NEJM H7N9

T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med nejm.org 1 original article Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus Rongbao Gao, M.D., Bin Cao, M.D., Yunwen Hu, M.D., Zijian Feng, M.D., M.P.H., Dayan Wang, M.D., Wanfu Hu, M.D., Jian Chen, M.D., Zhijun Jie, M.D., Haibo Qiu, M.D., Ph.D., Ke Xu, M.D., Xuewei Xu, M.D., Hongzhou Lu, M.D., Ph.D., Wenfei Zhu, M.D., Zhancheng Gao, M.D., Nijuan Xiang, M.D., Yinzhong Shen, M.D., Zebao He, M.D., Yong Gu, M.D., Zhiyong Zhang, M.D., Yi Yang, M.D., Ph.D., Xiang Zhao, M.D., Lei Zhou, M.D., Xiaodan Li, M.D., Shumei Zou, M.D., Ye Zhang, M.D., Xiyan Li, M.D., Lei Yang, M.D., Junfeng Guo, M.D., Jie Dong, M.D., Qun Li, M.D., Libo Dong, M.D., Yun Zhu, M.D., Tian Bai, M.D., Shiwen Wang, M.D., Pei Hao, M.D., Weizhong Yang, M.D., Yanping Zhang, M.D., Jun Han, M.D., Hongjie Yu, M.D., Dexin Li, M.D., George F. Gao, Ph.D., Guizhen Wu, M.D., Yu Wang, M.D., Zhenghong Yuan, Ph.D., and Yuelong Shu, Ph.D. The authors’ affiliations are listed in the Appendix. Address reprint requests to Dr. Shu at the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Preven- tion, Key Laboratory for Medical Virolo- gy, National Health and Family Planning Commission, 155 Changbai Rd., Beijing, 102206, China, or at yshu@cnic.org.cn; or to Dr. Yuan at the Key Lab of Medical Molecular Virology, School of Basic Med- ical Sciences, Shanghai Medical College of Fudan University, Shanghai City 200032, or at zhyuan@shmu.edu.cn. *Drs. R. Gao, Cao, Y. Hu, Feng, D. Wang, W. Hu, Chen, Jie, and Qiu contributed equally to this study. This article was published on April 11, 2013, at NEJM.org. N Engl J Med 2013. DOI: 10.1056/NEJMoa1304459 Copyright © 2013 Massachusetts Medical Society. A BS TR AC T Background Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus. Methods We obtained and analyzed clinical, epidemiologic, and virologic data from these pa- tients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase–polymerase-chain-reaction assays, viral culturing, and sequence analyses. Results A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respira- tory specimens obtained from all three patients and was identified as H7N9. Sequenc- ing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 vi- rus. A T160A mutation was identified at the 150-loop in the HA gene of all three vi- ruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed dif- fuse opacities and consolidation. Complications included acute respiratory distress syn- drome and multiorgan failure. All three patients died. Conclusions Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.) The New England Journal of Medicine Downloaded from nejm.org on April 14, 2013. For personal use only. No other uses without permission. Copyright © 2013 Massachusetts Medical Society. All rights reserved. T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med nejm.org2 Sporadic human infections with avian influenza A viruses, which usually occur after recent exposure to poultry, have caused a wide spectrum of illness, ranging from conjunctivitis and upper respiratory tract disease to pneumonia and multiorgan failure. Low pathogenic avian influenza A (H7N2, H7N3, H9N2, or H10N7)1-4 virus infections have caused lower respiratory tract illness that is mild (con- junctivitis or uncomplicated influenza-like ill- ness) to moderate in severity. Most human infec- tions with highly pathogenic avian influenza (HPAI) A (H7) viruses have resulted in conjuncti- vitis (H7N3) or uncomplicated influenza illness, but one case of fatal acute respiratory distress syndrome (ARDS) was reported in a patient with H7N7 virus infection during an outbreak in the Netherlands.1,5 In contrast, the cumulative case fatality rate since 2003 for reported cases of HPAI H5N1 virus infection is approximately 60%.6-8 The transmission of H7 viruses to mammals has been reported only rarely9 in Asia. Human infections with N9 subtype viruses had not been documented anywhere in the world. In February and March 2013, three patients were hospital- ized with severe lower respiratory tract disease of unknown cause. We report the identification of a novel avian-origin reassortant influenza A (H7N9) virus associated with these infections. Me thods Surveillance, Reporting, and Data Collection Throat-swab specimens obtained from three adult Chinese patients (two from Shanghai City and one from Anhui Province) who were hospi- talized with severe bilateral pneumonia, leuko- penia, and lymphocytopenia were sent to Shang- hai Public Health Clinical Center, the Shanghai Centers for Disease Control