Epidemiological and clinical characterization of human infections with H9N2 avian influenza virus in Changsha city,China,2015-2025

Introduction

Avian influenza virus (AIV) belongs to the type A influenza viruses. Based on their pathogenicity in chickens and molecular characteristics of the HA protein cleavage site, AIVs can be classified into highly pathogenic avian influenza viruses (HPAIV) and low pathogenic avian influenza viruses (LPAIV). ¹ In Chinese poultry farming and trading environments, prevalent AIV subtypes primarily include H3Ny, H5Ny, H6Ny, H7Ny, and H9Ny. ² The H9N2 subtype is widely circulating among poultry and is recognized as one of the most devastating LPAIV subtype, causing severe economic losses to the poultry industry. ³

The public health significance of H9N2 virus extends far beyond its infection in avian species. The H9N2 virus possesses a complex internal gene lineage and plays a prominent role as a “gene donor.” It contributes internal gene segments to reassort with influenza viruses from wild birds or domestic poultry, participating in the generation of various novel reassortant viruses, including the H7N9 and H10N8 viruses. Consequently, the H9N2 virus is regarded as an “incubator” or “parental virus” for potential pandemic influenza viruses. ⁴

In recent years, the cross-species transmissibility of the H9N2 subtype AIV has significantly increased. Since the first recorded human case of avian influenza A (H9N2) in southern China in 1998, ⁵ the number of human H9N2 infections has gradually increased globally over the past decade. According to WHO data, from December 2015 to September 12, 2025, 135 human infections with H9N2 subtype AIV have been reported in the Western Pacific Region, with 132 cases reported in China. ⁶

Currently, systematic research or reports on human H9N2 infections remain limited. Most existing studies are individual case reports, and investigations into its epidemiological characteristics, clinical features, and molecular virological properties are neither in-depth nor comprehensive. This study systematically collects and analyzes epidemiological data and clinical information from laboratory-confirmed human H9N2 cases in Changsha City between 2015 and 2025, with laboratory confirmation performed by the Changsha CDC on throat swabs. It aims to comprehensively delineate the epidemiological and clinical characteristics of these infections, providing crucial scientific evidence for optimizing clinical diagnosis and treatment protocols, as well as for formulating precise surveillance and prevention strategies.

Materials and methods

Results

Clinical characteristics

Clinical information was collected for 10 cases (Tables 1 and 2). Among them, 8 patients presented with fever, with peak body temperatures exceeding 38.5°C; one case had a maximum temperature surpassing 40°C. Other symptoms included cough (n=4), vomiting (n=1), and abdominal pain with diarrhea (n=1). One adult case was a cancer patient. Two cases were co-infected with other pathogens; one severe case was co-infected with Pseudomonas aeruginosa and Burkholderia cepacia. Analysis of routine blood test parameters from 7 cases revealed that white blood cell counts were generally normal or slightly elevated. Elevated C-reactive protein was observed in 2 cases, increased monocyte percentage in 1 case, elevated lymphocyte percentage in the case co-infected with respiratory syncytial virus, and raised neutrophil percentage in the case with bacterial co-infection.

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Table 1.

Symptoms in human H9N2 avian influenza cases in Changsha City.

Symptoms	n (%)
Fever	8 (80.00%)
Cough	4 (40.00%)
Abdominal pain	1 (10.00%)
Diarrhea	1 (10.00%)
Vomiting	1 (10.00%)

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Table 2.

Clinical and laboratory characteristics of human H9N2 avian influenza cases.

Case number	Co-infection	White blood cell count	C-reactive protein	Neutrophil percentage	Lymphocyte percentage	Monocyte percentage
2	None	14.94×109/L	Normal	Normal	19.70%	Normal
3	None	10.31×109/L	Normal	45.90%	Normal	Normal
6	None	12.41×109/L	97.10mg/L	69.60%	24.30%	Normal
7	None	Normal	Normal	Normal	Normal	15.80%
9	Respiratory Syncytial Virus	9.09×109/L	Normal	18.60%	72.90%	Normal
10	Pseudomonas aeruginosa, Burkholderia cepacia	10.86×109/L	87.34 (mg/L)	82.20%	10.10%	Normal
11	None	Normal	Normal	Normal	Normal	Normal

Exposure history

Detailed investigations were conducted for 10 cases. Among these, 7 cases (70%) had a history of exposure to live poultry or live poultry markets before symptom onset, while 3 cases had no clear exposure history (Table 3). Trace-back sampling was performed at exposure sites linked to the 7 cases with confirmed live poultry or market exposure. H9 nucleic acid fragments were detected in live poultry or environmental samples from 6 of these exposure sites, with positive detection rates ranging from 66.67% to 100%. Except for one site where N2 testing was not performed, N2 nucleic acid fragments were detected in samples from the remaining 5 sites, with positive rates between 92.31% and 100%. Cumulatively, 49 samples were collected, of which 37 were positive for H9 (positive rate: 75.51%) and 35 were positive for N2 (positive rate: 76.09%).

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Table 3.

Detection results from exposure sites of human H9N2 avian influenza cases.

