Journal of Medical Virology 87:1641–1648 (2015)

Human Infection With an Avian Influenza A (H9N2) Virus in the Middle Region of China Yiwei Huang,1† Xiaodan Li,2† Hong Zhang,1† Bozhong Chen,3 Yonglin Jiang,3 Lei Yang,2 Wenfei Zhu,2 Shixiong Hu,1 Siyu Zhou,1 Yunli Tang,3 Xingyu Xiang,1 Fangcai Li,1 Wenchao Li,1 and Lidong Gao1 1

Hunan Provincial Center for Disease Control and Prevention, Changsha, China National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China 3 Yongzhou City Center for Disease Control and Prevention, Yongzhou, China 2

During the epidemic period of the novel H7N9 viruses, an influenza A (H9N2) virus was isolated from a 7-year-old boy with influenza-like illness in Yongzhou city of Hunan province in November 2013. To identify the possible source of infection, environmental specimens collected from local live poultry markets epidemiologically linked to the human case in Yongzhou city were tested for influenza type A and its subtypes H5, H7, and H9 using realtime RT-PCR methods as well as virus isolation, and four other H9N2 viruses were isolated. The real-time RT-PCR results showed that the environment was highly contaminated with avian influenza H9 subtype viruses (18.0%). Sequencing analyses revealed that the virus isolated from the patient, which was highly similar (98.5–99.8%) to one of isolates from environment in complete genome sequences, was of avian origin. Based on phylogenetic and antigenic analyses, it belonged to genotype S and Y280 lineage. In addition, the virus exhibited high homology (95.7–99.5%) of all six internal gene lineages with the novel H7N9 and H10N8 viruses which caused epidemic and endemic in China. Meanwhile, it carried several mammalian adapted molecular residues including Q226L in HA protein, L13P in PB1 protein, K356R, S409N in PA protein, V15I in M1 protein, I28V, L55F in M2 protein, and E227K in NS protein. These findings reinforce the significance of continuous surveillance of H9N2 influenza viruses. J. Med. Virol. 87:1641–1648, 2015. © 2015 Wiley Periodicals, Inc. KEY WORDS:

avian influenza virus; H9N2; human infection; phylogenetic analysis

C 2015 WILEY PERIODICALS, INC. 

INTRODUCTION Avian influenza A (H9N2) viruses are widely endemic in domestic poultry throughout Eurasia, and mainly circulate in wild birds in North America [Alexander, 2007; Hossain et al., 2008]. Similar to all the other influenza A viruses, H9N2 viruses have natural reservoirs as wild birds. More and more studies indicated that H9N2 viruses had been adapted well and were prevalent in gallinaceous poultry in Asia since the early 1990s [Alexander, 2000]. Various reassortments among different H9N2 strains or lineages resulted in a variety of genotypes. Among these genotypes, three distinct lineages, represented by prototype viruses A/Quail/Hong Kong/G1/97 (G1), A/Duck/ Hong Kong/Y280/97 (Y280), and A/Duck/Hong Kong/ Y439/97 (Y439), respectively, had been recognized as three predominant H9N2 lineages in birds. Of them, G1 and Y280 lineages were considered to be dominant in poultry [Fusaro et al., 2011]. Another lineage of A/chicken/Shanghai/F/98 (F98) emerged in eastern China since 1998 and reassorted with other lineages, resulting in multiple distinct genotypes, one of which was genotype S that bore the backbone of A/chicken/ Shanghai/F/98-like viruses by acquiring A/quail/HongKong/G1/97-like PB2 and M genes [Zhang et al., 2009; Gu et al., 2014]. Furthermore, H9N2 can be occasionally isolated from mammalian species such as pigs, and even humans [Guo et al., 1999; Butt et al., 2005; Cheng et al., 2011; Yu et al., 2011].



