Antigenicity and genetic properties of an Eurasian avian-like H1N1 swine influenza virus in Jiangsu Province, China

Highlights

• •Scientific question

• Cross-species transmission of influenza A viruses from swine to humans occurs occasionally because their tracheal epitheliums possess both sialic acid α-2,6-Gal and α-2,3-Gal receptors. In 2011, the first human case of swine influenza virus infection in the mainland of China was detected in Jiangsu Province. Subsequently, the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIV) had sporadically crossed the host barrier and infected humans, raising public concern for its pandemic potential.

• •Evidence before this study

• A/Jiangsu/1/2011 (H1N1v) was first discovered in 2011 and belongs to the G1 genotype. The G4 and G5 genotypes that appeared successively in 2013 are recombinant H1N1 swine influenza viruses. The EAH1N1 SIVs from 2016 to the present are dominated by the G4 genotype, with hemagglutination (HA) and neuraminidase (NA) genes derived from the EAH1N1 SIVs, non-structural protein (NS) genes derived from the triple-origin reassortant swine influenza viruses, and the rest of the internal genes from influenza A (H1N1) pdm09 virus.

• •New findings

• This study investigated a case of the EAH1N1 SIV infection in a child. This is the first case of the EAH1N1 SIV genotype G4 infection in a child in Jiangsu Province. This virus maintained the genetic properties of the EAH1N1 SIV but differed significantly in HA protein antigens.

• •Significance of the study

• This new human case of the EAH1N1 SIV infection indicates the pandemic potential of avian influenza viruses.

Abstract

Pigs are vital genetic mixing vessels for human and avian influenza viruses because their tracheal epitheliums possess both sialic acid α-2,6-Gal and α-2,3-Gal receptors. Cross-species transmission of influenza A viruses from swine to humans occurs occasionally. The first case of human infection with the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) genotype G4 was detected in Jiangsu Province, China, in February 2023, and backtracking epidemiological investigations did not reveal a clear source of the infection. The hemagglutination (HA) and neuraminidase (NA) amino acid variant sites, antiviral drug susceptibility, and antigenic variation of the isolated A/Jiangsu/27271/2023 (JS/27271/23) virus were analyzed, and we evaluated the protective effect of sera collected from occupationally exposed populations in 2024 against the virus. Compared with the vaccine strain, the nucleotide sequence similarities of JS/27271/23 HA and NA were 96.5 % and 95.2 %, respectively. JS/27271/23 was sensitive to polymerase inhibitors (favipiravir and baloxavir), and the antigenicity of its HA protein was 8-fold different from that of the vaccine strain. The percentage of occupationally exposed population with antibody titers of ≥ 40 against A/Hunan/42443/2015 (HN/42443/15) and A/Jiangsu/1/2011 (JS/1/11) were 7.25 % and 2.25 %, respectively, and the geometric mean titers (GMT) were 6.24 and 5.34, respectively. Out of 400 serum samples examined, none had antibody titers of ≥ 40 against JS/27271/23. This suggests that low serum levels of antibodies to EAH1N1 SIVs in occupationally exposed populations may not provide adequate protection because of significant differences in amino acid sites and antigenicity between this virus and the current vaccine strain of EAH1N1 SIVs. There is no evidence of human-to-human transmission of EAH1N1 SIVs. Therefore, surveillance for EAH1N1 SIVs and the development of new vaccine strains are required.

