Accepted Manuscript Human infection with a novel highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings Ming Pan, Rongbao Gao, Qiang Lu, Shunhe Huang, Zhonghui Zhou, Lei Yang, Xiaodan Li, Xiang Zhao, Xiaohui Zou, Wenbin Tong, Suling Mao, Shumei Zou, Hong Bo, Xiaoping Zhu, Lei Liu, Heng Yuan, Minghong Zhang, Daqing Wang, Zumao Li, Wei Zhao, Maoli Ma, Yaqiang Li, Tianshu Li, Huiping Yang, Jianan Xu, Lijun Zhou, Xingyu Zhou, Wei Tang, Ying Song, Tao Chen, Tian bai, Jianfang Zhou, Dayan Wang, Guizhen Wu, Dexin Li, Zijian Feng, George F. Gao, Yu Wang, Shusen He, Yuelong Shu PII:
S0163-4453(15)00220-0
DOI:
10.1016/j.jinf.2015.06.009
Reference:
YJINF 3561
To appear in:
Journal of Infection
Received Date: 23 April 2015 Revised Date:
24 June 2015
Accepted Date: 25 June 2015
Please cite this article as: Pan M, Gao R, Lu Q, Huang S, Zhou Z, Yang L, Li X, Zhao X, Zou X, Tong W, Mao S, Zou S, Bo H, Zhu X, Liu L, Yuan H, Zhang M, Wang D, Li Z, Zhao W, Ma M, Li Y, Li T, Yang H, Xu J, Zhou L, Zhou X, Tang W, Song Y, Chen T, bai T, Zhou J, Wang D, Wu G, Li D, Feng Z, Gao GF, Wang Y, He S, Shu Y, Human infection with a novel highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings, Journal of Infection (2015), doi: 10.1016/j.jinf.2015.06.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Human infection with a novel highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings 3
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Ming Pan1†, Rongbao Gao2†, Qiang Lu1†, Shunhe Huang †, Zhonghui Zhou †, Lei Yang2
3
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Xiaodan Li2, Xiang Zhao2, Xiaohui Zou2, Wenbin Tong1, Suling Mao1, Shumei Zou2, Hong Bo2, 4
4
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Xiaoping Zhu1, Lei Liu1, Heng Yuan1, Minghong Zhang , Daqing Wang , Zumao Li , Wei Zhao , 6
7
Maoli Ma , Yaqiang Li , Tianshu Li1, Huiping Yang1, Jianan Xu1, Lijun Zhou1, Xingyu Zhou1, 3
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Wei Tang , Ying Song8, Tao Chen2, Tian bai2, Jianfang Zhou2, Dayan Wang2, Guizhen Wu2,
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Dexin Li2, Zijian Feng9, George F Gao9, Yu Wang9, Shusen He1*, Yuelong Shu2*
1
Sichuan Provincial Disease Control and Prevention, Chengdu 610041, China.
2
National Institute for Viral Disease Control and Prevention, China CDC, Key
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Laboratory for Medical Virology, National Health and Family Planning Commission, Beijing 102206, China.
Nanchong City Disease Control and Prevention, Nanchong 637000, China.
4
Affiliated Hospital of Chuanbei Medical School, Nanchong 637000, China.
5
Nanbu County Disease Control and Prevention, Nanchong 637300, China.
6
Nanbu County Hospital, Nanchong 637300, China.
7
Dongba Center Hospital of Nanbu County, Nanchong 637300, China.
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Department of Pathology, Beijing Renhe Hospital, Beijing 102600, China.
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Chinese Center for Disease Control and Prevention, Beijing 102206, China.
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†These first authors contributed equally in this study
ACCEPTED MANUSCRIPT *Correspondence footnote: Address reprint requests to Dr. Yuelong Shu, National Institute for Viral Disease Control and Prevention, China CDC, Key Laboratory for Medical Virology, National
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China. E-mail: [email protected], tel:8610-58900850;
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Health and Family Planning Commission. 155 Changbai Road, Beijing, 102206, P. R
Reassortment, Pathology
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Key words: Human infection, Highly pathogenic avian influenza virus, H5N6,
Summary: A fatal human infection with a novel reassortant H5N6 avian influenza
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virus was first identified. The virus was with backbone of H5N1 virus acquired NA gene from H6N6 virus.
Key words: Highly pathgenic avian influenza virus, H5N6, Reassortment, Emerging
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infectious diseases
ACCEPTED MANUSCRIPT Abstract Objectives Human severe infection with avian influenza A (H5N6) virus infection was identified firstly in 2014 in China. It was unknown or unclear on the disease or the pathogen by
human case with H5N6 virus infection. Methods
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people. This study would illustrate the virological and clinical findings of a fatal
We obtained and analyzed the clinical, epidemiological and virological data from the
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patient. RT-PCR, viral culture and sequencing were conducted for determination of causative pathogen.
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Results
The patient who presented with fever, severe pneumonia, leucopenia and lymphopenia, developed septic shock and ARDS, and died on day 10 after illness onset. A novel reassortant avian-origin influenza A (H5N6) viruswas isolated from the throat swab or trachea aspirate of the patient. The virus was reassorted with HA
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gene of Clade 2.3.4.4 H5, internal genes of Clade 2.3.2.1 H5 and NA gene of H6N6 avian viruses. The cleavage site of HA gene contained multiple basic amino acids indicating the novel H5N6 virus was highly pathogenic in chicken.
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Conclusions
A novel highly pathogenic avian influenza H5N6 virus with the backbone of H5N1
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virus acquired NA gene from H6N6 virus was first identified, and caused human infection with severe respiratory disease.
ACCEPTED MANUSCRIPT Text The threat posed by avian influenza A viruses to humans remains significant since these viruses raised sporadic human infections, underwent gene evolution and
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potential reassortment with human virus (1). Of these viruses, H5N1 and the recently emerged H7N9 in China have raised the global concerns due to high
mortality and limited human-to-human transmission (2, 3). Highly pathogenic
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avian influenza (HPAI) H5N1 virus caused first human infection in 1997 in Hong
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Kong (4), and reemerged in 2003. So far, this virus had resulted into more than 650 human infections with around 60% fatality in 16 countries since 2003(5). The HA gene of H5 virus has been undergoing evolution, generating multiple clades. The currently circulating clades in poultry include 1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4
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and 7 in the world.Clade 2.3.4 and 2.3.2 of these clades were predominantly circulating in China (6-8). And a total of 46 human H5N1 cases have been identified in China since 2003(5).
