. Methods Prevalence of influenza type A, H5 and H9 virus infection was esti-


mated from the pooled samples at the market and province levels,
2.1. Sample collections, molecular screening, and virus isolations using a maximum likelihood modeling approach described in Supple-
mentary Appendix A. We used an influenza A Matrix gene Ct threshold
Samples used in this study were collected at 10 time points from Oc- of 35 as the standard cut-off for positivity. For subtype specific preva-
tober to December 2014, representing one discrete period of surveil- lence of H5 and H9, the threshold for positivity was set at Ct ≤38. Evi-
lance activity in the FAO-supported programme for early detection of dence for interaction between H9 and H5 from pooled samples was
H7N9 viruses. This surveillance activity focused on sampling LBM in tested by a likelihood ratio test as described in the Supplementary Ap-
northern provinces believed to play a key role in cross-border poultry pendix B.
trade with China. The sampling frame was based on a stratified sam-
pling design, and entailed collections in 7 northern provinces, with a 2.5. Nucleotide accession numbers
primary focus on market trade routes along the Vietnam/China border.
Five district markets were sampled at weekly intervals for approximate- The complete and partial genome sequences of the viruses
ly 10 weeks between October 30 and December 31 of 2014. At each sequenced in this study were submitted to the GenBank database
sampling timepoint, oropharyngeal (OP) swabs of 35 chickens were (Table S2).
sampled individually from 7 different vendors per market, and aggre-
gated into pools (5 swabs per pool). The pooled OP swabs were stored 3. Results
in 2 mL viral transport medium (PBS pH 7.2 supplemented with antibi-
otics, gentamicin, and buffering agents), transported by cold chain to 3.1. Prevalence of influenza A in market chickens
the National Center for Veterinary Diagnostics (NCVD) and stored at -
80 °C prior to further processing. RNA of pooled swab samples were ex- A total of 1207 of 2450 pooled swabs (49%) screened positive for in-
tracted using a tacoTM Nucleic Acid Automatic Extraction System fluenza A by matrix gene real time RT-PCR. Using a maximum likelihood
(GeneReach Biotechnology Corp., Taiwan). Detection of influenza A modeling approach that accounts for pooling (Suppl Appendix A), this
and subtype-specific monoplex real time RTPCR were performed using detection level corresponded to an overall influenza A prevalence of
standardized protocols (Supplementary T2). Allantoic fluid from 5.45% (95% Confidence Interval [CI] 5.4–6.0%). Fig. 1(a) presents
532 D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540

Fig. 1. (a) Influenza A prevalence estimates in 5 district markets each for 7 northern provinces from Oct.–Dec. 2014; (b) map showing provinces surveyed by the H7F programme.
Yellow = Lao Cai province; orange = Ha Giang province; red = Cao Bang province; green = Ha Noi province; purple = Bac Giang province.

Table 1
estimated prevalence of influenza A per weekly sampling round (x-
H5 and H9 subtyping by RTPCR of H7F surveillance swabs, Oct.–Dec. 2014. axis), depicted by province (rows) and by market (individual graphs).
None of the 1207 influenza positive pools were found positive for H7
AIV+ AIV+ swabs H5+ H9+ Dual H5+ H5–H9−
subtype. Further molecular subtyping to detect H5 and H9 viruses was
screened (%)⁎ (%) (%) H5+ only
H9+ conducted on all pooled swabs with an influenza A matrix gene
Ct b 26 (n = 468) (Table 1). Detection rates for H9 and H5 subtype var-
Bac 230 97 (20.7) 2 62 1 1 34
Giang (0.4) (63.9%) ied dramatically between provinces and individual markets, with H9 ac-
Bac 220 82 (17.5) 0 80 0 0 2 counting for 64–100% of influenza A positive pools; H5 was detected in
Ninh (97.6%) 4.7% (22/468) of positive pools; 3.8% (18/468) pools screened positive
Cao 254 93 (19.9) 1 92 1 0 1 for both H5 and H9 subtypes; and 8.9% (42/468) were classified as ‘sub-
Bang (0.2) (98.9%)
type unknown’. The maximum likelihood prevalence (accounting for
Ha 167 68 (14.5) 6 60 3 3 5
Giang (1.3) (88.2%) pooling) for H9 and H5 was 3.7% (95%CI 3.3–4.1%) and 1.09% (95%CI
Ha Noi 85 34 (7.3) 0 34 0 0 0 0.7–1.6%), respectively. We found no evidence of significant interaction
(100%) between H5 and H9 when using a maximum likelihood modeling ap-
Lao Cai 246 94 (20.1) 13 94 13 0 0
proach (Suppl Appendix B). Virus isolation in embryonated eggs was
(2.8) (100%)
Hưng 5 0 0 0 0 0 0 attempted on selected representative H9 positive pools with Ct b 24
Yên (n = 25), and all H5 positive pools and pools the were ‘unknown sub-
Total 1207 468 (100) 22 422 18 (3.8) 4 42 (8.9) type’, regardless of Ct value (n = 22 and 42, respectively). We isolated
(4.7) (90.1) (0.85) 25 H9 viruses, however none of the H5 positive pools or the pools of ‘un-
⁎ Only swabs with AIV matrix gene RTPCR Ct b 26 were selected for screening. known subtype’ yielded a virus isolate.
D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540 533

