Skip to main content
Download PDF
  • Research article
  • Open access
  • Published:

Genetic and molecular characterization of a novel reassortant H3N2 influenza virus from a sick pig in Eastern China in 2019

Veterinary Research volume 56, Article number: 38 (2025) Cite this article

Abstract

Swine influenza viruses (SIVs) cause clinical respiratory symptoms associated with high mortality rates among pigs. Because pigs can be a “mixing vessel” for influenza viruses, the SIV might pose a serious threat to animal and human health. In this study, an H3N2 SIV [A/swine/Zhejiang/19/2019(H3N2) (ZJ-SW19)] was isolated from a sick pig in Eastern China in 2019, and its molecular genetics were characterized. Phylogenetic analysis demonstrated the hemagglutinin (HA) and neuraminidase (NA) segments of ZJ-SW19 are highly homologous with those of H3N2 SIVs, belonging to human-like lineages; in contrast, the remaining six SIV segments (PB2, PB1, PA, NP, M, and NS) demonstrate the highest similarity with H1N1 SIVs isolated in East Asia during 2014–2020. The in vitro analysis of the virus’s growth kinetics revealed that ZJ-SW19 can replicate efficiently in various mammalian and avian cell lines (including MDCK, A549, and DF-1). The receptor-binding analysis results indicated that ZJ-SW19 can bind to human-like receptors (α-2,6-linked sialic acid) and avian-like receptors (α-2,3-linked sialic acid). Moreover, ZJ-SW19 demonstrated significant differences compared with avian- and human-origin H3N2 influenza viruses in the antigenic analysis. Finally, in the pathogenicity test, ZJ-SW19 effectively replicated in the mouse lungs with moderate virulence. Therefore, continuous circulation of novel reassortant H3N2 SIVs indicates the need for long-term, close surveillance of influenza viruses in pig herds.

Introduction

Influenza A viruses are enveloped viruses containing eight RNA segments, which can infect several host species, including poultry, wild birds, and marine and terrestrial mammals [1]. Swine influenza virus (SIV) is highly contagious, with the ability to infect pigs and cause various respiratory symptoms, such as runny nose, cough, and fever [2]. Although SIV-related clinical signs are typically mild, co-infection of SIVs and other pathogens increases mortality in infected pigs or leads to stillbirths in infected pregnant sows [3, 4]. In pigs, the respiratory tract carries both N-acetylneuraminic acid-α-2,3-galactose (an avian-like receptor) and N-acetylneuraminic acid-α-2,6-galactose (a human-like receptor) [5]. Therefore, pigs are generally considered “mixing vessels” between birds and humans during the evolution, reassortment, and transmission of influenza viruses [6]. Therefore, SIVs circulating in a pig herd can reassort with avian or human influenza viruses to produce novel strains, which can transmit back to humans or pigs, potentially leading to an epidemic or pandemic [7].

Based on multiple antigenic determinants, SIVs isolated from pigs worldwide have been classified into three main subtypes: H1N1, H1N2, and H3N2 [8]. The human-origin H3N2 SIV was first simultaneously identified in pig and human populations in China in 1968 [9]. Subsequently, a reassortant swine-origin H1N1 strain, pdm/2009, first isolated in North America, caused a pandemic in humans; its evolution involved multiple reassortments of avian-, swine- and human-origin viruses [10]. Reassortant SIVs containing segments of pdm/2009 have appeared widely in Southern China, North America, and Eurasia. After the 2009 pandemic, the H1N1 pdm/2009 virus continued to transmit to pigs in many regions and reassort with local viruses, significantly increasing and enriching the diversity of SIV gene sources among pig populations [11]. As such, since the emergence and circulation of the 2009 H1N1 pandemic virus, SIV surveillance has been strengthened globally [12,13,14]. In 2011, the first known human-origin H3N2 SIV was confirmed in the United States; it caused > 400 human infections over 2011–2018 [15]. In China, H3N2 SIVs were also detected in humans and caused sporadic human infections [16]. Therefore, the widespread transmission of the aforementioned H3N2 SIVs indicates that the virus may be a serious threat to global public health.