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Besides H5N1 virus, other HPAI H5 subtypes such as H5N2, H5N8, H5N5 viruses
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had been detected in poultry(9). And H5N8 outbreaks have been reported in poultry in Korea, Japan, and recently Europe. In addition, serologic evidence of human infection with H5N2 was reported in poultry workers in Japan (10). In April 2014, a fatal case infected with a novel H5N6 virus was identified in China. And total three H5N6 infected cases were identified so far in China. Research on the disease or the pathogen is necessary. In this study, the virological and clinical findings of the first fatal case were illustrated.
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Methods Surveillance, reporting, and data collection
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Throat swab and tracheal aspirate were obtained from a severe pneumonia case on day 8 and 9 after illness onset of fever, respectively. The samples were tested as influenza A (for M gene) and H5 positive with real-time RT-PCR. A standardized surveillance
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reporting form was used to collect epidemiologic and clinical data, including
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demographic characteristics; underlying medical conditions; recent exposures to swine, poultry, or other animals; recent visits to live poultry markets; clinical signs and symptoms; chest radiographic findings; clinical laboratory testing results; antiviral treatment; clinical complications; and outcomes. Environmental samples
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were collected in epidemiological linked live poultry markets. Close contacts of the patient were defined as those who had provided care to, had been living with, or had potentially been exposed directly to respiratory secretions or body fluids of the patient
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from the 14th day before illness onset to the day of death. Two weeks of medical
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monitor was conducted among all contacts and throat samples were collected. Virus isolation
The throat swab and tracheal aspirate samples collected from the patient were performed virus isolation on embryonated eggs. Briefly, 0.2ml of throat swab or tracheal aspirate samples were injected into the allantoic cavity of 9 days old specific-pathogen-free chicken eggs respectively, and were propagated for 48 hours at 37°C. The allantoic fluids were tested for haemagglutination activity with turkey red blood cells.,Positive isolates were subjected to RT-PCR and sequencing. Three blind passages were performed on haemagglutination-negative samples to confirm the
ACCEPTED MANUSCRIPT absence of infectious virus. All steps were conducted in biosafty level-3 containment laboratory. RNA extraction and RT-PCR for subtyping RNA was extracted from all collected specimens and viral isolates using QIAamp
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Viral RNA Mini Kit (Qiagen, Germany), according to the manufacturer’s instructions. Specific real-time RT-PCR or conventional RT-PCR assays with self-designed
specific primer and probe sets for detecting seasonal influenza viruses (H1, H3, or B)
Genome sequencing and phylogenetic analysis
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and avian influenza viruses (H5, H7, H9, H10 and N1-N9) (11-13).
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The full genome of the virus was amplified with the use of Qiagen OneStep RT-PCR Kit for sequencing. PCR products were purified from agarose gel with the use of the QIAquick Gel Extraction Kit (Qiagen, Germany). We performed the sequencing using an ABI 3730xl automatic DNA analyzer (Life Technologies, USA) and the ABI
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BigDye Terminator v3.1 cycle sequencing kit (Life Technologies, USA), according to the manufacturer’s recommendations. Phylogenetic analysis was conducted with
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MEGA5.1 using neighbor-joining tree for HA gene as recommended by World Health Organization( WHO) (7) and maximum likelihood tree for other 7 genes.
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Deep sequencing and data analysis Total RNA was extracted from throat swab of the patient, reverse transcribed to double-strand DNA. After quantification, 200ng DNA was subjected to fragmentation and adapter ligation using Ion Xpress™ Plus Fragment Library Kit (Life technologies, Carlsbad, USA). The 200bp DNA library was selected by size with E-Gel® iBase™ unit and applied to emulsion-PCR with Ion PGM™ Template OT2 200 Kit (Life technologies, Carlsbad, USA). After enrichment, the
ACCEPTED MANUSCRIPT template-positive Ion Sphere™ Particles were loaded on Ion 318™ v2 chip (Life technologies, Carlsbad, USA) and sequenced with Ion PGM™ Sequencing 200 Kit v2(Life technologies, Carlsbad, USA). The Next Generation Sequencing (NGS)
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reads were analyzed with a pipeline process using the CLC Genomics workbench 7.0.4 (Qiagen, Germany). Briefly, the raw reads were trimmed and de novo
assembled under CLC standard settings. The contigs with coverage over 30 were
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extracted and locally blasted in the non-redundant nucleotide database downloaded
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from GenBank in CLC with default parameters. Base on the Blast results, the taxonomic composition (at the species level) of each dataset was identified with MEGA(14) software (version 5.2.3). The percentages of the identified microbial species were estimated according to the numbers of the sequencing reads of each
Case report
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Results
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species followed by normalization with their genome sizes(15).
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The patient was a 49 years old male without underlying medical conditions, who was a dealer in a local live poultry market for previous year. By report, he presented with fever, cough, headache, myalgia and stomach discomfort as initial clinical symptoms on April 13, 2014. He was treated as an upper respiratory infection in a rural clinic, and developed cough with blood-tinged sputum on day 7 after illness onset of fever. He was hospitalized with fever (axillary temperature in 37.5
) on April 20, 2014 in
Nanbu county hospital (Table 1, Figure 1). The chest radiography showed that the
ACCEPTED MANUSCRIPT patient had consolidation in left lung and patchy shadows in right lung on day 7 after illness onset of fever (Figure 2-A). The patient was transferred to the hospital affiliated with north Sichuan medical university on April 21, 2014. The chest
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radiography and computed tomography showed patchy shadows of both left and right lung with ground-glass opacities and consolidation on day 8 after illness onset of fever (Figure 2-B,C).
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The white blood cells (WBC) and platelet counts, and the ratio of lymphocytes were
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decreased on admission. The absolute neutrophil counts were decreased on day 8 but returned back to normal range on day 9, however, the ratio of neutrophils was elevated.Both absolute counts and ratio of lymphocytes were decreased. The eosinophil granulocytes in WBC was not detectable. The levels of c-reaction protein,
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procalcitonin, spartate aminotransferase (AST), lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH) and plasma Fibrin/Fibrinogen Degradation Products (P-FDP) were increased, while decreased levels of total
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protein, albumin and prealbumin were observed (Table 2).
