Surveillance and biological characterization of H3 subtype avian influenza viruses in Eastern China

ABSTRACT

H3 subtype avian influenza viruses (AIVs) are frequently detected in poultry and wild birds, however, systematic characterization of contemporary isolates remains limited. We aimed to investigate the genetic evolution, pathogenicity, and transmission characteristics of H3 subtype AIVs circulating in Eastern China. Seven H3 subtype AIVs isolated between 2014 and 2021, including five H3N2, one H3N3, and one H3N6 strain, were analyzed. Phylogenetic analysis showed that all isolates belonged to the Eurasian lineage. Evidence of extensive reassortment with other AIV subtypes, as well as adaptive mutations associated with pathogenicity, and cross-species transmission, particularly in H3N2 subtype AIVs, was identified. Notably, H3N2 subtype AIVs exhibited dual receptor-binding properties, recognizing both SA α-2,3-Gal and SA α-2,6-Gal receptors. Although all isolates demonstrated low pathogenicity in chickens, mice, and guinea pigs, variations in transmission efficiency were observed. The H3N2 strain A/Duck/Anhui/LY/2021 showed the highest capacity for cross-species and aerosol transmission among guinea pigs. Overall, these findings indicate that H3 subtype AIVs have the potential for cross-species transmission and highlight the importance of continued surveillance of H3 subtype AIVs circulating in nature.

Introduction

The avian influenza virus (AIV) poses a significant threat to the poultry industry and public health. In chickens, AIVs are classified into highly pathogenic AIVs (HPAIVs) and lowly pathogenic AIVs (LPAIVs) [1]. Although HPAIVs cause high mortality rates in both poultry and humans, the potential threat of LPAIVs to human health should not be underestimated. Unlike HPAIVs, LPAIVs typically do not cause high mortality or severe lesions during infection. Consequently, they are often assigned lower priority in animal disease control programs, allowing them to circulate and evolve silently in nature [2]. Moreover, several LPAIV subtypes, including H6N1, H9N2, and H10N8, have been associated with human infection in recent years [3]. Notably, LPAIVs are widely recognized as important donors of internal genes to HPAIVs [4]. For example, the H7N9 subtype, which has caused human infections and fatalities in China, contains six internal genes derived from H9N2 LPAIVs [5].

Most reassorted viruses have been isolated from live poultry markets (LPMs). The geographic distribution of these markets, which are predominantly located along wild bird migration routes, suggests that avian migration plays an important role in the dissemination of AIVs [6]. For example, the PA gene of H3N2 AIVs isolated from ducks in China shows a high degree of homology with that of H7N9 HPAIVs, isolated from wild ducks in Korea [7]. Phylogenetic analyses have demonstrated that the internal genes of certain H5 HPAIVs are closely related to those of contemporary H3N2 AIVs [8], indicating the occurrence of genetic reassortment. Eastern China, characterized by an extensive water network, serves as a key hub for migratory bird routes and contains a high density of LPMs. These markets, with their diverse and high animal density, provide an ideal environment for viral reassortment and cross-species transmission (CST). Recent surveillance studies have reported the widespread detection of H3 subtype AIVs in LPMs across Eastern China [9,10]. Therefore, the potential risk of LPAIVs to humans and the possibility of reassortment between HPAIVs and LPAIVs warrant careful consideration.

H3 subtype viruses have a broad host range, including wild birds, poultry, and various mammalian species, such as humans, pigs, dogs, and seals [11]. Historically, H3 subtype viruses were responsible for the 1968 H3N2 Hong Kong pandemic and have continued to circulate with their internal genes undergoing ongoing reassortment [12]. Evolutionary studies suggest that H3 subtype AIVs exhibit relatively high evolutionary rates [13], contributing to the emergence of diverse subtypes in recent years, including H3N2, H3N3, H3N6, and H3N8 [14]. The first reported human infection with H3N8 subtype AIV occurred in Henan Province, China, in April 2022 [15]. Since then, an increasing number of human H3N8 infections have been reported in China, some of which have resulted in severe illnesses and death [16]. Most affected individuals had a history of exposure to live poultry, suggesting that H3N8 AIVs have the capacity for avian-to-human transmission. Notably, an H3N8 virus isolated from a human case was shown to transmit between ferrets via respiratory droplets, indicating adaptation to human-type receptors and the presence of the PB2-E627K substitution, which is associated with enhanced airborne transmissibility [17]. Recently, H3N2 subtype AIVs become the predominant subtype in avian species in China and have undergone frequent reassortment with other influenza virus subtypes in LPMs [18]. Over the past two decades, seasonal human H3N2 influenza viruses have evolved its HA receptor specificity toward both avian-type and human-type receptors [19]. Although previous studies have demonstrated the potential of avian-origin H3N2 AIVs for interspecies transmission in guinea pigs and ferrets [2], systematic characterization of contemporary avian-origin H3N2 isolates, particularly comparative analysis of their CST from avian to mammalian hosts remains limited. Therefore, we aimed to investigate the genetic evolution, receptor binding characteristics, and pathogenicity of H3N2 subtype AIVs. Additionally, cross-species transmission models using chickens and guinea pigs were established to evaluate the transmission dynamics of H3 subtype AIVs, particularly H3N2 strains, isolated in Eastern China between 2014 and 2021. This study provides experimental data that may inform the risk assessment of H3N2 subtype AIVs.

Materials and methods

Ethics statement

All animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China. Experimental protocols were approved by the Administrative Committee for Laboratory Animals of Yangzhou University, Jiangsu Province (permission numbers: SYXKSU-2017–0044, SYXKSU-2021–0027, and SYXKSU-2021–0026), in accordance with the Jiangsu Laboratory Animal Welfare and Ethics Regulations. All procedures involving anesthesia and anesthesia were performed following the guidelines of the American Veterinary Medical Association (AVMA).

Virus isolation and purification

All viral isolates used in this study were obtained directly by our research team from samples collected in Eastern China between 2014 and 2021. Samples were collected from LPMs and diseased avians across several provinces, including Jiangsu and Anhui, as part of routine and project-specific AIV surveillance. Background information for the seven isolates is provided in Table 1. The LPMs primarily sell chickens and ducks. Oropharyngeal (OP) and cloacal (CL) swabs were randomly collected from chickens and ducks at the LPMs every 3 months, with the cooperation of market management. All sample collection procedures were conducted according to animal study protocols approved by the Administrative Committee for Laboratory Animals of Yangzhou University.

Table 1.

Background information of seven H3 subtype AIVs.

