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 hospitalized with severe bilateral pneumonia, leukopenia, and lymphocytopenia were sent to Shanghai 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; underlying 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 influenza and other respiratory viruses; antiviral treatment; clinical complications; and outcomes. A confirmed case of human infection with avian-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 medium. The specimens were propagated in the allantoic 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 instructions. Specific real-time reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays for seasonal influenza viruses (H1, H3, or B), H5N1, severe acute respiratory syndrome coronavirus (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 performed 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 performed the sequencing using an ABI 3730xl automatic DNA analyzer (Life Technologies) and the ABI BigDye Terminator v3.1 cycle sequencing kit (Life Technologies), according to the manufacturer’s recommendations. Full genome sequences of the viruses from these patients were deposited 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 with the ClustalW program using MEGA software, version 5.05. Phylogenetic trees were constructed by means of the neighbor-joining method with the use of MEGA software, version 5.05, to estimate the viral gene relationship with selected influenza A virus strains obtained from GenBank.

Patients

Patient 1 was an 87-year-old man with chronic obstructive pulmonary disease (COPD) and hypertension who reported a cough and sputum production at the onset of illness. High fever and dyspnea developed 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 history 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 before the onset of illness. Both Patient 1 and Patient 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 depression, 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 characteristics of the three patients are summarized in Table 1Table 1Demographic, Epidemiologic, and Virologic Characteristics and Complications, Treatment, and Clinical Outcomes of Three Patients Infected with Avian-Origin Influenza A (H7N9) Virus..

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 clinical samples obtained from all three patients were confirmed, by means of real-time RT-PCR, to be positive for H7N9 and negative for seasonal influenza viruses (H1, H3 or B), H5N1, SARS-CoV, and HCoV-Erasmus Medical Center (EMC). Influenza 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 (Figure 1Figure 1Phylogenetic Trees of Genes of H7N9 Influenza A Viruses., and Figure 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 neuraminidase (NA) protein was most closely related to A/wild bird/Korea/A14/2011 (H7N9, subtype KO14); however, the HA gene from the H7N9 viruses 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) (Figure 1). Phylogenetic results indicated that it was a triple reassortant H7N9 virus (Figure 2Figure 2Hypothetical Host and Lineage Origins of the Gene Segments of the Novel Reassortant Human Influenza A (H7N9) Viruses.).

In all three viruses, the HA cleavage site possesses 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 viruses. 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 2Table 2Molecular Analysis of Three of the 2013 H7N9 Viruses.). Five amino acids were deleted in the stalk region of NA residue 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 additional 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 Appendix. 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 consolidation were detected on chest radiography (Figure 3Figure 3Chest Radiographs.).

Several complications of the illness were observed. All the patients had ARDS. Patient 3 had septic shock and acute renal damage. Carbapenem-resistant Acinetobacter baumannii was cultured from lower respiratory tract specimens obtained from two of the patients after the initiation of mechanical ventilation. Combination antibiotic therapy, glucocorticoids, and intravenous 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 after the onset of illness. She was admitted to the ICU, and extracorporeal membrane oxygenation was initiated. She died on April 9.