In China, the rapid, intensive growth of swine and poultry farming industries may increase influenza virus reassortment frequencies and infection risks [17]. However, available data regarding the genetic and molecular characteristics of H3N2 SIVs in Eastern China are scant; therefore, monitoring influenza among pig populations in Eastern China is crucial. In the current study, we conducted SIV surveillance in Zhejiang, Eastern China; we isolated and characterized the SIV A/swine/Zhejiang/19/2019(H3N2) (ZJ-SW19) to understand the genetic evolution and biological characteristics of SIVs further.

Materials and methods

Sample treatment and virus isolation

From December 2018 to January 2019, we performed SIV surveillance in Zhejiang, Eastern China. We collected 125 lung tissue samples from clinically ill pigs and stored them in 1 mL of phosphate-buffered saline (PBS, pH 7.4) containing penicillin G and streptomycin sulfate.

Virus isolation was conducted in embryonated specific-pathogen-free (SPF) eggs, as described elsewhere [18]. In brief, all virus samples were centrifuged; the supernatants were collected, filtered, and injected into 9-day-old SPF eggs, followed by incubation at 37 °C for 3 days. Finally, the allantoic fluid was harvested and evaluated using a hemagglutination inhibition (HI) assay [19]. All positive allantoic fluids were stored and subsequently subjected to genome sequencing.

RNA extraction and genome sequencing

TRIzol reagent (Life Technology, USA) was used for viral RNA extraction, according to the manufacturer’s instructions, as described previously [20]. In brief, allantoic fluid was mixed with TRIzol reagent for protein lysis. Chloroform was then added to separate the organic and inorganic fractions. After centrifugation, the upper inorganic phase was aspirated and treated with isopropanol for precipitation. The precipitate was washed twice with 75% ethanol and eluted with RNase-free water. Finally, RNA concentrations were detected based on absorbance measured on NanoDrop.

The reverse transcription polymerase chain reaction (RT-PCR) with Uni12 primers, 5′-AGCAAAAGCAGG-3′, was used to synthesize cDNA, as described previously [21]. Eight segments of SIV were amplified separately using specific segment primers [22]. The RT-PCR products were sequenced using the Sanger method. Sequences of each segment of the isolated H3N2 virus were aligned using Basic Local Alignment Search Tool (BLAST), analyzed on BioEdit, and submitted to GISAID (EPI3528504- EPI3528511).

Phylogenetic study and molecular analysis

Classical reference viruses were selected based on previous reports [8,9,10,11, 13, 14]. Moreover reference sequences of the strains used in this study were obtained from the Influenza Sequences Database. Molecular Evolutionary Genetics Analysis was used to construct phylogenetic trees by using the distance-based neighbor-joining method and 1000 bootstrap replications [23].

Immunofluorescence assay

To evaluate virus activity in MDCK cells, we performed an immunofluorescence assay (IFA), as described previously [24]. We cultured MDCK cells in six-well plates until they formed a confluent monolayer and washed them with PBS twice. Next, we infected the cells with ZJ-SW19 [multiplicity of infection (MOI) = 0.1] for 16 h and washed them twice with PBS. The infected MDCK cells were then fixed with 4% paraformaldehyde and permeabilized using 0.5% Triton X-100. The cells blocked by 3% bovine serum albumin (BSA) were incubated with monoclonal antibodies against NP (GeneTex, USA) at 4 °C overnight. Next, the cells were incubated with Alexa Fluor 488 goat antimouse antibodies (Abcam, USA) at 37 °C for 1 h. The fluorescent signal was detected under an EVOS M7000 Microscope Imaging System (Invitrogen, USA).

Analysis of virus replication in vitro

We evaluated the growth kinetics of H3N2 SIVs in MDCK, A549, and DF-1 cells [25]. Monolayer confluent MDCK, A549, or DF-1 cells were grown in six-well plates and infected with ZJ-SW19 (MOI = 0.01). The cells were cultured in Dulbecco’s modified Eagle medium containing BSA and tosyl phenylalanyl chloromethyl ketone-treated trypsin. The cultures were centrifuged at 0, 12, 24, 36, 48, 60, and 72 h post-infection (hpi), and their supernatants were collected. Finally, the viral titers were measured using the 50% tissue culture infective dose (TCID50) assay [26].