Virus	Subtype	Year	Host	Source	Location	Abbreviation
A/duck/Wuxi/W7-4/2014(H3N3)	H3N3	2014	Duck	LPM	Wuxi	W7-4(H3N3)
A/duck/Jiangsu/J84610/2017(H3N6)	H3N6	2017	Duck	LPM	Jiangsu	J84610(H3N6)
A/chicken/Jiangsu/W23910/2017(H3N2)	H3N2	2017	Chicken	LPM	Jiangsu	W23910(H3N2)
A/swan/Yangzhou/901084/2018(H3N2)	H3N2	2018	Swan	Dead avian	Yangzhou	901084(H3N2)
A/duck/Jiangsu/JY020416/2019(H3N2)	H3N2	2019	Duck	LPM	Jiangsu	JY020416(H3N2)
A/duck/Gaoyou/4D1-1/2021(H3N2)	H3N2	2021	Duck	LPM	Gaoyou	4D1-1(H3N2)
A/duck/Anhui/LY/2021(H3N2)	H3N2	2021	Duck	LPM	Anhui	LY(H3N2)

Strains were initially identified using standard hemagglutination inhibition (HI) tests with chicken polyclonal antiserum, following the methods recommended by the Office International des Epizooties (OIE) https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.03.04_AI.pdf. The isolates were purified through three rounds of plaque assays [20] and subsequently propagated in 9-day-old specific-pathogen-free (SPF) chicken embryos before being stored at −70°C. Viral RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized via reverse transcription using the Uni12 primer. Gene segments were amplified by PCR using primers targeting conserved promoters and noncoding regions specific to each segment [21]. PCR products were sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, CA, USA) on an ABI 3500xL Genetic Analyzer (Applied Biosystems), and nucleotide sequences were edited using the SeqMan module of the DNASTAR software suite. No mutations were detected in the viral genomes before or after purification.

Cell culture and growth kinetics

Madin-Darby canine kidney (MDCK) cells (ATCC, CCL-34) and human non-small cell lung cancer (A549) cells (ATCC, CCL-185) were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C and 5% CO₂. Chicken embryonic fibroblasts (CEF) were prepared from 10-day-old embryos and cultured in M199 medium (Gibco, USA) supplemented with 4% fetal bovine serum (Gibco, USA) under the same conditions. Monolayers of MDCK, A549, and CEF cells were infected with the virus at a multiplicity of infection (MOI) of 0.01 for 1 hour, followed by incubation in maintenance medium containing 1 μg/mL tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich, USA) for 72 hours at 37°C and 5% CO₂. Culture supernatants were collected at 12, 24, 48, and 72 hours post-infection (h.p.i.), and viral titers were determined using the Reed–Muench method [22].

Genetic and phylogenetic analysis

A total of 195 HA sequences were aligned using MAFFT software (version 7.505), with reference segments obtained from GISAID (http://www.gisaid.org). The optimal nucleotide substitution model (GTR+F+I+G4) was selected using IQ-TREE software (version 1.6.12). The best-fit combinations of the molecular clock model (the uncorrelated relaxed log-normal [UCLN] clock model) and the coalescent demographic model (Bayesian skyline) were determined via path sampling and stepping-stone sampling (PS/SS). The Bayesian stochastic search variable selection (BSSVS) model was used to estimate host jumping events, divergence times, and root state posterior probability (RSPP). Parameters were estimated using the Markov chain Monte Carlo (MCMC) approach, implemented in BEAST software (version 1.10.4) [23]. Six separate Bayesian analyses were conducted, each consisting of 200 million MCMC steps with sampling parameters and trees at every 20,000-step interval. The convergence of parameters was verified via Tracer software (version 1.7.1) [24] with a 10% burn-in of the total chain length. The Effective Sample Size (ESS) values for all estimated parameters were confirmed to be >200, ensuring sufficient sampling. All tree files and log files were [25] amalgamated via LogCombiner (version 2.6.6) [25]. TreeAnnotator software (version 1.10.4) [23] was used to summarize the maximum clade credibility (MCC) tree after discarding the initial 10% burn-in. SpreaD3 (version 0.9.7.1) [26] was used to calculate Bayes factors (BFs) and posterior probabilities (PPs) from the BSSVS analysis results. BF >3 and PP >0.5 were used as statistically significant thresholds to assess the route of virus transmission between hosts. MCC tree visualization was achieved via FigTree software (version 1.4.4) [24]. The RSPP for the ancestral states was also summarized from the posterior density of trees via TreeAnnotator software (version 1.10.4) [23].

Receptor binding analysis

The receptor-binding specificity of H3-subtype AIVs was evaluated using a solid-phase binding assay with two glycopolymers: an α-2,3-sialylglycopolymer [Neu5Acα2-3Galβ1-4GlcNAcβ1-pAP (para-aminophenyl)-alpha-polyglutamic acid (α-PGA)] (SA α-2,3-Gal) and an α-2,6-sialylglycopolymer [Neu5Acα2-6Galβ1-4GlcNAcβ1-pAP (para-aminophenyl)-alpha-polyglutamic acid (α-PGA)] (SA α-2,6-Gal), as previously described [27]. A/California/04/2009(H1N1), which binds exclusively to SA α-2,6-Gal receptor, and A/Mallard/Huadong/S/2005(H5N1), which binds exclusively to SA α-2,3-Gal receptor, were used as control. Chicken antisera raised against homologous or same-clade viruses served as the primary antibody. The glycan analogs were serially diluted in PBS and added to 96-well streptavidin-coated microtiter plates. Plates were blocked with 5% BSA overnight, thenincubated with 64 HA titers of virus per well. Chicken antiviral serum corresponding to each virus was added, followed by incubation with the primary antibody. Enzyme-labeled sheep anti-chicken antibody and a tetramethylbenzidine substrate solution were successively applied. After 10 minutes, the reaction was stopped with 1M H₂SO₄, and absorbance at 450 nm was measured to generate binding curves.

Pathogenicity in chickens

To assess the pathogenicity of the seven isolates in chickens, 24 healthy 3-week-old SPF chickens (Lihua Corporation, China) were randomly assigned to eight groups (three chickens per group), including seven H3 subtype virus-infected groups and one uninfected control group (PBS). Each virus-infected group was inoculated intranasally with 10⁶ EID₅₀/100 μL of the corresponding H3 subtype AIV, while the control group received 100 μL of PBS. The survival status of each chicken, including drinking, feeding, and death status, was observed every day. All chickens were euthanized by CO₂ asphyxiation at 3 days post-infection (d.p.i.). The euthanasia chamber was pre-filled with CO₂ to achieve a high initial concentration (70% displacement rate, well above ambient levels). Chickens were promptly placed into the chamber, ensuring rapid exposure to CO₂ concentrations sufficient to induce loss of consciousness within seconds. One lobe of each lungs and the nasal cavity were collected and fixed with 4% paraformaldehyde for confocal laser scanning microscopy (CLSM) and hematoxylin–eosin (H&E) staining. CLSM was used to detect the viral distribution by observing the expression of the NP protein (Abcam, UK) labeled indirectly with Alexa Fluor 649 secondary antibody (Abcam, UK). Histopathological changes were scored by a certified pathologist according to the established criteria [28]. The sample size was determined based on previously published studies on H3 subtype AIV pathogenicity [29].

Pathogenicity in mice

To evaluate the pathogenicity of the seven isolates in mice, 40 healthy 6-week-old female BALB/c mice (Yangzhou University Laboratory Animal Center, Yangzhou, China) were randomly assigned to eight groups (five mice per group), including seven H3 subtype virus-infected groups and one uninfected control group (PBS). Mice in the virus-infected groups were anesthetized with ketamine (20 mg/kg body weight) and xylazine (1 mg/kg) and inoculated intranssally with 10⁶ EID₅₀/50 μL of the corresponding H3 subtype AIV. The control group was similarly anesthetized and inoculated with 50 μL of PBS. To minimize the potential confounders, two persons who were not aware of this experiment were designed to record the daily weight, mortality, and morbidity of the infected mice in each group. Animals were monitored daily for mortality over a period of 14 days.

