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Virus Research journal homepage: www.elsevier.com/locate/virusres
Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China
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Yanjun Zhang a,1 , Haiyan Mao a,1 , Juying Yan a,1 , Lei Zhang a,1 , Yi Sun a,1 , Xinying Wang a,1 , Yin Chen a , Yiyu Lu a , Enfu Chen a , Huakun Lv a , Liming Gong a , Zhen Li a , Jian Gao a , Changping Xu a , Yan Feng a , Qiong Ge a , Baoxiang Xu a , Fang Xu a , Zhangnv Yang a , Guoqiu Zhao b , Jiankang Han c , Koch Guus e , Hui Li f , Yuelong Shu d , Zhiping Chen a,∗ , Shichang Xia a,∗ a
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang 310051, China Center for Disease Control and Prevention of Hangzhou, Hangzhou, Zhejiang 310021, China Center for Disease Control and Prevention of Huzhou, Huzhou, Zhejiang 313000, China d National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Beijing 102206, China e Central Veterinary Institute, Part of Wageningen UR, Virology Department, 8200 AB Lelystad, The Netherlands f Shanghai Huirui Biotechnology Co. Ltd., Shanghai 200002, China b c
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a b s t r a c t
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Article history: Received 30 October 2013 Received in revised form 27 March 2014 Accepted 1 May 2014 Available online xxx
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Keywords: Influenza A H7N9
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1. Introduction
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In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E−3 (95% Highest posterior density: 3.8218E−3 to 7.7873E−3) while the NA gene showed 2.243E−3 (4.378E−4 to 3.79E−3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation. © 2014 Elsevier B.V. All rights reserved.
The avian influenza virus, H7N9, previously unknown in humans, was first detected in three urban residents in Shanghai and Anhui, China, in year 2013, who presented with rapidly progressing lower respiratory tract infections (Gao et al., 2013). It was reported
∗ Corresponding authors at: Zhejiang Provincial Center for Disease Control and Prevention, 630 Xincheng Road, Hangzhou, Zhejiang 310051, China. Tel.: +86 571 87115198; fax: +86 571 87115198. E-mail address: [email protected] (S. Xia). 1 These authors contributed equally to this article.
that most persons with confirmed H7N9 virus infection were critically ill and epidemiologically unrelated. Laboratory-confirmed human-to-human H7N9 virus transmission was not documented to have occurred through close contact (Li et al., 2013). Previous reports indicate that the virus does not appear to cause serious disease in birds, but has the potential for epidemiological and public health implications in humans (Spackman et al., 2010; Liu et al., 2013a,b). It was suspected that the H7N9 virus could establish a reservoir of infection that would lead to frequent sporadic human infections that appear without warning. Enhanced surveillance was implemented, resulting in the identification of additional H7N9 clinical cases in Zhejiang Province, which is located between Shanghai and Anhui.
http://dx.doi.org/10.1016/j.virusres.2014.05.002 0168-1702/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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We describe the isolation and characterization of two novel strains of the H7N9 avian influenza A virus (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) that were isolated from throat swabs of two H7N9 infected patients. To trace the origins of the virus and to understand its lineage and evolutionary dynamics, we sequenced the two genomes using next generation sequencing (NGS) methods. Our aims were to understand: (1) variation and mutations in the novel reassortants in H7N9; (2) determine the evolution/migration history based on phylogenetic methods from the hemagglutinin and neuraminidase lineage by comparing it with other viruses of avian origins; and (3) the role that live poultry markets have played in H7N9 circulation.
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2. Materials and methods
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2.1. Ethics statement and clinical samples
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This study was approved by the ethics committee of Zhejiang Provincial Center for Disease Control and Prevention (CDC), China. From April 2013 to May 2013, with permission of the patients, forty pharyngeal swabs were collected from patients suspected to be infected with the avian influenza virus H7N9. Fifty environmental samples comprising live poultry stools and water from the treatment of live poultry, were collected from the markets visited by the H7N9-infected patients (Han et al., 2013). The samples were stored in 3–5 ml of preservation solution (Hanks solution containing 100 U/ml penicillin and 100 g/ml streptomycin) at −70◦ C until analysis.
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2.2. Viral RNA extraction and RT-PCR detection
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Viral RNA was extracted using the RNeasy Mini kit (Qiagen, CA, USA) according to the manufacturer’s instructions. Multiplex Real time RT-PCR reactions were performed by using the AgPath-IDTM One-Step RT-PCR Kit (Life technologies). Procedures for the RT-PCR reactions followed the instructions of the manufacturer.
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2.3. Virus isolation and hemagglutination test
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Specific pathogen-free embryonated chicken eggs were used for virus isolation. Six 9-to-11-day-old chicken embryos were each inoculated with 200 l of sample by the chorioallantoic sac route. The eggs were incubated for 48 h at 35◦ C, after which allantoic fluid was tested for hemagglutination of 0.5% turkey red blood cells in a phosphate buffered saline (PBS) solution. Samples negative for hemagglutination were processed a second time.
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2.4. Electron microscopy identification
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The chorioallantoic fluid was fixed with 2.5% glutaraldehyde for 2 h at room temperature. The influenza virus was observed under transmission electron microscope (Hitachi H-600, Japan) (Malenovska, 2013).