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Despite combination antibiotic therapy, mechanical ventilation, glucocorticoids, and antiviral therapy were administered, the patient developed severe pneumonia, septic shock, and acute respiratory distress syndrome (ARDS), and died soon on day 10 after illness onset (Table 1, Figure 1). Determination of causative pathogen Throat swab and trachea aspirate obtained from the patient on April 20 and 21 respectively, and were confirmed on April 22 (Figure-1), by means of real-time
ACCEPTED MANUSCRIPT RT-PCR, conventional RT-PCR and sequencing, to be positive for influenza A (H5N6) virus but negative for seasonal influenza viruses (H1, H3 or B) or avian influenza viruses(H5N1, H7N9, H9N2 or H10N8). The virus isolated from the throat
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swab sample was designated as A/Sichuan/26221/2014(H5N6) (SC26221). No pathogenic bacteria and fungus were detected in sputum samples collected on day 7 and 8 after illness onset by culture. The deep sequencing data of throat swab
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revealed that the avian influenza A (H5N6) virus was overwhelmingly dominant
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(>73%) in microbial species. The genetic characterization of the virus
Full genome sequences of SC26221 were deposited in the Global Initiative on Sharing Avian Influenza Data (GISAID) database (Accession No. EPI533583-90). The virus
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showed a high similarity to the recently reported virus
A/environment/Zhenjiang/C13/2013 (H5N6) detected in live poultry market (16). Phylogenetic analysis showed that the HA gene of SC26221 belonged to clade 2.3.4.4
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H5 viruses nominated recently(17), while the NA gene belonged to an H6N6 subclade
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of Eurasian lineage viruses (Figure 3). All of the six internal genes of SC26221 were closely related to clade 2.3.2.1 H5 viruses circulating in duck in China (Figure S1 in Supplementary Appendix). The HA cleavage site possesses multiple basic amino acids motif (REKRRKR↓G), indicating potential high pathogenicity in chicken (Table S1 in Supplementary Appendix). The amino acid motif QSG at positions 226-228 (H3 numbering) of the HA protein indicated that the virus was avian-like (α2,3-SA) receptor binding
ACCEPTED MANUSCRIPT preference. The S137A and T160A mutations which resulted in the absence of the 158N glycosylation were detected in the HA protein of SC26221 virus, indicating increased human-like (α2,6-SA) receptor recognition of the virus(18, 19). The L89V,
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I495V, D701N, G309D and R477G mutations in PB2 protein, which were reported to increase viral pathogenic, virulence, and replication of H5N1 virus in mice, were
observed in the SC26221(20-22). No mutation associated with reduced sensitivity to
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NA inhibitors and adamantanes was detected in NA or M2 respectively.
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Epidemiological investigation
The patient was a dealer in a local live poultry market for one year. He would take the unsold birds to temporarily breed in the backyard of his house. One goose, 2 ducks and 5 chickens died during April 13-17th. A total of 144 environmental
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samples associated were collected and detected by real-time RT-PCR.Only 3 samples were influenza A PCR positive but H5 negative. However, H5N6 virus was detected in the dead poultries by report(23) , indicating that the source of
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infection may be from infected poultries. A total of 47 close contacts, including 12
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health care providers, 7 family members, 2 patients in same ward and 26 others were indentified. Medical observations on all contacts showed that no influenza like illness (ILI) symptom was observed during the investigation period (2 weeks). Meanwhile, throat swabs were collected from 46 contacts during April 21-23 and negative for influenza virus by real-time RT-PCR test.
Discussion
ACCEPTED MANUSCRIPT A fatal human infection with a novel reassortant influenza A (H5N6) virus was firstly identified. Phylogenetic analysis showed that the virus is a triple reassortant avian influenza virus contained an HA from clade 2.3.4.4 H5 viruses, a NA from H6N6
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viruses, and 6 internal genes from clade 2.3.2.1 H5 viruses (Figure S2 in Supplementary Appendix). Both H5N1 and H6N6 viruses have been circulating in
poultry in China, increasing the possibility for their reassortments. The novel H5N6
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viruses could be generated by two independent reassortments or from a common
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H5N6 progenitor virus. Our study showed that the HA genes of the H5N6 viruses shared only 95% homology, and located in the different subclades of HA phylogenetic tree. Two divergent N6 NAs with or without 59-69aa deletion in NA stalk existed in poultry viruses before the H5N6 case reported. NA genes of these H5N6 viruses
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distributed into two subclades in phylogenetic trees, whereas SC26221 and A/environment/Zhenjiang/C13/2013(H5N6) locating in one subclade without 59-69aa deletion in NA stalk. In hence, we proposed that these two “groups” of H5N6 viruses
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may be derived from independent reassortments. However, more sequences and
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surveillance data were needed to verify the origination of the H5N6 viruses. Consisted with the chest radiographs of severely viral pneumonia, our microbiologic studies demonstrated that the avian influenza A (H5N6) virus was overwhelmingly dominant in microbial species in throat swab, and no other pathogens was detected although the prior antibiotics treatments may influence the possibility of secondary infection detection. Several factors may contribute to the fatal outcome of the patient. Similar to H5N1 infection, the pathological damage in lung may link to
ACCEPTED MANUSCRIPT cytokine dysregulation triggered by H5N6 virus infection and resultant inflammatory damage to the lungs as the inflammation cells infiltration and diffuse hemorrhage observed in lung (24). And the patient presented liver and heart damage
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besides respiratory function failure, supported by increased levels of AST, LDH and HBDH, and decreased levels of total protein, albumin and prealbumin (Table.2)(25, 26). Previous reports showed no beneficial effect of corticosteroids in patients with
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ARDS secondary to influenza pneumonia, and that very early corticosteroids
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therapy may be harmful in 2009 pandemic H1N1 infection (27-29). In this study, systemic glucocorticoid therapy was performed subsequently three days on the patient after hospitalization on day 7 after illness onset. The effect of glucocorticoid therapy for the disease development of the patient might not be excluded. In
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addition, Oseltamivir therapy starting on day 7 of illness did not prevent death, although the NA sequence data predicted susceptibility. It may be related to the delay in starting antiviral therapy as antiviral therapy can shorten the duration of
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viral shedding and reduced mortality in hospitalized patients with severe influenza if
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prescribed during the first 3 days of illness(30-32). Human infection with different avian influenza viruses had been reported, and some have presented limited human-to-human transmission such as H5N1 (2) and H7N9 (3). Of note, the mutations associated with mammalian adaptation were detected in the H5N6 virus, and three cases have been identified since 2014. Taken together with continuous emergence of avian influenza A (H7N9 and H10N8) viruses caused human infections, it is important to enhance surveillance to monitor mutations and
ACCEPTED MANUSCRIPT reassortment of avian influenza viruses for pandemic preparedness and response.
Acknowledgments
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We thank the Chinese influenza surveillance network for providing access to their collection of clinical samples and data of the patient.This work was supported by the National Basic Research Program (973) of China (2011CB504704, to Dr. Yuelong
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Shu), Emergency Research Project on human infection with avian influenza H7N9
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virus from the National Ministry of Science and Technology (no. KJYJ-2013-01-01 to Dr. Yuelong Shu), and National Mega-projects for Infectious Diseases (2013ZX10004-101 to Dr. Dexin Li and 2012ZX10004215 to Dr. Biao Kan), the Chinese National Influenza Center– Centers for Disease Control and Prevention
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(CDC) collaborative project 5U51IP000334-03 from the CDC China- U.S. Collaborative Program on Emerging and Re-emerging Infectious Diseases, and the
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National Natural Science Foundation of China (81373107 to Dr. Rongbao Gao).