These authors contributed equally to this article. Correspondence to: Lidong Gao, Hunan Provincial Center for Disease Control and Prevention, No.450, Section 1, Furong Rd, Changsha, China. E-mail: [email protected] Received in original form 22 December 2014; revised form 6 March 2015; Accepted 8 April 2015 

DOI 10.1002/jmv.24231 Published online 12 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

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The first five laboratory confirmed cases infected with avian influenza A (H9N2) viruses were recorded in Southern China in 1998, and the patients suffered from acute respiratory disease and recovered finally [Guo et al., 1999]. Additional three human cases were reported in Hong Kong in the following four years [Gou et al., 2000; Butt et al., 2005]. All these patients were children with uncomplicated influenzalike illness and fully recovered. In 2008 and 2009, two more H9N2 cases were reported [Cheng et al., 2011]. In 2011, a human infected with an avian influenza A (H9N2) virus that contained multiple mammalian molecular characteristics was identified in Bangladesh [Shanmuganatham et al., 2013]. One additional case was laboratory confirmed at the end of 2013, during the second wave of H7N9 epidemic in China [http://www.scmp.com/news/hong-kong/article/ 1393266/86-year-old-man-infected-h9n2-avian-flu]. Emerging interspecies transmission of H9N2 viruses highlight their persistent threat to human health. Since March 2013, several novel avian influenza A viruses including H7N9 and H10N8 viruses which we had never known before had attacked humans in China [Gao et al., 2013; Chen et al., 2014]. What they had in common was that they both had six internal genes of H9N2 origin. The H9N2 viruses had also been well known to donate their internal genes to the highly pathogenic H5N1 avian influenza viruses in human in Hong Kong [Guan et al., 1999]. Therefore, continuing surveillance of genetic evolution of H9N2 viruses especially in human infections are very important for understanding the behavior of these viruses. During the epidemic periods of novel avian influenza H7N9 spread, we isolated an H9N2 virus from a 7-year-old boy with influenza-like illness (ILI) in Yongzhou city, Hunan province, located in the middle of China, in 2013 through Chinese national ILI surveillance network, and explored its possible sources of infection as well as its genetic evolution and molecular characteristics.

using 10-day-old SPF embryonated chicken eggs based on standard procedures as recommended by the World Health Organization (WHO). The viral RNA was extracted from the harvested liquids using QIAamp Vrial RNA Mini Kit (QIAGEN, Hilden, Germany). Genomic sequences were amplified using Qiagen OneStep RT-PCR Kit (QIAGEN). Primer sequences are available from the authors on request. PCR products were sequenced by using the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) on Applied Biosystems 3730xl DNA analyzer (Applied Biosystems). Phylogenetic Analysis The complete genome sequence alignments were performed with MegAlign of Lasergene 7.01 software package. Phylogenetic analysis was based on complete genome sequences of the eight segments. Phylogenetic trees were constructed by using neighbor-joining method by means of bootstrap analysis with 1000 replications in the MEGA software (Version 5.10). Antigenic Analysis Antigenic relationships between the A/HunanLengshuitan/11197/2013 and H9N2 viruses of different lineages were investigated by hemagglutination inhibition (HI) assay with post-infection ferret antisera as recommended by the WHO. Nucleotide Sequence Accession Numbers All eight sequences of the five isolates in our study were submitted to the GenBank database and the accession numbers were KM455869 to KM455876 and KP289291 to KP289322. RESULTS Patient

MATERIALS AND METHODS Sampling and Testing A throat swab of the patient was collected from the Yongzhou City Central Hospital on day 1 after illness onset. To determine the possible source of infection, 742 environmental samples involving poultry drinking water, sewage, poultry feces, cage swabs, and chopping board swabs were collected from 12 live poultry trade and slaughter markets epidemiologically linked to the human H9N2 infection case in Yongzhou city from November of 2013 to May of 2014. Samples were tested by real-time RT-PCR assays for influenza A, its subtype H5, H7, and H9. Sequences of primers and probes are available on request. Virus Isolation and Genome Sequencing Virus isolation was conducted from samples which were tested positive for influenza H9 subtype J. Med. Virol. DOI 10.1002/jmv