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2.5. Creating and sequencing the library
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Building and sequencing the H7N9 library was completed in the Life Technologies Demo Center. Because of the low concentration of nucleic acid, we used PathAmp FluA Pre-Amplification Reagents (Life technologies) to enrich the eight segments in each sample for better performance in the ABI Ion Torrent. We then constructed two libraries consisting of random fragments averaging 200 bp in length. Each sample was assigned a different barcode in the Ion Xpress Plus Fragment Library Kit (Life technologies). In the final step, we pooled the two libraries evenly into 314 chips after
running them through quality control. The barcoded libraries were sequenced together in the Ion Torrent. 2.6. Data analysis Basic bioinformatic analysis, such as alignment and pathogen analyzer, were conducted after passing the data through quality checks. Both full genome virus sequences from the patients were deposited in the Global Initiative on Sharing Avian Influenza Data (GISAID) database. We then checked for variant positions in the amino acid sequences using MEGA 5.0 (www.mega.software. informer.com/5.0/) and calculated the identical index between isolates from humans and isolates from both human and birds, such as sequences infected chickens downloaded from GenBank (A/chicken/Zhejiang/DTZD-ZJU01/2013(H7N9)), for each segment in DNAStar Lasergene. v7.1 (www.dnastar.com). Phylogenetic trees were constructed using maximum likelihood (ML) based on raxml v7.2.8 (http://sco.h-its.org/exelixis/ software.html). We performed two multiple sequence alignments with data matrixes of other sequences downloaded from the public databases GISAID and GenBank according to the HA gene and NA gene for H7NX and HXN9 (X indicates the number of all possible combinations), respectively, using Geneious 4.8.3 (www.geneious.com). For the 115 sequences of HA and the 26 sequences of the NA segment partitions of the total evidence, an analysis using dataset-specific models was conducted by using the Akaike Information Criterion (AIC) in Modeltest 3.7 (Posada and Crandall, 1998). Model parameters were estimated in all analyses. The ML analysis for each data set was performed using the GTR model with a gamma distribution for site variation and setting the parameter values as ‘estimated’ using raxmlHPC (Stamatakis, 2006). BEAST 1.6 (http://beast.bio.ed.ac.uk/Main Page) was used to compute the substitution rate with uncorrelated lognormal distributed relaxed-clock model for our sample H7N9 virus segments HA and NA. Sequences in all analyses were downloaded from GenBank. We took two measures, as follows, to ensure the accuracy of our data and results: (1) we performed five simulations on BEAST with different sets of parameters; no significant differences were found among the final results from different runs; (2) because of the random sampling for each subtype and from different hosts (birds and humans), the rates would differ due to different sample matrices. We therefore re-sampled and tested several matrices. No significant differences were found. Therefore, for each virus subtype, we used the constant population size coalescent as the tree prior and data-specific model for substitution. Two replicates were run for 100,000,000 generations with tree and parameter sampling every 1000 generations. A burn-in of 10% was used and the convergence of all parameters assessed using the software TRACER within the BEAST package.
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3. Results
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3.1. Patients
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Table 1 showed the demographic and epidemiologic characteristics of both two patients. Patient 1 was a 37-year-old cook with chronic hepatitis B, working in Suzhou and living in Hangzhou, reported cough and diarrhea. He bought meals and avian birds from the local living poultry market 2 miles away every morning, prepared lunch and dinner for about 30 company staff. He died of acute respiratory distress syndrome, disseminated acute respiratory distress syndrome (ARDS) and intravascular coagulation, and multiorgan distress syndrome on March 27. Laboratory tests showed that the patient’s blood white cell count obviously
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Patient 1
Patient 2
Age Gender Occupation Area of origin Underlying conditions Exposure to live poultry markets in past 7 days Date of illness onset Date of admission Date of specimen collection Date of lab confirmation of virus Viral isolation
37 Male Cook Hangzhou in Zhejiang Hepatitis B Yes
64 Male Retired Huzhou in Zhejiang Chronic bronchitis Yes
March 7, 2013 March 19, 2013 March 24, 2013 April 1, 2013
March 29, 2013 March 31, 2013 April 3, 2013 April 3, 2013
A/Zhejiang/1/2013 (H7N9)
A/Zhejiang/2/2013 (H7N9)
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decreased in the early period to the lowest of 1.6 × 109 /L, then slowly climbed to the normal level after treatment, and abnormally elevated in the later period. He was confirmed as avian influenza A (H7N9) virus case by analyzing 8 segments performed in China CDC on April 1st, 2013, as the first confirmed case in Zhejiang Province. Patient 2 was a 64-year-old retried worker suffering long time chronic bronchitis. He visited market including living poultry every morning. He did not have high fever and cough at the onset of the illness, but he had severe pneumonia and respiratory distress syndrome in the later period. He was confirmed as H7N9 case by Zhejiang Provincial CDC on April 3rd, 2013.
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Pharyngeal swabs and environmental samples were first applied for H7N9 virus RNA detection. One-step multiplex qRT-PCR was performed to detect the H7N9 virus. A total of 36 samples (40%) were positive for H7N9. Fever and coughing were the most common symptoms presented in patients with H7N9 virus infections. Acute respiratory distress syndrome, respiratory failure, co-infection with bacteria, shock and congestive heart failure were also observed in these positive cases. The positive samples were then used for virus isolation through embryonated chicken eggs. After incubation, the allantoic fluid was used for the hemagglutination (HA) test. The HA test results showed 26 (29%) positive samples. Transmission electron microscopy (×106 ) was then used to observe the influenza virus in the positive allantoic fluid (Fig. 1).