Receptor-binding analysis

To analyze the receptor-binding specificity of H3N2 SIVs, we performed the HI assay by using receptor-specific red blood cells (RBCs) [27]. In brief, normal chicken RBCs containing both α-2,6 and α-2,3 receptors were prepared, and they were treated with α-2,3-specific neuraminidase (NA) to induce the production of sialidase-treated chicken RBCs, which only contain the α-2,6 receptor. Finally, to define the HA titer, we performed the HI assay by incubating two-fold serially diluted viruses with RBCs in 96-well microtiter plates.

Antigenic study

The antigenic relationship between ZJ-SW19 and other H3N2 influenza viruses (including those of swine, human, and avian origin) was evaluated using cross-reactive HI assays, as described previously [28]. Mouse antisera for ZJ-SW19, A/swine/Zhejiang/6/2010(H3N2) (ZJ-SW6), A/Hong Kong/2671/2019 (H3N2) (HK-2671), A/Texas/50/2012(H3N2) (Tex-50), A/duck/Zhejiang/4613/2013(H3N2) (ZJ-4613), and A/chicken/Zhejiang/139/2016 (H3N2) (ZJ-139) were generated and stored in our laboratory [22, 29].

Animal study

To evaluate SIVs’ pathogenicity and replication ability in mammalian hosts, we used 15 6-week-old female BALB/c mice and inoculated them with 50 µL of suspension containing 105.0 or 106.0 50% egg infectious dose (EID50) of ZJ-SW19 via nasal drip [28]. At 3, 6, and 9 days post-infection (dpi), we euthanized three mice, collected their tissues (including lungs, brains, hearts, livers, kidneys, and spleens), and determined host SIV distribution by using the TCID50 assay. Moreover, we measured the daily survival rate and weight loss in the remaining six mice over 14 dpi, as reported previously [28]. For histopathological analysis, half of the lung tissue specimens were fixed with formalin solution fixation, embedded in paraffin for 3 days, sectioned, and stained with hematoxylin and eosin (H&E) dye.

Results

Virus isolation and molecular analysis results

We first isolated the SIV ZJ-SW19 from grow-finish pigs with clinical respiratory symptoms in Hangzhou, Zhejiang, China, and sequenced its eight segments. Our results indicated that the HA gene is 1701 bp long and encodes 566 amino acid residues (Figure 1). The motif of the HA cleavage site is PEKQTR↓G, indicating that ZJ-SW19 has low pathogenicity [30]. Compared with A/swine/Guangxi/2518/2011(H3N2) (the closest reference), ZJ-SW19 demonstrates 13 amino acid mutations located in HA1 protein (F19S, M41V, N47D, M184I, K184E, A198V, A212T, D241G, P243T, I246V, I252V, I258M, and R285K) and seven in HA2 protein (L397M, V429I, M478I, D505N, E509D, S513N, and Q517R). The amino acid mutation A212T is located in the antigenic site B of HA, which can increase human H3N2 influenza virus replication in eggs [31]. Moreover, the HA protein of ZJ-SW19 contains eight potential N-glycosylation sites: NGT(38–40), NAT(54–56), NCT (79–81), NES(138–140), NWT(142–144), NST(262–264), NGS(301–303), and NGT(499–501). None of these substitutions related to ZJ-SW19’s virulence and host range were observed in PB2 (E627K and D701N) or PA (T97I), and none of the substitutions in NA (H274Y) were associated with oseltamivir resistance [32].

Figure 1
figure 1

Amino acid comparison at the HA domains of H3N2 influenza viruses. Dots denote amino acids similar to the consensus, and squares denote conserved amino acid residues at the receptor-binding site. The asterisk denotes ZJ-SW19. N-glycosylation sites are presented in boxes. Amino acid residues mapped at previously defined antigenic sites A, B, C, D, and E are presented in blue, purple, yellow, pink, and green, respectively.