1. Introduction

The influenza virus causes influenza, which is an acute respiratory infectious disease. Different influenza virus subtypes exhibit evident species-specificity. Cross-species transmission has continued in recent years, with human cases of infection with animal influenza viruses such as H5N6, H7N9, and H10N8. Swine can be considered a “mixed container of influenza viruses” with the potential to be infected with different subtypes of influenza viruses. Among these, the H1N1 subtype is the most prevalent in swine populations [1], [2]. It can be classified as Eurasian avian-like H1N1, classical swine H1N1 (CS H1N1), and 2009 pandemic H1N1 (2009/H1N1). Since its emergence, the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) have spread rapidly in pig populations in Europe and Asia, progressively replacing the CS H1N1 swine influenza virus that was previously circulating. In some areas, different subtypes of swine influenza viruses or genotypes of EAH1N1 SIVs have co-circulated, particularly following the emergence of 2009/H1N1 influenza viruses. This provided more selectable gene fragments for recombination, increasing the probability of recombination. A study that collected nasal swabs from 103,110 pigs in 22 provinces in China between October 2013 and December 2019 revealed that EAH1N1 SIVs formed eight different genotypes through reassortment with viruses of other lineages circulating in humans and pigs, and two of these genotypes (G4 and G5) were widely distributed in pigs [3]. G4 and G5 accounted for 53.6 % and 42.6 % of EAH1N1 SIVs isolated from pigs between 2013 and 2019 [4]. EAH1N1 SIVs of the G4 genotype exhibited enhanced adaptability in mammals and became the dominant genotype [5]. EAH1N1 SIVs have undergone evolutionary changes [6], including increased efficiency of viral replication, enhanced virulence, breach of the species barrier to transmission, and the development of drug resistance. These changes had rendered the vaccine’s cross-protection less effective, which can potentially lead to the next influenza pandemic. The World Health Organization (WHO) established the Global Influenza Surveillance and Response System (GISRS) in response to the potential for an influenza pandemic. The influenza surveillance network, a pivotal element of the GISRS system, continuously monitors fever outpatient clinics and hospitalized patients with pneumonia in designated sentinel hospitals throughout the year. In February 2023, a case of EAH1N1 SIV infection was identified by the National Influenza Surveillance Network laboratory in Jiangsu Province; the antigenic and genetic characteristics of the strain are discussed in this paper.

2. Materials and methods

2.1. Virus isolation and identification

A throat swab was taken from the patient and tested by real-time reverse transcription-polymerase chain reaction (RT-PCR) for human subtypes H1, 2009/H1N1, H3, and avian H5, H7, H9, and H10 viruses. The sample was genotyped in real-time RT-PCR assays with specific primers to identify the Eurasian avian-like H1N1 subtype. Madin-Darby canine kidney (MDCK) cells were maintained in dulbecco's modified eagle medium with TPCK-trypsin (2 μg/mL). Pharyngeal swabs were inoculated into MDCK cells and adsorbed at 35 °C for one hour. A cell maintenance solution was added, and the cells were observed for several days until they became diseased.

2.2. Genomic sequencing and phylogenetic analysis

Viral RNAs were extracted using a MagMAX^TM CORE Nucleic Acid Purification kit, and the primer Uni12 (5′-AGCGAAAGCAGG-3′) was used for reverse transcription. PCR was performed using the SuperScript® Ⅲ One-step RT-PCR system with Platinum® Taq High Fidelity. The PCR reaction system was as follows: DEPC-treated ddH₂O 12 μL, 2 × Reaction buffer 25 μL; 10 μmol/L (μM) Uni-12/Inf1 (primer A: 5′-GGGGGGAGCAAAAGCAGG-3′) 0.4 μL; 10 μM Uni-12/Inf3 (primer B: 5′-GGGGGGAGCGAAAGCAGG-3′) 0.6 μL; 10 μM Uni-13/Inf1 (primer C: 5′-CGGGTTATTAGTAGAAACAAGG-3′) 1 μL; RT/HiFi enzyme mix 1; and RNA 8 μL. The PCR reaction conditions were as follows: 45 °C for 60 min, 94 °C for 2 min; 94 °C for 30 s, 44 °C for 30 s, 68 °C for 3 min, 5 cycles; 94 °C for 30 s, 57 °C for 30 s, 68 °C for 3 min, 31 cycles; 68 °C for 7 min; and 4 °C hold. PCR products were purified using a QIAquick PCR purification and sequenced using an Illumina Analyzer (Miniseq).

Nucleotide BLASTn analysis was used to identify related reference sequences. The sequences were first aligned with cluster muscle, and the maximum likelihood (ML) trees for each of the eight gene segments were constructed using MEGA 11.0. Sequence-based antigenic variation distance prediction analysis was performed using PREDAV-FluA online (computationalbiology.cn/home/index.html). For comparison, representative isolates from the following H1N1 clades were selected as reference sequences: Eurasian avian-like swine, Eurasian avian, classical swine, North American avian, 2009/H1N1, and seasonal human influenza viruses. The reference sequences were obtained from the GISAID and the National Center for Biotechnology Information (NCBI) databases, and the accession numbers for the reference sequences have been listed in Table S1.