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Conflicts of Interest
No potential conflicts in this study.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 The timeline of the clinical course of the patient and identification of the
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pathogen
Figure 2 Chest radiographs.
Chest radiograph showed that consolidation of left lung and bilateral ground-glass
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opacity were clearly seen on day 7 after illness onset (Panel A). White lung in left
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and developed patchy shadows of right lung were observed on day 8 (Panel B). Computed tomographic scan of the chest of the patient, obtained on day 8, was shown in Panel C. Consolidation of left lung lower lobe, increased density of right
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lung can be seen in upper, middle and lower lobes.
Figure 3 Phylogenetic analysis of HA (A) and NA (B) genes of SC26221. The novel H5N6 virus isolated from the patient was highlighted in red, other H5N6
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viruses isolated from poultry in China in blue. Supporting bootstrap values of greater
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than 0.6 were shown (1000 replicates).
Supplementary figure legends
Figure S1. Phylogenetic trees of HA and internal genes of H5 influenza viruses
ACCEPTED MANUSCRIPT The novel H5N6 isolate was highlighted in red, the clade 2.3.4 H5 viruses in blue, and the clade 2.3.2.1 viruses in pink. Supporting bootstrap values of greater than 0.6
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were shown (1000 replicates).
Figure S2 Hypothetical origins of the gene segments of the novel reassortant influenza A (H5N6) viruses
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Virus particles were represented by colored ovals containing horizontal bars that
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represented the eight gene segments (from top to bottom: PB2, PB1, PA, HA, NP, NA, M and NS) Arrowheads pointed to the resulting reassortants. The broken line
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represented NA gene with 59-69aa deletion.
ACCEPTED MANUSCRIPT Table 1 Clinical features and treatments of the patient Characteristics Treatment Mechanical ventilation Initiated on day 9 after illness onset Treatments (Days after illness onset) Antibiotic therapy Day 7
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Day 8
Glucocorticoids Day 7
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Day 9
Methylprednisolone (two 80mg doses given intravenously) Methylprednisolone (one 40mg dose given intravenously) Methylprednisolone (one 40mg dose given intravenously)
Day 8 Day 9
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Antiviral therapy Day 7 Day 8 Anti-bleeding therapy Day 7
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Imipenem (two 1g doses given intravenously) Imipenem (1g doses given intravenously every 8h), Moxifloxacin Hydrochloride (one 0.4g doses given intravenously) Vancomycin Hydrochloride(one 1g dose given intravenously), Moxifloxacin Hydrochloride (one 0.4g dose given intravenously)
Tamiflu (two150mg doses given ) Tamiflu (two 75mg doses given) Pantoprazole sodium (40mg I.V. twice), Carbazochrome Sodium Sulfonate (80mg I.V. once) Pantoprazole sodium (40mg I.V. q8h)
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WBC counts (×109/L) Lymphocytes counts (×109/L) Ratio of lymphocytes (%) Neutrophils counts (×109/L) Ratio of Neutrophils (%) Lymphomonocyte Numbering (×109/L) Ratio of lymphomonocyte (%) Eosinophile granulocyte counts (×109/L) Ratio of Eosinophile granulocyte (%) (×109/L)
3.0-10
-
0.02-0.52
0
0
0
-
0
0
91
91
92
8.5-11.8
14.1
12.6
13
0.00-8.00 0.00-0.50 5.0-40.0
162.30 -
252.00 3.60 -
49.0
84.4
76.5
1.10-3.20
20-50 1.8-6.3
40-75
0.4-8 100-300
Mean platelet volume (fL) C-reaction protein(mg/L) Procalcitonin (ng/ml) Aspartate aminotransferase (U/L)
1.48
3.05
0.19
0.73
12.8
23.9
1.22
2.25
82.4
73.8
0.06
0.03
4.1
1
35.0-125.0
66.0
78.1
63.0
Alpha-L-fucosidase (U/L)
0.0-40.0
-
41.9
32.3
Prealbumin(mg/dL) Total protein (g/L) Albumin(g/L) Globulin(g/L) apoA1 (g/L) LACT(mmol/L) LDH (U/L) HBDH(U/L) CK(U/L) CK-MB(U/L) APTT (s) TT(s) FIB(g/L) P-FDP (Ug/ml)
150.0-400.0 65.0-85.0 40.0-55.0 20.0-40.0 1.00-1.60 0.50-2.20 114.0-240.0 60.00-180.00 25.00-180.00 1.0-25.0 18.0-30.0 14.0-21.0 1.80-3.60 0.0-5.0
111.0 61.1 36.1 25.0 -
62.7 58.4 32.4 26.0 0.72 2.54 895.4 703.43 128.47 25.5 35.0 19.7 4.65 114.3
49.4 54.5 26.5 28 -
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Alkaline phosphatase(U/L)
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2 3 4 5
0.1-0.6
1.2 23.9 70.3 -
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platelet counts
4.0-10.0
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Variable
Table.2 The results of clinical blood biochemistry tests Sample collected time (Days after Normal illness onset) range 7 8 9
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Note: “-” means data unavailable. LACT= lactic acid; LDH= lactate dehydrogenase; HBDH =alpha-hydroxybutyrate dehydrogenase; CK= creatine kinase; CK-MB=creatine kinase MB isoenzyme. APTT= Activated partial thromboplastin time; TT= Thrombin time; FIB=Fibrinogen; P-FDP= Plasma Fibrin/Fibrinogen Degra-dation Products
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MANUSCRIPT Supplementary Figure ACCEPTED 1
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Supplementary Figure 2
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Table 1 Selected molecular markers detected in avian influenza A (H5N6) viruses A/duck/Guangdong/GD01/2014(H5N6) A/Anhui/1/2005(H A/Sichuan/2622 Gene Phenotypic consequences Mutations A/duck/Jiangxi/95/2014(H5N6) 5N1) 1/2014(H5N6) A/environment/Shenzhen/25-24/2013(H5N6) Altered receptor specificity S128P S T P Increased α2,6-SA S137A S A A recognition Removal of the 158N HA$ T160A T A A glycosylation Q226L Q Q Q RBS S227N S R R G228S G G G Linkpeptide RERRRKR↓G REKRRKR↓G RERRRKR↓G N6 59-69 del NA* No Yes Increased path in mice L89V L V V Increased virulence and G309D, T339K GTRI DMGV DTGV PB2 replication in mice R477G, I495V Increased path in mice E627K E E E Enhanced transmission D701N D N D PB1Increased Pathogenicity 90AA 57AA 57AA F2 Increased virulence in mice D92E D E E NS1 and pigs PDZ-motif ESEV ESEV ESEV Note: $, H3 numbering; *, Not Applicable.