Full size image

Phylogenetic analysis results

We determined sequence similarity through BLAST analysis and selected sequences with high homogeneity. The results indicated that the segments of the major capsid proteins (HA and NA) of ZJ-SW19 are closely related to swine H3N2 viruses, whereas the other six segments have the highest homogeneity with H1N1 SIVs isolated in East Asia over 2014–2020 (Table 1). The PB2, PB1, PA, NP, and M segments of ZJ-SW19 were assigned to the H1N1 pdm/2009 lineage, whereas the HA gene of ZJ-SW19 demonstrated the highest homology with A/swine/Guangxi/2518/2011(H3N2). The phylogenetic analysis revealed that the HA segment of ZJ-SW19 has a human-like lineage (Figure 2), and that the NA segment of ZJ-SW19 has the highest homology with A/swine/Shandong/15/2018(H3N2). Taken together, these results demonstrated that ZJ-SW19 is a reassortant strain derived from other local SIVs, particularly H1N1 and H3N2 SIVs; moreover, the internal genes of H1N1 pdm/2009 influence H3N2 SIV prevalence in pig populations.

Table 1 Comparison of ZJ-SW19 genomes with reference virus sequences available in the GenBank database
Full size table
Figure 2
figure 2

Phylogenetic trees for the HA(A), NA(B), PB2(C), PB1(D), PA(E), NP(F), M(G), and NS(H) genes in ZJ-SW19 isolated in this study. Red dots denote ZJ-SW19, and the black box indicates human influenza viruses originating from SIVs. The scale bar represents the distance unit between the sequence pairs.

Full size image

Growth kinetics

The replication ability of ZJ-SW19 was evaluated in MDCK, A549, and DF-1 cells. The results of in vitro growth kinetics showed that ZJ-SW19 equivalently replicates efficiently in mammalian and avian cells (Figure 3A). At 12 hpi, live viruses were detected in the cell supernatant. Between 12 and 48 hpi, virus production gradually increased; thereafter, the virus content in cell supernatants remained stable or decreased slightly. Finally, we assessed the infection potential of ZJ-SW19 using an IFA, and the results demonstrated that ZJ-SW19 can effectively replicate in MDCK cells (Figure 3B).

Figure 3
figure 3

ZJ-SW19 replication potential in vitro. A Growth curves of ZJ-SW19 in MDCK, A549, and DF-1 cells (MOI = 0.01). B Infection potential of ZJ-SW19 in MDCK cells using IFA.

Full size image

Receptor-binding analysis outcomes

The receptor-binding specificity assay results suggested that ZJ-SW19 efficiently binds to both human- and avian-like receptors (α-2,6 and α-2,3, respectively). Thus, ZJ-SW19 can infect via either of these two receptor types (Table 2).

Table 2 Virus receptor binding specificity of ZJ-SW19
Full size table

Antigenic study results

Our analysis of the antigenic relationship between ZJ-SW19 and other H3N2 influenza viruses (including those of swine, human, and avian origins) demonstrated that viruses with the same host origin have similar antigenicity, whereas swine-origin H3N2 and human-origin H3N2 viruses have significantly different antigenicity (Table 3).

Table 3 Analysis of ZJ-SW19 antigenic characteristics using HI assay
Full size table

Pathogenicity in mice

BALB/c mice, the conventional animal model widely used to study influenza virus pathogenesis [20, 26,27,28], was used to predict SIV pathogenicity and replication potential in mammals. At 3, 6, and 9 dpi, ZJ-SW19 was detected in the lungs of infected mice, with the highest titer being noted on day 3, indicating that ZJ-SW19 can effectively replicate in the lungs without prior adaptation (Figure 4A). In contrast, ZJ-SW19 was not detected in other organs such as the brain, heart, liver, kidneys, and spleen. Moreover, over 5–8 dpi, the infected mice experienced moderate weight loss (4–8%) with 100% survival (Figure 4B). Finally, histopathological analysis revealed multiple plaque lesions in the mouse lung tissue on 6 dpi, indicating mild or moderate interstitial inflammatory congestion and exudative lesions, accompanied by the infiltration of many inflammatory cells and RBCs (Figure 5).

Figure 4
figure 4

ZJ-SW19 pathogenicity in vivo. A Viral titers in organs in the group of 106.0 EID50 identified using the TCID50 assay. B Daily weight loss and survival rate in six mice in each experimental group infected with 50 µL of viruses at 105.0 or 106.0 EID50 over 14 dpi.