To investigate viral replication in vivo, 48 healthy 6-week-old female BALB/c mice (Yangzhou University Laboratory Animal Center, Yangzhou, China) were randomly assigned to eight groups (six mice per group), including seven H3 subtype virus-infected groups and one uninfected control group (PBS). Inoculation was performed as described above. Three mice from each group were euthanized at 3 and 5 d.p.i. via intraperitoneal injection of sodium pentobarbital (150 mg/kg). Death was confirmed by the absence of respiratory effort and, if necessary, a secondary physical method such as bilateral thoracotomy or exsanguination. The nasal cavities, lungs, ileums, and brains were collected aseptically. 0.1 g of tissues from each organs was homogenized at 3 and 5 d.p.i. and inoculated into chicken embryos to assess the viral distribution. One lobe of lungs and nasal cavities collected at 3 d.p.i. were fixed with 4% paraformaldehyde for CLSM and H&E staining. CLSM was used to detect the virus distribution by observing the expression of the NP protein (Abcam, UK) indirectly labeled with Alexa Fluor 649 secondary antibody (Abcam, UK). Histopathological changes were scored by a certified pathologist according to the established criteria [28]. Sample size was determined based on previously published studies on H3 subtype AIV pathogenicity [29].

Pathogenicity in Guinea pigs

To evaluate the pathogenicity of the LY strain in guinea pigs, six healthy female guinea pigs (Laifu Corporation, China), weighing approximately 300 g were randomly assigned to two groups (three guinea pigs per group) including LY strain group and PBS (uninfected control) group. No maternal antibodies against influenza viruses were detected in any of the animals. Three guinea pigs were anesthetized with ketamine (20 mg/kg of body weight) and xylazine (1 mg/kg), and inoculated with 10⁶ EID₅₀/300 μL of the LY(H3N2) strain. Three guinea pigs were anesthetized with ketamine (20 mg/kg of body weight) and xylazine (1 mg/kg), and inoculated with 300 μL of PBS as the control group. The survival status of each guinea pig, including drinking water, eating, activity, and death, was observed. All guinea pigs were euthanized by intraperitoneal injection of a sodium pentobarbital overdose (150 mg/kg) at 3 d.p.i. Death was confirmed by the absence of respiratory effort and, if necessary, a secondary physical method such as bilateral thoracotomy or exsanguination. The nasal cavities, tracheas, lungs and ileums were collected aseptically. 0.1 g of each tissue was homogenized and inoculated into chicken embryos to assess the viral distribution. One lobe of lungs and nasal cavities were fixed with 4% paraformaldehyde for CLSM and H&E staining. CLSM was used to detect the virus distribution by observing the expression of the NP protein (Abcam, UK) indirectly labeled with Alexa Fluor 649 secondary antibody (Abcam, UK). Sample size was determined based on previously published studies on H3 subtype AIV pathogenicity [2].

Contact and cross-species transmission of H3 subtype AIVs

A total of 24 healthy 3-week-old SPF chickens (Lihua Corporation, China) were randomly assigned to eight groups (three chickens per group), including seven H3 subtype virus-infected groups and one uninfected control group (PBS). Chickens in the virus-infected groups were inoculated intranasally with 10⁶ EID₅₀/100 μL of the corresponding virus, whereas the uninfected control group was inoculated with 100 μL of PBS.

For the contact transmission, additional 24 healthy 3-week-old SPF chickens (Lihua Corporation, China) were introduced into the respective groups 24 hours after inoculation (three chickens per group). Each group consisted of three inoculated chickens co-housed with three naïve contact chickens in individually ventilated cages (IVCs). The cage had a stainless steel wire mesh floor (1 cm × 1 cm grid) to minimize direct contact with feces. Three inoculated chickens and three naïve contact chickens were co-housed in the same cage for the duration of the experiment. The IVCs were supplied with HEPA-filtered air at a rate of 60 air changes per hour, with directional airflow from the cage interior to exterior to prevent cross-contamination. The housing room was maintained at 22 ± 1°C and 50 ± 5% relative humidity. A schematic diagram of the transmission cage setup is provided in Figure S13(A).

For the cross-species transmission, 24 healthy female guinea pigs (Laifu Corporation, China) weighing approximately 300 g were randomly introduced into the corresponding groups 24 hours after inoculation (three guinea pigs per group). Three inoculated chickens and three naïve guinea pigs were co-housed in each IVC. Guinea pigs had free access to the entire cage floor and could have direct contact with the chickens, including contact with chicken feces and respiratory secretions. A schematic diagram of the transmission cage setup is provided in Figure S13(B).

OP and CL swabs were collected from inoculated chickens at 1, 3, 5, 7, 9, and 11 d.p.i., while nasal washes from contact guinea pigs and OP and CL swabs from contact chickens were collected at 2, 4, 6, 8, 10, and 12 d.p.i. to assess viral shedding. Serums from inoculated chickens, contact chickens, and contact guinea pigs were collected at 21 d.p.i. to evaluate seroconversion. To prevent inadvertent physical transmission of virus by the investigators, the exposure guinea pigs were always handled first, and gloves, implements, and napkins on the work surface were changed between animals. The sample size (n = 3 per group for transmission experiments) was determined based on published research on H3 subtype AIV and consistent with standard practices in the field, where n = 3 is widely used for influenza virus transmission studies in guinea pigs and chickens [2,30,31].

Transmission studies among guinea pigs

Nine healthy female guinea pigs (Laifu Corporation, China) weighing approximately 300 g were randomly assigned to three groups (three guinea pigs per group), including the inoculated group, contact group, and exposure group. The transmission cage was divided into two compartments by a double-layered net divider (4 cm apart) to prevent direct contact between animals in adjacent sections. Ambient conditions for these studies were set at 20 to 22°C and 30 to 40% relative humidity. The airflow in the isolator was horizontal with a speed of 0.1 m/s, and the airflow direction was from the inoculated group toward the exposed group as previously described [32,33]. Guinea pigs in the inoculated group, were anesthetized with ketamine (20 mg/kg of body weight) and xylazine (1 mg/kg), and inoculated with 10⁶ EID₅₀/300 μL of the LY(H3N2) strain. At 24 hours post-inoculation, naïve guinea pigs were introduced: the contact group was co-housed with the inoculated animals in the same compartment, while the exposure group was placed in the adjacent compartment separated by the barrier. A schematic diagram of the transmission cage setup is provided in Figure S13(C). Nasal washes from three inoculated guinea pigs were collected at 1, 3 5, 7, 9, and 11 d.p.i. to assess viral shedding. The nasal washes of three guinea pigs in the contact group and exposure group were collected at 2, 4, 6, 8, 10, and 12 d.p.i. to detect virus shedding. Serum samples from guinea pigs in the inoculated, contact, and exposure groups were collected at 14 and 21 d.p.i. to detect seroconversion. To minimize inadvertent physical transmission by investigators, animals in the exposure group were handled first, and gloves, implements, and work surfaces were changed between handling of animals. A schematic diagram of the transmission cage setup is provided in Figure S13(D). The sample size (n = 3 per group for inoculated, contact, and exposure groups) was determined based on published research on H3 subtype AIV and consistent with standard practices in the field, where n = 3 is widely used for influenza virus transmission studies in guinea pigs [2,30,31].