3.2. Phylogenetic analysis and genotyping were selected for Nextera XT library preparations, and 50 were included
in the run. Next generation sequencing identified 17 H9N2 complete ge-
We utilized next generation sequencing using the Illumina Nextera nomes, 2 H5N6 complete genomes, 11 partial genomes. The HPAI H5
XT Sample Preparation Kit and the MiSeq platform as previously de- genomes detected from LBM will be presented within a separate publi-
scribed (Zaraket et al., 2015) using both direct sequencing of pooled cation and are only briefly discussed here. To determine relationships
surveillance swabs, as well as egg-grown virus isolates. Amplification among the novel A/H9N2 sequences, we performed Blastn on each
enrichment for influenza genomes was performed on a total of 96 sam- gene segment to identify closely related viruses within public datasets,
ples (19 egg isolates, 77 pooled surveillance swabs), from which 55 and included a number of additional references. All H9 HA genes fell

Fig. 2. Neighbor joining phylogenetic trees of Vietnamese H9N2 viruses (n = 17) and reference strains (n = 83 strains). Red = virus sequences generated in this study; Green = genotype
57 viruses as defined by Pu et al., PNAS 2015; Blue = H9 sequences from non-H9N2 subtype viruses; Pink = recent genotype 57 human H9N2 isolates from China; Black = additional
representative H9N2 viruses available from Genbank. Bootstrap values below 70% excluded.
534 D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540

into the BJ94/Y280-like lineage, the predominant H9 lineage found in


China. Segment 6 (NA) exhibited the most diversity with 15.7% nucleo-
tide differences between strains, and fell into 2 distinct clusters (Fig. 2).
In contrast, the other segments showed reduced diversity, with maxi-
mum pairwise nucleotide differences of 6.0%, 4.0%, 2.9%, 5.0%, 4.8%,
1.6% and 6.0% for segments 1–5, 7 and 8 respectively.
Each of the 17 whole genomes of H9N2 possessed the G57-like inter-
nal gene cassette, similar to zoonotic H7N9 and H10N8 viruses in main-
land China. For example, the genes most closely related to the PB2 and
PB1 segments of H7F-14-BN4-437 belonged to the human H10N8 and
H7N9 isolates, A/Jiangxi/IPB13/2013(H10N8) and A/Guangdong/04/
2013(H7N9) with percentage nucleotide homologies of 99.6% and
99.5% respectively. Additionally H7F-14-CB4-2, H7F-14-CB4-29, H7F-
14-CB4-31 and H7F-14-CB4-34 possessed PB1 and NS genes closely re-
lated to human and avian H7N9 isolates from 2013 with homologies of
over 99.9%, and their PA, NP and MP genes were related to H10N8 virus-
es with homologies N99.3%. Using an N98% nucleotide difference cutoff
for each segment, we determined patterns of clustering at the sub-ge-
notype level, and identified 9 distinct sub-genotypes, all of which ap-
peared to be reassortants between closely related H9N2 viruses (Fig.
3). (For reference, the comparable divergence level used for clade desig-
nations in H5 viruses is 1.5%) (WHO/OIE/FAO, 2012). Previous investi-
gations of nucleotide substitution rates of H9N2 viruses have
suggested that 2% divergence signifies approximately 4.5 to 9 years of
evolution (Fusaro et al., 2011).
Of the 18 surveillance swabs initially identified by RT-PCR as con-
taining both H5 and H9, only 8 were subjected to MiSeq (the other 10
samples were excluded during quality control steps), and 1 yielded se-
quence results indicating presence of both subtypes (H7F-CB4-35). Ad-
ditionally, there was one sample that tested positive for H5 but not H9
on initial RTPCR screening (H7F-HG4-604), however it yielded H9HA,
H5HA, H9-like PB2, PA, NP, NA, MP and NS genes, and an H5-like PB1.
The status of these samples as intersubtypic reassortants cannot be
established as they represented pooled swabs from multiple birds. Of
the samples designated as ‘influenza positive, unknown subtype’ by
RT-PCR, 3 of 9 samples sequenced by MiSeq yielded partial sequence
data of H9N2, and the remaining did not yield sequences of sufficient
coverage for analysis.