S0163-4453(15)00220-0
DOI:
10.1016/j.jinf.2015.06.009
Reference:
YJINF 3561
To appear in:
Journal of Infection
Received Date: 23 April 2015 Revised Date:
24 June 2015
Accepted Date: 25 June 2015
Please cite this article as: Pan M, Gao R, Lu Q, Huang S, Zhou Z, Yang L, Li X, Zhao X, Zou X, Tong W, Mao S, Zou S, Bo H, Zhu X, Liu L, Yuan H, Zhang M, Wang D, Li Z, Zhao W, Ma M, Li Y, Li T, Yang H, Xu J, Zhou L, Zhou X, Tang W, Song Y, Chen T, bai T, Zhou J, Wang D, Wu G, Li D, Feng Z, Gao GF, Wang Y, He S, Shu Y, Human infection with a novel highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings, Journal of Infection (2015), doi: 10.1016/j.jinf.2015.06.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Human infection with a novel highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings 3
4
Ming Pan1†, Rongbao Gao2†, Qiang Lu1†, Shunhe Huang †, Zhonghui Zhou †, Lei Yang2
3
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Xiaodan Li2, Xiang Zhao2, Xiaohui Zou2, Wenbin Tong1, Suling Mao1, Shumei Zou2, Hong Bo2, 4
4
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Xiaoping Zhu1, Lei Liu1, Heng Yuan1, Minghong Zhang , Daqing Wang , Zumao Li , Wei Zhao , 6
7
Maoli Ma , Yaqiang Li , Tianshu Li1, Huiping Yang1, Jianan Xu1, Lijun Zhou1, Xingyu Zhou1, 3
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Wei Tang , Ying Song8, Tao Chen2, Tian bai2, Jianfang Zhou2, Dayan Wang2, Guizhen Wu2,
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Dexin Li2, Zijian Feng9, George F Gao9, Yu Wang9, Shusen He1*, Yuelong Shu2*
1
Sichuan Provincial Disease Control and Prevention, Chengdu 610041, China.
2
National Institute for Viral Disease Control and Prevention, China CDC, Key
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Laboratory for Medical Virology, National Health and Family Planning Commission, Beijing 102206, China.
Nanchong City Disease Control and Prevention, Nanchong 637000, China.
4
Affiliated Hospital of Chuanbei Medical School, Nanchong 637000, China.
5
Nanbu County Disease Control and Prevention, Nanchong 637300, China.
6
Nanbu County Hospital, Nanchong 637300, China.
7
Dongba Center Hospital of Nanbu County, Nanchong 637300, China.
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Department of Pathology, Beijing Renhe Hospital, Beijing 102600, China.
9
Chinese Center for Disease Control and Prevention, Beijing 102206, China.
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†These first authors contributed equally in this study
ACCEPTED MANUSCRIPT *Correspondence footnote: Address reprint requests to Dr. Yuelong Shu, National Institute for Viral Disease Control and Prevention, China CDC, Key Laboratory for Medical Virology, National
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China. E-mail: [email protected], tel:8610-58900850;
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Health and Family Planning Commission. 155 Changbai Road, Beijing, 102206, P. R
Reassortment, Pathology
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Key words: Human infection, Highly pathogenic avian influenza virus, H5N6,
Summary: A fatal human infection with a novel reassortant H5N6 avian influenza
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virus was first identified. The virus was with backbone of H5N1 virus acquired NA gene from H6N6 virus.
Key words: Highly pathgenic avian influenza virus, H5N6, Reassortment, Emerging
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infectious diseases
ACCEPTED MANUSCRIPT Abstract Objectives Human severe infection with avian influenza A (H5N6) virus infection was identified firstly in 2014 in China. It was unknown or unclear on the disease or the pathogen by
human case with H5N6 virus infection. Methods
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people. This study would illustrate the virological and clinical findings of a fatal
We obtained and analyzed the clinical, epidemiological and virological data from the
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patient. RT-PCR, viral culture and sequencing were conducted for determination of causative pathogen.
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Results
The patient who presented with fever, severe pneumonia, leucopenia and lymphopenia, developed septic shock and ARDS, and died on day 10 after illness onset. A novel reassortant avian-origin influenza A (H5N6) viruswas isolated from the throat swab or trachea aspirate of the patient. The virus was reassorted with HA
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gene of Clade 2.3.4.4 H5, internal genes of Clade 2.3.2.1 H5 and NA gene of H6N6 avian viruses. The cleavage site of HA gene contained multiple basic amino acids indicating the novel H5N6 virus was highly pathogenic in chicken.
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Conclusions
A novel highly pathogenic avian influenza H5N6 virus with the backbone of H5N1
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virus acquired NA gene from H6N6 virus was first identified, and caused human infection with severe respiratory disease.
ACCEPTED MANUSCRIPT Text The threat posed by avian influenza A viruses to humans remains significant since these viruses raised sporadic human infections, underwent gene evolution and
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potential reassortment with human virus (1). Of these viruses, H5N1 and the recently emerged H7N9 in China have raised the global concerns due to high
mortality and limited human-to-human transmission (2, 3). Highly pathogenic
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avian influenza (HPAI) H5N1 virus caused first human infection in 1997 in Hong
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Kong (4), and reemerged in 2003. So far, this virus had resulted into more than 650 human infections with around 60% fatality in 16 countries since 2003(5). The HA gene of H5 virus has been undergoing evolution, generating multiple clades. The currently circulating clades in poultry include 1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4
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and 7 in the world.Clade 2.3.4 and 2.3.2 of these clades were predominantly circulating in China (6-8). And a total of 46 human H5N1 cases have been identified in China since 2003(5).
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Besides H5N1 virus, other HPAI H5 subtypes such as H5N2, H5N8, H5N5 viruses
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had been detected in poultry(9). And H5N8 outbreaks have been reported in poultry in Korea, Japan, and recently Europe. In addition, serologic evidence of human infection with H5N2 was reported in poultry workers in Japan (10). In April 2014, a fatal case infected with a novel H5N6 virus was identified in China. And total three H5N6 infected cases were identified so far in China. Research on the disease or the pathogen is necessary. In this study, the virological and clinical findings of the first fatal case were illustrated.