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3.3. Sequence analysis
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Two samples, A/Zhejiang/1/2013(H7N9) and A/Zhejiang/2/2013(H7N9), with different barcodes in ABI Ion Torrent, resulted in a total of 464,974 reads with a mean length of 163 bp, of which more than 39,600 reads passed quality filtering. The average coverage for both samples was 2551 and 2628. The figures in the supplemental materials show the average coverage for each segment in each individual. Two alignments were obtained for the HA gene of H7NX with 115 sequences and for the NA gene of HXN9 with 26 sequences. Both included five novel reassortant H7N9 viruses, with three from the National CDC and two from our own sequences. Complete sequences of the five novel reassortant H7N9 revealed that they were 97.3–100% identical in all 8 gene segments while they were 94.6–97.8% identical in isolates from humans and from chickens (Table 2). Table 3 shows the substitutions found in addition to the variations shown in the China CDC table (Gao et al., 2013). Mutations at positions T155Y and G186V in the HA gene in both Zhejiang viruses are associated with receptor binding (Masuda
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et al., 1999; Chen et al., 2013). A/Zhejiang/1/2013 had substitution Q226I while A/Zhejiang/2/2013 had the more common Q226L in the HA segment. Zhejiang/1 showed E627K changes, while Zhejiang/2 showed D701N changes in the PB2 segment. Both 627 and 701 are considered to be mutation sites necessary for mammalian adaptation (Chen et al., 2013). The two variations were found to coexist in highly pathogenic H5N1 isolates. Li et al. (2003) hypothesized that the both of these variations could facilitate effective transmission. V100A, K356R, S409N identified in polymerase PA protein can cause attenuation in mammals (Yamayoshi et al., 2013). The D66S mutation in the PB1-F2 protein, found in both Zhejiang isolates, demonstrated increased pathogenicity in the recombinant H5N1 virus by Korteweg and Gu (2008). Other substitutions were compared to the first three isolation obtained from China CDC (Shanghai/1, Shanghai/2 and Anhui/1), mutations in samples from Zhejiang Province displayed the similar patterns: motif PEIPKGR*G in amino acid positions 333–340 in the HA gene and conserved glycosylation motifs at positions 30, 46, 249, 421 and 493. Receptor binding site positions altered receptor specificity site G228S in HA was found in both Zhejiang virus. Drug resistant site (R294K in NA and S31N in M2), mutations in increasing virulence in mice (L89 V in PB2, N30D, T215A in M1 and P42S in NS1 segment), variation site for H5 subtype transmissible among ferrets (H99Y and I368V in PB1), were also detected in our Zhejiang isolations. Maximum parsimony, maximum likelihood and Bayesian analysis of the 115 H7NX HA sequences resulted in basically similar topological patterns for the phylogenetic tree (Fig. 2). All phylogenetic trees showed that all 115 HA segments diverged into two groups: a North American and a European-Asian group, with high bootstrap values (BS) in MP and Bayesian posterior probability (PP). Within the Asian clade, the five novel reassortant H7N9s showed a monophyletic relationship with virus A/duck/Zhejiang/2011 as sister group with 100% BS and PP support. Two H7N9 viruses from Zhejiang Province, A/Zhejiang/1/2013 and A/Zhejiang/2/2013, were most closely related to each other. Viruses from Shanghai and Anhui were their sister groups. The phylogenetic relationships of the sequences from the 26 HXN9s were nearly identical, since their alignment strongly supported a monophyletic relationship among the five novel reassortant H7N9s with high BS and PP values as well (Fig. 3). Viruses closely related to the H7N9 clade were A/Anas crecca/Spain/1460/2008, A/duck/Mongolia/119/2008 and A/wild duck/Korea/SH20-27/2008. All phylogenetic results indicate that the novel reassortant H7N9 originated from bird viruses. The Bayesian MCMC method estimated the mean rate of evolution of the HA gene in H7NX to be 5.74E−3 (95% HPD: 3.8218E−3 to 7.7873E−3) while the NA gene in the HxN9 matrix showed 2.243E−3 (4.378E−4 to 3.79E−3) substitutions per nucleotide site per year.
4. Discussion To confirm the origin of the H7N9 virus to be poultry, we analyzed whole genome sequences from two isolates taken from human patients in Zhejiang Province and used them for phylogenetic analysis. We compared our two viral amino acid sequences with the first three samples published by the Chinese CDC and found several new mutations in the novel reassortant H7N9. In both Zhejiang isolates, most mutations encoded for human/mammalian amino acid adaptations. Because of the high substitution rate, the HA segment is considered to be the most diverse segment in the influenza virus, not only at the nuclear level but also in amino acids (Worobey et al., 2014). Chen et al. (2013) reported that T155Y, found in all HA segments of 5 H7N9 isolates, could have played an important role in detecting ␣-2,6-linked sialic acids. Q226L in the HA protein is one of the receptor binding sites,
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Fig. 1. Shape of the influenza A (H7N9) virus observed by transmission electron microscopy.
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the same as G186V, which increases binding affinity for ␣-2,6linked sialic acid receptors. Glutamine converted to Isoleucine in A/Zhejiang/1/2013 may fail to bind to ␣-2,6 human-like receptors due to a mutation at position 226. We also suspect that the E627 K substitution in the PB2 segment is responsible for restoring the ability of the PB2 single gene reassortant to replicate in MDCK cells. Recent studies (Gabriel et al., 2005; Manz et al., 2013) suggest
that increases in the ability to replicate may be critical for allowing avian influenza viruses to replicate in mammals. Interestingly, A/Zhejiang/2/2013 virus has glutamic acid at the 627 site, which may facilitate bird/human infections and result in severe disease. Massin et al. (2001), Subbarao et al. (1993) and Tarendeau et al. (2008) showed that the PB2 of all bird isolates has glutamine at position 627, while mammalian viruses have lysine at this position.
Table 2 Information on each segment of each novel H7N9 of avian origin. Gene
PB2 PB1 PA HA NP NA MP NS
Nucleotide length (bp)
2280 2274 2151 1683 1497 1398 982 838
GISAID number
Identities to A/Anhui/1/2013
A/Zhejiang/1/2013
A/Zhejiang/2/2013
EPI443031 EPI443032 EPI443033 EPI443034 EPI443035 EPI443036 EPI443037 EPI443038
EPI443039 EPI443040 EPI443041 EPI443042 EPI443043 EPI443044 EPI443045 EPI443046
99.4–99.9 99.4–99.9 98.2–100.0 99.2–100.0 97.3–100.0 97.3–100.0 99.8–100.0 99.3–100.0
Identities to A/Chicken/Zhejiang/DTID-ZJU01/2013(H7N9) Genbank accession: KC899666–KC899673
97.8–99.7 97.3–99.7 94.6–99.4 96.9–99.5 97.3–99.1 97.0–99.7 97.6–99.9 96.1–99.7
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Fig. 2. Phylogenetic tree based on the HA gene in H7NX subtypes. An ML tree is shown with MP bootstrap and Bayesian posterior probability above (BS/PP value). Branches with BS/PP values were found in all phylogenetic analyses. (A) Whole maximum likelihood tree. (B) Enlargement of 5 novel avian-origin H7N9 with their sister groups.
Fig. 3. Phylogenetic tree based on the NA gene in HXN9 subtypes. An ML tree is shown with MP bootstrap and Bayesian posterior probability above (BS/PP value). Branches with BS/PP values were found in all phylogenetic analyses.