3.3. Molecular characteristics of the novel H9N2 genomes

3.3.1. Hemagglutinin (HA)


The 19 H9 sequences generated retained the classical BJ94/Y280-like
‘low-pathogenic’ dibasic RSSR cleavage motif that has previously shown
to allow cleavage by epithelial proteases such as matriptase and is
thought to facilitate systemic infections in poultry (Baron et al., 2013).
The receptor binding site of all sequences had leucine at position 216
(226 in H3 numbering), a residue associated with increased binding to
human-like α2,6 linked sialic acids in multiple subtypes including
H9N2, and consequently associated with mammalian adaptation and
transmission (Sorrell et al., 2009). This is a change from previously de-
tected Vietnamese H9N2 viruses which tended to have a glutamine at
Fig. 2 (continued).
this position which preferentially binds to the ‘avian’ α2.3 linked sialic
acid (Jadhao et al., 2009). Position 217 (227, H3 numbering) has been
implicated in both receptor binding and mammalian adaptation in a 2005; Sun et al., 2013; Zhong et al., 2014a, 2014b) (Table S1). All viruses
number of subtypes, however the significance of methionine at this po- contained conserved N-linked glycosylation sequence (N-X-S/T, where
sition (as observed in the H9 Vietnamese sequences here) remains un- X =/=P) at positions 11–13, 123–125, 280–282, 287–289 and 295–
known (Sang et al., 2015; Stevens et al., 2006). Additionally position 180 297, and more than half of the sequences had an additional glycosyla-
(190 H3 numbering) is implicated in receptor binding specificity in the tion sequence at position 200–202 (Table S1). Finally, by analyzing
closely related H1 hemagglutinins (Matrosovich et al., 2001; known H9N2 antigenic residues from the literature (Kaverin et al.,
Matrosovich et al., 2000) the Vietnamese viruses have either alanine 2004; Okamatsu et al., 2008; Peacock et al., 2016; Ping et al., 2008;
or valine, which are very common in recent Chinese H9s, or threonine, Wan et al., 2014; Zhu et al., 2015), we predict there are at least two an-
which is more rarely found, at this position. The HA sequences also tigenically variant sub-groups circulating in northern Vietnam: those
contained a number of previously described markers with the potential antigenically similar to H7F-14-CB4-2 and those similar to HF7-LC4-26
to confer mammalian transmission potential, as well as the chicken air- which vary from each other at 11 antigenically important residues
borne transmission marker K363 (Rudneva et al., 2005; Kaverin et al., (Fig. 4).
D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540 535

Fig. 2 (continued).