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Methods Surveillance, reporting, and data collection
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Throat swab and tracheal aspirate were obtained from a severe pneumonia case on day 8 and 9 after illness onset of fever, respectively. The samples were tested as influenza A (for M gene) and H5 positive with real-time RT-PCR. A standardized surveillance
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reporting form was used to collect epidemiologic and clinical data, including
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demographic characteristics; underlying medical conditions; recent exposures to swine, poultry, or other animals; recent visits to live poultry markets; clinical signs and symptoms; chest radiographic findings; clinical laboratory testing results; antiviral treatment; clinical complications; and outcomes. Environmental samples
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were collected in epidemiological linked live poultry markets. Close contacts of the patient were defined as those who had provided care to, had been living with, or had potentially been exposed directly to respiratory secretions or body fluids of the patient
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from the 14th day before illness onset to the day of death. Two weeks of medical
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monitor was conducted among all contacts and throat samples were collected. Virus isolation
The throat swab and tracheal aspirate samples collected from the patient were performed virus isolation on embryonated eggs. Briefly, 0.2ml of throat swab or tracheal aspirate samples were injected into the allantoic cavity of 9 days old specific-pathogen-free chicken eggs respectively, and were propagated for 48 hours at 37°C. The allantoic fluids were tested for haemagglutination activity with turkey red blood cells.,Positive isolates were subjected to RT-PCR and sequencing. Three blind passages were performed on haemagglutination-negative samples to confirm the
ACCEPTED MANUSCRIPT absence of infectious virus. All steps were conducted in biosafty level-3 containment laboratory. RNA extraction and RT-PCR for subtyping RNA was extracted from all collected specimens and viral isolates using QIAamp
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Viral RNA Mini Kit (Qiagen, Germany), according to the manufacturer’s instructions. Specific real-time RT-PCR or conventional RT-PCR assays with self-designed
specific primer and probe sets for detecting seasonal influenza viruses (H1, H3, or B)
Genome sequencing and phylogenetic analysis
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and avian influenza viruses (H5, H7, H9, H10 and N1-N9) (11-13).
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The full genome of the virus was amplified with the use of Qiagen OneStep RT-PCR Kit for sequencing. PCR products were purified from agarose gel with the use of the QIAquick Gel Extraction Kit (Qiagen, Germany). We performed the sequencing using an ABI 3730xl automatic DNA analyzer (Life Technologies, USA) and the ABI
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BigDye Terminator v3.1 cycle sequencing kit (Life Technologies, USA), according to the manufacturer’s recommendations. Phylogenetic analysis was conducted with
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MEGA5.1 using neighbor-joining tree for HA gene as recommended by World Health Organization( WHO) (7) and maximum likelihood tree for other 7 genes.
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Deep sequencing and data analysis Total RNA was extracted from throat swab of the patient, reverse transcribed to double-strand DNA. After quantification, 200ng DNA was subjected to fragmentation and adapter ligation using Ion Xpress™ Plus Fragment Library Kit (Life technologies, Carlsbad, USA). The 200bp DNA library was selected by size with E-Gel® iBase™ unit and applied to emulsion-PCR with Ion PGM™ Template OT2 200 Kit (Life technologies, Carlsbad, USA). After enrichment, the
ACCEPTED MANUSCRIPT template-positive Ion Sphere™ Particles were loaded on Ion 318™ v2 chip (Life technologies, Carlsbad, USA) and sequenced with Ion PGM™ Sequencing 200 Kit v2(Life technologies, Carlsbad, USA). The Next Generation Sequencing (NGS)
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reads were analyzed with a pipeline process using the CLC Genomics workbench 7.0.4 (Qiagen, Germany). Briefly, the raw reads were trimmed and de novo
assembled under CLC standard settings. The contigs with coverage over 30 were
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extracted and locally blasted in the non-redundant nucleotide database downloaded
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from GenBank in CLC with default parameters. Base on the Blast results, the taxonomic composition (at the species level) of each dataset was identified with MEGA(14) software (version 5.2.3). The percentages of the identified microbial species were estimated according to the numbers of the sequencing reads of each
Case report
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Results
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species followed by normalization with their genome sizes(15).
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The patient was a 49 years old male without underlying medical conditions, who was a dealer in a local live poultry market for previous year. By report, he presented with fever, cough, headache, myalgia and stomach discomfort as initial clinical symptoms on April 13, 2014. He was treated as an upper respiratory infection in a rural clinic, and developed cough with blood-tinged sputum on day 7 after illness onset of fever. He was hospitalized with fever (axillary temperature in 37.5
) on April 20, 2014 in
Nanbu county hospital (Table 1, Figure 1). The chest radiography showed that the
ACCEPTED MANUSCRIPT patient had consolidation in left lung and patchy shadows in right lung on day 7 after illness onset of fever (Figure 2-A). The patient was transferred to the hospital affiliated with north Sichuan medical university on April 21, 2014. The chest
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radiography and computed tomography showed patchy shadows of both left and right lung with ground-glass opacities and consolidation on day 8 after illness onset of fever (Figure 2-B,C).
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The white blood cells (WBC) and platelet counts, and the ratio of lymphocytes were
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decreased on admission. The absolute neutrophil counts were decreased on day 8 but returned back to normal range on day 9, however, the ratio of neutrophils was elevated.Both absolute counts and ratio of lymphocytes were decreased. The eosinophil granulocytes in WBC was not detectable. The levels of c-reaction protein,
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procalcitonin, spartate aminotransferase (AST), lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH) and plasma Fibrin/Fibrinogen Degradation Products (P-FDP) were increased, while decreased levels of total
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protein, albumin and prealbumin were observed (Table 2).
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Despite combination antibiotic therapy, mechanical ventilation, glucocorticoids, and antiviral therapy were administered, the patient developed severe pneumonia, septic shock, and acute respiratory distress syndrome (ARDS), and died soon on day 10 after illness onset (Table 1, Figure 1). Determination of causative pathogen Throat swab and trachea aspirate obtained from the patient on April 20 and 21 respectively, and were confirmed on April 22 (Figure-1), by means of real-time
ACCEPTED MANUSCRIPT RT-PCR, conventional RT-PCR and sequencing, to be positive for influenza A (H5N6) virus but negative for seasonal influenza viruses (H1, H3 or B) or avian influenza viruses(H5N1, H7N9, H9N2 or H10N8). The virus isolated from the throat
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swab sample was designated as A/Sichuan/26221/2014(H5N6) (SC26221). No pathogenic bacteria and fungus were detected in sputum samples collected on day 7 and 8 after illness onset by culture. The deep sequencing data of throat swab
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revealed that the avian influenza A (H5N6) virus was overwhelmingly dominant
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(>73%) in microbial species. The genetic characterization of the virus
Full genome sequences of SC26221 were deposited in the Global Initiative on Sharing Avian Influenza Data (GISAID) database (Accession No. EPI533583-90). The virus
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showed a high similarity to the recently reported virus
A/environment/Zhenjiang/C13/2013 (H5N6) detected in live poultry market (16). Phylogenetic analysis showed that the HA gene of SC26221 belonged to clade 2.3.4.4
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H5 viruses nominated recently(17), while the NA gene belonged to an H6N6 subclade
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of Eurasian lineage viruses (Figure 3). All of the six internal genes of SC26221 were closely related to clade 2.3.2.1 H5 viruses circulating in duck in China (Figure S1 in Supplementary Appendix). The HA cleavage site possesses multiple basic amino acids motif (REKRRKR↓G), indicating potential high pathogenicity in chicken (Table S1 in Supplementary Appendix). The amino acid motif QSG at positions 226-228 (H3 numbering) of the HA protein indicated that the virus was avian-like (α2,3-SA) receptor binding
ACCEPTED MANUSCRIPT preference. The S137A and T160A mutations which resulted in the absence of the 158N glycosylation were detected in the HA protein of SC26221 virus, indicating increased human-like (α2,6-SA) receptor recognition of the virus(18, 19). The L89V,
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I495V, D701N, G309D and R477G mutations in PB2 protein, which were reported to increase viral pathogenic, virulence, and replication of H5N1 virus in mice, were
observed in the SC26221(20-22). No mutation associated with reduced sensitivity to
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NA inhibitors and adamantanes was detected in NA or M2 respectively.
