Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome

Abstract

Background: Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China.

Methods: Between March 7 and April 8, 2013, we included hospital inpatients if they had new-onset respiratory symptoms, unexplained radiographic infiltrate, and laboratory-confirmed H7N9 virus infection. We recorded histories and results of haematological, biochemical, radiological, and microbiological investigations. We took throat and sputum samples, used RT-PCR to detect M, H7, and N9 genes, and cultured samples in Madin-Darby canine kidney cells. We tested for co-infections and monitored serum concentrations of six cytokines and chemokines. We collected cloacal swabs from 86 birds from epidemiologically linked wet markets and inoculated embryonated chicken eggs with the samples. We identified and subtyped isolates by RT-PCR sequencing. RNA extraction, complementary DNA synthesis, and PCR sequencing were done for one human and one chicken isolate. We characterised and phylogenetically analysed the eight gene segments of the viruses in the patient's and the chicken's isolates, and constructed phylogenetic trees of H, N, PB2, and NS genes.

Findings: We identified four patients (mean age 56 years), all of whom had contact with poultry 3-8 days before disease onset. They presented with fever and rapidly progressive pneumonia that did not respond to antibiotics. Patients were leucopenic and lymphopenic, and had impaired liver or renal function, substantially increased serum cytokine or chemokine concentrations, and disseminated intravascular coagulation with disease progression. Two patients died. Sputum specimens were more likely to test positive for the H7N9 virus than were samples from throat swabs. The viral isolate from the patient was closely similar to that from an epidemiologically linked market chicken. All viral gene segments were of avian origin. The H7 of the isolated viruses was closest to that of the H7N3 virus from domestic ducks in Zhejiang, whereas the N9 was closest to that of the wild bird H7N9 virus in South Korea. We noted Gln226Leu and Gly186Val substitutions in human virus H7 (associated with increased affinity for α-2,6-linked sialic acid receptors) and the PB2 Asp701Asn mutation (associated with mammalian adaptation). Ser31Asn mutation, which is associated with adamantane resistance, was noted in viral M2.

Interpretation: Cross species poultry-to-person transmission of this new reassortant H7N9 virus is associated with severe pneumonia and multiorgan dysfunction in human beings. Monitoring of the viral evolution and further study of disease pathogenesis will improve disease management, epidemic control, and pandemic preparedness.

Funding: Larry Chi-Kin Yung, National Key Program for Infectious Diseases of China.

Figure 1

Representative radiographic findings of H7N9 influenza Chest radiograph of patient 1 taken 19 days after onset of symptoms, showing bilateral pulmonary infiltrates of airspace consolidation (A); CT of patient 1 taken 13 days after onset of symptoms, showing consolidation of right middle lobe (B); chest radiograph of patient 2 taken 14 days after onset of symptoms, showing bilateral interstitial infiltrate (C); and serial CTs of patient 4 taken 20 (D), 27 (E), and 35 (F) days after onset of symptoms, showing interval radiological improvement and resolution of bilateral ground glass changes.

Figure 2

Serum cytokine and chemokine profile of patients 2 (A) and 3 (B) Normal ranges: interferon γ (0·01–13·64 pg/mL); interleukin 2 (0·01–10·67 pg/mL); interleukin 4 (0·01–2·25 pg/mL); interleukin 6 (0·01–8·86 pg/mL); interleukin 10 (2·42–16·33 pg/mL); and tumour necrosis factor α (0·82–11·05 pg/mL).

Figure 3

Phylogenetic trees for the haemagglutinin (HA1) (A) and neuraminidase (N) (B) genes of H7N9 viruses isolated from a patient and a chicken in Zhejiang, China Sequences of H7N9 viruses characterised in our study are red—A/Zhejiang/UTID-ZJU01/2013 (H7N9) is the human isolate and A/chicken/Zhejiang/DTID-ZJU01/2013 (H7N9) is the epidemiologically linked chicken isolate. H7N7 viruses that were reported to cause human infections are blue. Human isolates of H7N9 viruses described in a 2013 report are green. The other sequences (black) were derived from other subtypes of influenza viruses that were available in Genbank. The triangle represents viruses of North American (ie, Canadian, Mexican, and US) lineage.

Figure 4

Phylogenetic trees for the PB2 (A) and NS (B) genes of H7N9 viruses isolated from a patient and a chicken in Zhejiang, China Sequences of H7N9 viruses characterised in our study are red. Human isolates of H7N9 viruses described in a 2013 report are green. The other sequences (black) were derived from H9N2 influenza viruses characterised at different times and from different places.