2.3. Hemagglutination-inhibition (HI) assay

Sera were incubated with receptor-destroying enzyme (RDE) at a ratio of 1:3 serum-RDE dilution for 18 to 20 h at 37 °C, followed by inactivation of the RDE at 56 °C for 60 min. Immediately after heat inactivation, serum-RDE were incubated with phosphate buffered saline (PBS) in a ratio of 2:3, resulting in RDE-treated, PBS-diluted serum samples at a dilution of 1:10. The viral reagents were diluted for use at four hemagglutinating units (HU) in 25 μL. To perform the HI assay, pretreated sera were serially 2-fold diluted from the initial 1:10 dilution, mixed with the diluted virus in a ratio of 1:1 (25 μL diluted sera and 25 μL diluted virus), and incubated at room temperature for 60  min. Next, 50 μL of 1 % turkey red blood cells (TRBC) was added, followed by incubation at room temperature for 30 min. Assay plates were tilted to read, and the titer was reported as the reciprocal of the highest serum dilution in which agglutination was completely inhibited. HI was used for antigenicity analysis and antibody titer determination.

2.4. Serological survey of EAH1N1 SIVs infection

Serum samples obtained from the occupationally exposed populations in 2024 were assessed using the HI and micronutrient neutralization (MN) assays for antibody titer against the EAH1N1 SIVs (HN/42443/15, JS/1/11, JS/27271/23). To determine the risk of EAH1N1 SIVs infection, we conducted 400 serological surveillance tests for antibodies against EAH1N1 SIVs among occupationally exposed populations in Yancheng (n = 102), Huai'an (n = 90), Yangzhou (n = 48), Changzhou (n = 80), and Suzhou (n = 80). HI and MN tests were done under WHO guidelines.

2.5. Plaque reduction assay

MDCK cells were seeded in 96-well culture plates at 3 × 10⁵ cells/mL and incubated at 35 °C with 5 % CO₂ and in saturated humidity. Cells were washed two times with virus growth medium (VGM) after reaching > 90 % confluence. Cells were incubated for 1 h with the virus at 35 °C and 5 % CO₂. A 200 μL of microcrystalline cellulose (MCC; Avicel®) and favipiravir/baloxavir mixture was used to cover the cells. Then, the plates were incubated at 35 °C with 5 % CO₂ overnight. Viral plaques were counted on the last day. The test was done following WHO guidelines.

2.6. Statistical analysis

Statistical significance was determined using chi-square tests, and the GraphPad Prism 9 software package was used for statistical analyses. A P-value of < 0.05 was considered statistically significant.

3. Results

3.1. Case investigation

The patient was an 18-month-old girl with a fever of 39 °C who began coughing on 30 January 2023. The patient was managed at a hospital whose laboratory is a part of the National Influenza Network of laboratories. Her sample tested positive for influenza A virus by real-time RT-PCR but negative for human subtypes H1, 2009/H1N1, H3, and avian H5, H7, H9, and H10 influenza viruses. Jiangsu CDC classified the sample as EAH1N1 SIVs by real-time RT-PCR.

Through the mother of the child, it was ascertained that the patient had no history of traveling abroad two months before the onset of the disease and no contact history with live poultry or swine. The only individuals present at the residence during the Spring Festival were the child’s grandfather, uncle, and aunt, who exhibited no unusual symptoms. The family did not keep any pets, and the domestic and external environments were observed to be clean and tidy. The mother often goes to the live poultry farmers’ market approximately two kilometers away from her home, where live poultry are sold, mainly chickens and pigeons. However, she had not purchased any live poultry within the past month.

Throat swabs were taken on February 8 from the patient, and her mother and grandmother were all negative for influenza virus nucleic acid. On February 14, 36 poultry, pig, and environmental samples were collected from live poultry markets, pig farms, and slaughterhouses, which were all negative for influenza virus nucleic acid. A review of hospitalized pneumonia cases in the treating hospitals since January 24 revealed no similar cases. The results of the traceability survey have yet to clarify the source of the virus and failed to identify the mode of transmission.