and Prevention (CDC), and the Anhui CDC, respectively. After preliminary detection of respiratory pathogens, the samples were sent to the Chinese National Influenza Center (CNIC) on March 25, 2013. A standardized surveillance reporting form was used to collect epidemiologic and clinical data, including demographic characteristics; un- derlying medical conditions; history of seasonal influenza vaccination; recent exposures to swine, poultry, or other animals; recent visits to a live animal market; clinical signs and symptoms; chest radiographic findings; laboratory testing results, including diagnostic testing for influ- enza and other respiratory viruses; antiviral treatment; clinical complications; and outcomes. A confirmed case of human infection with avi- an-origin influenza A (H7N9) virus was defined as evidence of pneumonia with H7N9 viral RNA or isolation of H7N9 virus from respiratory specimens at the CNIC. Isolation of the Virus Throat-swab specimens obtained from all three patients were maintained in a viral-transport me- dium. The specimens were propagated in the al- lantoic sac and amniotic cavity of 9-to-11-day-old specific pathogen-free embryonated chicken eggs for 48 to 72 hours at 35°C. RNA Extraction and Real-time RT-PCR RNA was extracted from throat-swab samples with the use of the QIAamp Viral RNA Mini Kit (Qiagen), according to the manufacturer’s in- structions. Specific real-time reverse-transcrip- tase–polymerase-chain-reaction (RT-PCR) assays for seasonal influenza viruses (H1, H3, or B), H5N1, severe acute respiratory syndrome corona- virus (SARS-CoV), and novel coronavirus were used. Real-time RT-PCR assays with self-designed specific primer and probe sets for detecting H1 to H16 and N1 to N9 subtypes were then per- formed to verify the viral subtypes. Genome Sequencing and Phylogenetic Analysis A total of 198 primer sets were used to amplify the full genome for sequencing, with the use of Qiagen OneStep RT-PCR Kit. PCR products were purified from agarose gel with the use of the QIAquick Gel Extraction Kit (Qiagen). We per- formed the sequencing using an ABI 3730xl auto- matic DNA analyzer (Life Technologies) and the ABI BigDye Terminator v3.1 cycle sequencing kit (Life Technologies), according to the manufac- turer’s recommendations. Full genome sequenc- es of the viruses from these patients were depos- ited in the Global Initiative on Sharing Avian Influenza Data (GISAID) database on March 29, 2013 (accession numbers are provided in Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). We performed multiple sequence alignments The New England Journal of Medicine Downloaded from nejm.org on April 14, 2013. For personal use only. No other uses without permission. Copyright © 2013 Massachusetts Medical Society. All rights reserved. Human Infection with an Avian-Origin Influenza A Virus n engl j med nejm.org 3 with the ClustalW program using MEGA soft- ware, version 5.05. Phylogenetic trees were con- structed by means of the neighbor-joining meth- od with the use of MEGA software, version 5.05, to estimate the viral gene relationship with se- lected influenza A virus strains obtained from GenBank. R esult s Patients Patient 1 was an 87-year-old man with chronic ob- structive pulmonary disease (COPD) and hyperten- sion who reported a cough and sputum production at the onset of illness. High fever and dyspnea de- Table 1. Demographic, Epidemiologic, and Virologic Characteristics and Complications, Treatment, and Clinical Outcomes of Three Patients Infected with Avian-Origin Influenza A (H7N9) Virus.* Characteristic Patient 1 Patient 2 Patient 3 Age (yr) 87 27 35 Sex Male Male Female Occupation Retired Butcher Housewife Underlying conditions COPD, hypertension Hepatitis B Depression, hepatitis B, obesity Area of origin Shanghai Shanghai Anhui Exposure to chicken market in past 7 days No Yes Yes Date of illness onset February 18, 2013 February 27, 2013 March 13, 2013 Date of admission February 25, 2013 March 4, 2013 March 19, 2013 Admission to ICU None March 6, 2013 March 20, 2013 Date of specimen collection February 26, 2013 March 5, 2013 March 20, 2013 Date of laboratory confirmation of virus March 30, 2013 March 30, 2013 March 30, 2013 Viral isolation A/Shanghai/1/2013 (H7N9) A/Shanghai/2/2013 (H7N9) A/Anhui/1/2013 (H7N9) Complications Septic shock No No Yes ARDS Yes Yes Yes Acute renal damage No No Yes Encephalopathy Yes No Yes Rhabdomyolysis No Yes Yes Secondary infections No Yes† Yes† Oxygen therapy Mask‡ Mechanical ventilation Mechanical ventilation Extracorporeal membrane oxygenation No