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Epidemiological investigation
The patient was a dealer in a local live poultry market for one year. He would take the unsold birds to temporarily breed in the backyard of his house. One goose, 2 ducks and 5 chickens died during April 13-17th. A total of 144 environmental
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samples associated were collected and detected by real-time RT-PCR.Only 3 samples were influenza A PCR positive but H5 negative. However, H5N6 virus was detected in the dead poultries by report(23) , indicating that the source of
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infection may be from infected poultries. A total of 47 close contacts, including 12
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health care providers, 7 family members, 2 patients in same ward and 26 others were indentified. Medical observations on all contacts showed that no influenza like illness (ILI) symptom was observed during the investigation period (2 weeks). Meanwhile, throat swabs were collected from 46 contacts during April 21-23 and negative for influenza virus by real-time RT-PCR test.
Discussion
ACCEPTED MANUSCRIPT A fatal human infection with a novel reassortant influenza A (H5N6) virus was firstly identified. Phylogenetic analysis showed that the virus is a triple reassortant avian influenza virus contained an HA from clade 2.3.4.4 H5 viruses, a NA from H6N6
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viruses, and 6 internal genes from clade 2.3.2.1 H5 viruses (Figure S2 in Supplementary Appendix). Both H5N1 and H6N6 viruses have been circulating in
poultry in China, increasing the possibility for their reassortments. The novel H5N6
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viruses could be generated by two independent reassortments or from a common
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H5N6 progenitor virus. Our study showed that the HA genes of the H5N6 viruses shared only 95% homology, and located in the different subclades of HA phylogenetic tree. Two divergent N6 NAs with or without 59-69aa deletion in NA stalk existed in poultry viruses before the H5N6 case reported. NA genes of these H5N6 viruses
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distributed into two subclades in phylogenetic trees, whereas SC26221 and A/environment/Zhenjiang/C13/2013(H5N6) locating in one subclade without 59-69aa deletion in NA stalk. In hence, we proposed that these two “groups” of H5N6 viruses
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may be derived from independent reassortments. However, more sequences and
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surveillance data were needed to verify the origination of the H5N6 viruses. Consisted with the chest radiographs of severely viral pneumonia, our microbiologic studies demonstrated that the avian influenza A (H5N6) virus was overwhelmingly dominant in microbial species in throat swab, and no other pathogens was detected although the prior antibiotics treatments may influence the possibility of secondary infection detection. Several factors may contribute to the fatal outcome of the patient. Similar to H5N1 infection, the pathological damage in lung may link to
ACCEPTED MANUSCRIPT cytokine dysregulation triggered by H5N6 virus infection and resultant inflammatory damage to the lungs as the inflammation cells infiltration and diffuse hemorrhage observed in lung (24). And the patient presented liver and heart damage
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besides respiratory function failure, supported by increased levels of AST, LDH and HBDH, and decreased levels of total protein, albumin and prealbumin (Table.2)(25, 26). Previous reports showed no beneficial effect of corticosteroids in patients with
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ARDS secondary to influenza pneumonia, and that very early corticosteroids
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therapy may be harmful in 2009 pandemic H1N1 infection (27-29). In this study, systemic glucocorticoid therapy was performed subsequently three days on the patient after hospitalization on day 7 after illness onset. The effect of glucocorticoid therapy for the disease development of the patient might not be excluded. In
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addition, Oseltamivir therapy starting on day 7 of illness did not prevent death, although the NA sequence data predicted susceptibility. It may be related to the delay in starting antiviral therapy as antiviral therapy can shorten the duration of
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viral shedding and reduced mortality in hospitalized patients with severe influenza if
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prescribed during the first 3 days of illness(30-32). Human infection with different avian influenza viruses had been reported, and some have presented limited human-to-human transmission such as H5N1 (2) and H7N9 (3). Of note, the mutations associated with mammalian adaptation were detected in the H5N6 virus, and three cases have been identified since 2014. Taken together with continuous emergence of avian influenza A (H7N9 and H10N8) viruses caused human infections, it is important to enhance surveillance to monitor mutations and
ACCEPTED MANUSCRIPT reassortment of avian influenza viruses for pandemic preparedness and response.
Acknowledgments
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We thank the Chinese influenza surveillance network for providing access to their collection of clinical samples and data of the patient.This work was supported by the National Basic Research Program (973) of China (2011CB504704, to Dr. Yuelong
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Shu), Emergency Research Project on human infection with avian influenza H7N9
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virus from the National Ministry of Science and Technology (no. KJYJ-2013-01-01 to Dr. Yuelong Shu), and National Mega-projects for Infectious Diseases (2013ZX10004-101 to Dr. Dexin Li and 2012ZX10004215 to Dr. Biao Kan), the Chinese National Influenza Center– Centers for Disease Control and Prevention
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(CDC) collaborative project 5U51IP000334-03 from the CDC China- U.S. Collaborative Program on Emerging and Re-emerging Infectious Diseases, and the
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National Natural Science Foundation of China (81373107 to Dr. Rongbao Gao).
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Conflicts of Interest
No potential conflicts in this study.
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ACCEPTED MANUSCRIPT 14. Huson DH, Auch AF, Qi J, Schuster SC. MEGAN analysis of metagenomic data. Genome Res. 2007 Mar;17(3):377-86. 15. Yang J, Yang F, Ren L, Xiong Z, Wu Z, Dong J, et al. Unbiased Parallel Detection of Viral Pathogens in Clinical Samples by Use of a Metagenomic Approach. Journal of Clinical Microbiology. 2011 October 1, 2011;49(10):3463-9.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 The timeline of the clinical course of the patient and identification of the
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pathogen
Figure 2 Chest radiographs.