Full size image
Figure 5
figure 5

H&E staining for histopathological analysis of lung tissues of mice infected with 106.0 EID50 of ZJ-SW19 on 3, 6, and 9 dpi. Mice displayed mild or moderate interstitial inflammatory congestion and exudative lesions, accompanied by inflammatory cells (diamond) and RBC infiltration (triangle). A, B, C, and D show sections from the control, 3 dpi, 6 dpi, and 9 dpi groups, respectively.

Full size image

Discussion

Influenza is the most widely distributed acute zoonotic infectious disease caused by influenza virus. The range of influenza virus hosts is extremely wide, including humans and most animals (e.g., pigs and birds). The H3N2 SIV is one of the most predominant influenza virus strains in pig herds. At large-scale pig farms, swine influenza is a common and difficult-to-eradicate mass disease; it not only causes the death of affected pigs directly but also reduces their production performance, increases their feeding costs, affects the health status and quality of pig herds directly, and poses a great threat to the pig farming industry [33, 34]. H3N2 SIVs are globally endemic, and serological surveillance has revealed that the H3N2 SIV-positivity rate in pig populations is 7.9% [35]. In China, workers coming into contact with pigs have an extremely high risk of epidemic SIV infections. Serological studies have demonstrated that approximately 31.5% of pigs have been infected with at least one SIV type (H1N1 or H3N2 SIV) [36]. In a ferret model, seasonal trivalent inactivated influenza vaccine was noted to be ineffective at preventing H3N2 SIV infection and transmission [37]. Therefore, intensive surveillance is essential for reducing SIV infection and pandemic risk.

In the present study, the H3N2 SIV ZJ-SW19 was isolated, and its antigenicity appeared to differ from that of current human H3N2 viruses. The binding of surface HA protein to the host cell sialic acid can initiate influenza virus infection, and several single amino acid substitutions on HA can affect the host species. The single amino substitution A196T can increase virus replication in eggs without affecting virus antigenicity [31]. The mutations E190D and Q226L in H3 HA and G225D/E in H1 HA are essential for alternations in viral receptor-binding properties during the adaptation of avian viruses to humans and pigs [38, 39]. Here, we noted that ZJ-SW19 binds strongly to the human-like sialic acid receptor but weakly to the avian-like sialic acid receptor. In addition, considering the differences in their biological characteristics, novel reassortant influenza virus strains may also demonstrate considerable differences in pathogenicity and replicability [40]. Finally, we also noted that ZJ-SW19 effectively replicates only in the respiratory tract and causes mild weight loss in mice.

Pigs, crucial influenza virus hosts, are susceptible to avian and human influenza viruses, making them “mixing vessels” that produce new types of pandemic influenza viruses. Moreover, the SIV subtypes H1N1, H1N2, and H3N2 are prevalent in pig herds and can cause the emergence of various reassortant isolates. Moreover, H3N2 influenza viruses containing the H1N1 internal genes have been widely discovered in Southern China; these viruses, including completely human-origin viruses, early-stage or recent double-reassortant human viruses, and avian-origin viruses, include at least four H3N2 genotypes [41,42,43,44]. In the analysis of five H3N2 SIV strains isolated from Guangdong, Southern China, in 2005, the aforementioned virus strains were noted to originate from human-like H3N2 virus strains in the 1990s: H3N2 reassortant virus from humans and H1N1 virus from pigs (both extremely common among pig populations) [45]. Thus, pigs are intermediate hosts for avian-origin viruses before human infection, and they can be “mixing vessels” for genetically reassortant virus generation. We previously reported that the H1N2 virus circulating in pig herds in Zhejiang, Eastern China, is multireassortant with genomes derived from both H1N1 pdm/2009 and swine-origin H3N2 viruses [46]. In general, SIVs circulating in pig herds in Southern and Eastern China have undergone extensive reassorting processes over a long period.