The Chinese Centre for Disease Control and Prevention (China CDC) isolated the virus with hemagglutinating activity under the designation JS/27271/23 (The sequence data have been deposited in the Global Initiative on Sharing Avian Influenza Data; the accession numbers are EPI3597230-EPI3597237). Genome sequencing and nucleotide BLASTn analysis identified the isolate as the European avian-like swine H1N1 subtype G4.

3.2. Genetic analysis

The complete genome of JS/27271/23 has been sequenced, and evolutionary trees of its hemagglutination (HA) and neuraminidase (NA) sequences have been constructed (Fig. 1). Evolutionary trees of the polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic protein (PA), nucleoprotein (NP), matrix protein (M), non-structural protein (NS). genes are shown in Fig. S1. HN/42443/15, JS/1/11, and A/swine/Liaoning/TL5239/2020 were selected as representatives for analyzing nucleotide homology. JS/27271/23 was 94.4 % – 98.9 % homologous to HN/42443/15 and only 82.2 % – 96.0 % homologous to JS/1/11 (Table 1).

Fig. 1.

Phylogenetic relationships of the HA and NA genes using the maximum likelihood method with 1,000 bootstraps. Note: The JS/27271/23 is indicated using an orange dot, a blue triangle indicates the HN/42443/15 vaccine strain, and the Jiangsu strain JS/1/11 is indicated using a green square. Abbreviations: HA, hemagglutination; NA, neuraminidase.

Table 1.

Nucleotide sequence identity between JS/27271/23 and reference sequences.

Gene (JS/27271/23)		PB2	PB1	PA	HA	NP	NA	MP	NS
Identities (%)	LN/TL5239/20	99.0	98.9	98.8	98.7	99.3	98.3	99.3	97.7
	HN/42443/15	96.5	97.0	98.9	96.5	96.9	95.2	94.4	95.9
	JS/1/11	82.6	83.9	84.0	96.0	82.4	95.0	94.6	82.2

Abbreviations: PB2, polymerase basic 2; PB1, polymerase basic 1; PA, polymerase acidic protein; HA, hemagglutination; NP, nucleoprotein; NA, neuraminidase; MP, matrix protein; NS, non-structural protein.

Based on the deduced amino acid sequence, JS/27271/23 contained the amino acid motif PSVQSR↓G at its HA cleavage sites, characteristic of low-pathogenic influenza viruses. The receptor-binding specificity of HA has been proposed to be an essential determinant of the host range of a given influenza virus. Some researchers have shown that two amino acid mutations (E190D and G225D/E) can cause a shift in receptor binding specificity from the avian SA-α-2,3-Gal to the human SA-α-2,6-Gal [5]. In this study, JS/27271/23 possessed 190D and 225E, which may indicate that the viruses had the same receptor-binding preference as those of human viruses [6].

Amino acid substitutions (H274Y and N294S) were not observed in the NA proteins of JS/27271/23, suggesting that it is susceptible to oseltamivir and zanamivir [7], [8]. RNA polymerase inhibitor antivirals include baloxavir, favipiravir, and pimodivir [9], [10]. The JS/27271/23 did not produce PB1-K229R, PB2-S324I, or PA-I38M resistance mutations, suggesting that JS/27271/23 is sensitive to polymerase inhibitors [10]. In addition, our data showed that several amino acid residues of JS/27271/23 could enhance viral adaptation or virulence in mammals, such as 89 V, 251 K, 588I, 271A, 431 M and 591R in the PB2 [9], [10], [11], [12], [13], [14], [15], [16], [17]; 336 M, 356R, and 409 N in the PA [18], [19], [20]; 48 K, 98R, 99R, 305 K, 313 V, 357 K in the NP [17], [21], [22]; 41A and 215A in the M1 [23], [24]; 42S in the NS1 [25]. The matrix protein 2 (M2) proteins of the isolate had N instead of S at residue 31, conferring resistance to amantadine and rimantadine antivirals, a characteristic marker of European swine viruses (H1N1, H3N2, and H1N2) since 1987 [26] (Table 2).

Table 2.

Molecular analysis of JS/27271/23, JS/1/11, and HN/42443/15.