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Table 3 Substitutions in segments in each novel H7N9 of avian origin. Substitutions that are consisted with Chinese CDC isolations published in NEJM (Gao et al., 2013) are not present in below. Gene
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Site
Position
A/Zhejiang/1/2013
A/Zhejiang/2/2013
A/Anhui/1/2013
HA
Receptor binding site
T155Y G186V Q226L
Y V I
Y V L
Y V L
PB2
Replication of AIV in mammals
E627K D701N
K D
E N
K D
PB1-F2
H5N1 virus increased virulence in mice
D66S
S
S
S
PA
Attenuating in mammals
V100A K356R S409N
A R N
V R S
A R N
They concluded that residue 627 is host determinant and correlates with virulence of the influenza A viruses in primates. The mutation at site 701 in the PB2 segment of the A/Zhejiang/2/2012 genotype indicated that this strain may exhibit enhanced transmission in guinea pigs (Chen et al., 2013). These two variations were found to coexist in highly pathogenic H5N1 isolates and it is believed that they may facilitate effective transmission (Li et al., 2003). Moreover, a recent study by Conenello et al. (2007) of recombinant viruses demonstrated that a single mutation in the PB1-F2 protein D66S in H5N1 increased virulence in mice. Expression of the PB1-F2 protein may cause immune-mediated injury and a decline in the survival rate in mice (McAuley et al., 2013). V100A, K356R and S409N are considered to be host specific amino acid mutations in the PA segment that allow H7N9 to replicate in mammals rather than in avian cells (Yamayoshi et al., 2013). In the same study, Yamayoshi indicated that the PA protein has several variants that attenuate in mammals. Additional mutation sites, such as R65K in HA, need further analysis to determine their function and relationships with the phenotype. To determine the evolutionary history of amino acid changes in HA and NA segments, we produced phylogenic trees for both of them using maximum parsimony, maximum likelihood and Bayesian methods. Different viral strains associated with different geographic regions were seen in trees based on the HA and NA genes. All three methods for constructing trees showed similar phylogenetic topologies. The strains were separated into two large groups, a North American group and a European-Asian group. Viruses from the same country or region clustered together within each clade with strong support, indicating that the viruses within a region exhibit their own evolutionary lineage due to adaptations within the local area. The trees also showed multiple origins and rapid evolution with fascinating substitution rates in different countries and areas in the North American and European-Asian subgroups (Fig. 2). The human H7N9 isolates are likely the result of a reassortment of three viruses: A/duck/Zhejiang/2011(H7N3-like), A/wild bird/Korea/A14/2011 (KO14-like H7N9) and A/brambling/Beijing/16/2012 (BJ16-like H9N2). The genetic relationship of these five novel H7N9 strains that infect humans showed monophyly with high bootstrap values in the HA lineage. The identification of a novel reassortant influenza A (H7N9) virus, which has been associated with severe human infections in Shanghai and Anhui, is of great significance worldwide, especially in areas adjacent to known areas of infection. A reciprocal monophyletic pattern of HA trees of A/Shanghai/1/2013 and other strains indicated that the virus is more ancient than any of the other four virus and may have been one of the first avian viruses to infect humans. There is also indication of more than one origin of the virus in the Shanghai area according to the topological structure of the monophyletic clade. Because A/Zhejiang/1/2013(H7N9) was isolated from a 37-year-old cook working in Suzhou, Jiangsu Province, and living
in Hangzhou, Zhejiang Province, the virus could be considered to have originated in Jiangsu Province. The nesting of the two strains of viruses from Zhejiang as a derived clade within the Shanghai and Anhui groups may reflect past migrations to Zhejiang and Jiangsu from Shanghai. The findings indicate that the H7N9 virus was transmitted to Zhejiang Province within a short time period. The novel influenza A (H7N9) virus may have been circulating in urban areas of southeast China. Frequent contact with poultry, immune function suppression and elderly persons with underlying diseases were the most common characteristics of people who were infected (Chen et al., 2013). There is no strong evidence to prove transmission from person to person. Further analysis of birds, humans and the environment are needed to determine the relationships and migration routes of the vectors. Live poultry markets (LPMs) are popular in China, since Chinese people prefer to purchase freshly killed chickens for immediate cooking. One survey reported that 80% of households obtain poultry at LPMs at least once a year (Liao et al., 2009; Yu et al., 2014). Live poultry markets play an important role in the circulation of H7N9. They provide a perfect place for mixing different strains of avian influenza, and for transmission and infection, as both cases isolated in Zhejiang showed (Table 1; Li et al., 2013; Yu et al., 2013; Liu et al., 2013a,b). During our enhanced surveillance of H7N9 virus Q2 infections in March and April, 2013, in Zhejiang, positive infections obtained in LPMs were also identified. To control the spread of H7N9, the LPMs in Hangzhou and Huzhou, where H7N9 virus infections were found, were closed as a precautionary public health measure in April 2013 and disinfected by the government. Epidemiological studies indicate that exposure to live poultry in the LPMs is a major risk of H7N9 infection in humans, as closure of the LPMs reduced the mean daily number of infections by 99% (92–100%) in Hangzhou and by 97% (68–100%) in Huzhou (Yu et al., 2013). When the LPMs reopened during the last several months of 2013 due to economic and political pressure, the number of confirmed cases soared in the days before the spring festival of 2014. Recent studies in Zhejiang Province showed that risk factors contributing to H7N9 infection include a long history of smoking, chronic lung disease, immune-suppressive disorders and delayed oseltamivir treatment (Gao et al., 2013; Liu et al., 2013a,b). Centralized slaughter of live poultry would be an effective strategy to prevent susceptible populations from becoming infected. The Zhejiang provincial government, through collaboration among many departments, will permanently shut down all LPMs in July 2014. Authors’ contributions YZ, JY, HM, LZ, YS, XW contributed to the original draft of the manuscript, and approved the final version. GZ, JH, KG, HL, YS contributed to the concepts and design of the manuscript and in revising the manuscript. YC, YL, EC, HL, LM, ZL, JG, CX, YF, QG, BX, FX, ZY performed the experiments and provided information and
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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suggestions. ZC and SX are corresponding authors. All authors read and approved the final manuscript. Conflict of interest The authors declare that they have no competing interests. Uncited references Bush et al. (1999), Culter (2000), Drummond and Rambaut (2007), Glazko and Nei (2003) and Swofford (2002). Acknowledgements
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This work was supported by grants from the Provincial Medical Research Fund of Zhejiang, China (U201129691), the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents and Monitor Technology Platform of Infectious Diseases of the State Major Science and Technology Special Projects during China’s 12th five-year plan (2012ZX10004-210). David E. Boufford is gratefully acknowledged for editing the manuscript.