Date	Case number	Probable exposure	Number of samples	H9 positive detection rate	N2 positive detection rate
November 2017	4	Live poultry market	3	100.00% (3/3)	100.00% (3/3)
August 2021	6	Poultry	3	66.67% (2/3)	Not tested
April 2025	7	Live poultry market	13	92.31% (12/13)	92.31% (12/13)
April 2025	8	Poultry	9	0	0
April 2025	9	Live poultry market	2	100.00% (2/2)	100.00% (2/2)
August 2025	10	Live poultry market	5	100.00% (5/5)	100.00% (5/5)
December 2025	11	Live poultry market	14	92.86% (13/14)	92.86% (13/14)
Total	​	​	49	75.51% (37/49)	76.09% (35/46)

Environmental surveillance

From 2014 to 2024, a total of 3,034 environmental samples from large live poultry markets or wholesale markets were tested in Changsha City, with 1,601 testing positive for H9, yielding a positive rate of 52.77% (Table 4 and Figure 1). Across the study period from 2014 to 2024, the positive rate showed a year-on-year increasing trend (χ²=111.30, P<0.001), with the average rate being significantly lower in 2014-2019 than in 2020-2024 (47.43% vs. 77.11%; χ²=158.24, P<0.001).

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Table 4.

Environmental surveillance for H9N2 in live poultry markets (2014-2024).

Year	Number of positive specimens	Number of tests	Positivity rate
2014	178	578	30.80%
2015	337	564	59.75%
2016	434	707	61.39%
2017	133	436	30.50%
2018	42	74	56.76%
2019	56	129	43.41%
2020	69	108	63.89%
2021	80	114	70.18%
2022	87	108	80.56%
2023	95	108	87.96%
2024	90	108	83.33%
Total	1601	3034	52.77%

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Figure 1.

H9N2 positivity rate in environmental samples from live poultry markets (2014-2024).

Discussion

This study confirms that children under five years old are at high risk of H9N2 virus infection, a finding consistent with research from Anhui Province. ⁹ Cases were sporadically distributed throughout the year, and none of the close contacts developed abnormal symptoms of infection, indicating that H9N2 virus infection remains primarily zoonotic, mainly originating from poultry or the external environment, and has not yet acquired efficient and sustained human-to-human transmission capability. ¹⁰ The transmission of the virus is most likely through sporadic spillover events resulting from direct contact with infected poultry or exposure to contaminated live poultry markets and environments (such as feces, feathers, dust, etc.).

The results show that most H9N2 patients presented with acute respiratory symptoms such as fever and cough. The disease course was self-limiting with a favorable prognosis, aligning with most relevant domestic and international reports.^11,12 Hematological tests on seven cases revealed normal or slightly elevated total white blood cell counts, with some cases showing elevated C-reactive protein levels, consistent with the general characteristics of infectious diseases. Notably, the direct cause of the one severe case in this study was bacterial co-infection. This finding carries significant clinical implications: although the H9N2 virus itself is relatively low in pathogenicity, the respiratory mucosal damage and immune dysfunction it may cause may create opportunities for secondary bacterial invasion. Therefore, clinicians managing H9N2 cases, especially young children or patients with poor underlying health, should closely monitor for changes in condition and promptly conduct etiological testing. Once bacterial co-infection is confirmed, antibacterial therapy should be initiated immediately to prevent clinical deterioration.

Environmental surveillance data from live poultry markets indicate a persistently high detection rate of the H9N2 virus, suggesting that the virus has established a stable circulation among poultry hosts in Changsha city. This implies an objective increase in population exposure risk. Studies have found that internal gene segments of various AIV subtypes, including H5N1, H7N9, H10N8, and H10N3, originate from the H9N2 subtype.^13–15 AIVs carrying H9N2-derived internal genes may exhibit an enhanced ability to infect humans, suggesting that the H9N2 subtype may play a significant role in expanding the host range of other influenza viruses. Our viral sequencing results revealed diverse mutations in key sites and functional domains, indicating that the H9N2 virus is in an active state of evolution. Notably, Leu-to-Gln mutation at position 226 enhances the virus’s affinity for the human-type α-2,6-linked sialic acid receptor, and studies have shown that this mutation can act in concert with other antigenic site mutations to drive the emergence of new antigenic variants.^16,17 Loss of glycosylation at position 218 and gain at position 313 are typical of recent H9N2 strains and may enhance infectivity. ¹⁸ Although the current human-derived strains are highly homologous to isolates from poultry environments, confirming the infection source, these persistent minor variations may reflect ongoing adaptation to new hosts. While efficient human-to-human transmission has not yet occurred, the ongoing genetic evolution of the virus undoubtedly increases the potential risk of it crossing the species barrier and eventually acquiring efficient transmissibility among humans.

This study has several limitations. First, as a descriptive study based on sentinel surveillance data, the sample size was relatively small, and epidemiological investigations for some cases were incomplete, potentially failing to fully capture the entire clinical spectrum of the disease. Second, investigations into exposure history may be subject to recall bias. Third, due to the secondary nature of the surveillance data, complete HA and NA gene sequences required for constructing a reliable phylogenetic tree were not available. Future larger-scale studies are needed to gain a deeper understanding of the epidemiology and viral evolution of H9N2 avian influenza.

In summary, at the current stage, human infection with the H9N2 virus mostly causes mild illness, primarily affecting children, and no efficient human-to-human transmission has been observed. However, the high prevalence of the virus in the environment, its potential for continuous genetic evolution, and its capacity to cause severe cases collectively constitute a public health threat that cannot be ignored. The role of the H9N2 virus as a gene donor for multiple AIV subtypes may be a key driver in the emergence of potential pandemic influenza viruses. Therefore, establishing and maintaining sustained surveillance, conducting in-depth research, and ensuring adequate preparedness for this virus are of paramount importance for preventing the next influenza pandemic.