Gene	Function impact	Mutation	HN/42443/15	JS/1/11	JS/27271/23	References
HA	Increases the receptor-binding affinity to α2,6-linked sialosides	E190D	D	D	D	[6]
		D225E	E	E	E
NA	Confers antiviral resistance (Oseltamivir)	H274Y	H	H	H	[7], [8]
		N294S	N	N	N
PB2	Enhances polymerase activity	L89 V	V	V	V	[9], [10]
	Increases viral replication efficiency and pathogenicity	R251K	R	R	K	[11]
		T588I	I	A	I	[12]
	Increases viral replication efficiency, polymerase activity, and pathogenicity	T271A	A	T	A	[13]
	Enhances polymerase activity and virulence	T431M	M	M	M	[14]
	Enhances virus replication and adaptability in mammals	Q591R	R	Q	R	[15], [16], [17]
		E627 K	E	E	E
		D701 N	D	N	D
PB1	Confers resistance to favipiravir	K229R	K	K	K	[10]
PA	Confers resistance to baloxavir	I38M	I	I	I	[10]
	Enhances polymerase activity	L336 M	M	L	M	[18]
	Enhances virus replication and virulence	K356R	R	K	R	[19], [20]
		S409 N	N	N	N	[20]
NP	Breaking through the species barrier	Q48K	K	Q	K	[21]
		K98R	R	K	R
		K99R	R	K	R
	Mammalian adaptation molecular marker	R305K	K	R	K	[17]
		F313V	V	F	V
	Makes binding to human receptors easy	Q357K	K	Q	K	[22]
M1	Increases the efficiency of virus transmission	P41A	A	A	A	[23]
	Virulence enhancement	T215A	A	A	A	[24]
NS1	Increases replication efficiency and regulates host antiviral response	P42S	S	S	S	[25]
	Alters the antiviral response in the host	G210R	G	R	G	[17]
M2	Confers antiviral resistance (Amantadine)	S31 N	N	N	N	[26]

Abbreviations: HA, hemagglutination; NA, neuraminidase; PB2, polymerase basic 2; PB1, polymerase basic 1; PA, polymerase acidic protein; NP, nucleoprotein; MP, matrix protein; NS, non-structural protein; M1, matrix protein 1; M2, matrix protein 2.

3.3. Antigenicity analysis

Sequence-based analysis of antigenic variant distance prediction was conducted using the HA sequences of 13 EAH1N1 SIVs. This included the HA sequences of the WHO-recommended vaccine candidate strain HN/42443/15, the first EA strain emerging in Jiangsu Province (JS/1/11), and the JS/27271/23 strain obtained in this study. The antigenic relationships among the 13 strains were predicted using PREDAV-FluA [27]. JS/27271/23 underwent significant antigenic variation compared with the vaccine strain HN/42443/15 (Fig. 2). Additionally, it differed in antigenicity from previously identified EAH1N1 SIVs. The sequence alignment of the HA1 protein is shown in Fig. S2. Further biological functional validation was performed on the results of the aforementioned sequence-based analyses.

Fig. 2.

Prediction of the variation distance between the Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) antigens. Notes: light green, HN/42443/15; orange, JS/27271/23; pink, JS/1/11; dark green, 10 strains of other EAHlN1 SIVs; “-”, the antigenicity of the two viruses is similar.

The EAH1N1 SIVs strains (HN/42443/15, JS/1/11, and JS/27271/23) were tested for antigenicity, and the autoantibody titers were 1:1,280 for HN/42443/15C/E and 1:640 for HN/42443/15 RG. The antibody titers of HN/42443/15C/E antiserum against JS/1/11 and JS/27271/23 were 1:1,280 and 1:160, respectively, and those of HN/42443/15 RG antiserum against JS/1/11 and JS/27271/23 were 1:640 and 1:80, respectively (Table 3). The vaccine strain HN/42443/15 can effectively cover JS/1/11, but the antigenic difference between JS/27271/23 and HN/42443/15 was up to 8-fold, and they were mutually low-reactive strains.

Table 3.

Analysis of the antigenicity of Eurasian avian-like H1N1 swine influenza virus (EAH1N1 SIVs) found in Jiangsu Province.