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Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China
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Yanjun Zhang a,1 , Haiyan Mao a,1 , Juying Yan a,1 , Lei Zhang a,1 , Yi Sun a,1 , Xinying Wang a,1 , Yin Chen a , Yiyu Lu a , Enfu Chen a , Huakun Lv a , Liming Gong a , Zhen Li a , Jian Gao a , Changping Xu a , Yan Feng a , Qiong Ge a , Baoxiang Xu a , Fang Xu a , Zhangnv Yang a , Guoqiu Zhao b , Jiankang Han c , Koch Guus e , Hui Li f , Yuelong Shu d , Zhiping Chen a,∗ , Shichang Xia a,∗ a
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang 310051, China Center for Disease Control and Prevention of Hangzhou, Hangzhou, Zhejiang 310021, China Center for Disease Control and Prevention of Huzhou, Huzhou, Zhejiang 313000, China d National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Beijing 102206, China e Central Veterinary Institute, Part of Wageningen UR, Virology Department, 8200 AB Lelystad, The Netherlands f Shanghai Huirui Biotechnology Co. Ltd., Shanghai 200002, China b c
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Article history: Received 30 October 2013 Received in revised form 27 March 2014 Accepted 1 May 2014 Available online xxx
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Keywords: Influenza A H7N9
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1. Introduction
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In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E−3 (95% Highest posterior density: 3.8218E−3 to 7.7873E−3) while the NA gene showed 2.243E−3 (4.378E−4 to 3.79E−3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation. © 2014 Elsevier B.V. All rights reserved.
The avian influenza virus, H7N9, previously unknown in humans, was first detected in three urban residents in Shanghai and Anhui, China, in year 2013, who presented with rapidly progressing lower respiratory tract infections (Gao et al., 2013). It was reported
∗ Corresponding authors at: Zhejiang Provincial Center for Disease Control and Prevention, 630 Xincheng Road, Hangzhou, Zhejiang 310051, China. Tel.: +86 571 87115198; fax: +86 571 87115198. E-mail address: [email protected] (S. Xia). 1 These authors contributed equally to this article.
that most persons with confirmed H7N9 virus infection were critically ill and epidemiologically unrelated. Laboratory-confirmed human-to-human H7N9 virus transmission was not documented to have occurred through close contact (Li et al., 2013). Previous reports indicate that the virus does not appear to cause serious disease in birds, but has the potential for epidemiological and public health implications in humans (Spackman et al., 2010; Liu et al., 2013a,b). It was suspected that the H7N9 virus could establish a reservoir of infection that would lead to frequent sporadic human infections that appear without warning. Enhanced surveillance was implemented, resulting in the identification of additional H7N9 clinical cases in Zhejiang Province, which is located between Shanghai and Anhui.
http://dx.doi.org/10.1016/j.virusres.2014.05.002 0168-1702/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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We describe the isolation and characterization of two novel strains of the H7N9 avian influenza A virus (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) that were isolated from throat swabs of two H7N9 infected patients. To trace the origins of the virus and to understand its lineage and evolutionary dynamics, we sequenced the two genomes using next generation sequencing (NGS) methods. Our aims were to understand: (1) variation and mutations in the novel reassortants in H7N9; (2) determine the evolution/migration history based on phylogenetic methods from the hemagglutinin and neuraminidase lineage by comparing it with other viruses of avian origins; and (3) the role that live poultry markets have played in H7N9 circulation.
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2. Materials and methods
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2.1. Ethics statement and clinical samples
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This study was approved by the ethics committee of Zhejiang Provincial Center for Disease Control and Prevention (CDC), China. From April 2013 to May 2013, with permission of the patients, forty pharyngeal swabs were collected from patients suspected to be infected with the avian influenza virus H7N9. Fifty environmental samples comprising live poultry stools and water from the treatment of live poultry, were collected from the markets visited by the H7N9-infected patients (Han et al., 2013). The samples were stored in 3–5 ml of preservation solution (Hanks solution containing 100 U/ml penicillin and 100 g/ml streptomycin) at −70◦ C until analysis.
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2.2. Viral RNA extraction and RT-PCR detection
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Viral RNA was extracted using the RNeasy Mini kit (Qiagen, CA, USA) according to the manufacturer’s instructions. Multiplex Real time RT-PCR reactions were performed by using the AgPath-IDTM One-Step RT-PCR Kit (Life technologies). Procedures for the RT-PCR reactions followed the instructions of the manufacturer.
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2.3. Virus isolation and hemagglutination test
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Specific pathogen-free embryonated chicken eggs were used for virus isolation. Six 9-to-11-day-old chicken embryos were each inoculated with 200 l of sample by the chorioallantoic sac route. The eggs were incubated for 48 h at 35◦ C, after which allantoic fluid was tested for hemagglutination of 0.5% turkey red blood cells in a phosphate buffered saline (PBS) solution. Samples negative for hemagglutination were processed a second time.
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2.4. Electron microscopy identification
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The chorioallantoic fluid was fixed with 2.5% glutaraldehyde for 2 h at room temperature. The influenza virus was observed under transmission electron microscope (Hitachi H-600, Japan) (Malenovska, 2013).
