Clinical features and laboratory abnormalities of the patient
The patient was a 4-year-old boy living with his father, grandparents, brother and sister, who are otherwise healthy, in Shangcai County, Zhumadian City of Henan province, China (Supplemental Figure 1). On April 5, 2022, he developed a high fever of 39.3°C (Figure 1), with lethargy and anorexia appearing on April 6, and cough was present on April 8. Although treated with antipyretic drugs and other supportive therapy, his condition did not improve and respiratory symptoms worsened. On April 10, day 6 of his illness, he was admitted to a local hospital (Hospital A) for treatment. On admission, he had fever, shortness of breath, with the blood oxygen saturation values dropping to 80%. On the same day, severe respiratory signs and symptoms appeared, and the patient was transferred to the intensive care unit in hospital B (Zhumadian Central Hospital, Zhumadian city of Henan). On admission, physical extermination revealed a body temperature of 39.5°C, abnormal pupillary light reflex, moist rales, and cyanosis of lips, accompanied by lymphadenopathy. His blood pressure was 135/91 mmHg, tachycardic (pulse rate, 150/min), and transcutaneous oxygen saturation level was 80.0 % with mask oxygen inhalation. Blood tests showed leukopenia (2.63 109/L), hyperglycemia (blood sugar level 20.3 mmol/L), hyponatremia (129 mmol/L) and hypocalcemia (1.9 mmol/L). There was decreased concentrations of total protein (50.2 g/L), albumin (30.9 g/L), total bilirubin (1.9 μmol/L) and elevated concentrations of aspartate aminotransferase (AST, 138 U/L), and adenosine deaminase (ADA, 31.0 U/L), indicating liver dysfunction. Coagulation tests showed a significant prolongation of prothrombin time (PT, > 23.9 s), activated partial thromboplastin time (APTT, > 170 s), thrombin time (TT, > 160 s), and elevated D-dimer (D-D, 5.11 ug/ml) and fibrin degradation products (FDP, 17.36 ug/ml), indicating a coagulation dysfunction. Myocardial damage was displayed by high level of lactate dehydrogenase (LDH, 265 U/L), creatine kinase (CK, 603 U/L) and creatine kinase BB (CK-BB, 55.0 U/L). A chest computed tomographic (CT) scan showed multiple patchy high-density shadows in both lungs, especially in the lower lobe of the right lung and the left lung (Figure 2A-C). Antigen test for nine respiratory pathogens were all negative. The bronchoalveolar lavage fluid was positive for influenza A, while negative for the other 13 commonly seen respiratory pathogens. The same sample was simultaneously subject to high throughput sequencing.
Due to high fever of 41 °C, and exacerbation of respiratory symptoms, the patient was further transferred to a tertiary hospital in Zhengzhou City (Hospital C). Laboratory investigations revealed leukopenia, hyperglycemia, hyponatremia, liver dysfunction, myocardial damage, renal damage, and coagulation dysfunction, which contributed to multiple organ failure and sepsis (Figure 1). An inflammatory response characterized by elevated interleukin 6 (IL-6, 13.91 pg/mL), interferon-γ (IFN-γ, 45.55 pg/mL), IL-12P70 (5.63 pg/mL) was observed on day 8 after disease, indicating the development of systemic inflammatory response syndrome. The C-reactive protein (CRP) elevated to a remarkably high level on day-19 after disease, possibly due to bacterial coinfection. The respiratory symptoms exacerbated with laboratory abnormalities including platelet, albumin, Na+ remained abnormally low, while γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), APTT, FDP, D-D, CK and blood urea nitrogen (BUN) elevated above normal until recently (Supplemental Figure 2). Pseudomonas aeruginosa was determined in bronchoalveolar lavage fluid on day 8 of disease. Supportive care was prescribed on disease onset, in addition to this, extensive treatment, including antiviral therapy (Oseltamivir 30 mg twice a day and interferon α1b 30 ug twice a day), antibiotic therapy (linezolid, meropenem, and compound sulfamethoxazole), mechanical ventilation via endotracheal intubation, and extracorporeal membrane oxygenation (ECMO) was administered since day 6 of disease onset. (Supplemental Table 1). The patient was still hospitalized, and his condition improved with ECMO withdrawn on May 3, 2022.