On November 20th, 2013, a 7-year-old boy in Yongzhou city of Hunan province sought medical advice for his upper respiratory tract infection symptoms and high body temperature in pediatric specialty clinic of the Yongzhou City Central Hospital. Clinical features included high fever (39.2˚C on day 1 after illness onset), runny nose, sneezing, red throat, and rough but without rales lung breath. New symptoms including vomiting and diarrhea appeared on day 3. Blood test results on day 2 were 15.52  109 per liter of white blood cell count (WBC) (normal range: 8.0–10.0  109 per liter), 12.35  109 per liter of neutrophil count (normal range: 1.8–6.3  109 per liter), and 0.79 of percent neutrophils (normal range: 0.40–0.75). The elevated WBC may indicate a secondary bacterial infection. That neutrophil count and percent neutrophils elevated was common in influenza virus infection. His fever resolved 4 days without complication. Five days later, the symptoms

Human Infection With an Avian Influenza A (H9N2) Virus

disappeared and the patient recovered. Epidemiological investigation showed the case had no history of direct contact with birds. However two live poultry markets located 500 m far from his home to the east and west, respectively, and his family often bought slaughtered chickens and ducks from the markets. Environmental Sample Testing We collected and tested environmental samples from the two live poultry markets and ten large-scale live poultry trade and slaughter markets epidemiologically linked to the human case for 6 months since the discovery of the patient. Of 742 environmental samples, 185 samples (24.9%) were positive for influenza A viruses and 133 (18.0%) for H9 subtype, including 82 (11.1%) for single H9 and 51 (6.9%) for both H5 and H9 positive (Table I). An H9N2 virus from the patient and four from the environment were isolated and termed as A/Hunan-Lengshuitan/11197/2013 (H11197), A/Environment/Hunan/20502/2013 (E20502), A/Environment/ Hunan/26018/2014 (E26018), A/Environment/Hunan/ 28176/2014 (E28176), and A/Environment/Hunan/ 28184/2014 (E28184). Phylogenetic Analysis In order to explore the genetic relationships, full genome of the five strains were sequenced and deposited in GenBank. All eight gene segments of the patient’s isolate H11197 shared 98.5–99.8% nucleotide homology with E26018. Additionally, six internal genes had a 95.7–98% and 95.9–99.5% homology with the human H7N9 and H10N8 isolates -A/Changsha/1/ 2013 (H7N9) and A/Jiangxi-Donghu/346/2013 (H10N8), respectively. To facilitate the phylogenetic analysis, sequences of representative H9N2 viruses of G1, Y280, and Y439 lineages were downloaded from the Global Initiative on Sharing Avian Influenza Data (GISAID) and GenBank databases. Phylogenetic analysis revealed that all five isolates had the same gene constellations of genotype S, which had PB2 and M genes of G1 lineage and other 6 genes of F98 lineage [Gu et al.,

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2014]. All five viruses including H11197 fell into the cluster of Y280 lineage of hemagglutinin (HA) genes (Fig. 1A). Each internal gene of our H9N2 viruses fell into the cluster together with that of the 2013 H7N9 and H10N8 viruses (Fig. 1C–H). However they exhibited genetic diversity in eight gene segments except PB2, PB1 and NS genes. Specifically, in HA gene, H11197, E26018, and E20502 were in a same branch, in contrast E28176 and E28184 were closer to a patient’s isolate of Hong Kong (A/Hong Kong/308/2014) (Fig. 1A). In NA gene, E26018 and H11197 were in a same branch, otherwise the other three viruses in another one (Fig. 1B). In PA gene, E28176 and E28184 were similar to an H7N9 isolates (A/Shandong/01/2013(H7N9)) (Fig. 1E). In NP gene, E28176 and E28184 were highly homologous to an H7N9 isolates in our province (A/Changsha/1/2013 (H7N9)) (Fig. 1F). Additionally, in M gene, E20502 were in a separate branch (Fig. 1G). Antigenic Analysis The HI test showed the virus of the patient was clearly distinguishable from A/Hong Kong/1073/1999, the candidate vaccine virus of G1 lineage. The virus reacted well to antisera against both A/Chicken/HK/ G9/1997 (HI titers ¼ 5,120), the candidate vaccine virus of Y280 lineage, and A/Guinea Fowl/HK/NT101/ 2003 (HI titers  20,480) (Table II). Therefore the virus was of Y280 lineage, in correlated with the phylogenetic analysis. Molecular Characteristics Deduced amino acid sequences were compared to explore the molecular characterization of the virus isolated from the patient (Table III). Compared to the four strains from environment, we observed only three amino acids of consistent mutation whose functions were unknown: R258K, I392V of NA protein and I667V of PB1 protein. S43N, P45S, D208N, R338K, N342S of NA protein and V408I of NP protein were also found in both H11197 and E26018 compared to the other three viruses from environment. In addition,