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2.5. Creating and sequencing the library
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Building and sequencing the H7N9 library was completed in the Life Technologies Demo Center. Because of the low concentration of nucleic acid, we used PathAmp FluA Pre-Amplification Reagents (Life technologies) to enrich the eight segments in each sample for better performance in the ABI Ion Torrent. We then constructed two libraries consisting of random fragments averaging 200 bp in length. Each sample was assigned a different barcode in the Ion Xpress Plus Fragment Library Kit (Life technologies). In the final step, we pooled the two libraries evenly into 314 chips after
running them through quality control. The barcoded libraries were sequenced together in the Ion Torrent. 2.6. Data analysis Basic bioinformatic analysis, such as alignment and pathogen analyzer, were conducted after passing the data through quality checks. Both full genome virus sequences from the patients were deposited in the Global Initiative on Sharing Avian Influenza Data (GISAID) database. We then checked for variant positions in the amino acid sequences using MEGA 5.0 (www.mega.software. informer.com/5.0/) and calculated the identical index between isolates from humans and isolates from both human and birds, such as sequences infected chickens downloaded from GenBank (A/chicken/Zhejiang/DTZD-ZJU01/2013(H7N9)), for each segment in DNAStar Lasergene. v7.1 (www.dnastar.com). Phylogenetic trees were constructed using maximum likelihood (ML) based on raxml v7.2.8 (http://sco.h-its.org/exelixis/ software.html). We performed two multiple sequence alignments with data matrixes of other sequences downloaded from the public databases GISAID and GenBank according to the HA gene and NA gene for H7NX and HXN9 (X indicates the number of all possible combinations), respectively, using Geneious 4.8.3 (www.geneious.com). For the 115 sequences of HA and the 26 sequences of the NA segment partitions of the total evidence, an analysis using dataset-specific models was conducted by using the Akaike Information Criterion (AIC) in Modeltest 3.7 (Posada and Crandall, 1998). Model parameters were estimated in all analyses. The ML analysis for each data set was performed using the GTR model with a gamma distribution for site variation and setting the parameter values as ‘estimated’ using raxmlHPC (Stamatakis, 2006). BEAST 1.6 (http://beast.bio.ed.ac.uk/Main Page) was used to compute the substitution rate with uncorrelated lognormal distributed relaxed-clock model for our sample H7N9 virus segments HA and NA. Sequences in all analyses were downloaded from GenBank. We took two measures, as follows, to ensure the accuracy of our data and results: (1) we performed five simulations on BEAST with different sets of parameters; no significant differences were found among the final results from different runs; (2) because of the random sampling for each subtype and from different hosts (birds and humans), the rates would differ due to different sample matrices. We therefore re-sampled and tested several matrices. No significant differences were found. Therefore, for each virus subtype, we used the constant population size coalescent as the tree prior and data-specific model for substitution. Two replicates were run for 100,000,000 generations with tree and parameter sampling every 1000 generations. A burn-in of 10% was used and the convergence of all parameters assessed using the software TRACER within the BEAST package.
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3. Results
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3.1. Patients
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Table 1 showed the demographic and epidemiologic characteristics of both two patients. Patient 1 was a 37-year-old cook with chronic hepatitis B, working in Suzhou and living in Hangzhou, reported cough and diarrhea. He bought meals and avian birds from the local living poultry market 2 miles away every morning, prepared lunch and dinner for about 30 company staff. He died of acute respiratory distress syndrome, disseminated acute respiratory distress syndrome (ARDS) and intravascular coagulation, and multiorgan distress syndrome on March 27. Laboratory tests showed that the patient’s blood white cell count obviously
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Y. Zhang et al. / Virus Research xxx (2014) xxx–xxx Table 1 Demographic and epidemiologic characteristic of first two patients in Zhejiang province infected with H7N9 virus. Characteristic
Patient 1
Patient 2
Age Gender Occupation Area of origin Underlying conditions Exposure to live poultry markets in past 7 days Date of illness onset Date of admission Date of specimen collection Date of lab confirmation of virus Viral isolation
37 Male Cook Hangzhou in Zhejiang Hepatitis B Yes
64 Male Retired Huzhou in Zhejiang Chronic bronchitis Yes
March 7, 2013 March 19, 2013 March 24, 2013 April 1, 2013
March 29, 2013 March 31, 2013 April 3, 2013 April 3, 2013
A/Zhejiang/1/2013 (H7N9)
A/Zhejiang/2/2013 (H7N9)
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decreased in the early period to the lowest of 1.6 × 109 /L, then slowly climbed to the normal level after treatment, and abnormally elevated in the later period. He was confirmed as avian influenza A (H7N9) virus case by analyzing 8 segments performed in China CDC on April 1st, 2013, as the first confirmed case in Zhejiang Province. Patient 2 was a 64-year-old retried worker suffering long time chronic bronchitis. He visited market including living poultry every morning. He did not have high fever and cough at the onset of the illness, but he had severe pneumonia and respiratory distress syndrome in the later period. He was confirmed as H7N9 case by Zhejiang Provincial CDC on April 3rd, 2013.
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Pharyngeal swabs and environmental samples were first applied for H7N9 virus RNA detection. One-step multiplex qRT-PCR was performed to detect the H7N9 virus. A total of 36 samples (40%) were positive for H7N9. Fever and coughing were the most common symptoms presented in patients with H7N9 virus infections. Acute respiratory distress syndrome, respiratory failure, co-infection with bacteria, shock and congestive heart failure were also observed in these positive cases. The positive samples were then used for virus isolation through embryonated chicken eggs. After incubation, the allantoic fluid was used for the hemagglutination (HA) test. The HA test results showed 26 (29%) positive samples. Transmission electron microscopy (×106 ) was then used to observe the influenza virus in the positive allantoic fluid (Fig. 1).