Detection and identification of avian influenza A H3N8 virus
By high-throughput sequencing on the bronchoalveolar lavage fluid collected on April 10, we obtained the whole genome sequence of an influenza A/H3N8 virus (A/Henan/ZMD-22-2/2022(H3N8); GenBank accession number ON342803-ON342810). Phylogenetic analysis revealed ZMD-22-2 as a novel reassortant H3N8 influenza virus which is distinct from previously reported H3N8 viruses.
The closest related HA gene was in the clade of H3N2 and H3N8 viruses detected in ducks from Guangdong Province (A/duck/Guangdong/F352/2018 (A/H3N2) determined in 2018 with a nucleotide homology of 96.09% (Figure 3A). The highest similarity of NA segment was to H3N8 viruses in various species of birds in North America in 2014 and Japan in 2016 (A/northern pintail/Alaska/870/2014 (A/H3N8), with nucleotide homologies of 97.07% (Figure 3B). The nucleotide homologies of internal genes were closely related to H9N2 viruses that had been identified throughout China and isolated from humans, ducks or wild birds in recent years (Supplemental Figure 3). This indicates that the virus was a novel reassortant genotype which had undergone complicated mutation and reassortment events (Figure 4).
The amino acid sequence at cleavage site in HA protein is PEKQTR/GL, indicate that the virus was a low pathogenic avian influenza (LPAI) virus. Genetic features were evaluated based on a well-accepted correlation between receptor binding characteristics and host specificity of influenza viruses20, i.e., viruses isolated from wild aquatic birds bind strongly to α-2,3 sialyl glycan (SA 2,3) receptor, in contrast, human-adapted influenza viruses recognize and bind SA 2,6 receptors, which predominate in the human respiratory tract. For the novel H3N8 virus, the residue 226 and 228 of HA gene were glutamine (Q) and glycine (G), respectively. The key mutation Q226L and G228S in HA protein which rendered a strong binding to SA 2,6 receptors and increased transmission ability by air, are absent, thus indicating limited ability to bind to cells in the human respiratory tract.
The mutation of E627K was identified in PB2 gene. This mutation had been associated with increased virulence in mice and was reported to be associated with improved replication of avian influenza virus in mammals20, 21, 22. Besides, the N30D and T215A mutation in M gene and P42S mutation in NS gene were also observed, which mutations were associated with increased virulence in animal models23, 24.
Epidemiological investigation revealed the family had been raising 6 chickens in the back yard, which however, had been killed before the investigation was performed. The patient had intensive contact with a domesticated dog and a cat kept in the household before his illness onset, by feeding and playing with the dog and the cat. Five close-contact family members (grandfather, grandmother, father, eight-year-old brother, and six-year-old sister) who lived together with the patient, and the aunt who had close contact with the patient while he was ill were interviewed. None of them had bred livestock, or reported recent visit to poultry market, the grandfather and brother reported infrequent contact to the dog and cat, although less frequently than the patient. No other significant exposures, i.e., visiting poultry market or exposure to febrile patients were reported. There was a pond 27-meters distant from the household of the patient, which wild ducks inhabited, with frequent congregation with the domesticated poultry in the backyard (Supplemental Figure 1).
The molecular test on the 63 animal and environmental samples yielded 10 positive results for the novel H3N8, all from the patient’s household, including one nasopharyngeal swab of the dog; one anal swab of the cat; surface swabs of the boarding kennels of cat (1), dog (1), and chicken (1); drinking water (1) and food (1) of the dog; swab of the dining table (1), waste bin (1) and cabinet (1). No positive result was obtained in other 53 animal or environmental samples collected in the household (13) or the village (40).
No obvious illness was observed for the animals, although the dog was less active than usual and labored breathing as recalled by the family. Biochemistry test revealed elevated level of creatine kinase (CK, 323 U/L), lactate dehydrogenase (LDH, 1.29 g/mL) of the dog, Decreased LDH (0.96 g/mL) in the cat (Supplemental Table 2). Nucleotide sequence of full-length HA from the dog and cat were identical to sequence from the patient.
Detection for the novel A(H3N8), IFV, as well as 13 common respiratory pathogens were all negative for throat swabs collected from the 6 close contacts. The follow-up of the close contacts revealed influenza like illness developed until recently. Serological test on serum of the patient collected on April 10 revealed neutralizing antibody (Nab) titer of 1:121 against ZMD-22-2 virus by using a pseudotyped virus-based assay. Positive Nab was also determined from the grandparents of the patient (1:339 and 1:179, respectively) (Supplemental Table 3).