TABLE I. Prevalence of avian influenza viruses in specimens collected from poultry markets in Yongzhou city, Hunan province, China, determined by real-time RT-PCR No. of samples positive for subtypes Year/Month 2013/Nov 2013/Dec 2014/Jan 2014/Feb 2014/Mar 2014/Apr 2014/May Total

No. of samples

No. of Pos. (%)

H5

H7

H9

Other types

H5,H7

H5,H9

H7,H9

H5,H7,H9

30 20 60 194 180 200 58 742

3 (10) 4 (20) 2 (3.3) 38 (19.6) 41 (22.8) 80 (40) 17 (29.3) 185 (24.9)

0 0 0 9 8 17 2 36

0 0 1 0 1 0 0 2

3 4 0 14 13 36 12 82

0 0 0 0 0 1 0 1

0 0 0 0 2 0 0 2

0 0 0 14 12 22 3 51

0 0 1 1 1 2 0 5

0 0 0 0 4 2 0 6

J. Med. Virol. DOI 10.1002/jmv

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Fig. 1. Continued.

J. Med. Virol. DOI 10.1002/jmv

Human Infection With an Avian Influenza A (H9N2) Virus

the virus of H11197 carried amino acid substitution Q226L (H3 numbering), a mutation that correlated with a shift in affinity of the HA from the “avian” type sialic receptors to the human type and from a preference for a 2,3 link to a preference for a 2,6 link between the sialic acid residues and galactose [Matrosovich et al., 2001]. The HA cleavage site of H11197 had an R-S-S-R motif, representing its low pathogencity in chickens. The combination of amino acid at position 183, 189, 190, and 226 of the HA protein was essential for respiratory droplet transmission of an avian-human H9N2 reassortant [Sorrell et al., 2009]. In these four positions, H11197 had N183, T189, A190, and L226, and we identified only one change at position 190 compared to three of four environment strains. In NA protein it had a 3-aa deletion at positions 63-65 in the stalk region, indicating its potential terrestrial poultry adaptation. No oseltamivir or zanamivir resistance substitutions had been found in this virus, indicating its sensitivity to neuraminidase inhibitors [Shanmuganatham et al., 2013]. The analysis of the M2 protein sequence showed that the virus possessed S31N substitution which increased resistance to the adamantine [Scholtissek et al., 1998]. Some mammalian host-specific markers had been found in the PB1 (L13P), PA (K356R, S 409N), M (M1 V15I, M2 I28V and L55F), and NS1 protein (E227K) [Fusaro et al., 2011; Shanmuganatham et al., 2013]. DISCUSSION Chinese national ILI surveillance network has been originally constructed for vaccine virus selections. With its gradual maturity, the network has been proved to play an increasingly important role in pandemic preparedness as well. Since its establishment, Chinese national ILI surveillance network has hunted human infections with several un-human influenza viruses accurately, including Eurasian Avian-like Influenza A (H1N1) Virus [Wang et al., 2013], novel influenza A (H7N9) and H10N8 viruses [Ip et al., 2013; Chen et al., 2014; Wang et al., 2014]. Here, the 7-year-old child with H9N2 infection in Hunan province elaborated in our study was also identified through this national network. It has been a hot spot clamoring scientists’ attention that people infected with the avian influenza viruses, because of its potential possibilities that cause an influenza pandemic. Avian influenza viruses

