Evidence of an emerging triple-reassortant H3N3 avian influenza virus in China

doi:10.21203/rs.3.rs-4943745/v1

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Evidence of an emerging triple-reassortant H3N3 avian influenza virus in China

https://doi.org/10.21203/rs.3.rs-4943745/v1

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The H3 subtype of avian influenza virus (AIV) stands out as one of the most prevalent subtypes, posing a significant threat to public health, and a novel triple-reassortant H3N3 AIV, designated A/chicken/China/16/2023 (H3N3), was isolated from a chicken in northern China. The complete genome of the isolate was determined using next-generation sequencing, and the AIV-like particles were confirmed via transmission electron microscopy. Phylogenetic analyses revealed that eight genes of the H3N3 isolate clustered within the Eurasian lineage of AIVs, indicating the novel H3N3 isolate has various constellations and was generated by complex reassortment events involving H3N8, H9N2, and H10N3 subtype influenza viruses. Strikingly, the HA gene of the H3N3 isolate exhibited the closest evolutionary relationships to a human-derived H3N8 influenza virus, posing a potential threat to public health. Additionally, the presence of Q226 and T228 in the HA protein suggests the H3N3 virus preferentially binds to α-2,3-linked sialic acid receptors. While the HA cleavage site motif (PEKQTR/GIF) and the absence of E627K and D701N mutations in PB2 protein classify the virus as a characteristic low pathogenicity AIV. However, several mutations in internal genes raise concerns about potential increases in viral resistance, virulence, and transmission in mammalian hosts. Overall, this study provides valuable insights into the molecular and genetic characterization of the emerging triple-reassortant H3N3 AIVs, and continued surveillance of domestic poultry is essential for monitoring the H3N3 subtype evolution and potential spread.

Avian influenza virus

H3N3

reassortment

phylogenetics

genetic characterization

Avian influenza viruses (AIVs) are considered significant pathogens affecting a wide range of species, primarily birds and mammals[1–3]. AIVs belong to the genus Influenzavirus A of the family Orthomyxoviridae. As segmented negative-sense RNA viruses, AIVs exhibit high genetic diversity due to reassortment, enabling the emergence of novel subtypes [4]. Currently, AIVs are classified into 16 HA (H1-H16) and 9 NA (N1-N9) subtypes based on the antigenicity diversity and genetic evolution of the HA and NA proteins[4, 5]. Intrasubtypic reassortment between different AIVs makes it possible for them to exchange genes, thereby increasing genetic diversity and acquiring antigenic shift. Furthermore, multiple reassortment events may lead to the generation of novel AIVs with increased capacity to replicate and transmit in mammals[6, 7].

Although waterfowls and shorebirds are well known as the natural reservoirs of AIVs, these viruses have also established themselves in domestic poultry populations[5, 8]. Based on their pathogenicity in chickens, AIVs were categorized into highly pathogenic avian influenza viruses (HPAIVs) and low pathogenic avian influenza viruses (LPAIVs) by the World Organization for Animal Health (WOAH)[9]. While HPAIVs, primarily H5 and H7 subtypes, pose severe threats to poultry and public health, LPAIVs often cause mild or asymptomatic infections. However, LPAIVs play a crucial role in the evolution of AIVs, as they can serve as a reservoir for donating gene segments that can contribute to the emergence of novel and more virulent strains[2, 13, 14].

Among LPAIVs, the H3 subtype is one of the most prevalent, causing a range of symptoms from mild illness to severe disease in domestic poultry[10]. While most H3 subtype AIV infections in poultry are asymptomatic, their potential for cross-species transmission to humans poses a significant public health threat[10, 11]. Historically, H3 subtype AIVs have played a significant role in influenza pandemics, notably the 1968 Hong Kong H3N2 pandemic resulting from reassortment with human H2N2 influenza viruses[12, 13]. More recently, the emergence of H3N8 AIVs capable of infecting humans has raised concerns about the zoonotic potential of this subtype[14]. Three reported human cases in China, including a fatal case, have highlighted the severity of these infections[7, 14, 15]. Furthermore, H3 subtype AIVs exhibit a broad host range, encompassing wild and domestic birds, as well as mammals such as humans, swine, and seals[5, 16]. This extensive host diversity facilitates rapid viral evolution through antigenic drift and shift[17, 18]. Moreover, recent studies have documented the generation of novel H3 subtype AIVs through reassortment with H5, H7, and H10 subtypes, increasing genetic diversity and the potential for cross-species transmission[19–21].

It is noteworthy that over the past decades, H3 subtypes AIVs have established diverse lineages in poultry worldwide, including H3N1, H3N2, H3N3, H3N6, H3N7 and H3N8[10]. While H3N3 AIVs were historically uncommon, primarily isolated from ducks, a recent surge in detections in chickens from China, South Korea, and Vietnam has raised significant concerns[22–24]. These newly emerged H3N3 AIVs are pathogenic to chickens, causing histopathological damage to vital organs, and resulting in significant economic losses due to reduced egg production. Unfortunately, limited genetic data and inadequate monitoring have hindered our understanding of the evolutionary relationships and molecular characteristics of these novel H3N3 AIVs.