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Two samples, A/Zhejiang/1/2013(H7N9) and A/Zhejiang/2/2013(H7N9), with different barcodes in ABI Ion Torrent, resulted in a total of 464,974 reads with a mean length of 163 bp, of which more than 39,600 reads passed quality filtering. The average coverage for both samples was 2551 and 2628. The figures in the supplemental materials show the average coverage for each segment in each individual. Two alignments were obtained for the HA gene of H7NX with 115 sequences and for the NA gene of HXN9 with 26 sequences. Both included five novel reassortant H7N9 viruses, with three from the National CDC and two from our own sequences. Complete sequences of the five novel reassortant H7N9 revealed that they were 97.3–100% identical in all 8 gene segments while they were 94.6–97.8% identical in isolates from humans and from chickens (Table 2). Table 3 shows the substitutions found in addition to the variations shown in the China CDC table (Gao et al., 2013). Mutations at positions T155Y and G186V in the HA gene in both Zhejiang viruses are associated with receptor binding (Masuda
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et al., 1999; Chen et al., 2013). A/Zhejiang/1/2013 had substitution Q226I while A/Zhejiang/2/2013 had the more common Q226L in the HA segment. Zhejiang/1 showed E627K changes, while Zhejiang/2 showed D701N changes in the PB2 segment. Both 627 and 701 are considered to be mutation sites necessary for mammalian adaptation (Chen et al., 2013). The two variations were found to coexist in highly pathogenic H5N1 isolates. Li et al. (2003) hypothesized that the both of these variations could facilitate effective transmission. V100A, K356R, S409N identified in polymerase PA protein can cause attenuation in mammals (Yamayoshi et al., 2013). The D66S mutation in the PB1-F2 protein, found in both Zhejiang isolates, demonstrated increased pathogenicity in the recombinant H5N1 virus by Korteweg and Gu (2008). Other substitutions were compared to the first three isolation obtained from China CDC (Shanghai/1, Shanghai/2 and Anhui/1), mutations in samples from Zhejiang Province displayed the similar patterns: motif PEIPKGR*G in amino acid positions 333–340 in the HA gene and conserved glycosylation motifs at positions 30, 46, 249, 421 and 493. Receptor binding site positions altered receptor specificity site G228S in HA was found in both Zhejiang virus. Drug resistant site (R294K in NA and S31N in M2), mutations in increasing virulence in mice (L89 V in PB2, N30D, T215A in M1 and P42S in NS1 segment), variation site for H5 subtype transmissible among ferrets (H99Y and I368V in PB1), were also detected in our Zhejiang isolations. Maximum parsimony, maximum likelihood and Bayesian analysis of the 115 H7NX HA sequences resulted in basically similar topological patterns for the phylogenetic tree (Fig. 2). All phylogenetic trees showed that all 115 HA segments diverged into two groups: a North American and a European-Asian group, with high bootstrap values (BS) in MP and Bayesian posterior probability (PP). Within the Asian clade, the five novel reassortant H7N9s showed a monophyletic relationship with virus A/duck/Zhejiang/2011 as sister group with 100% BS and PP support. Two H7N9 viruses from Zhejiang Province, A/Zhejiang/1/2013 and A/Zhejiang/2/2013, were most closely related to each other. Viruses from Shanghai and Anhui were their sister groups. The phylogenetic relationships of the sequences from the 26 HXN9s were nearly identical, since their alignment strongly supported a monophyletic relationship among the five novel reassortant H7N9s with high BS and PP values as well (Fig. 3). Viruses closely related to the H7N9 clade were A/Anas crecca/Spain/1460/2008, A/duck/Mongolia/119/2008 and A/wild duck/Korea/SH20-27/2008. All phylogenetic results indicate that the novel reassortant H7N9 originated from bird viruses. The Bayesian MCMC method estimated the mean rate of evolution of the HA gene in H7NX to be 5.74E−3 (95% HPD: 3.8218E−3 to 7.7873E−3) while the NA gene in the HxN9 matrix showed 2.243E−3 (4.378E−4 to 3.79E−3) substitutions per nucleotide site per year.
4. Discussion To confirm the origin of the H7N9 virus to be poultry, we analyzed whole genome sequences from two isolates taken from human patients in Zhejiang Province and used them for phylogenetic analysis. We compared our two viral amino acid sequences with the first three samples published by the Chinese CDC and found several new mutations in the novel reassortant H7N9. In both Zhejiang isolates, most mutations encoded for human/mammalian amino acid adaptations. Because of the high substitution rate, the HA segment is considered to be the most diverse segment in the influenza virus, not only at the nuclear level but also in amino acids (Worobey et al., 2014). Chen et al. (2013) reported that T155Y, found in all HA segments of 5 H7N9 isolates, could have played an important role in detecting ␣-2,6-linked sialic acids. Q226L in the HA protein is one of the receptor binding sites,
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Fig. 1. Shape of the influenza A (H7N9) virus observed by transmission electron microscopy.
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the same as G186V, which increases binding affinity for ␣-2,6linked sialic acid receptors. Glutamine converted to Isoleucine in A/Zhejiang/1/2013 may fail to bind to ␣-2,6 human-like receptors due to a mutation at position 226. We also suspect that the E627 K substitution in the PB2 segment is responsible for restoring the ability of the PB2 single gene reassortant to replicate in MDCK cells. Recent studies (Gabriel et al., 2005; Manz et al., 2013) suggest
that increases in the ability to replicate may be critical for allowing avian influenza viruses to replicate in mammals. Interestingly, A/Zhejiang/2/2013 virus has glutamic acid at the 627 site, which may facilitate bird/human infections and result in severe disease. Massin et al. (2001), Subbarao et al. (1993) and Tarendeau et al. (2008) showed that the PB2 of all bird isolates has glutamine at position 627, while mammalian viruses have lysine at this position.
Table 2 Information on each segment of each novel H7N9 of avian origin. Gene
PB2 PB1 PA HA NP NA MP NS
Nucleotide length (bp)
2280 2274 2151 1683 1497 1398 982 838
GISAID number
Identities to A/Anhui/1/2013
A/Zhejiang/1/2013
A/Zhejiang/2/2013
EPI443031 EPI443032 EPI443033 EPI443034 EPI443035 EPI443036 EPI443037 EPI443038
EPI443039 EPI443040 EPI443041 EPI443042 EPI443043 EPI443044 EPI443045 EPI443046
99.4–99.9 99.4–99.9 98.2–100.0 99.2–100.0 97.3–100.0 97.3–100.0 99.8–100.0 99.3–100.0
Identities to A/Chicken/Zhejiang/DTID-ZJU01/2013(H7N9) Genbank accession: KC899666–KC899673
97.8–99.7 97.3–99.7 94.6–99.4 96.9–99.5 97.3–99.1 97.0–99.7 97.6–99.9 96.1–99.7
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Fig. 2. Phylogenetic tree based on the HA gene in H7NX subtypes. An ML tree is shown with MP bootstrap and Bayesian posterior probability above (BS/PP value). Branches with BS/PP values were found in all phylogenetic analyses. (A) Whole maximum likelihood tree. (B) Enlargement of 5 novel avian-origin H7N9 with their sister groups.
Fig. 3. Phylogenetic tree based on the NA gene in HXN9 subtypes. An ML tree is shown with MP bootstrap and Bayesian posterior probability above (BS/PP value). Branches with BS/PP values were found in all phylogenetic analyses.