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from live poultry markets have been considered one of the main sources of human infection. Although there was no evidence of direct contact, we found that H9 subtype of avian influenza viruses were widely distributed in poultry markets and isolated a highly homology virus which may be the source of infection. In neighboring Guangxi province, it was a similar monitoring result that 10.8% samples (336/3121) were low pathogenic avian influenza virus positive in live bird markets [Peng et al., 2013]. It seemed that most people were not susceptible to avian influenza viruses as live poultry markets were high contaminated with avian influenza viruses while only a handful of patients were found. However we have to closely monitor the evolution of the viruses, especially in human cases. Our analyses showed that the human H9N2 virus as well as the viruses isolated from the environment in Yongzhou city had evolved to acquire multiple mammalian host-specific mutations, including changes in the hemagglutinin, matrix, nonstructural and polymerase proteins. Receptor specificity of the HA protein is important in determining host range. All five strains had the amino acid L at position 226 instead of Q at the receptor-binding site of the HA protein, which facilitated preferential binding to human a-2,6-linked sialic acid receptors [Matrosovich et al., 2001; Wan and Perez, 2007; Wan et al., 2008]. However, they had different combinations from an avian-human H9N2 reassortant (H183, A189, E190, and L226) of the four positions in the HA protein which were critical for respiratory droplet transmission in ferrets [Sorrell et al., 2009]. These multiple mammalian host-specific mutations imply that these H9N2 viruses may pose a potential threat to human health, nevertheless how the mutations contribute to human infection have yet to be determined. In fact, a more powerful ability that should be concerned for H9N2 viruses comparing to other subtypes is their wild-range distribution and their high gene compatibility with other subtypes of influenza A viruses. H9N2 viruses are widely spread in China and eastern Asia. They are also the most abundant influenza viruses isolated in live poultry market system [Choi et al., 2004; Ge et al., 2009], with the Y280 lineage of H9N2 viruses having been prevalent in poultry in central and southern China [Ji et al., 2010; Chu et al., 2011]. Therefore it is not surprised that the H9N2 virus isolated from the mild case of

3

Fig. 1. Phylogenetic analysis of the full genes of the human avian influenza H9N2 virus in Yongzhou city, China. Phylogenetic trees of the nucleotide sequences for the eight genes (HA, NA, PB2, PB1, PA, NP, M, NS (A–H), respectively) of the human avian influenza H9N2 virus in this study (in red) compared with nucleotide sequences of representative H9N2 viruses of G1, Y280, and Y439 lineages. Novel H7N9 and H10N8 viral sequences were supplies by Chinese National Influenza Center and added as well for assaying the phylogenetic relationship of these two subtype viruses with our H9N2 virus. Multiple alignments were constructed by using the MEGA 5.10 software. Phylogenetic trees were constructed by using the neighbor-joining method with bootstrap analyses of 1,000 replications. Bootstrap values over 60% were shown in the nodes. Solid triangles and hollow triangle showed the H9N2 viruses isolated from the patient and the live poultry markets in this study, respectively. J. Med. Virol. DOI 10.1002/jmv

J. Med. Virol. DOI 10.1002/jmv

North American Ck/BJ/1/94 Y280 Y280 Y280 G1 G1 Y280

Lineage

5 20 160 40 80 80 80

160

183 N N N N N

Virus

A/Hunan-Lengshuitan/11197/2013 A/Environment/Hunan/20502/2013 A/Environment/Hunan/26018/2014 A/Environment/Hunan/28176/2014 A/Environment/Huna /28184/2014

According to the H3 numbering.