Reference Virus	Passage	Reference serum
		HN/42443/15C	HN/42443/15 E	HN/42443/15 RG
HN/42443/15	C4	1,280	1,280	640
HN/42443/15	E4	1,280	1,280	640
HN/42443/15 RG	E2/E7	1,280	1,280	640
JS/1/11	E5	1,280	1,280	640
JS/27271/23	C2	160	160	80

Note: Homologous hemagglutination-inhibition titers are in bold (hemagglutination-inhibition method). Titers were determined as the reciprocal of the highest serum dilution in which hemagglutination is completely inhibited.

3.4. Levels of antibodies to EAH1N1 SIVs in occupationally exposed populations

The percentages of occupationally exposed individuals in Jiangsu Province with serum antibody titers to HN/42443/15, JS/1/11, and JS/27271/23 viruses of ≥ 40 were 7.25 % (χ² = 8.887, P = 0.020), 2.25 % (χ² = 8.415, P = 0.072), and 0, respectively. The geometric mean titers (GMT) of the antibodies were 6.24 (F = 8.699, P = 0.069), 5.34 (F = 8.349, P = 0.080), and 5, respectively. The results showed that antibodies to JS/27271/23 had not reached the antibody level for effective protection in the serum of the occupationally exposed population in 2024. Specific trends in the prevalence of the disease among cities are shown in Table 4.

Table 4.

Levels of antibodies to EAH1N1 SIVs in occupationally exposed populations.

Virus	Antibody titers ≥ 40 (%)						GMT
	YC	HA	YZ	CZ	SZ	P-value	YC	HA	YZ	CZ	SZ	P-value
HN/42443/15	0.98	7.78	10.42	8.75	11.25	0.020	5.17	6.65	6.67	6.21	7.13	0.069
JS/1/11	0.98	4.44	0.00	0.00	5.00	0.072	5.17	5.74	5.00	5.00	5.69	0.080
JS/27271/23	0.00	0.00	0.00	0.00	0.00	-	5.00	5.00	5.00	5.00	5.00	-

Abbreviations: EAH1N1 SIV, the Eurasian avian-like H1N1 swine influenza virus; GMT, geometric mean titers; YC, Yancheng; HA, Huai'an; YZ, Yangzhou; CZ, Changzhou; SZ, Suzhou.

3.5. Inhibitory effect of polymerase inhibitors

Influenza virus polymerase inhibitors are the most promising types of drugs for treating this disease. Favipiravir and baloxavir are approved for the treatment of influenza in Japan and the United States. Favipiravir effectively and selectively inhibits the RNA-dependent RNA polymerase (RdRp) of RNA viruses, while baloxavir specifically targets the cap-dependent endonuclease PA of influenza viruses [28]. Plaque reduction assays have shown that the 50 % inhibitory concentration (IC₅₀) of favipiravir in inhibiting the RdRp of JS/27271/23 was 3,403 nmol/L (nM), while the IC₅₀ of baloxavir in inhibiting the cap-dependent endonuclease PA was 0.876 nM. Meanwhile, the IC₅₀ of favipiravir against the representative strain of the 2009 influenza pandemic, A/California/07/2009 (H1N1 pdm), was 4,584 nM, and the IC₅₀ of baloxavir against H1N1 pdm was 1.429 nM (Fig. 3). This indicates that JS/27271/23 is sensitive to the polymerase inhibitors.

Fig. 3.

Inhibitory effect of polymerase inhibitors. A) A/Jiangsu/27271/2023(EAH1N1v). B) A/California/7/2009(H1N1pdm). Fitting curve to calculate IC₅₀, with the abscissa representing the concentration of favipiravir and baloxavir and the ordinate representing the plaque inhibition rate (%). Plaque inhibition rate (%) = (average plaque numbers in the virus control group − average plaque numbers in the drug-treated group) / average plaque numbers in the virus control group. Notes: red, baloxavir; blue, favipiravir; nM, nmol / L.