No Yes Continuous renal-replacement therapy No No Yes Antibiotic therapy Imipenem, moxifloxacin, and vancomycin Cefoperazone–sulbactam, levofloxacin, and linezolid Imipenem and vancomycin Antiviral agent§ Oseltamivir (started on day 7) Oseltamivir and amantadine (started on day 7) Oseltamivir (started on day 8) Glucocorticoid therapy Yes Yes Yes Intravenous immune globulin therapy Yes Yes Yes Length of stay in hospital 6 days 6 days 19 days Date of death March 4, 2013 March 10, 2013 April 9, 2013 * ARDS denotes acute respiratory distress syndrome, and COPD chronic obstructive pulmonary disease. † Patients 2 and 3 were infected with carbapenem-resistant Acinetobacter baumannii. ‡ This patient refused intubation and mechanical ventilation. § Oseltamivir was administered in Patient 1 on February 25, 2013; the viral sample was obtained after two doses of oseltamivir were adminis- tered. Oseltamivir was administered in Patient 2 on March 6, 2013, and in Patient 3 on March 21, 2013. The New England Journal of Medicine Downloaded from nejm.org on April 14, 2013. For personal use only. No other uses without permission. Copyright © 2013 Massachusetts Medical Society. All rights reserved. T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med nejm.org4 veloped 1 week after the onset of illness. He had no known history of exposure to live birds during the 2 weeks before the onset of symptoms. Patient 2 was a 27-year-old man with a his- tory of hepatitis B virus infection with positive hepatitis B surface antigen who presented to the hospital with high fever and cough. This patient was a butcher who worked at a market where there were transactions involving live birds. He sold pork but had not butchered bird meat be- fore the onset of illness. Both Patient 1 and Pa- tient 2 lived in Min-hang District, Shanghai, and were admitted to the Fifth People’s Hospital. Patient 3 was a 35-year-old woman who lived in Anhui Province. She had a history of depres- sion, hepatitis B virus infection, and obesity. Patient 3 also had high fever and cough at the onset of the illness. She had visited a chicken market 1 week before the onset of symptoms. The demographic and epidemiologic characteris- tics of the three patients are summarized in Table 1. Determination of Causative Pathogens We confirmed, by means of real-time RT-PCR, viral isolation, and full genome sequencing, that all three patients were infected with a novel avian- origin influenza A (H7N9) virus. Original clini- cal samples obtained from all three patients were confirmed, by means of real-time RT-PCR, to be positive for H7N9 and negative for seasonal in- fluenza viruses (H1, H3 or B), H5N1, SARS-CoV, and HCoV-Erasmus Medical Center (EMC). Influ- enza viruses A/Shanghai/1/2013 (H7N9), A/ Shanghai/2/2013 (H7N9), and A/Anhui/1/2013 (H7N9) were isolated from Patients 1, 2, and 3, respectively. Complete sequences of the three H7N9 influenza viruses showed that they were 97.7 to 100% identical in all eight gene segments (see Table S1 in the Supplementary Appendix). Phylogenetic analysis of all genes of the isolates showed that each gene was of avian origin (Fig. 1, and Fig. S1 in the Supplementary Appendix). The gene encoding hemagglutinin (HA) shared the highest identity with A/duck/Zhejiang/12/2011 (H7N3, subtype ZJ12). The gene encoding neur- aminidase (NA) protein was most closely related to A/wild bird/Korea/A14/2011 (H7N9, subtype KO14); however, the HA gene from the H7N9 vi- ruses in our three patients was highly divergent from that in the KO14 virus. All six internal genes shared the highest similarity with A/brambling/ Beijing/16/2012-like viruses (H9N2) (Fig. 1). Phylo- genetic results indicated that it was a triple reas- sortant H7N9 virus (Fig. 2). In all three viruses, the HA cleavage site pos- sesses only a single amino acid R (arginine), indicating low pathogenic effects in poultry. A T160A mutation was identified at the 150-loop (H3 numbering) in the HA gene of all three vi- ruses. Substitution Q226L at the 210-loop in the HA gene was found in both the A/Anhui/1/2013 and A/Shanghai/2/2013 viruses but not in the A/ Shanghai/1/2013 virus (Table 2). Five amino ac- ids were deleted in the stalk region of NA resi- due 69 to 73. The M2 protein contained the S31N substitution, indicating resistance to amantadine. Other mutations — 89V and E627K in PB2 and 42S in NS1 — were also identified (Table 2). The amino acids in A/Shanghai/1/2013, which differed from those in A/Anhui/1/2013 and A/Shanghai/2/2013, are shown in Table S2 in the Supplementary Appendix. To date, five