N1 of HPAI H5N1 viruses, has been shown experimentally to increase


the pathogenicity of H9N2 viruses in chickens and mice, and has be-
come predominant in Chinese H9N2 circulation (94.8% of viruses since
2013) (Sun et al., 2013). Although the Vietnamese NA genes had a num-
ber of additional molecular markers of virulence and airborne transmis-
sion in mammals and poultry (Lv et al., 2015; Z. Zhang et al., 2011)
(Table S1), they did not have any known molecular markers of
oseltamivir resistance, and thus probably remain susceptible to the
drug (Gubareva, 2004).
Fig. 2 (continued).
3.3.3. NS1 and NEP
Similar to divergence of NA, segment 8 also segregated phylogenet-
ically into two groups within the G57-like lineage, with a major group
3.3.2. Neuraminidase (NA) represented by H7F-14-CB4-2 and a minor group represented by H7F-
The N2 genes of the Vietnamese viruses fell into two distinct clades 14-BN4-315 (Fig. 2). Interestingly, the minor group contained a NS1
(Fig. 2) within the G57 lineage, clade 0 and 1 (Pu et al., 2014). These dis- C-terminal elongation from 217 to 237 amino acids. This elongation
tinct clades vary in regards to a 3 amino acid deletion in the NA stalk has been shown to influence inflammation in poultry and mammalian
(clade 1 NAs have the deletion, clade 0 do not). This deletion, although experimental models, and to increase transmissibility among poultry
minor compared to the 20+ amino acid deletions found recently in the (Kong et al., 2015). Similar to the Chinese human H9N2 isolates, the
536 D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540

Fig. 2 (continued).

important in modulating virulence (Chen et al., 2001; Gao, 2015;


Jagger et al., 2012). All sequenced PA segments coded for a complete
PA-X protein, shown to increase mammalian replication and pathoge-
nicity in H9N2 viruses (Gao, 2015). Length of the PB1-F2 protein, how-
ever, varied between different isolates. Of the H9N2-like PB1s, 18
Fig. 2 (continued). isolates contained a full length PB1-F2 (90aa), 5 had a minor truncation
(76aa), and a single isolate had a major truncation (27aa). 76aa long
PB1-F2s from H9N2 viruses has previously been implied to increase
the virulence of H7N9 viruses in mice but are thought not to interfere
NS1 proteins of these virus contain a large number of mammalian with function (Gibbs et al., 2003; Su et al., 2015). PB1-F2 functional
markers (Dankar et al., 2011; Ping et al., 2011; Z. Zhang et al., 2011) knockouts, similar to the 27aa variant seen, have also been shown to in-
(Table S1). crease virulence of avian influenza viruses (Gibbs et al., 2003). The PB1-
F2 of the mixed isolate H7F-HG4–604 was H5-like and had a length of
57 amino acids, which like the 27aa variants is often considered a func-
3.3.4. Accessory proteins (PB1-F2, PA-X) tional knockout (Gibbs et al., 2003). All PB1-F2s of over 76aa also had
In addition to the 10 well characterized and almost universally several molecular markers associated with mammalian virulence and
expressed core proteins of influenza, some virus strains also code for ac- predisposition to secondary bacterial pneumonia (Alymova et al.,
cessory proteins such as PB1-F2 and PA-X, which are thought to be 2014) (Table S1).
D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540 537

Fig. 2 (continued).
Fig. 2 (continued).

3.3.5. Polymerase subunits and nucleoprotein


The polymerase subunits of each of the H9N2 isolates, PB2, PB1, PA,
and NP, displayed multiple mammalian adaptation markers (Chen et al., 3.3.6. Matrix proteins
2006; Mei et al., 2016; Park et al., 2015; Sun et al., 2013; Xiao et al., 2016; The M1 and M2 proteins, encoded as alternative splice products of
Yao et al., 2013) (Table S1). In all sequenced isolates the PA subunit had segment 7 of the influenza genome, similarly to the other segments,
a marker of airborne transmission in chickens, L672 (Zhong et al., 2014a, contained a number of known markers of mammalian adaptation
2014b). However, none of the viruses had any of the polymerase (Song et al., 2009; Yao et al., 2013; Zhang et al., 2011) (Table S1). All vi-
markers most well associated with mammalian adaptation of avian in- ruses had an asparagine (N) at amino acid position 31 of M2, a mutation
fluenza viruses, i.e. PB2 E627K or D701N (Herfst et al., 2012; Imai et associated with resistance to antivirals amantadine and rimantadine
al., 2012; Mehle and Doudna, 2009; Subbarao et al., 1993). (Belshe et al., 1988).
538 D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540

Fig. 3. Genotype constellations for H9N2. Segments of different colours represent N2% nucleotide difference. Different genotypes were assigned an arbitrary ‘Vietnam genotype #’ (VNG#).