4. Discussion

Although human infections with EAH1N1 SIVs currently cause mainly mild disease, the number of EAH1N1 SIVs infections has increased in recent years, mainly among children aged 1 to 9 years [29]. It is, therefore, essential to pay more attention to EAH1N1 SIVs in routine influenza surveillance. EAH1N1 SIVs are capable of infecting swine, avian, and human populations and can potentially mutate into pandemic influenza strains. The cross-reactivity of antibodies to EAH1N1 SIVs in swine herds in China was observed to be 15 % in 2000, with a slight increase to 26 % by 2004. This was observed as a replacement of the then prevalent classical swine influenza and triple-array swine influenza viruses [30]. In 2011, the first human case of swine influenza virus infection in the mainland of China was detected in Jiangsu Province [31]. An eight-year swine influenza testing program initiated in 2011 revealed that EAH1N1 SIVs constituted the predominant subtype of influenza viruses circulating among pigs in China, accounting for 92.2 % of the 179 swine influenza viruses identified. The surface of porcine tracheal epithelial cells is dominated by SA-α-2,6-Gal with less expression of SA-α-2,3-Gal. The Jiangsu strains (JS/1/11 and JS/27271/23) and the vaccine strain (HN/42443/15) in this study both exhibited the 190D and 225E mutations in the HA protein, indicating that the Jiangsu strain and the vaccine strain predominantly bind to the human SA-α-2,6-Gal. Additionally, the EAH1N1 SIVs demonstrated a high propensity for infecting humans, presenting a significant public health concern.

The report of JS/27271/23 infection is the first report of human infection with EAH1N1 SIVs of the G4 genotype isolated in Jiangsu Province, which has multiple amino acid antigenic site differences in the HA and NA amino acid sites compared to those of the WHO-recommended vaccine candidate for EAHIN1 SIVs (HN/42443/15). This was also confirmed in the antigenicity analyses. Therefore, the assessment of the antigenic variation of isolated EAH1N1 SIVs strains should be strengthened in the future. H. Sun et al. [5] and E. Vandoorn et al. [32] revealed a deficiency in suitable antibodies within swine herds and populations. The study results showed that HN/42443/15 had a lower GMT in the serum of the occupationally exposed population. In comparison, JS/1/11 had a slightly lower GMT in the serum of the same population than that of HN/42443/15. JS/27271/23, which was identified in this study, has not yet caused infection in the occupationally exposed population in 2024. The seroprevalence of EAHIN1 SIVs in the population is very low, suggesting little pre-existing immunity. Z. Li et al. [29] showed that antibodies produced after seasonal influenza vaccination did not provide adequate protection against the G4 genotype EAH1N1 virus, and the results suggest the need to develop new vaccine strains. A study showed that two-thirds of EAH1N1 viruses reacted poorly with ferret serum antibodies induced by the currently used H1N1 human influenza vaccine, also suggesting that existing immunity may not prevent the transmission of EA H1N1 viruses in humans [3].

Antiviral drugs represent a crucial strategy for combating influenza virus infections, particularly when vaccination is ineffective or unavailable. Anti-influenza drugs that function as M2 ion channel blockers (amantadine and rimantadine) are no longer in clinical use due to the widespread emergence of drug-resistant influenza A viruses and their lack of therapeutic efficacy against influenza B viruses. In contrast, NA inhibitors (oseltamivir, zanamivir, peramivir, and laninamivir) remain in clinical use. However, their efficacy against the influenza virus has diminished due to mutations in the NA protein of the viruses [33]. The polymerase-associated resistance site remained unaltered in JS/27271/23, JS/1/11, and HN/42443/15. The results of the biological characterization of resistance demonstrated that the EAH1N1 SIVs remained sensitive to polymerase inhibitors, indicating that the utilization of polymerase inhibitors in clinical treatment, particularly during the initial stages of the disease, could prove beneficial in the management of EAH1N1 SIVs.

We must continue to enhance our capacity to forecast and alert the public about influenza outbreaks and promptly and efficiently assess the potential risks of emerging influenza viruses, such as EAH1N1 SIVs. Furthermore, we must improve the quality and accessibility of data related to the recommendation of vaccine strains for EAH1N1 SIVs in terms of animal models, viral transmissibility, viral virulence, and the monitoring of antibody levels to recommend better-matched strains and reduce the potential risk that EAH1N1 SIVs pose to humans.

Ethics statement

The Ethics Committee of the Jiangsu Provincial Center for Disease Control and Prevention approved this study (No. JSJK2024-B012-02). Informed consent was obtained from each participant enrolled in the study.

Supplementary data

The following are the Supplementary data to this article: