Since the first human infection of low pathogenicity avian influenza A (H9N2) virus was detected in Hong Kong, China in 1999 (1), this strain has been reported to intermittently infect humans. In April 2021, a case of H9N2 was reported in Huizhou City, Guangdong Province, China. The patient was sampled by throat swabs, which were sent to Huizhou CDC for quantitative reverse transcription-polymerase chain reaction (RT-PCR) testing. An investigation group carried out epidemiological and environmental investigations to find other avian influenza cases.
Emergency sampling of environment was conducted; both the viral RNA from the throat swab of the patient and environment were extracted using the QIAamp® Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions, and RT-PCR assays with specific primers and probe sets were used for detecting the avian influenza A virus(H9N2) M gene. The virus gene segments were obtained through metagenomic sequencing. Genome sequences were aligned with the reference genomes downloaded from GenBank using the basic local alignment search tool (BLAST). The avian influenza virus activity in live poultry markets in Huizhou was analyzed using IBM SPSS Statistics for Windows (version 22.0. Armonk, NY: IBM Corp, USA).
The case occurred in a 30-year-old female living in Huicheng District of Huizhou City. On April 12, 2021, she experienced symptoms of sore throat but no fever. After medical treatment, her symptoms were alleviated. On April 20, 2021, she had symptoms of fever with an auxiliary temperature of 38.5 ℃ and was admitted to the Huizhou Central People’s Hospital for treatment. Her respiratory symptoms were paroxysmal cough, mainly dry cough, and wheezing. Other symptoms included chills, headache, and general malaise. The blood routine examination confirmed that the absolute value of neutrophils increased and that of lymphocytes decreased. The patient refused X-ray check as she thought it was unnecessary due to her perceived mild symptoms. On April 21, the patient’s throat swabs were collected and sent to the Huizhou CDC as a sample for routine influenza surveillance. On April 23, she was found to be positive for H9N2.
The patient had a history of travel to Dali Bai Minority Region, Yunnan Province from April 14 to 18 but denied having any direct contact history with live birds or other poultry. Photographs indicated that the patient did not wear a mask as required in a park. Before she went to Yunnan Province, she left her car in a surface parking lot, which was surrounded by many trees and birds. Her son was in a kindergarten near a live poultry market and was picked up by his grandmother. Their shoes may have been contaminated by sewage, poultry manure, and feathers when they passed through the live poultry market.
Through RT-PCR assays, the cycle threshold value (Ct value) of the patient’s throat swab was 32, which indicated the viral load of the sample was fairly low. As a result, the 8 sequences of H9N2 were unobtainable and only 9 gene reads were obtained (2 HA gene reads, 2 NA gene reads, 4 PB2 gene reads, 1 M gene reads), with the length being 50 bp for each read. The BLAST results showed that these gene reads had high homology with chicken H9N2 strains isolated from Guangdong Province, Zhejiang Province, and Shandong Province. The molecular characterization of the gene reads was analyzed, and the amino acid residues in proteins of these gene reads were the same or similar to reference strains A/chicken/China/GD2021/2021(H9N2) and A/Zhongshan/201501/2015(H9N2), but different to reference strain A/chicken/Hong Kong/G9/1997(H9N2) as shown in the Table 1. A total of 40 environment samples of the live poultry market near the patient’s son’s kindergarten were collected. The nucleic acid positive rate of avian influenza A virus was detected, the H9 subtype positive rate was 50% (20/40), but H5 and H7 subtype were not detected.
Virus HA NA PB2 334* 501 432 434 299 327 334 336 381 A/chicken/Hong Kong/G9/1997(H9N2) A K K T K G K F M A/chicken/China/GD2021/2021(H9N2) S Q H P R G S S L A/Zhongshan/201501/2015(H9N2) S Q Q P R G S S L The H9N2 gene reads from Huizhou City F Q H P R C S S L Abbreviations: N=Aspara; T=Threonine; L=Leucine; M=Methionine; G=Glycine; S=Serine; R=Arginine; K=Lysine; P=Proline; F=Phenylalanine.
* The mutation sites found in the detected 9 gene reads.
Table 1. The molecular characteristics of the H9N2 gene reads from Huizhou City.
A total of 386 environment samples were collected in poultry markets in Huizhou. The nucleic acid positivity rate of avian influenza A was detected in 176 of these samples from December 2020 to August 2021. The nucleic acid positive rate of subtype H9 fluctuated between 30.00% and 65.38% among different months, without significant difference (χ2=56.00, P>0.05) (Figure 1). The highest positivity rate of avian influenza A virus was in August 2021, and the lowest was in April 2021.