In this study, we isolated and sequenced a novel H3N3 avian influenza virus (AIV), designated A/chicken/Liaoning/16/2022 (H3N3), from poultry in Liaoning Province, China. A systematic investigation of its molecular and genetic characterization was carried out to enrich our understanding of the emerging H3N3 AIVs, which are also essential in the prevention and control of the H3N3 avian influenza in domestic poultry.

Virus isolation and transmission electron microscope

The oropharyngeal and cloacal samples from sick chickens were identified as positive for influenza virus by both hemagglutination assay (HA) and RT-PCR. Further characterization revealed the novel influenza virus as H3N3 AIV, as confirmed by Sanger sequencing of the HA and NA genes. Moreover, Transmission electron microscopy (TEM) analysis of the allantoic fluid samples from infected SPF chicken embryos revealed the presence of influenza virus-like particles, characterized by their typical vesicular and spiked morphology. (Fig. 1).

In this study, we comprehensively analyzed all available influenza virus strain information from the GenBank and GISAID EpiFlu™ database. Despite a relatively small total number (n = 67) of H3N3 influenza virus isolates recorded since its initial detection in 1972, the virus exhibited a broad host range, including swine, humans, seals, and avian species (Fig. 2A). Furthermore, avian species, particularly chickens, ducks, geese and other birds were the primary hosts for H3N3 viruses. Notably, ducks constituted the largest host group, representing over half (49.3%) of all the H3N3 virus hosts, followed by the environment isolates (13.4%). In contrast, the isolation rate from chickens was relatively low (9.0%) (Fig. 2B).

Genomic structure and comparative analysis

The complete genome of A/chicken/China/16/2023 (H3N3) was sequenced by next-generation sequencing (NGS). It comprised eight fragments encoding PB2 (2280 nucleotides, nt), PB1 (2274 nt), PA (2151 nt), HA (1707 nt), NP (1497 nt), NA (1410 nt), M (982 nt), and NS (876 nt). The genome organization was consistent with other members of the Influenzavirus A genus (Table 1). The amino acid sequences of the encoded proteins were similar in length and number to those of other AIVs. Additionally, the complete coding sequence (CDS) of this novel H3N3 AIV isolate was annotated (Table 1).

Table 1

Genomic features of A/chicken/China/16/2023(H3N3) strain.
Segment number	Gene	Abbreviation	Coding sequence (from-to)^a	Total length/nt	Protein length/aa^b
1	Polymerase PB2	PB2	1-2280	2280	759
2	Polymerase PB1 and PB1-F2 protein	PB1	1-2274; PB1-F2: 95–367	2274	847
3	Polymerase PA and PA-X protein	PA	1-2151; PA-X: 1-570, 572–760	2151	968
4	Hemagglutinin	HA	1-1701	1707	566
5	Nucleocapsid protein	NP	1-1497	1497	498
6	Neuraminidase	NA	1-1410	1410	469
7	Matrix protein 2 and matrix protein 1	M	M1: 1-759; M2: 1–26, 715–982	982	97
8	Nuclear export protein and nonstructural protein 1	NS	NS1: 21–674; NS2: 21–50, 523–858	876	338
^aIncluding stop codon; ^bWithout stop codon.

Comparative nucleotide sequence analysis of each gene was performed using BLAST against the GenBank database. As shown in Table 2, comparative nucleotide sequence analysis revealed high identity between the H3N3 AIV genome segments and their closest influenza virus counterparts: 98.03% (PB2), 98.24% (PB1), 98.19% (PA), 98.77% (HA), 98.33% (NP), 98.72% (NA), 99.19% (M), and 98.40% (NS), respectively. Intriguingly, the H3N3 strain exhibited a complex reassortment pattern. Three segments (PB2, PB1, and NP) shared the highest nucleotide similarity with H9N2 subtype AIVs, while another three segments (HA, M, and NS) were most closely related to H3N8 subtype AIVs. The NA gene was most similar to H10N3, and the PA gene showed high homology to both H9N2 and H3N8 subtypes (Table 2). These findings strongly suggest that the H3N3 AIV is a novel triple reassortant derived from H9N2, H3N8, and H10N3 influenza viruses.

Table 2

Sequence identity of novel H3N3 AIV with closest homologous Influenza Viruses.
Gene	Viruses with greatest homology	Nucleotide identity (%)	GenBank accession No.
PB2	A/chicken/Shanghai/11/2018(H9N2)	98.03	MK053869
PB2	A/chicken/China/71/2019(H9N2)	97.98	MN263225
PB1	A/environment-air/Kunshan/NIOSH-BL20/2018(H9N2)	98.24	MN606218
PB1	A/swine/Shandong/TA009/2019(H9N2)	98.01	MT265024
PA	A/chicken/China/2-3-1/2022(H9N2)	98.19	OR528343
PA	A/China/ZMD-22-2/2022(H3N8)	98.19	ON342808
HA	A/China/ZMD-22-2/2022(H3N8)	98.77	ON342803
HA	A/chicken/China/NXFWB5/2022(H3N8)	98.70	OP897716
NP	A/chicken/China/HP-1/2018(H9N2)	98.33	OR528592
NP	A/chicken/Anhui/12.25_YHZGS004-O/2018(H9N2)	98.33	MW102497
NA	A/Environment/China/04940NA/2021(H10N3)	98.72	OM802522
NA	A/chicken/China/189-6/2021(H10N3)	98.58	OL638128
M	A/chicken/Huizhou/104/2022(H3N8)	99.19	OQ291992
M	A/chicken/Dongguan/364/2022(H3N8)	99.19	OQ291664
NS	A/Duck/Jiangxi/447/2022(H3N8)	98.40	OQ826115
NS	A/chicken/Shandong/3424/2016(H9N2)	97.94	MH667576