*

1280 640 640 1280 80 20 2560

10 320 2560 1280 10240 40 20 5120

5 320 1280 2560 2560 40 20 1280

10 160 80 160 320 2560 1280 640

5

A/Chicken/ A/Chicken/ A/Guangzho/ A/HK/ Beijing/1/1994 HK/G9/1997 333/1999 1073/1999

10 40 80 320 2560 1280 640

5

A/Quail/Hong Kong/G1/97

T T T T T

189 A A T T T

190 L L L L L

226 M M M M M

227

Receptor binding site

HA*

G G G G G

228 RSSR RSSR RSSR RSSR RSRR

Cleavage site

K R R R R

258

V I I I I

392

Yes Yes Yes Yes Yes

63-65

Stalk deletions

NA

I I I I I

15

M1

V V V V V

28

F F F F F

55

M2

K K K K K

227

NS

P P P P P

13

V I I I I

667

PB1

R R R R R

356

N N N N N

409

PA

160 2560 1280 20480 20 10 20480

5

A/Guinea Fowl/HK/ NT101/2003

TABLE III. Comparison of critical amino acid residues in proteins of influenza A (H9N2) viruses from Yongzhou city, Hunan province, China

Homologous titers are indicated in bold. * The H9N2 influenza virus analyzed in this study were highlighted in italic.

A/Chicken/Beijing/1/1994 A/Chicken/HK/G9/1997 A/Guangzhou/333/1999 A/Guinea Fowl/HK/NT101/2003 A/HK/1073/1999 A/Quail/Hong Kong/G1/97 A/Hunan-Lengshuitan/11197/2013

A/Shore bird/DE/249/2006

Reference virus

A/Shore bird/DE/249/06

HI titers with post-infection ferret antisera*

TABLE II. Antigenic analysis of H9N2 avian influenza viruses

1646 Huang et al.

Human Infection With an Avian Influenza A (H9N2) Virus

7-year-old boy in our study is of Y280 lineage and a pure avian-origin virus without reassortment. Despite of this, different subtypes of avian influenza viruses co-circulating widely in poultry raise a concern about creation of multiple reassortants. H9N2 viruses exhibit strong gene compatibility with other subtypes of influenza A viruses and reassortants. As we mentioned previously, human H5N1, H7N9, and H10N8 viruses possessed internal gene cassettes of poultry H9N2 viruses. Moreover, the novel H7N9 viruses are still continuously resorted with different H9N2 internal genes [Wang et al., 2014]. Experimentally, H9N2 viruses exhibited high compatibility with pandemic H1N1/2009 influenza viruses and seasonal human H3N2 and reassortant viruses have shown higher virulence or efficient transmission in mammalian models [Sorrell et al., 2009; Kimble et al., 2011; Sun et al., 2011]. Therefore, wild-distributed H9N2 provided an opportunity for these viruses to reassort. Reassortments between the wild birds-origin influenza viruses and poultry H9N2 viruses could generate a more domestic hostadapted influenza virus. Meanwhile, high gene compatibility could cause reassortments between H9N2 and other mammalian influenza viruses more workable and make the influenza evolve to adapt to the mammalian host. Since 1998, when the first human infection with H9N2 avian influenza viruses was detected, isolation of the viruses from humans has been reported occasionally in Hong Kong and Mainland China [Guo et al., 1999; Peiris et al., 1999]. Similar to all previously reported H9N2 human cases, the associated disease symptoms of the case in our study were mild. However, internal genes of diverse H9-lineage viruses have been showed high fitness for replication in both mammalian models and human cells [Group, 2013]. Thus, internal genes of H9N2 would still have the potential to generate the pandemic isolates, exemplified as the novel H7N9 and H5N1 viruses. Therefore, close monitoring of H9N2 avian influenza viruses in both human populations and their intimate animals are essential for timely discovery of potential pandemic influenza viruses.

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J. Med. Virol. DOI 10.1002/jmv

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Human infection with an avian influenza A (H9N2) virus in the middle region of China.

During the epidemic period of the novel H7N9 viruses, an influenza A (H9N2) virus was isolated from a 7-year-old boy with influenza-like illness in Yo...
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