4. Discussion cassette is thought to contribute to the pathogenicity of H7N9 viruses in


mammals, to confer zoonotic and poultry enzootic potential to other
Our results confirm the hyperendemicity of avian influenza viruses subtypes (e.g. H10N8), and is at least partly responsible for the extreme-
in chickens sampled in LBM of northern Vietnam, where influenza ly high transmissibility of H9N2 viruses in chickens (Pu et al., 2014;
H9N2 viruses are by far the dominant subtype in circulation. Our detec- Zhang et al., 2014). The H9N2 viruses circulating in northern Vietnam
tion rates are comparable to those reported from China, where overall also closely resembled recent H9N2 strains isolated from human clinical
prevalence of AIV in chickens has ranged from 21 to 36% in Hubei, 34– cases in China (Huang et al., 2015b). Like its more temperate neighbor,
46% in Zhejiang province (Chen et al., 2016), and 15.7% in Hunan China, Vietnam has seasonal fluctuations in AIV prevalence, with peak
(Huang et al., 2015a), and where H9N2 is the dominant AIV subtype de- incidence in HPAI the winter months of November to February
tected throughout all provinces (Shi et al., 2014). Although a plethora of (Durand et al., 2015; Nguyen et al., 2014), and a similar pattern for
AIV subtypes are known to circulate in wild and domestic Vietnamese LPAI (Pu et al., 2014).
ducks, including H3, H4, H5, H6, H7 and H10 viruses (Hotta et al., Although the current surveillance system provides an excellent op-
2012; Jadhao et al., 2009; D. C. Nguyen et al., 2005; Nomura et al., portunity to monitor diversity of co-circulating HPAI H5 and LPAI
2012; Okamatsu et al., 2013; Takakuwa et al., 2013), our deep sequenc- H9N2, the process of combining swabs from multiple birds (i.e. pooling)
ing data confirm that the only detectable AIV subtypes circulating en- complicates the inference of co-infection rates (and definitely decrease
demically within chickens are H5 and H9. Additionally, although no its power), and complicates analysis of intersubtypic reassortment.
Chinese-like H7N9 viruses have presently been found in Vietnam, the Among the deep sequencing data generated directly from surveillance
H9N2 viruses sequenced here shared the H7N9-like G57/genotype S in- swabs, although we could not assess intersubtypic reassortment be-
ternal gene cassette (Gu et al., 2014; Pu et al., 2014). This internal gene tween H5 and H9N2 viruses, the genomic analysis suggested high levels
of intrasubtypic reassortment between different subclades of H9N2 (Liu
et al., 2014a, 2014b; Sun et al., 2010).
The genomic data demonstrated that most gene segments were
closely related to Chinese viruses, which likely reflects proximity of
our sampling sites to the Chinese-Vietnam border. Importation of live
chickens from China has been illegal since May 2013; hence, these find-
ings suggest that despite regulations to curtail illegal traffic in live poul-
try, there remains potential for cross-border spread. The pattern of
phylogenetic clustering of the H9N2 sequenced here revealed two dis-
tinct clusters for each gene segment (or sometime 3 clusters, as in the
case of the HA phylogeny), suggesting there may have been separate in-
troduction events. However, H9N2 viruses are known to be highly
transmissible between chickens and wild terrestrial birds (e.g. spar-
rows) (Iqbal et al., 2013), and thus cross-border wild bird migration
may also have played a role in geographical spread of H9 viruses. Inter-
estingly, the N2 sequences generated here were predominantly from
the BJ94-like lineage, and, unlike all other gene segments, were most
similar to Vietnamese N2s that have been circulating in Vietnam for at
least a decade suggesting the possibility of underlying continual, in
situ evolution of H9N2 viruses within Vietnam.
Given the evidence that prevalence and diversity of AIV in neighbor-
Fig. 4. Structural diagram showing antigenically important amino acid substitutions
ing China continues to increase at alarming rates (Chen, 2016; Huang
observed in H9HA. Made using pymol, amino acids mapped onto the HA structure of A/ 2015), and given that Vietnam ranks as one of the countries with
Swine/Hong Kong/9/98 (H9N2) PDBID:1JSD. highest clade diversity for HPAI H5 (Shi et al., 2014), the public health
D.M. Thuy et al. / Infection, Genetics and Evolution 44 (2016) 530–540 539

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