Notably, the HA and PA gene of the H3N3 isolate exhibited the highest nucleotide homology with A/China/ZMD-22-2/2022(H3N8) strain, isolated from a human[25], while the NS gene was most similar to A/Duck/Jiangxi/447/2022(H3N8) isolated from duck. The PB2, NP and M genes showed the highest homology to these AIVs isolated from chickens. These results indicate that the H3N3 virus likely originated from multiple host species, including humans, ducks, and chickens, highlighting the complex reassortment dynamics occurring within the avian influenza virus population.

Phylogenetic analysis of HA and NA genes

To explore the evolutionary relationships of the novel H3N3 AIV, phylogenetic trees were constructed by using the full-length nucleotide sequences of the surface genes (HA gene and NA gene). The best-fit models for the phylogenetic trees based on HA and NA genes were determined by using MEGA software. As listed in supplementary Table S1, the HA genetic tree was performed by using the GTR + G + I model, and the TN93 + G model was utilized for the phylogenetic tree of the NA gene. Phylogenetic analyses of the HA and NA genes revealed a clear division of the reference AIV isolates into Eurasian and North American lineages. The newly characterized A/chicken/China/16/2023(H3N3) strain was confidently assigned to the Eurasian lineage (Fig. 3).

H3 subtype influenza viruses are typically classified based on their host range, including human, avian, swine and equine. Phylogenetic analysis of the HA gene further supports this classification. As shown in Fig. 3, the maximum likelihood (ML) tree of the HA gene definitively places the novel H3N3 strain within the Eurasian lineage of avian-origin H3 AIVs and confirms its derivation from H3N8 AIVs.

Furthermore, the novel H3N3 strain had the closest genetic evolutionary relationship to A/chicken/China/NXFWB5/2022(H3N8) isolated from chicken in 2022 (Fig. 3A). Strikingly, within the Eurasian lineage of the HA gene-based evolutionary tree, both human-derived H3N8 strains, A/China/ZMD-22-2/2022(H3N8) and A/China/CSKFQ-22-5/2022(H3N8), clustered closely with the novel H3N3 strain. These strains shared high maximum inter-lineage sequence identities of 98.8% and 97.6%, respectively (Fig. 3A). Interestingly, the novel identified H3N3 AIV occupied a basal position within a subclade containing A/chicken/China/NXFWB5/2022(H3N8) and A/China/ZMD-22-2/2022(H3N8), suggesting a potential evolutionary origin of the HA genes in these H3N8 isolates from the novel H3N3 strain.

Phylogenetic analysis of the NA gene, as depicted in Fig. 3B, revealed a clear division into American and Eurasian lineages. The novel H3N3 isolate clustered within the Eurasian lineage of H3 subtype AIVs. Within the subclade containing the novel H3N3 AIV, four viruses were closely related. A/Environment/China/04940NA/2021(H10N3) exhibited the closest relationship to the H3N3 isolate (Fig. 3B). Additionally, a human-derived H10N3 strain, A/China/0428NA/2021, shared a high 98.2% sequence identity with the H3N3 virus. Two other chicken-derived H10N3 isolates, A/chicken/China/189-6/2021 and A/chicken/China/146-6/2021, also demonstrated high sequence similarity (98.5%) to the H3N3 isolate. These findings strongly support a common ancestral origin for the H3N3 and these four H10N3 viruses, further solidifying the phylogenetic placement of the H3N3 isolate based on the neuraminidase (NA) gene.

Phylogenetic analysis of internal genes

To further elucidate the evolutionary history of this novel H3N3 AIV, phylogenetic analyses were conducted on the full-length nucleotide sequences of the remaining six internal genes. The optimal substitution model and its corresponding parameters for tree construction are detailed in Supplementary Table S1. The GTR + G + I model was used for the phylogenetic analysis of PB2, PB1, PA and NP genes, and the phylogenetic trees based on M and NS genes were performed using the HKY + G model. Overall, among the internal genes of the novel H3N3 isolate, four genes (PB2, PB1, PA and NP) were classified in the Eurasian lineage of H9N2 AIVs, as well as two genes (M and NS) were categorized into the Eurasian lineage of H3N8 AIVs.

In the ML tree of the PB2 gene (Supplementary Fig. S1), the H3N3 AIV isolated in this study were closely related to prevalent H9N2 AIVs, specifically A/chicken/China/71/2019 and A/chicken/China/1102/2019, circulating in Chinese poultry. These findings suggest that the PB2 gene of the novel H3N3 strain may have originated from chicken-derived H9N2 AIVs in 2019. In contrast, analysis of the PB1 gene (Supplementary Fig. S2) indicated a closer genetic relationship between the H3N3 strain and A/environment-air/Kunshan/NIOSH-BL20/2018 (H9N2) and A/swine/Shandong/TA009/2019 (H9N2). Strong bootstrap support for these relationships suggests a common ancestor for these viruses. Importantly, a human-derived H7N9 strain (A/Changsha/41/2017(H7N9)) identified in 2017 shares 97.7% genetic similarity with the novel H3N3 strain within the same subclade. This close relationship suggests that the PB1 gene of human-origin H7N9 influenza viruses may have originated from the prevalent H9N2 AIVs. Furthermore, it highlights the potential for the PB1 gene of the novel H3N3 strain to recombine with human-origin H7N9 influenza viruses.