Statistical analysis

Data are presented as means ± standard deviations of three independent replicates derived from representative experiments. Descriptive statistics were used to summarize the data, and a graphical visualization was employed to illustrate trends and variability among groups. All graphs were generated using Prism version 9 (GraphPad Software, San Diego, CA, USA).

Results

Virus isolation and growth kinetics of H3 subtype AIVs

Between 2014 and 2021, seven H3-subtype AIVs were isolated, including five H3N2 subtype AIVs, one H3N3 subtype AIV, and one H3N6 subtype AIV. Details of their geographic origin, hosts, years of isolation, and abbreviations are listed in Table 1. Sequencing analysis confirmed that no adaptive mutations were detected after three rounds of plaque purification. All sequences have been uploaded to GenBank of NCBI. The complete genome sequences of the seven isolates have been deposited in GenBank under the following accession numbers: for A/duck/Gaoyou/4D1-1/2021 (H3N2): PV124803–PV124810; for A/duck/Anhui/LY/2021(H3N2): PV124795–PV124802; for A/duck/Jiangsu/JY020416/2019(H3N2): PV124763–PV124770; for A/duck/Jiangsu/J84610/2017(H3N6): PV124755–PV124762; for A/chicken/Jiangsu/W23910/2017(H3N2): PV124747–PV124754; for A/swan/Yangzhou/901084/2018(H3N2): PV124739–PV124746; for A/duck/Wuxi/W7-4/2014(H3N3): PV124731–PV124738 (each accession number represents a single gene segment, with eight segments per isolate). As shown in Figure 1(A) and S1, the seven H3 subtype AIVs exhibited distinct plaque morphologies in MDCK cells, suggesting differences in replication efficiency in mammalian cells. Notably, except for the W7-4 (H3N3) and J84610(H3N6) AIVs, the H3N2 isolates produced larger and smoother plaques. Growth kinetics analysis (Figure 1(B)) demonstrated that all isolates replicated efficiently in avian-origin CEF cells, reaching peak titers at 12 and 24 h.p.i. In mammalian-origin MDCK cells (Figure 1(C)), viral titers increased initially but declined after 24 h.p.i., indicating less stable replication. In other mammalian-origin A549 cells (Figure 1(D)), replication was generally lower, with the exception of the H3N2 subtype AIV (JY020416 strain), titers of the remaining isolates were below 10⁴ lgTCID₅₀/mL, which was lower than those observed in MDCK and CEF cells. Overall, H3N2 subtype AIVs showed more efficient replication in both CEF and MDCK cells, compared with H3N3 and H3N6 isolates, suggesting greater adaptability of H3N2 subtype AIVs to mammalian cell lines.

Figure 1.

Replication kinetics of H3 subtype AIVs in vitro.

(A) Plaque morphologies of the seven isolates in MDCK cells. Viral growth in CEF (B), MDCK (C), and A549 (D) cells was determined according to the TCID₅₀ values at 12, 24, 36, 48, 60, and 72 h.p.i. with an infection dose of 0.01 MOI. The TCID₅₀ titers were determined in MDCK cells. Data are presented as means ± SD from three independent experiments.

Adaptation mutations occurred in H3 subtype AIVs

Analysis of the HA gene revealed that all seven isolates possessed a low-pathogenicity cleavage site motif containing a single basic amino acid (PEKQTR↓GLF), consistent with LPAIVs [34]. Analysis of potential HA glycosylation sites identified five conserved sites (³⁸NGT⁴⁰, ⁵⁴NAT⁵⁶, ¹⁸¹NVT¹⁹³, ³⁰¹NGS³⁰³, and ⁴⁹⁹NGT⁵⁰¹) across the seven isolates. Additionally, two extra glycosylation sites, ¹⁸NLS²⁰ and ²⁴NST²⁶, were identified in W23910 (H3N2) and 901084 (H3N2), respectively. An I155T substitution was detected in the HA protein of all isolates (Table 2), which has been associated with increased binding affinity for α-2,6-linked sialic acid receptor, characteristic of human-like receptor. Several mutations associated with enhanced mammalian adaptation and virulence were identified in the polymerase genes. In PB2, mutations I333T [46], A683T, I675L, and Q508R [37] were detected in all isolates. In PB1, mutations H436Y, R198K [38], V171M [37], and C38Y [39] were observed, which have been previously associated with increased virulence in both mice and chickens. Mutations L295P [40], F666L [47], A100V, R356K [48], and S409N [42], which have been demonstrated to be associated with increased polymerase activity in mammalian cells and contribute to high adaptability and virulence in mammals, were also found in the PA protein of the seven isolates. Additionally, mutations N30D and 215A [41] in M1 and P42S [8] in NS1, previously associated with increased virulence in mice were detected. Notably, M2 substitutions L69P and P64S [37], which are associated with reduced sensitivity to the M2 ion channel blocker (amantadine), were identified, suggesting potential antiviral resistance in these isolates.

Table 2.

Key amino acid mutations in seven H3 subtype AIVs.

Functional category	Mutation	Gene	Reported effect	Presence in isolates
Receptor binding	I155T	HA	Associated with increased α-2,6 receptor binding [9]	All seven isolates
	G225D	HA	Associated with increased thermostability [35]	None
	Q226L, G228S	HA	Associated with increased α-2,6 receptor binding [36]	None
Mammalian adaptation & virulence	I333T	PB2	Associated with increased adaptability in mice [36]	All seven isolates
	A683T, I675L, Q508R	PB2	Associated with increased virulence in mice [37]	All seven isolates
	H436Y, R198K, V171M, M317I	PB1	Associated with increased virulence in mice/ducks [38]	All seven isolates
	C38Y	PB1	Associated with increased pathogenicity in chickens [39]	All seven isolates
	L295P, F666L, A100V, R356K	PA	Associated with increased polymerase activity in mammalian cells [40]	All seven isolates
	N30D, T215A	M1	Associated with increased virulence in mice [41]	All seven isolates
	P42S	NS1	Associated with increased replicative ability in mice [8]
Enhanced transmission	E382D	PA	Associated with increased polymerase activity in mammalian cells [42]	LY only
	S524G	PB1	Associated with enhanced airborne transmission [43]	None
Drug resistance	L69P, P64S	M2	Associated with reduced amantadine sensitivity [37]	All seven isolates
	V27I, S31N	M2	Associated with reduced amantadine sensitivity [44]	JY020416 (S31N)
Increased pathogenicity in chickens	M105V	NP	Associated with increased virulence in chickens [45]	4D1-1 only

Cross-species transmission and reassortment of H3 subtype AIVs

To investigate the ancestral hosts and cross-species transmission of H3 subtype AIVs, the seven isolates in this study, together with H3 strains obtained from the influenza virus database were analyzed using Bayesian phylodynamics. As shown in Figure 2(A), three major CSTs were inferred during the evolutionary history of H3 subtype viruses. The first CST occurred around in 1837 (95% highest posterior density [HPD], 1732–1937) (RSPP = 0.5364) and spread from equine to avian (BF = 8 and PP = 0.79). The second CST occurred around in 1945 (95% HPD, 1923–1966), with transmission from avians to swine and humans (BF = 19 and PP = 0.89). The last CST occurred around in 2000 (95% HPD, 1997–2003), representing transmission from avians to canines (BF = 319 and PP = 0.99). Phylogenetic analysis further showed that H3 subtype AIVs diverged into Eurasian and North American lineages around 1916 (95% HPD, 1880–1949). All seven isolates in this study belonged to the Eurasian lineage. Within the Eurasian lineage, frequent reassortment and host switching occur between wild birds and poultry, but the major cross-species transmission events identified remain avian-to-mammal in nature. Rather than being isolated events, these historical CSTs underscore the inherent evolutionary propensity of H3 subtype viruses to repeatedly breach species barriers. Because two major CSTs occurred within the Eurasian lineage, the contemporary H3 subtype AIVs identified here require urgent evaluation for their ongoing potential for cross-species transmission to mammals.

Figure 2.

Phylogenetic analysis of the HA genes of H3 subtype AIVs.

The MCC tree of the HA gene was constructed via the package BEAST (v1.10.4). (A) Time tree showing the evolutionary relationships and timescale of H3 subtype viruses. The branches shown in different colors in the tree represent different hosts. The seven isolates in this study are denoted by orange circles. The RSPP for species jumps are shown in the inset panel. The arrows point to the main evolutionary species jumps. (B) The possible origins of each gene segment are indicated by different colored bars.

Analysis of internal genes segments revealed substantial genetic diversity among the seven isolates (Figure 2(B)). At the nucleotide level, sequence identities ranged from 86.1% to 97.6% for PB2, 88.7% to 98.3% for PB1, 88.7% to 98.0% for PA, 93.5% to 98.6% forNP, 89.0% to 98.3% for M and 93.7% to 99.8% forNS genes. These findings indicate frequent reassortment events involving multiple AIV subtypes. The internal genes of most isolates were primarily derived from poultry in central and eastern China. However, in the 4D1-1(H3N2) strain, all gene segments except PB2 and NA were closely related to viruses from other Asian countries (Figure S2-S10). Overall, phylogenetic clustering showed that the internal genes of H3 subtype AIVs were associated with multiple HA subtype viruses, including H1, H3, H4, H5, H6, H7, H9, H10, and H11, highlighting extensive reassortment among diverse influenza virus lineages.

Receptor binding characteristics of H3 subtype AIVs

To evaluate the sialic acid receptor-binding properties of H3 subtype AIVs, a solid-phase binding assay was performed to assess receptor-binding specificity [49]. Human influenza virus (A/California/04/2009 (H1N1)), which binds exclusively to α-2,6-Gal receptor, and AIV (A/Mallard/Huadong/S/2005(H5N1)), which binds exclusively to the SA α-2,3-Gal receptor, were used as controls. As shown in Figure 3(A-I), with the exception of the H3N6 subtype AIV (J84610 strain), which bound only to SA α-2,3-Gal receptor, the remaining six isolates exhibited dual binding to both SA α-2,3-Gal receptor and SA α-2,6-Gal receptor. Notably, two H3N2 subtype AIVs (JY020416 strain and LY strain) showed strong binding to SA α-2,3-Gal receptor, with binding signals of 1.46 ± 0.03 and 1.50 ± 0.01, respectively, comparable to or slightly higher than that of the avian control virus (A/Mallard/Huadong/S/2005/H5N1) (1.24 ± 0.13) (Figure 3(E,G)). Although the affinity of H3N2 subtype AIVs for SA α-2,3-Gal receptors was greater than that for SA α-2,6-Gal receptors, the threat of human receptor-binding characteristics in H3N2 subtype AIVs should receive more attention.

Figure 3.

Receptor binding characteristics of H3 subtype AIVs.

The binding signals of the seven isolates to two different glycans (α-2,3-glycans and α-2,6-glycans) were assessed. Two viruses, A/Mallard/Huadong/S/2005(H5N1), which only bind to the SA α-2,3-Gal receptor, and A/California/04/2009(H1N1), which only bind to the SA α-2,6-Gal receptor, were used as control viruses. The data shown represent the means ± SDs from three independent experiments.

H3 subtype AIVs exhibited low pathogenicity in chickens

To evaluate the pathogenicity of the seven isolates in chickens, 3-week-old SPF chickens were inoculated intranasally at a dose of 10⁶ EID₅₀/100 μL. Notably, none of the infected chickens exhibited overt clinical symptoms throughout the infection period. Histopathological analysis revealed lung lesions in all infected groups (Figure 4(A)) with the 4D1-1(H3N2) strain causing the highest lesion scores among all groups (Figure S11(A)). In the JY020416(H3N2), LY(H3N2), and W7-4(H3N3) infection groups, lung lesions were characterized by proliferation of alveolar epithelial cells and dilation and hemorrhage of alveolar wall capillaries. In contrast, the W23910(H3N2), 901084(H3N2), and J84610(H3N6) infection groups exhibited milder lesions, characterized byslight infiltration of neutrophils, lymphocytes, and macrophages, along with widened alveolar spaces.

Figure 4.

Pathogenicity of H3 subtype AIVs in chickens.

(A) H&E staining of lungs. Scale: 50 μm. (B–C) The viral distributions in the lungs and nasal cavities of infected chickens were observed via confocal laser scanning microscopy. Nuclei (DAPI; blue); NP protein of the virus (Alexa Fluor 649; red). The virus distribution in organs is marked with white arrows. Scale: 20 μm.

Furthermore, CLSM was used to examine viral distribution in the nasal mucosa and lungs by detecting NP protein expression labeled with Alexa Fluor 649 secondary antibody. As shown in Figure 4(B,C), viral distribution was observed in the nasal mucosa epithelium of all the infection groups. However, virus distribution in the lungs was observed only in JY020416(H3N2), 4D1-1(H3N2), LY(H3N2), and W7-4(H3N6) infection groups. These findings indicated that replication of the H3N2 subtype AIVs isolated in this study is largely restricted to the upper respiratory tract, consistent with their low pathogenicity in chickens.

H3 subtype AIVs exhibited low pathogenicity in mice

To assess the pathogenicity of H3N2 subtype AIVs in mice, 6-week-old BALB/c mice were anesthetized and inoculated intranasally at a dose of 10⁶ EID₅₀/50 μL. No mortality was observed in any infection group during the whole infection period. However, mice in the 4D1-1 (H3N2), JY020416(H3N2), and LY(H3N2) infection groups exhibited transient clinical signs, including loss of appetite, lethargy, and ruffled fur within the first 3 d.p.i. In particular, the body weight of the mice infected with the JY020416(H3N2) strain had decreased to 8.9% of their body weight at 3 d.p.i., and that of the 4D1-1(H3N2) infection group had decreased to 10.5% of their body weight at 2 d.p.i., and that of the LY(H3N2) infection group had decreased to 6.7% of their body weight at 2 d.p.i. No mortality was observed in any group during the 14-day observation period (Figure 5(A,B)). Histopathological examination of the lungs via H&E staining revealed severe lesions in the 4D1-1 (H3N2), JY020416(H3N2), LY(H3N2), and W7-4 (H3N3) infection groups. These lesions were characterized by extensive infiltration of neutrophils, lymphocytes, and macrophages, dilation and hemorrhage of alveolar wall capillaries, hyperplasia of alveolar epithelial cells, and pronounced widening of alveolar spaces. In contrast, the W23910(H3N2), 901084(H3N2), and J84610(H3N6) infection groups, exhibited mild alveolar widening with limited immune cell (Figure 5(C), S11(B)). Viral replication in murine organs was assessed by endpoint titration in embryonated eggs at 3 and 5 d.p.i. Viruses were widely detected in the nasal cavities, lungs, and brains in all groups except 901084(H3N2) and J84610(H3N6) infection groups. No virus was detected in the ileum of any group (Table 3).

Figure 5.

Pathogenicity of H3 subtype AIVs in mice.

The infected mice were observed continuously for 14 days. (A) Body weight and (B) survival of the mice were recorded every day. The data represent the average body weight of each group (n = 5/group). (C) H&E staining of lungs. Scale: 50 μm. (D–E) The viral distributions in the lungs and nasal cavities of infected chickens were observed via confocal laser scanning microscopy. Nuclei (DAPI; blue); NP protein of the virus (Alexa Fluor 649; red). The virus distribution in organs is marked with white arrows. Scale: 20 μm.

Table 3.

Viral titers in tissues of mice infected with seven H3 subtype AIVs.

Virus	Viral titers ± SD (lg EID50/mL)
	3 d.p.i.				5 d.p.i.
	Nasal cavity	Lung	Ileum	Brain	Nasal cavity	Lung	Ileum	Brain
A/duck/Wuxi/W7-4/2014(H3N3)	5.00 ± 2.50a (3/3)	3.39 ± 1.25 (3/3)	0	2 (1/3)	6.39 ± 0.67 (3/3)	6.30 ± 0.82 (3/3)	0	2.25 ± 0.35 (2/3)
A/duck/Jiangsu/J84610/2017(H3N6)	0	0	0	0	0	0	0	0
A/chicken/Jiangsu/W23910/2017(H3N2)	0	0	0	0	0	1.74 ± 0.42 (3/3)	0	0
A/swan/Yangzhou/901084/2018(H3N2)	0	0	0	0	0	0	0	0
A/duck/Jiangsu/JY020416/2019(H3N2)	3.23 ± 0.49 (3/3)	4.06 ± 0.59 (3/3)	0	2 (1/3)	1.83 ± 0.28 (3/3)	1 (1/3)	0	0
A/duck/Gaoyou/4D1-1/2021(H3N2)	3.08 ± 1.00 (3/3)	4.56 ± 1.26 (3/3)	0	0	2.44 ± 2.80 (2/3)	6.09 ± 1.94 (2/3)	0	0
A/duck/Anhui/LY/2021(H3N2)	3.61 ± 0.82 (3/3)	3.72 ± 1.20 (3/3)	0	0	4 ± 1.20 (3/3)	5.06 ± 0.58 (3/3)	0	0

a: The results indicate the mean virus titer from three independent mice ± SDs.

At 5 d.p.i., W7-4(H3N3) infection group showed the highest viral titers in nasal cavities and lungs (6.39 ± 0.67 lgEID₅₀/mL and 6.30 ± 0.82 lgEID₅₀/mL, respectively). High viral titers were also observed in the lungs of 4D1-1(H3N2) and LY(H3N2) infection groups at 5 d.p.i. (6.09 ± 1.94 lgEID₅₀/mL and 5.06 ± 0.58 lgEID₅₀/mL, respectively). In JY020416(H3N2) and 4D1-1(H3N2) infection groups, viral titers in the nasal cavity peaked 3 d.p.i., (3.23 ± 0.49 lgEID₅₀/mL and 3.08 ± 1.00 lgEID₅₀/mL, respectively), with JY020416(H3N2) reaching 4.06 ± 0.59 lgEID₅₀/mL in the lungs at the same time point. Consistent with these findings, CLSM results revealed viral distribution in both nasal cavities and lungs only in the JY020416 (H3N2), 4D1-1(H3N2), LY(H3N2), and W7-4(H3N3) infection groups, which was consistent with the viral titer results observed in murine lungs (Figure 5(D,E)), aligning with the viral titer data. Overall, although H3N2 subtype AIVs exhibited low pathogenicity in mice, the isolates from 2019 to 2021 (JY020416, 4D1-1, and LY) demonstrated a degree of replication competence in murine lungs, indicating partial mammalian adaptation and highlighting the need for continued surveillance and further investigation.

H3 subtype AIVs exhibit interspecies and cross-species transmission

To assess interspecies and cross-species transmission ability, contact chickens or guinea pigs were introduced into the clean cages containing inoculated chickens 24 hours after inoculation. As shown in Figure 6, viral shedding was detected in the OP swabs of all infection groups. CL swabs showed viral shedding only in three H3N2 subtype AIV infection groups: the W23910, 901084, and JY020416 infection groups. The duration of shedding varied among groups, with the shortest period in the JY020416(H3N2) infection group (3 days), and the longest period in the 901084(H3N2) infection group (11 days). Among the contact chickens, viral shedding was detected in the OP swabs of W23910(H3N2), 901084(H3N2), JY020416(H3N2), LY(H3N2), and W7-4(H3N3) contact groups, whereas CL swab shedding was observed only in W23910(H3N2) and JY020416(H3N2) group. These findings indicate that interspecies contact transmission occurs among chickens. Moreover, with the exception of the JY020416(H3N2) infection group, the other six 901084(H3N2), W23910(H3N2), 4D1-1(H3N2), LY(H3N2), W7-4(H3N3), and J84610(H3N6) infection groups possessed the ability to transmit the virus from chickens to guinea pigs, resulting in virus shedding in nasal wash samples. Specifically, W23910(H3N2), 4D1-1(H3N2), and J84610(H3N6) infection groups transmitted the virus to only one of the three contact guinea pigs. Furthermore, 901084(H3N2), LY(H3N2), and W7-4(H3N3) infection groups transmitted the virus to two or three contact guinea pigs, with the highest titer of virus shedding (4 lgEID₅₀/mL) observed in guinea pigs exposed to the LY(H3N2) strain. Notably, virus shedding in guinea pigs exposed to the LY(H3N2) strain lasted for 8 days. These results strongly indicated the potential of H3N2 subtype AIV especially LY(H3N2) strain for cross-species transmission.

Figure 6.

Interspecies and cross-species transmission of H3 subtype AIVs.

For the transmission study, inoculated chickens (n = 3) were intranasally inoculated with 10⁶ EID₅₀ of the test virus, and three uninfected chickens and guinea pigs were introduced into the same cages at 2 d.p.i. OP and CL swabs from inoculated chickens at 1, 3, 5, 7, 9, and 11 d.p.i. as well as nasal washes from all guinea pigs and OP and CL swabs from contact chickens at 2, 4, 6, 8, 10, and 12 d.p.i. were collected to detect viral shedding. Each colored bar represents the virus titer from the swab of an individual animal. The horizontal dashed lines indicate the lowest limit of detection (<0.67 lgEID₅₀/mL).

Serological analysis at 21 days post-inoculation revealed that all inoculated chickens were seropositive with HI titers ranging from 4 to 8 log₂ (Figure 7). Among contact chickens, all groups except J84610(H3N6) contact group tested positive. Specifically, two chickens in the W23910(H3N2) group, two in the 901084(H3N2) group, three in the JY020416(H3N2) group, one in the 4D1-1(H3N2) group, three in the LY(H3N2) group, and three in the W7-4(H3N3) group, were seropositive. HI titers of the contact chickens were generally lower than those of the inoculated chickens. For contact guinea pigs, seroconversion was absent in all individuals from the JY020416(H3N2) and 4D1-1(H3N2) groups. In contrast, two guinea pigs in the W23910(H3N2) group, three in the 901084(H3N2) group, and three in the LY(H3N2) group were seropositive. Notably, HI titers in the LY(H3N2) contact guinea pigs reached 6 log₂, whereas HI titers in other guinea pigs remained at or below 4 log₂.

