Jishou Municipal Center for Disease Control and Prevention (Jishou CDC) received a phone call at 7:30 a.m. on April 24, 2020 from Xiangxi Tujia and Miao Autonomous Prefectural CDC (Xiangxi CDC) that avian influenza A/H9N2 virus was detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR) from the throat swab sample of an influenza-like illness in an outpatient in the Xiangxi Traditional Chinese Medicine Hospital.
At 09:00 on April 24, the county and municipal CDCs carried out epidemiological investigations to show that the suspected case was found in a 5-year-old girl. At 03:00 on April 20, the girl developed initial symptoms of fever (37.3 ℃), fatigue, abdominal pain, and nausea with vomiting 3 times, and the top body temperature during the course of the disease was 38.6 ℃. In the morning of April 20, she was diagnosed with gastrointestinal cold in Xiangxi Traditional Chinese Medicine Hospital, and her throat swab samples were collected by the attending doctor. The fever disappeared by 17:00 the same day. On April 23, Xiangxi CDC carried out a RT-PCR test and the result illustrated that the patient was positive for avian influenza A/H9N2 virus, which verified by retesting on April 24. The patient was isolated to receive treatment in the hospital since April 25, and was released on April 30 based on the 2 consecutive negative results of RT-PCR test on April 28 and 29 (Figure1).
Figure 1. Timeline for the epidemiological investigation of the avian influenza A/H9N2 case in Hunan Province, 2020.
According to the investigation, the patient had no contact history with similar patients, live poultry, or poultry markets. On April 16, her grandmother bought a live duck from Stall 38 in a market and slaughtered it in Stall 14 before returning home to cook it. The patient’s parents and the attending doctor were judged to be close contacts and were placed under medical observation. On April 25, these 3 contacts were sampled and their RT-PCR test results were negative for avian influenza A/H9N2 virus. Up to May 6, none of them showed symptoms. Throat swab samples were collected from the patient’s grandparents and hosts of Stall 38, Stall 14, and adjacent stalls in the market and tested for avian influenza A/H9N2 virus, and all results were negative. About 30 live ducks were sold per day in Stall 38, and these ducks were bought from Huaihua City, Hunan Province. No more H9N2 cases were reported in Xiangxi and Huaihua by the end of May.
To identify the possible source of infection, 172 swab samples from live poultry’s throat and anus and 26 environmental samples from the market were tested by RT-PCR for subtype H9 influenza virus. A total of 101 live poultry swab samples and 10 environmental samples were influenza A H9 positive, including swabbed samples of cage surface in Stall 38, swabbed samples of chopping boards, and refuse water collected after cleaning poultry in Stall 14. Due to the lack of neuraminidase (NA) test kits, Xiangxi CDC could not identify whether the subtype H9 positive samples were N2 positive or not.
The full genomes of A/Hunan/11173/2020(H9N2) (HN11173/20) were sequenced. The unrooted phylogenetic tree was generated by the maximum likelihood method using Molecular Evolutionary Genetics Analysis (MEGA, version 7.0.26). The molecular phylogenetic analysis showed that the hemagglutinin (HA) and NA genes of HN11173/20 belonged to the A/Duck/HongKong/Y280/97(H9N2)-like lineage of Eurasian branch (Figure 2). The nucleotide sequences homology of 8 segments of HN11173/20 were analyzed with the online Basic Local Alignment Search Tool (BLAST) (Table 1), and matrix protein (M) segment was found to have highly homology with Human H7N9 strain isolated in Changsha (99.51%); polymerase acidic protein (PA) gene was 98.90% similar to that of H9N2 strain isolated from wildfowl (Accipiter gentilis schvedowi) in Tianjin. HA, NA, nucleoprotein (NP), nonstructural protein (NS), polymerase basic protein 1 (PB1) , and polymerase basic protein 2 (PB2) segments were highly homologous with chicken H9N2 strains isolated from Guangdong Province, Shandong Province, and Shanghai Municipality.
Segment Sequence length
The highest nucleotide identity of
H9N2 virus in GenBank
Collection date Collecting
Identities HA 1,742 A/chicken/China/C7/2018(H9N2) MN384772 Jul-2018 Guangdong 98.50% NA 1,442 A/chicken/China/63/2019(H9N2) MN263217 05-Jun-2019 Shanghai 98.54% NP 1,565 A/chicken/China/1103/2019(H9N2) MN918142 20-Nov-2019 Shanghai 98.27% NS 890 A/chicken/Shandong/3424/2016(H9N2) MH667576 02-Jun-2016 Shandong 98.76% MP 1,027 A/Hunan Changsha/26/2017(H7N9) MF370250 04-Feb-2017 Hunan 99.51% PA 2,175 A/Accipiter gentilis schvedowi/ Tianjin/22/2017(H9N2) MH114054 15-Jun-2017 Tianjin 98.90% PB1 2,341 A/chicken/China/C7/2018(H9N2) MN384772 Jul-2018 Guangdong 98.76% PB2 2,339 A/chicken/Shandong/3424/2016(H9N2) MH667576 02-Jun-2016 Shandong 97.52% Abreviation: HA=hemagglutinin; NA=neuraminidase; NP=nucleoprotein; NS=nonstructural protein; MP=matrix protein; PA=polymerase acidic protein; PB=polymerase basic protein.
* The date of this BLAST search was Jun 12, 2020.
Table 1. Homology of 8 segments of HN11173/20 analyzed with the online Basic Local Alignment Search Tool.*
Figure 2. Phylogenetic trees of hemagglutinin (HA) and neuraminidase (NA) gene segments of HN11173/20 isolated from Hunan Province. (A) HA gene segments; (B) NA gene segments.
The molecular characterization of the HN11173/20 strain were analyzed, whose critical amino acid residues in proteins were the same/similar to those of 3 strains of human H9N2 virus previously isolated from Hunan Province (A/Hunan/42088/2017(H9N2), A/Hunan/3728/2017 (H9N2), and A/Hunan/43517/2016(H9N2)), as shown in the Table 2. The cleavage site of HA protein HA1 and HA2 were SRSSRGL (H3 numbering 334–340), contained the basic amino acid arginine at positions 335 and 338 (R335, R338), which indicated low pathogenicity to poultry. The amino acid of the HA protein of this strain at positions 235–237 (H3 numbered 226–228) was LMG, which susceptibly binded to human-like α2,6-linked sialic acid receptors and can effectively replicate in mammalian cells (1). NA protein were deleted at the stalk region (positions 63–65), and no oseltamivir associated resistance mutations in amino acid residues were found.
Virus HA (H3 Numbering) NA M1 M2 NS1 PB1 PA Receptor binding site Cleavage site deletion 158 183 190 226 227 228 334-340 63-65 15 28 92 227 13 356 409 A/Hunan/11173/2020(H9N2) N N T L M G SRSSRGL Yes I V D K P R N The other three H9N2 viruses previously isolated from Hunan Province N N T L M G SRSSRGL Yes I V/I D K P R N Abreviation: N=Aspara; T=Threonine; L=Leucine; M=Methionine; G=Glycine; S=Serine; R=Arginine; I=Isoleucine; V=Valine; D=Aspartic acid; K=Lysine; P=Proline.
Table 2. The molecular characteristics of the H9N2 influenza viruses isolated from Hunan Province.