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
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Table 3 Substitutions in segments in each novel H7N9 of avian origin. Substitutions that are consisted with Chinese CDC isolations published in NEJM (Gao et al., 2013) are not present in below. Gene
280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
Site
Position
A/Zhejiang/1/2013
A/Zhejiang/2/2013
A/Anhui/1/2013
HA
Receptor binding site
T155Y G186V Q226L
Y V I
Y V L
Y V L
PB2
Replication of AIV in mammals
E627K D701N
K D
E N
K D
PB1-F2
H5N1 virus increased virulence in mice
D66S
S
S
S
PA
Attenuating in mammals
V100A K356R S409N
A R N
V R S
A R N
They concluded that residue 627 is host determinant and correlates with virulence of the influenza A viruses in primates. The mutation at site 701 in the PB2 segment of the A/Zhejiang/2/2012 genotype indicated that this strain may exhibit enhanced transmission in guinea pigs (Chen et al., 2013). These two variations were found to coexist in highly pathogenic H5N1 isolates and it is believed that they may facilitate effective transmission (Li et al., 2003). Moreover, a recent study by Conenello et al. (2007) of recombinant viruses demonstrated that a single mutation in the PB1-F2 protein D66S in H5N1 increased virulence in mice. Expression of the PB1-F2 protein may cause immune-mediated injury and a decline in the survival rate in mice (McAuley et al., 2013). V100A, K356R and S409N are considered to be host specific amino acid mutations in the PA segment that allow H7N9 to replicate in mammals rather than in avian cells (Yamayoshi et al., 2013). In the same study, Yamayoshi indicated that the PA protein has several variants that attenuate in mammals. Additional mutation sites, such as R65K in HA, need further analysis to determine their function and relationships with the phenotype. To determine the evolutionary history of amino acid changes in HA and NA segments, we produced phylogenic trees for both of them using maximum parsimony, maximum likelihood and Bayesian methods. Different viral strains associated with different geographic regions were seen in trees based on the HA and NA genes. All three methods for constructing trees showed similar phylogenetic topologies. The strains were separated into two large groups, a North American group and a European-Asian group. Viruses from the same country or region clustered together within each clade with strong support, indicating that the viruses within a region exhibit their own evolutionary lineage due to adaptations within the local area. The trees also showed multiple origins and rapid evolution with fascinating substitution rates in different countries and areas in the North American and European-Asian subgroups (Fig. 2). The human H7N9 isolates are likely the result of a reassortment of three viruses: A/duck/Zhejiang/2011(H7N3-like), A/wild bird/Korea/A14/2011 (KO14-like H7N9) and A/brambling/Beijing/16/2012 (BJ16-like H9N2). The genetic relationship of these five novel H7N9 strains that infect humans showed monophyly with high bootstrap values in the HA lineage. The identification of a novel reassortant influenza A (H7N9) virus, which has been associated with severe human infections in Shanghai and Anhui, is of great significance worldwide, especially in areas adjacent to known areas of infection. A reciprocal monophyletic pattern of HA trees of A/Shanghai/1/2013 and other strains indicated that the virus is more ancient than any of the other four virus and may have been one of the first avian viruses to infect humans. There is also indication of more than one origin of the virus in the Shanghai area according to the topological structure of the monophyletic clade. Because A/Zhejiang/1/2013(H7N9) was isolated from a 37-year-old cook working in Suzhou, Jiangsu Province, and living
in Hangzhou, Zhejiang Province, the virus could be considered to have originated in Jiangsu Province. The nesting of the two strains of viruses from Zhejiang as a derived clade within the Shanghai and Anhui groups may reflect past migrations to Zhejiang and Jiangsu from Shanghai. The findings indicate that the H7N9 virus was transmitted to Zhejiang Province within a short time period. The novel influenza A (H7N9) virus may have been circulating in urban areas of southeast China. Frequent contact with poultry, immune function suppression and elderly persons with underlying diseases were the most common characteristics of people who were infected (Chen et al., 2013). There is no strong evidence to prove transmission from person to person. Further analysis of birds, humans and the environment are needed to determine the relationships and migration routes of the vectors. Live poultry markets (LPMs) are popular in China, since Chinese people prefer to purchase freshly killed chickens for immediate cooking. One survey reported that 80% of households obtain poultry at LPMs at least once a year (Liao et al., 2009; Yu et al., 2014). Live poultry markets play an important role in the circulation of H7N9. They provide a perfect place for mixing different strains of avian influenza, and for transmission and infection, as both cases isolated in Zhejiang showed (Table 1; Li et al., 2013; Yu et al., 2013; Liu et al., 2013a,b). During our enhanced surveillance of H7N9 virus Q2 infections in March and April, 2013, in Zhejiang, positive infections obtained in LPMs were also identified. To control the spread of H7N9, the LPMs in Hangzhou and Huzhou, where H7N9 virus infections were found, were closed as a precautionary public health measure in April 2013 and disinfected by the government. Epidemiological studies indicate that exposure to live poultry in the LPMs is a major risk of H7N9 infection in humans, as closure of the LPMs reduced the mean daily number of infections by 99% (92–100%) in Hangzhou and by 97% (68–100%) in Huzhou (Yu et al., 2013). When the LPMs reopened during the last several months of 2013 due to economic and political pressure, the number of confirmed cases soared in the days before the spring festival of 2014. Recent studies in Zhejiang Province showed that risk factors contributing to H7N9 infection include a long history of smoking, chronic lung disease, immune-suppressive disorders and delayed oseltamivir treatment (Gao et al., 2013; Liu et al., 2013a,b). Centralized slaughter of live poultry would be an effective strategy to prevent susceptible populations from becoming infected. The Zhejiang provincial government, through collaboration among many departments, will permanently shut down all LPMs in July 2014. Authors’ contributions YZ, JY, HM, LZ, YS, XW contributed to the original draft of the manuscript, and approved the final version. GZ, JH, KG, HL, YS contributed to the concepts and design of the manuscript and in revising the manuscript. YC, YL, EC, HL, LM, ZL, JG, CX, YF, QG, BX, FX, ZY performed the experiments and provided information and
Please cite this article in press as: Zhang, Y., et al., Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.05.002
331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373
374
375 376 377 378 379
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380 381
382
383
384 Q3
385 386
387
suggestions. ZC and SX are corresponding authors. All authors read and approved the final manuscript. Conflict of interest The authors declare that they have no competing interests. Uncited references Bush et al. (1999), Culter (2000), Drummond and Rambaut (2007), Glazko and Nei (2003) and Swofford (2002). Acknowledgements
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This work was supported by grants from the Provincial Medical Research Fund of Zhejiang, China (U201129691), the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents and Monitor Technology Platform of Infectious Diseases of the State Major Science and Technology Special Projects during China’s 12th five-year plan (2012ZX10004-210). David E. Boufford is gratefully acknowledged for editing the manuscript.
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