Figure 7.

HI antibody of chickens and Guinea pigs in the inoculated and contacted groups.

For the transmission study, inoculated chickens (n = 3) were intranasally inoculated with 10⁶ EID₅₀ of the test virus, and three uninfected chickens and guinea pigs were introduced into the same cages at 2 d.p.i. Serum was collected at 21 d.p.i. for seroconversion detection. Each colored bar represents the HI antibody titer from the serum of an individual animal.

The LY(H3N2) strain exhibits low pathogenicity in guinea pigs

Based on its ability to replicate in the nasal cavities and lungs of mice, induce severe lung lesions, and transmit from chickens to guinea pigs, the LY (H3N2) strain was selected to assess pathogenicity in guinea pigs. Guinea pigs in the inoculated group showed no clinical signs of influenza infection. Viral replication was detected exclusively in the nasal cavity at 3.91 ± 0.80 lgEID₅₀/mL (Table 4). No pathological changes were observed in the lungs compared with the PBS control group (Figure S12(A)). The CLSM further confirmed that viral distribution was present only in the nasal cavity, and absent in the lung (Figure S12(B, C)). Collectively, these findings indicate that the LY(H3N2) strain exhibits low pathogenicity in guinea pigs.

Table 4.

Viral titers in tissues of guinea pigs inoculated with LY(H3N2).

Virus	Viral titers (lg EID50/mL)
	Nasal cavity	Lung	Trachea	Intestine	Liver
A/duck/Anhui/LY/2021(H3N2)	3.91 ± 0.8 (3/3)a	0	0	0	0

a: The results indicate the mean virus titer from three independent guinea pigs ± SDs.

The LY(H3N2) strain exhibited contact and aerosol transmission among guinea pigs

Although the LY (H3N2) strain exhibited low pathogenicity in guinea pigs, its potential for contact and aerosol transmission was further evaluated. As shown in Figure 8(A), viral shedding was detected in the nasal washes of all inoculated, contact, and exposure groups. In the inoculated group, viral shedding peaked at 1 d.p.i., with a titer of 10⁸ lgEID₅₀/mL, 10^6.23 lgEID₅₀/mL, and 10⁵ lgEID₅₀/mL in individual animals, and persisted for 5 days. Virus shedding in the contact group was first detected at 2 d.p.i., with a titer of 10⁴ lgEID₅₀/mL, 10^3.5 lgEID₅₀/mL, and 10³ lgEID₅₀/mL in each guinea pig, respectively. The peak virus shedding occurring at 4 d.p.i. in one guinea pig with the titer of 10⁶ lgEID₅₀/mL. Moreover, the virus shedding lasted for 5 days. The virus shedding of guinea pigs in the exposure group began at 6 d.p.i., with a titer of 10⁵ lgEID₅₀/mL in one guinea pig. The peak virus shedding occurring at 10 d.p.i., with a titer of 10^7.5 lgEID₅₀/mL, 10⁷ lgEID₅₀/mL, and 10^1.5 lgEID₅₀/mL in each guinea pig, respectively. The duration of virus shedding was 5 days. HI antibodies were detected in the serum of all guinea pigs in the inoculated, contact, and exposure groups (Figure 8(B)). The HI antibody titer of each guinea pig at 21 d.p.i. was greater than that at 14 d.p.i., with the highest HI antibody titer reaching 10 log₂ in the inoculated and exposure groups.

Figure 8.

Contact and aerosol transmission of the LY(H3N2) strain in guinea pigs.

Three guinea pigs were inoculated with 10⁶ EID₅₀ of the LY(H3N2) strain. At 24 h.p.i., three uninfected guinea pigs were placed in the same cage as the contact transmission group, and three uninfected guinea pigs were placed in the other side cage as the aerosol transmission group. (A) At 1, 3,5, 7, 9, and 11 d.p.i., the nasal washes of three guinea pigs in the inoculated group were collected. At 2, 4, 6, 8, 10, and 12 d.p.i., the nasal washes of the guinea pigs in the contact and aerosol transmission groups were collected to test virus shedding. (B) Serum was collected at 14 and 21 d.p.i. for seroconversion detection. Each colored bar in (A) represents the virus titer from the swab of an individual animal, and each colored bar in (B) represents the HI antibody titer from the serum of an individual animal. The horizontal dashed lines indicate the lowest limit of detection (<0.67 lgEID50/mL).

Discussion

In April 2022, the first human infection with the H3N8 virus was reported in Henan Province, China [50]. In May 2022, a second case was identified in a 5-year-old boy in Hunan Province, China [16]. These events have raised substantial concerns regarding the potential public health impact of emerging H3N8 subtype AIVs. In addition to H3N8 subtype AIVs, H3N2 subtype AIVs are continuously detected in poultry throughout the year in southern China, according to epidemiological surveys [51]. Previous studies have demonstrated that H3N2 subtype AIVs can be transmitted between guinea pigs and ferrets via respiratory droplets [2]. Furthermore, recent isolates have acquired mutations associated with increased binding affinity for human-type receptors and enhanced transmissibility in ferrets, posing a potential risk to human health [52]. In China, H3N2 subtype AIVs are widely detected in LPMs [44], increasing the likelihood of cross-species transmission from avians to mammals. Therefore, the cross-species transmission potential of H3N2 subtype AIVs, particularly from avians to mammals, warrants close attention. Phylogenetic analysis in this study showed that all seven H3 subtype AIVs belong to the Eurasian lineage, within which multiple cross-species transmission events have historically occurred. Notably, H3N2 subtype AIVs (LY strain), presented potential cross-species transmission characteristics from avians to mammals, which should be more attention.

Phylogenetic analysis also revealed extensive reassortment among H3 subtype AIVs, resulting in diverse genotypes. H3 viruses have been detected in combination with multiple neuraminidase (NA) subtypes (N1–N8) circulating in poultry and wild birds in mainland China, often without causing apparent disease [8]. In this study, the NA gene of the J84610(H3N6) strain was closely related to that of H5N6 subtype AIVs, which have caused multiple human infections in recent years, suggesting a potential reassortment with H5N6 subtype AIV [53]. Additionally, the classical H1N1 swine influenza virus, known to transmit between pigs and ferrets [54], has contributed NP, M, and NS gene segments to triple-reassortant H3N2 influenza virus identified in swine [55]. In our study, the PB2, M, and NS genes of the LY(H3N2) strain were closely related to those of H1 subtype influenza viruses. This genetic composition may partly explain the observed avian-to-mammalian and mammalian-to-mammalian transmission potential of the LY(H3N2) strain.

A notable observation from this study is the distinct phenotypic profiles of different H3N2 isolates, particularly the contrast between the 4D1-1 strain (which caused severe lung lesions in chickens) and the LY strain (which exhibited aerosol transmission in guinea pigs). These divergent phenotypes provide an opportunity to explore potential genetic correlates. The 4D1-1 strain was the only isolate harboring the NP-M105V mutation, which has been previously associated with increased virulence in chickens [45]. This mutation may contribute to the enhanced pathogenicity observed in avian hosts, although other genetic factors likely also play a role. In contrast, the 4D1-1 strain did not transmit efficiently in guinea pigs, suggesting that mutations favorable for avian virulence may not confer mammalian adaptation.