Chest radiograph showed that consolidation of left lung and bilateral ground-glass
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opacity were clearly seen on day 7 after illness onset (Panel A). White lung in left
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and developed patchy shadows of right lung were observed on day 8 (Panel B). Computed tomographic scan of the chest of the patient, obtained on day 8, was shown in Panel C. Consolidation of left lung lower lobe, increased density of right
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lung can be seen in upper, middle and lower lobes.
Figure 3 Phylogenetic analysis of HA (A) and NA (B) genes of SC26221. The novel H5N6 virus isolated from the patient was highlighted in red, other H5N6
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viruses isolated from poultry in China in blue. Supporting bootstrap values of greater
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than 0.6 were shown (1000 replicates).
Supplementary figure legends
Figure S1. Phylogenetic trees of HA and internal genes of H5 influenza viruses
ACCEPTED MANUSCRIPT The novel H5N6 isolate was highlighted in red, the clade 2.3.4 H5 viruses in blue, and the clade 2.3.2.1 viruses in pink. Supporting bootstrap values of greater than 0.6
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were shown (1000 replicates).
Figure S2 Hypothetical origins of the gene segments of the novel reassortant influenza A (H5N6) viruses
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Virus particles were represented by colored ovals containing horizontal bars that
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represented the eight gene segments (from top to bottom: PB2, PB1, PA, HA, NP, NA, M and NS) Arrowheads pointed to the resulting reassortants. The broken line
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represented NA gene with 59-69aa deletion.
ACCEPTED MANUSCRIPT Table 1 Clinical features and treatments of the patient Characteristics Treatment Mechanical ventilation Initiated on day 9 after illness onset Treatments (Days after illness onset) Antibiotic therapy Day 7
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Day 8
Glucocorticoids Day 7
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Methylprednisolone (two 80mg doses given intravenously) Methylprednisolone (one 40mg dose given intravenously) Methylprednisolone (one 40mg dose given intravenously)
Day 8 Day 9
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Antiviral therapy Day 7 Day 8 Anti-bleeding therapy Day 7
Day 9
Imipenem (two 1g doses given intravenously) Imipenem (1g doses given intravenously every 8h), Moxifloxacin Hydrochloride (one 0.4g doses given intravenously) Vancomycin Hydrochloride(one 1g dose given intravenously), Moxifloxacin Hydrochloride (one 0.4g dose given intravenously)
Tamiflu (two150mg doses given ) Tamiflu (two 75mg doses given) Pantoprazole sodium (40mg I.V. twice), Carbazochrome Sodium Sulfonate (80mg I.V. once) Pantoprazole sodium (40mg I.V. q8h)
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WBC counts (×109/L) Lymphocytes counts (×109/L) Ratio of lymphocytes (%) Neutrophils counts (×109/L) Ratio of Neutrophils (%) Lymphomonocyte Numbering (×109/L) Ratio of lymphomonocyte (%) Eosinophile granulocyte counts (×109/L) Ratio of Eosinophile granulocyte (%) (×109/L)
3.0-10
-
0.02-0.52
0
0
0
-
0
0
91
91
92
8.5-11.8
14.1
12.6
13
0.00-8.00 0.00-0.50 5.0-40.0
162.30 -
252.00 3.60 -
49.0
84.4
76.5
1.10-3.20
20-50 1.8-6.3
40-75
0.4-8 100-300
Mean platelet volume (fL) C-reaction protein(mg/L) Procalcitonin (ng/ml) Aspartate aminotransferase (U/L)
1.48
3.05
0.19
0.73
12.8
23.9
1.22
2.25
82.4
73.8
0.06
0.03
4.1
1
35.0-125.0
66.0
78.1
63.0
Alpha-L-fucosidase (U/L)
0.0-40.0
-
41.9
32.3
Prealbumin(mg/dL) Total protein (g/L) Albumin(g/L) Globulin(g/L) apoA1 (g/L) LACT(mmol/L) LDH (U/L) HBDH(U/L) CK(U/L) CK-MB(U/L) APTT (s) TT(s) FIB(g/L) P-FDP (Ug/ml)
150.0-400.0 65.0-85.0 40.0-55.0 20.0-40.0 1.00-1.60 0.50-2.20 114.0-240.0 60.00-180.00 25.00-180.00 1.0-25.0 18.0-30.0 14.0-21.0 1.80-3.60 0.0-5.0
111.0 61.1 36.1 25.0 -
62.7 58.4 32.4 26.0 0.72 2.54 895.4 703.43 128.47 25.5 35.0 19.7 4.65 114.3
49.4 54.5 26.5 28 -
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Alkaline phosphatase(U/L)
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2 3 4 5
0.1-0.6
1.2 23.9 70.3 -
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platelet counts
4.0-10.0
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Variable
Table.2 The results of clinical blood biochemistry tests Sample collected time (Days after Normal illness onset) range 7 8 9
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Note: “-” means data unavailable. LACT= lactic acid; LDH= lactate dehydrogenase; HBDH =alpha-hydroxybutyrate dehydrogenase; CK= creatine kinase; CK-MB=creatine kinase MB isoenzyme. APTT= Activated partial thromboplastin time; TT= Thrombin time; FIB=Fibrinogen; P-FDP= Plasma Fibrin/Fibrinogen Degra-dation Products
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Table 1 Selected molecular markers detected in avian influenza A (H5N6) viruses A/duck/Guangdong/GD01/2014(H5N6) A/Anhui/1/2005(H A/Sichuan/2622 Gene Phenotypic consequences Mutations A/duck/Jiangxi/95/2014(H5N6) 5N1) 1/2014(H5N6) A/environment/Shenzhen/25-24/2013(H5N6) Altered receptor specificity S128P S T P Increased α2,6-SA S137A S A A recognition Removal of the 158N HA$ T160A T A A glycosylation Q226L Q Q Q RBS S227N S R R G228S G G G Linkpeptide RERRRKR↓G REKRRKR↓G RERRRKR↓G N6 59-69 del NA* No Yes Increased path in mice L89V L V V Increased virulence and G309D, T339K GTRI DMGV DTGV PB2 replication in mice R477G, I495V Increased path in mice E627K E E E Enhanced transmission D701N D N D PB1Increased Pathogenicity 90AA 57AA 57AA F2 Increased virulence in mice D92E D E E NS1 and pigs PDZ-motif ESEV ESEV ESEV Note: $, H3 numbering; *, Not Applicable.