In the Eurasian lineage of the PA gene-based phylogenetic tree (Supplementary Fig. S3), the novel H3N3 strain was rooted as a subclade that included some chicken-derived H9N2 or H3N8 isolates, as well as a human-derived H3N8 isolate. This suggests a potential evolutionary relationship where the PA genes of these H9N2 and H3N8 isolates may have originated from the novel H3N3 strain. Conversely, all other strains within this subclade were isolated in 2022, excluding the novel H3N3 strain. This suggests that the PA gene of the newly isolated H3N3 strain likely originated from circulating H9N2 AIVs present before 2022. Evolutionary analysis further indicates that the most probable source is A/chicken/Shanghai/11/2018 (H9N2), identified in 2018.

The ML tree of the NP gene reveals that the novel H3N3 strain had the closest evolutionary affiliation with A/chicken/China/HP-1/2018(H9N2), followed by A/chicken/Anhui/12.25_YHZGS004-O/2018(H9N2) (Supplementary Fig. S4), proved that the NP gene of the novel H3N3 strain might also be provided by H9N2 AIVs prevalent in China. The M gene of the newly isolated H3N3 strain clustered within the Eurasian lineage of AIVs, specifically among H3N8 AIVs isolated from chickens (Supplementary Fig. S5). This suggests that the M gene of the novel H3N3 strain originated from chicken-derived H3N8 AIVs. For the phylogenetic analysis of NS gene, the novel H3N3 strain demonstrates its closest genetic connection with A/silkie chicken/Huizhou/411/2022(H3N8), followed by A/Duck/Jiangxi/447/2022(H3N8) and A/chicken/Huizhou/2542/2021(H3N8) (Supplementary Fig. S6). Meanwhile, the novel H3N3 strain and the three isolates belonged to a distinct subclade, the H3N3 strain being the root of the subclade, suggesting that the NS gene of the other three H3N8 AIVs may have originated from it.

Overall, Comprehensive phylogenetic analysis of all the novel H3N3 AIV isolate’s genes revealed a complex reassortment history. This virus emerged from the reassortment of H3N8 (from 2022), H9N2 (from 2018–2022), and H10N3 (from 2021) influenza virus subtypes. These parental viruses were isolated from chickens, humans, and the environment and were widely distributed in China, highlighting the intricate reassortment events leading to the novel H3N3 AIV (Fig. 4).

Molecular characterization

To assess the potential risk of the novel H3N3 strain spilling over to mammals, we conducted a molecular characterization of its amino acid sequences. The H3N3 AIV cleavage site between HA1 and HA2 contains the amino acid motif PEKQTR/GIF, typical of avian H3 subtype influenza viruses. This monobasic cleavage site characteristic classifies the novel isolate as a low pathogenic avian influenza virus (LPAIV). It is well known that the receptor binding sites (RBSs) motif of HA protein are critically crucial for cellular receptor specificity and determine the host range of influenza viruses[26, 27]. We analyzed the amino acid sequences at the RBSs of the HA protein in the novel H3N3 strain and other referenced H3N3 and H3N8 influenza viruses. According to Table 3, the amino acids at the RBSs of the novel H3N3 strain were well conserved and did not show variation compared to the related H3N8 viruses isolated from humans and chickens. Consistent with other H3N3 AIVs, the conserved amino acids Q226 and T228 (H3 numbering) were identified in the RBS of this novel H3N3 isolate (Table 3), suggesting that this H3N3 isolate also binds to the α-2,3-linked sialic acid receptor, which is generally considered to be the primary receptor in avian species.

Table 3

Amino acid comparison of cleavage and receptor binding sites in the HA protein of the novel H3N3 isolate and reference influenza viruses.
Strains	Cleavage sites	Receptor-binding sites (H3 numbering)
Strains	Cleavage sites	98	138	153	155	183	190	194	195	226	228
A/chicken/China/16/2023(H3N3)	PEKQTR/GIF	E	T	S	C	T	F	Y	I	Q	T
A/China/ZMD-22-2/2022(H3N8)^a	PEKQTR/GLF	E	T	S	C	T	F	Y	I	Q	T
A/China/CSKFQ-22-5/2022(H3N8)^a	PEKQTR/GLF	E	T	S	C	T	F	Y	I	Q	T
A/Iran/Clinical Sample/2019(H3N3)^a	PEKQTR/GIF	K	N	S	C	T	F	Y	I	Q	A
A/bantam/Nanchang/9-366/2000(H3N3)	PEKQTR/GLF	E	T	G	C	T	F	Y	I	Q	T
A/duck/Zhejiang/D16/2013(H3N3)	PEKQTR/GLF	E	T	N	C	T	F	Y	I	Q	T
A/chicken/China/NXFWB5/2022(H3N8)	PEKQTR/GLF	E	T	S	C	T	F	Y	I	Q	T
A/chicken/Shantou/481/2022(H3N8)	PEKQTR/GLF	E	T	S	C	T	F	Y	I	Q	T
A/duck/Hunan/199/2014(H3N8)	PEKQTR/GLF	E	T	G	C	T	F	Y	I	Q	T
^aThe strain was isolated from human.

The specific polypeptide sequence for N-linked potential glycosylation was defined by the amino acid configuration of Asn-X-Ser/Thr, where X can represent any amino acid apart from proline[28]. The N-linked potential glycosylation of HA protein is associated with viral pathogenicity and affinity for the influenza virus receptor[29, 30]. For the novel H3N3 isolated in this study, the HA protein shared six N-linked potential glycosylation sites (PGSs) at positions 38, 54, 61, 181, 301 and 499, which were highly conserved with these referenced H3N8 viruses isolated from chickens and humans (Supplementary Table S2). Notably, eight PGSs of NA protein were detected at the positions of 14, 57, 66, 72, 146, 308, 387, and 401 (Supplementary Table S3). Comparative to the most similar H10N3 isolate at the NA gene level, the novel H3N3 strain isolated in this study has two additional PGSs at positions 387 and 401, respectively; whether this will alter the viral characteristic needs to be further investigated.

Furthermore, a detailed amino acid analysis of the gene segment of the novel H3N3 virus isolated in this study was carried out. As shown in Table 4, several mutations were observed including in the NA protein (M26I), PB2 protein (L89V, K251R, G309D, T339K, Q368R, R389K, H447Q, R477G and I495V), PB1 protein (D/A3V, L13P, R207K, K328N, S375N/T, H436Y, L473V and D622G), PA protein (H266R, K356R, N383D, S409N and S/A515T), NP protein (I353V), M1 protein (V15I, N30D and T215A), and NS1 protein (P/A42S, D97E and V149A). These mutations were reported to be related to an increase in the viral replication ability and virulence of AIVs in mice or mammalian cells (Table 4). Besides, mutations of PB1 protein (I368V) and PA protein (F277S, C278Q and L653P), which may facilitate the adaptation of AIVs to mammalian hosts, were also found (Table 4).

Table 4

Molecular characterization of the novel H3N3 isolate presented in this study.
Viral protein	Amino acid	A/chicken/China /16/2023(H3N3)	Comments	References
HA	T160A	A	Increased binding to human-type influenza receptor	[1]
HA	Q226L	Q	Increased binding to human-type influenza receptor	[2]
NA	M26I	I	Increased virulence in mice	[3]
	V116A	V	Resistance to neuraminidase inhibitors	[4]
	I117T	T	Reduces susceptibility to oseltamivir and zanamivir	[5]
	E119V	E	Resistance to neuraminidase inhibitors	[6, 7]
	Q136L	Q	Resistance to neuraminidase inhibitors	[8]
	R152W	R	Resistance to neuraminidase inhibitors	[6, 9]
PB2	L89V	V	Enhanced polymerase activity and virulence in mammals	[10]
	E158G	E	Enhanced polymerase activity and virulence in mice	[11]
	K251R	R	Increased virulence in mice	[12]
	G309D	D	Enhanced polymerase activity and virulence in mice	[10]
	T339K	K	Enhanced polymerase activity and virulence in mice	[10]
	Q368R	R	Increased polymerase activity and virulence in mice	[13, 14]
	R389K	K	Enhanced polymerase activity and virulence in mice/Mammalian host adaptation	[15]
	H447Q	Q	Increased polymerase activity and virulence in mammals	[13, 14]
	R477G	G	Enhanced polymerase activity and virulence in mice	[10]
	I495V	V	Enhanced polymerase activity and virulence in mice	[10]
	Q591K	Q	Enhanced replication efficiency and virulence in mice	[11]
	E627K	E	Mammalian host adaptation	[10, 16, 17]
	D701N	D	Increased polymerase activity and viral replication in mammalian	[18]
PB1	D/A3V	V	Increased polymerase activity and virulence in mice	[14]
	L13P	P	Increased polymerase activity and virulence in mammals, Mammalian host marker	[19]
	H99Y	H	Increased transmissibility in ferrets	[11, 15]
	R207K	K	Increased polymerase activity in mammalian cells	[20]
	K328N	N	Increased polymerase activity and virulence in mammals	[14]
	I368V	V	Mammalian host adaptation	[11]
	S375N/T	N	Increased polymerase activity and virulence in mammals, human host marker	[14]
	H436Y	Y	Increased polymerase activity and virulence in mallards, ferrets and mice	[20]
	L473V	V	Increased polymerase activity and replication efficiency	[21]
	D622G	G	Increased polymerase activity and virulence in mice	[22]
PB1-F2	N66S	N	Increased replication, virulence and antiviral response in mice/Increased virulence in mammals	[23]
PB1-F2	T68I	T	Increased virulence in mice	[24]
PA	S37A	S	Increased polymerase activity and viral replication in mammalian cells	[25]
	T97I	T	Enhances polymerase and virulence	[7, 11]
	V100A	V	Contributed to the virulence and mammalian adaptation	[26, 27]
	H266R	R	Increased polymerase activity and virulence in mammals and birds	[13]
	F277S	S	Mammalian host adaptation	[13]
	C278Q	Q	Mammalian host adaptation	[13]
	K356R	R	Enhanced virulence and mammalian adaptation	[28]
	N383D	D	Increased polymerase activity and mammalian adaptation	[29]
	A404S	A	Human host marker	[30]
	S409N	N	Increased polymerase activity, viral replication and virulence to mammalian; enhanced transmission	[31]
	S/A515T	T	Increased polymerase activity and virulence in mammals and birds	[20]
	L653P	P	Mammalian host adaptation	[13]
NP	F253I	I	Results in attenuated pathogenicity of the virus in mice	[25]
	A286V	A	Affect the pathogenicity of the virus in mice	[32]
	N319K	N	Increased virulence in mammalian cells	[7, 33]
	I353V	V	Increased virulence in mice	[12]
	T437M	T	Affect the pathogenicity of the virus in mice	[32]
M1	V15I	I	Increased virulence in mammals	[15, 21, 34]
	N30D	D	Increased pathogenicity to mice	[35]
	A166V	A	Increased polymerase activity and virulence in mammals	[21]
	T215A	A	Increased virulence in mice	[35]
M2	L26P	L	Reduced susceptibility to amantadine and rimantadine	[36]
	V27A/I	V	Reduced susceptibility to amantadine and rimantadine	[36]
	A30T	A	Reduced susceptibility to amantadine and rimantadine	[36]
	S31N	N	Reduced susceptibility to amantadine and rimantadine	[36, 37]
	L55F	F	Mammalian host marker	[38]
NS1	P/A42S	S	Increased virulence in mice; Antagonism of IFN induction	[39]
	T/D92E	D	Increased virulence and antiviral response in mammals	[40]
	D97E	E	Increased pathogenicity to mice	[15]
	V149A	A	Pathogenicity in mice; Antagonism of IFN induction	[41]
NEP/NS2	M31I	M	Increased virulence in mice	[7, 42]

Moreover, the detection of a T160A substitution in the HA protein suggests enhanced binding affinity of the H3N3 isolate to human-type influenza receptors [37]. In contrast, the absence of Q591K, E627K, and D701N mutations in the PB2 protein indicates a low potential for cross-species transmission to mammals [38–42]. The NA protein's I117T mutation may reduce susceptibility to oseltamivir and zanamivir [43], while P/A42S and V149A substitutions in the NS1 protein could enhance interferon resistance [44, 45]. Additionally, the S31N mutation in the M2 protein confers resistance to amantadine and rimantadine [46, 47].

Overall, molecular characterization reveals that the novel H3N3 isolate predominantly binds to avian-type receptors despite possessing several mutations in internal genes that could potentially increase viral resistance, virulence, and transmission in mammalian hosts.

Sampling

Tracheal, oropharyngeal, and cloacal swab samples were collected from a single chicken flock in Shenyang City, Liaoning Province, China (41°48′11.75″N, 123°25′31.18″E) in June 2023. These samples tested positive for Avian Influenza Virus (AIV) using a universal RT-qPCR assay, but negative for the specific H5, H7, and H9 subtypes by RT-qPCR [48]. These samples were sent to the Key Laboratory of Animal Disease and Public Health, Henan University of Science and Technology for further subtype identification and pathogen investigation. Briefly, the samples were placed into a 2 mL microcentrifuge tube of cold phosphate-buffered saline (PBS) containing penicillin (2000 U/mL) and streptomycin (2000 µg/mL). Thereafter, the samples were centrifuged at 4000 xg for 20 min at 4℃, and the supernatant was filtered through 0.45-µm hydrophilic polyether sulfone membrane filters (Millipore, USA). The filtered solution was utilized for subsequent virus isolation and identification.

Virus isolation and identification

For virus isolation, 100 µL of the sample supernatant was inoculated onto the allantoic cavity of 10-day-old SPF chicken embryos obtained from the SPF Experimental Animal Center of Xinxing Dahua Agricultural, Poultry and Egg Co., Ltd. The inoculated SPF chicken embryos were incubated at 37°C and monitored every 12 hours. After 72h of post-inoculation, allantoic fluid was collected and tested for hemagglutination (HA) activity using 1% chicken red blood cells. Thereafter, viral RNA was extracted from allantoic fluids using a MiniBEST Viral RNA/DNA Extraction Kit (Takara, Beijing, China) according to the manufacturer’s instructions. Reverse transcription-PCR (RT-PCR) was performed using primers targeting the H3 subtypes’ hemagglutinin (HA) gene and neuraminidase (NA) gene (Primer sequences are available upon request) to confirm the presence of the virus in HA-positive samples. Subsequently, these positive samples underwent three rounds of purification by serial dilution inoculation into 10-day-old SPF chicken embryos.

Transmission electron microscope

Viral particles in the purified AIV allantoic fluid were visualized by transmission electron microscope (Wuhan Servicebio Technology Co., Ltd, China). Briefly, 20 µL of the sample was placed onto 150 meshes cuprum grids with carbon film and followed by settlement at room temperature for 5 minutes. The excess fluid was removed using filter papers, and the dried grid was stained with 2% phosphotungstic acid for 60 seconds. After removing the excess stain, the grid was examined using an HT7800 transmission electron microscope (Hitachi, Japan) at 80 kV acceleration voltage to observe negatively stained viral particles.

RNA extraction and Next-generation sequencing

Viral RNA was extracted from allantoic fluids containing purified virus using MiniBEST Viral RNA/DNA Extraction Kit (Takara, Beijing, China) according to the manufacturer’s instructions for complete genome sequencing. Next-generation sequencing was undertaken by Shanghai Tanpu Biotechnology Co., Ltd (Shanghai, China) to obtain the whole genome sequence data of the novel AIV isolate. Briefly, the sequencing libraries were prepared using TruSeq DNA Sample Prep Kit (Illumina, San Diego, CA, USA) as recommended by the manufacturer. PCR amplification was performed after adapter ligation for sequencing target enrichment. The library was normalized and pooled in equimolar quantities, denatured and diluted to optimal concentration before sequencing. The Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used for sequencing with read length of 150 bp paired end.

Assembly protocol

The resulting 10,586,382 paired raw sequence reads from NovaSeq 6000 were used to obtain the whole genome of the novel AIV isolate. Initial quality control of all raw reads was generated, and the raw reads were processed by fastp (version 0.20.0)[31] for filtering to remove adapters, ambiguous bases, polyclonal reads and low-quality reads, including those reads scored below Q20. Reads trimmed of adaptor sequences shorter than 50 nt were also discarded. The trimmed sequence reads are filtered by read-mapping using the BBMap program (Version 38.51) to remove likely contamination from the host organism, ribosomal RNAs and bacteria genome[32]. Unmapped reads were used as input data for de novo assembly using SPAdes (Version 3.14.1)[33] and SOAPdenovo (Version 2.04)[34]. Twenty-one contigs (108–2328 bp) were generated corresponding to AIV sequences according to searches of the GenBank database by BLAST[35]. Eventually, the complete genome sequence of the novel isolated AIV was obtained by splicing these contigs using Geneious Prime software (Version 10.2.2 Biomatters, Auckland, New Zealand).

Genome annotations, comparative and molecular analysis

The open reading frames (ORFs) and protein-coding sequence (CDS) of the newly identified AIV strain were annotated based on the available AIV whole genome sequences in the GenBank database[36]. Subsequently, the AIV isolates showing the highest homology in nucleotide sequence to the individual genes of the novel AIV strain isolated in this study were obtained through the NCBI database with BLAST[35], and the multiple sequences alignment was performed using MAFFT software (Version 7.490)[37]. The amino acid sequences at the receptor binding site of HA protein were identified as described by Weis et al.[38]. Potential N-linked glycosylation sites of HA and NA protein were predicted using the NetNGlyc 1.0 server[39].

Phylogenetic analysis

Phylogenetic analysis of the novel H3N3 AIV strain identified in this study was performed with other referenced AIV genome sequences available in the GenBank database[36]. The nucleotide sequences of each gene (PB2, PB1, PA, HA, NP, NA, M and NS) in the AIVs genome were aligned by MAFTT (Version 7.490) with default parameter (Gap open penalty 1.53; Offset value 0.123)[37]. Subsequently, the best-fit model testing of the AIV's gene for maximum-likelihood (ML) analysis was predicted in MEGA software (Version 11.0.11)[40]. Phylogenetic analysis for nucleotide sequences of eight genes of the novel AIV isolate was performed based on the best-fit model with 1000 bootstrap replicates in MEGA software (Version 11.0.11), and the phylogenetic trees were further modified by using tvBOT[41].

Data availability

The newly A/chicken/China/16/2023 (H3N3) complete genome sequence and the associated data sets that were generated during this study were deposited in GenBank under the accession number OR497659-OR497666 (Table 1).

This study conducted a comprehensive molecular and genetic analysis of a newly emerged, triple-reassortant H3N3 avian influenza virus (AIV) isolated from poultry in China. Since the initial identification of the H3N3 influenza virus strain in 1972, the GenBank database currently houses over 50 recorded H3N3 isolates. While the majority of these isolates originated from ducks, only five have been identified in chickens (Fig. 2A and 2B)[54]. Recent surveillance in Chinese chicken flocks has revealed an increasing prevalence of H3N3 subtype avian influenza virus (AIV) infections. However, limited whole genome sequence data and a lack of understanding of their evolutionary relationship with other subtypes hinder our comprehensive characterization of this novel reassortant H3N3 strain [13, 60].

In this study, the whole genome sequence of the novel H3N3 isolate was determined by utilizing next-generation sequencing on the secretion samples collected from the oropharynx and cloaca of chickens, and the Genome were annotated based on the sequence characterization of the reported AIV isolates. The resulting genome structure, including size, gene count, and location, closely resembled those of other known AIVs in the GenBank database[54]. However, the novel H3N3 strain exhibited a distinct genomic composition compared to other H3 subtype AIVs, displaying high similarity to H3N8, H9N2, and H10N3 influenza viruses. These findings classify the H3N3 isolate as a novel triple-reassortant AIV (Table 2). Meanwhile, phylogenetic analysis based on its genes revealed that all segments within the novel H3N3 virus genome were clustered in the Eurasian lineage of AIVs. The HA protein, crucial for viral entry into host cells and inducing the production of neutralizing antibodies production[61], exhibited a PEKQTR/GIF cleavage site characteristic of low pathogenic avian influenza viruses (LPAIVs). Additionally, the presence of Q226 and T228 amino acids at the receptor-binding site (RBS) suggested a preference for avian-like (α-2,3-linked sialic acid) receptors (Table 3)[31, 61, 62].

However, the results of both nucleotide sequence homology and evolutionary analyses based on the HA gene indicate that this newly H3N3 strain is closely genetically related to a human-derived H3N8 influenza virus (A/China/ZMD-22-2/2022(H3N8)), which was isolated in April 2022 from a four-year-old boy in China experiencing recurrent fever and severe pneumonia[25]. Further studies showed that this human-derived H3N8 influenza virus was likely generated by reassortment of the H3N2, H3N8 and H9N2 AIVs[7, 14, 25]. The PB2 gene is known to significantly influence influenza virus pathogenicity, with the E627K mutation in the PB2 protein associated with zoonotic potential[41]. Previous studies have speculated that it is likely that H3N8 AIVs gained the ability to spill over to humans by recombining with H9N2 viruses with the amino acid mutation at position 627 (E627K) in the PB2 fragment[19, 25]. Notably, the absence of the E627K mutation in the PB2 protein of the novel H3N3 strain suggests a reduced risk to public health (Table 4).

The segment exchange by reassortment between different subtypes of influenza viruses is a critical feature driving the evolution of influenza viruses and contributing to the emergence of novel potential pandemic and zoonotic strains[42–44]. Reassortment between H3 subtype AIVs and other influenza viruses can generate new pathogenic influenza viruses that may lead to human infections[10, 14]. Notably, the 1968 Hong Kong pandemic was triggered by reassortment with avian-derived H3 subtype and human-derived H2N2 influenza viruses[45]. Herein, by comprehensively analyzing the homology and genetic evolution results of the segments constituting the novel H3N3 AIV genome sequenced in this study, we postulate that this virus was likely formed by combining these influenza viruses (H3N8 from 2022, H9N2 from 2018 to 2022, and H10N3 from 2021) (Fig. 4B). The reassortment results revealed that the novel H3N3 subtype AIVs was likely to emerge in Chinese chicken flocks in 2022 and remained circulating until now. Moreover, as shown in Fig. 4A, the predicted influenza viruses with these provided genes were widely distributed in China, suggesting that this novel H3N3 AIV isolate had undergone a complex process of reassortment.

AIVs are considered to be of high economic importance due to their significant impact on poultry production and reproduction globally, coupled with the looming threat of zoonotic diseases[46, 47]. Although the H3 subtype AIV was classified as LPAIV in poultry, recent epidemiological studies have demonstrated that this emerging H3N3 AIVs had enhanced pathogenicity in chickens, with the ability to cause massive reductions in egg production in infected flocks[22, 48]. Meanwhile, the presence of the novel H3N3 AIVs in poultry allows them to reassort with multiple subtypes of AIVs to generate various novel AIV strains that have the potential to cause pandemics and zoonotic diseases. Therefore, continued surveillance of H3N3 AIVs in domestic poultry is essential and effective control strategies, including vaccination, are also urgently needed.

This paper presents a comprehensive genomic analysis of a newly emerged triple-reassortant H3N3 avian influenza virus (AIV) strain, designated A/chicken/China/16/2023 (H3N3), isolated from a chicken in China during 2023. Phylogenetic analysis of eight viral genes placed the strain within the Eurasian lineage of influenza viruses. Comparative genomic studies revealed that this novel H3N3 virus originated from a triple reassortment event involving H3N8, H9N2, and H10N3 influenza viruses. Notably, the close evolutionary relationship between this H3N3 strain and human-derived H3N8 influenza viruses highlights a potential public health risk. This study expands our understanding of triple-reassortant H3N3 AIVs and underscores the critical need for continued surveillance of these emerging viruses in domestic poultry.

Availability of data and materials

Funding

This research was supported by the science and technology research project of Henan province (nos. 222102110012 and 232102110078) funded by the Chinese Government.

Ethics approval and consent to participate

Samples collected were approved by the Ethics Committee of Henan University of Science and Technology (20230515032).

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Authors’ contributions

HL and SC designed the study. KS and KS performed the experiments. ZY, YW and CY performed the bioinformatics and data analysis. HL and YZ wrote the initial manuscript. All authors read, edited and approved the final manuscript.

Acknowledgements

Dr. Sarker is the recipient of an Australian Research Council Discovery Early Career Researcher Award (grant number DE200100367) funded by the Australian Government. The Chinese and Australian Governments had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Consent to participate

Not applicable.

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Evidence of an emerging triple-reassortant H3N3 avian influenza virus in China

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Abstract

Figures

Introduction

Results

Virus isolation and transmission electron microscope

Genomic structure and comparative analysis

Phylogenetic analysis of HA and NA genes

Phylogenetic analysis of internal genes

Molecular characterization

Methods

Sampling

Virus isolation and identification

Transmission electron microscope

RNA extraction and Next-generation sequencing

Assembly protocol

Genome annotations, comparative and molecular analysis

Phylogenetic analysis

Data availability

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1