The LY strain uniquely possessed several mutations associated with mammalian adaptation: PA-E382D, which has been linked to increased influenza A virus polymerase activity in mammalian cells, and PB1-M317R (where other isolates had M317I), which may alter polymerase complex function. Notably, while all isolates contained the HA-I155T mutation (associated with human-type receptor binding) and PB2-I333T (associated with mouse adaptation), only LY achieved aerosol transmission in guinea pigs. This indicates that these common mutations are necessary but not sufficient for the enhanced transmission phenotype. Instead, the combination of PA-E382D, PB1-M317R, and potentially other yet-unidentified mutations in the LY strain may create a permissive genetic constellation that enables aerosol transmission. Future studies using reverse genetics to introduce specific mutations into isogenic backgrounds are needed to definitively establish the functional contributions of these individual residues.

The mutations I333T in the PB2 protein; H436Y (H436R in LY(H3N2)), R198K, V171M, and M317I (M317R in LY(H3N2)) in the PB1 protein; N30D and T215A in the M1 protein; and L69P and P64S in the M2 protein have been reported to enhance virulence and adaptability in mice [37,41,56]. These mutations may partly explain the observed replication and host adaptation of several isolates in mice, including W7-4(H3N3), W23910(H3N2), JY020416(H3N2), 4D1-1(H3N2), and LY(H3N2). However, despite the presence of these mutations, all isolates exhibited low pathogenicity in mice, indicating that additional genetic or host factors likely modulate virulence. Receptor-binding preference for human-type receptors is a key factor facilitating the transmission of AIVs from avian species to humans [18]. The HA 155T substitution, which has been associated with increased affinity for the human-type receptor [9], was identified in all seven isolates. Consistent with this, most isolates exhibited dual receptor-binding specificity, recognizing both SA α-2,3-Gal receptor and the SA α-2,6-Gal receptor, with the exception of J84610(H3N6) strain. Previous studies have shown that H3N2 subtype AIVs have gradually adapted to bindto human-type receptors since the 1968 pandemic [57], supporting the potential for ongoing host adaptation.

LPMs provide a favorable environment for AIV transmission, thereby facilitating the transmission of H3 subtype AIVs. In this study, contact chickens became infected after exposure to inoculated chickens carrying W7-4(H3N3), W23910(H3N2), JY020416(H3N2), 901084(H3N2), and LY(H3N2)-strains. These findings demonstrate efficient transmission of H3 subtype AIVs among chickens, and highlight the potential for widespread dissemination in poultry within LPM settings. Such transmission dynamics may also contribute to the increased detection of H3 subtype AIVs in poultry over the last decade [8]. Notably, LPMs are major sites of human exposure to diverse AIVs. Although human infections with H3N2 subtype AIVs have not yet been reported, phylogenetic evidence indicates that H3 subtype AIVs, particularly those of the Eurasian lineage have undergone cross species transmission. This suggests a potential for spillover from poultry to mammalian hosts, including humans. Supporting this concern, human infections with H3N8 subtype AIVs were reported in April and May 2022 [16,50]. Epidemiological investigations further revealed that infected individuals had a history of exposure to live poultry in LPMs [16], underscoring the public health relevance of these environments. Collectively, these findings emphasize the importance of continued surveillance of H3-subtype AIVs in LPMs to monitor their evolution and assess their potential risk to human health.

In this study, seven H3 subtype AIVs were evaluated for cross-species transmission from chickens to guinea pigs using a model designed to simulate poultry–humans interactions in LPMs. Viral titers were detected in the nasal washes of contact guinea pigs for all isolates except for the JY020416(H3N2) strain, indicating that six isolates were capable of cross-species transmission from chickens to guinea pigs. Notably, the H3N2 subtype AIV (LY strain) was transmitted not only from chickens to guinea pigs but also among guinea pigs, demonstrating both avian-to-mammalian and mammalian-to-mammalian transmission potential. The PA E382D mutation, detected exclusively in the LY(H3N2) strain, has been reported to enhance influenza A virus polymerase activity in mammalian cells [42], which may contribute to its relatively high transmission efficiency in guinea pigs. Although this study did not experimentally validate the functional role of this mutation, future research will focus on a broader panel of H3 isolates, and employ comparative and mutational analyses to confirm its contribution. In addition, the PA 672L residue, previously implicated in facilitating aerosol transmission of AIVs [58], was also identified in the LY(H3N2) strain. Consistent with this, the LY(H3N2) strain exhibited efficient aerosol transmission among guinea pigs, accompanied by robust replication in the respiratory tract. Overall, this study demonstrates that certain H3N2 subtype AIVs possess the capacity for both avians-to-mammalian and mammalian to mammalian transmission under conditions simulating LPMs exposure. These results provide valuable insights for risk assessment and inform strategies for the prevention and control of H3 subtype AIVs.

This study has some limitations. First, the number of H3 subtype AIV isolates was relatively small, and all isolates were obtained from a limited geographic region, which may not fully reflect the global diversity of these viruses. Second, guinea pigs, although useful for assessing transmission, do not perfectly model human infection. Third, the functional roles of specific mutations were inferred from previous studies rather than experimentally confirmed in this study. Fourth, laboratory simulations of LPMs may not fully capture the complexity of natural avian-to-mammal transmission.

Future studies should include a larger number of diverse H3 isolates, use complementary mammalian models, and employ reverse genetics to validate the effects of key mutations on pathogenicity and transmission. Such research will provide a more comprehensive understanding of the adaptation, interspecies transmission, and potential public health risk of H3 subtype AIVs.

In conclusion, H3N2 subtype AIV, particularly the LY strain exhibited both interspecies and cross-species transmission characteristics, suggesting an increased potential for transmission from avian to mammalian hosts. These findings imply that continued surveillance of H3N2 AIVs in LPMs is critical, as they may pose an emerging concern to public health, and monitoring key molecular markers associated with mammalian adaptation could help in predicting and preventing future cross-species transmission events [59].

Funding Statement

This work was funded by the National Key Research and Development Program of China [2024YFC2310301], the National Natural Science Foundation of China [32573459, 32503076], the Jiangsu Provincial Natural Science Fund for Distinguished Young Scholar [BK20240045], the “Jie Bang Gua Shuai” Project at Yangzhou University [YZUXK202316], the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province [CX(23)3071], the National Postdoctoral Program for Innovative Talents [BX20240300], the China Postdoctoral Science Foundation [2024M762746], the Key Research and Development Program of Social Development of Jiangsu Province [BE2022774], the 111 Project [D18007], International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions [NO.7], a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the High-Performance Computing Cluster of the College of Veterinary Medicine, Yangzhou University.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The raw sequence data of this study are openly available on Figshare at https://doi.org/10.6084/m9.figshare.30344920.

Statement of adherence to ARRIVE guidelines

In this study, we followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines to ensure transparency, rigor, and integrity in the design, conduct, and reporting of experiments. The ARRIVE guidelines aim to enhance the quality of scientific research, justify the use of experimental animals, and promote the reliability and reproducibility of findings. We adhered to these guidelines and have uploaded a completed checklist as a supplementary file.

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2026.2673657