ad- ditional H7N9 viruses have been isolated from five patients. Sequencing analysis indicates that all five viruses are highly similar to both A/ Shanghai/2/2013 and A/Anhui/1/2013. Some variability is observed, such as Q226L in HA and R292K in NA. On the basis of these data, diagnostic tests for the novel reassortant H7N9 viruses have been developed. The specific sequences are available on the website of the World Health Organization (www.who.int/influenza/gisrs_ laboratory/a_h7n9/en/). Clinical Features and Outcomes of the Patients The clinical characteristics of the patients are shown in Table S3 in the Supplementary Appen- Figure 1 (facing page). Phylogenetic Trees of Genes of H7N9 Influenza A Viruses. Shown are phylogenetic trees of full-length hemagglutinin (HA; Panel A) and neuraminidase (NA; Panel B) genes of H7N9 influenza A viruses. The trees were generated by means of the distance-based neighbor-joining method with the use of MEGA software, version 5.05. The reliabil- ity of the trees was assessed by means of bootstrap analy- sis with 1000 replications. Horizontal distances are pro- portional to the genetic distance. The novel reassortant H7N9 viruses are shown in red. The viruses in green pre- sented the highest similarity with the novel H7N9 viruses. Other H7N9 and human H7 subtype are shown in blue and pink, respectively. Phylogenetic trees of the other six genes are included in the Supplementary Appendix. The New England Journal of Medicine Downloaded from nejm.org on April 14, 2013. For personal use only. No other uses without permission. Copyright © 2013 Massachusetts Medical Society. All rights reserved. Human Infection with an Avian-Origin Influenza A Virus n engl j med nejm.org 5 A B H A N A 0. 02 0. 02 Eu ra si an Eq ui ne A m er ic an N or th A m er ic an A vi an Eu ra si an / O ce an ia n A vi an I Eu ra si an / O ce an ia n A vi an II A us tr al ia n The New England Journal of Medicine Downloaded from nejm.org on April 14, 2013. For personal use only. No other uses without permission. Copyright © 2013 Massachusetts Medical Society. All rights reserved. T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med nejm.org6 dix. Fever and cough were the most common symptoms. The white-cell count was normal or slightly decreased. Elevated levels of aspartate aminotransferase, creatine kinase, and lactate dehydrogenase were observed in all the patients. Bilateral ground-glass opacities and consolida- tion were detected on chest radiography (Fig. 3). Several complications of the illness were ob- + A/Anhui/1/2013 (H7N9)–like BJ16-like (H9N2) Reassortant avian-origin (H7N9) Bramblings Ducks Wild birds KO14-like (H7N9) ZJ12-like (H7N3) Avian Influenza A Virus (H7N9) Unknown host Neuraminidase Envelope Matrix protein (M1) M2 ion channel NS M NA NP HA PA PB1 PB2 NS M NA NP HA PA PB1 PB2 Hemagglutinin Viral polymerase genes NS-1 Possible intermediate host? Figure 2. Hypothetical Host and Lineage Origins of the Gene Segments of the Novel Reassortant Human Influenza A (H7N9) Viruses. The colors of the gene segments in the ovals indicate their origin. BJ16 denotes A/brambling/Beijing/16/2012, KO14 A/wild bird/Korea/ A14/2011, and ZJ12 A/duck/Zhejiang/12/2011. The New England Journal of Medicine Downloaded from nejm.org on April 14, 2013. For personal use only. No other uses without permission. Copyright © 2013 Massachusetts Medical Society. All rights reserved. Human Infection with an Avian-Origin Influenza A Virus n engl j med nejm.org 7 served. All the patients had ARDS. Patient 3 had septic shock and acute renal damage. Carba- penem-resistant Acinetobacter baumannii was cul- tured from lower respiratory tract specimens obtained from two of the patients after the ini- tiation of mechanical ventilation. Combination antibiotic therapy, glucocorticoids, and intrave- nous immunoglobulin were administered in all three patients. Antiviral therapy was initiated 6 to 7 days after the onset of illness (Table 1). Patient 1 declined admission to the intensive care unit (ICU) and intubation. He died from refractory hypoxemia 13 days after the onset of illness. Patient 2 was admitted to the ICU and intubated 48 hours after admission owing to progressive dyspnea. He died from refractory hypoxemia after 4 days in the ICU. ARDS and septic shock developed in Patient 3 on day 6 af- ter the onset of illness. She was admitted to the ICU, and extracorporeal membrane oxygenation was initiated. She died on April 9. Discussion We have identified a novel reassortant influenza A (H7N9) virus that is associated with severe hu- man infe