The respiratory tracts of pigs have both avian- and human-like receptors and thus can become infected with either avian or human influenza viruses. Therefore, one of the most common, conventional cross-species transmission routes for avian influenza involves pigs. Moreover, pigs may be naturally infected with avian-origin influenza viruses, including the H4N6 virus, the H9N2 virus, and the highly pathogenic H5 viruses [47,48,49,50]. Considering the large scale of poultry and pig farming, various SIV and avian influenza virus subtypes have been widely discovered in China, highlighting that the potential threats of SIVs to human health may continue to exist. In the current study, ZJ-SW19 was noted to carry five pmd/2009 internal segments, suggesting that influenza virus reassortment occurs in pig populations in Eastern China. Multiple antigenic drifts have introduced H3N2 SIVs to humans over a long period [51]. Thus, further continual monitoring and research are warranted.

In summary, we genetically and biologically characterized the novel reassortant H3N2 SIV isolated from pig farms. Our results suggested that this SIV can bind to human- and avian-like receptors along with some mammal-adapted mutations; it can effectively replicate in the lungs in mice, exhibiting moderate virulence. Taken together, our results highlight the necessity for continued surveillance of H3N2 SIVs in Eastern China to prevent any related public health emergencies.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

References

  1. Yoon SW, Webby RJ, Webster RG (2014) Evolution and ecology of influenza A viruses. Curr Top Microbiol Immunol 385:359–375

    PubMed  Google Scholar 

  2. Takemae N, Tsunekuni R, Uchida Y, Ito T, Saito T (2018) Experimental infection of pigs with H1 and H3 influenza A viruses of swine by using intranasal nebulization. BMC Vet Res 14:115

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lin X, Huang C, Shi J, Wang R, Sun X, Liu X, Zhao L, Jin M (2015) Investigation of pathogenesis of H1N1 influenza virus and swine Streptococcus suis serotype 2 co-infection in pigs by microarray analysis. PLoS One 10:e0124086

    Article  PubMed  PubMed Central  Google Scholar 

  4. Nakharuthai C, Boonsoongnern A, Poolperm P, Wajjwalku W, Urairong K, Chumsing W, Lertwitcharasarakul P, Lekcharoensuk P (2008) Occurrence of swine influenza virus infection in swine with porcine respiratory disease complex. Southeast Asian J Trop Med Public Health 39:1045–1053

    PubMed  Google Scholar 

  5. Ma W, Kahn RE, Richt JA (2008) The pig as a mixing vessel for influenza viruses: Human and veterinary implications. J Mol Genet Med 3:158–166

    PubMed  PubMed Central  Google Scholar 

  6. Van Reeth K (2007) Avian and swine influenza viruses: our current understanding of the zoonotic risk. Vet Res 38:243–260

    Article  PubMed  Google Scholar 

  7. McDonald SM, Nelson MI, Turner PE, Patton JT (2016) Reassortment in segmented RNA viruses: mechanisms and outcomes. Nat Rev Microbiol 14:448–460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jung K, Song DS, Kang BK, Oh JS, Park BK (2007) Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea. Prev Vet Med 79:294–303

    Article  PubMed  Google Scholar 

  9. Yu H, Zhang GH, Hua RH, Zhang Q, Liu TQ, Liao M, Tong GZ (2007) Isolation and genetic analysis of human origin H1N1 and H3N2 influenza viruses from pigs in China. Biochem Biophys Res Commun 356:91–96

    Article  CAS  PubMed  Google Scholar 

  10. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, Sessions WM, Xu XY, Skepner E, Deyde V, Okomo-Adhiambo M, Gubareva L, Barnes J, Smith CB, Emery SL, Hillman MJ, Rivailler P, Smagala J, de Graaf M, Burke DF, Fouchier RAM, Pappas C, Alpuche-Aranda CM, Lopez-Gatell H, Olivera H, Lopez I, Myers CA, Faix D, Blair PJ, Yu C, et al. (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325:197–201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Watson SJ, Langat P, Reid SM, Lam TT, Cotten M, Kelly M, Van Reeth K, Qiu Y, Simon G, Bonin E, Foni E, Chiapponi C, Larsen L, Hjulsager C, Markowska-Daniel I, Urbaniak K, Durrwald R, Schlegel M, Huovilainen A, Davidson I, Dan A, Loeffen W, Edwards S, Bublot M, Vila T, Maldonado J, Valls L, Brown IH, Pybus OG, Consortium E, et al. (2015) Molecular epidemiology and evolution of influenza viruses circulating within European swine between 2009 and 2013. J Virol 89:9920–9931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zeller MA, Ma J, Wong FY, Tum S, Hidano A, Holt H, Chhay T, Sorn S, Koeut D, Seng B, Chao S, Ng GGK, Yan Z, Chou M, Rudge JW, Smith GJD, Su YCF (2023) The genomic landscape of swine influenza A viruses in Southeast Asia. Proc Natl Acad Sci U S A 120:e2301926120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nonthabenjawan N, Chanvatik S, Chaiyawong S, Jairak W, Boonyapisusopha S, Tuanudom R, Thontiravong A, Bunpapong N, Amonsin A (2015) Genetic diversity of swine influenza viruses in Thai swine farms, 2011–2014. Virus Genes 50:221–230

    Article  CAS  PubMed  Google Scholar 

  14. Zell R, Groth M, Krumbholz A, Lange J, Philipps A, Durrwald R (2020) Cocirculation of swine H1N1 influenza A virus lineages in Germany. Viruses 12:762

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kaplan BS, Kimble JB, Chang J, Anderson TK, Gauger PC, Janas-Martindale A, Killian ML, Bowman AS, Vincent AL (2020) Aerosol transmission from infected swine to ferrets of an H3N2 virus collected from an agricultural fair and associated variant infections with human. J Virol 94:e01009-20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhu W, Yang S, Dong L, Yang L, Tang J, Zou X, Chen T, Yang J, Shu Y (2015) The repeated introduction of the H3N2 virus from human to swine during 1979–1993 in China. Infect Genet Evol 33:20–24

    Article  PubMed  Google Scholar 

  17. Ding F, Li Y, Huang B, Edwards J, Cai C, Zhang G, Jiang D, Wang Q, Robertson ID (2021) Infection and risk factors of human and avian influenza in pigs in south China. Prev Vet Med 190:105317

    Article  PubMed  Google Scholar 

  18. Wu HB, Guo CT, Lu RF, Xu LH, Wo EK, You JB, Wang YT, Wang QG, Wu NP (2012) Genetic characterization of subtype H1 avian influenza viruses isolated from live poultry markets in Zhejiang Province, China, in 2011. Virus Genes 44:441–449

    Article  CAS  PubMed  Google Scholar 

  19. Klimov A, Balish A, Veguilla V, Sun H, Schiffer J, Lu X, Katz JM, Hancock K (2012) Influenza virus titration, antigenic characterization, and serological methods for antibody detection. Methods Mol Biol 865:25–51

    Article  CAS  PubMed  Google Scholar 

  20. Wu H, Lu R, Peng X, Peng X, Chen B, Cheng L, Wu N (2017) Molecular characterization of a novel reassortant H7N6 subtype avian influenza virus from poultry in Eastern China, in 2016. Arch Virol 162:1341–1347

    Article  CAS  PubMed  Google Scholar 

  21. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275–2289

    Article  CAS  PubMed  Google Scholar 

  22. Wu H, Wu N, Peng X, Jin C, Lu X, Cheng L, Yao H, Li L (2014) Molecular characterization and phylogenetic analysis of H3 subtype avian influenza viruses isolated from domestic ducks in Zhejiang Province in China. Virus Genes 49:80–88

    Article  CAS  PubMed  Google Scholar 

  23. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhu H, Li X, Chen H, Qian P (2022) Genetic characterization and pathogenicity of a Eurasian avian-like H1N1 swine influenza reassortant virus. Virol J 19:205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Plant EP, Ye Z (2015) Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus. J Gen Virol 96:752–755

    Article  CAS  PubMed  Google Scholar 

  26. Yang F, Xiao Y, Lu R, Chen B, Liu F, Wang L, Yao H, Wu N, Wu H (2020) Generation of neutralizing and non-neutralizing monoclonal antibodies against H7N9 influenza virus. Emerg Microbes Infect 9:664–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yang F, Xiao Y, Liu F, Yao H, Wu N, Wu H (2021) Molecular characterization and antigenic analysis of reassortant H9N2 subtype avian influenza viruses in Eastern China in 2016. Virus Res 306:198577

    Article  CAS  PubMed  Google Scholar 

  28. Yang F, Xiao Y, Liu F, Cheng L, Yao H, Wu N, Wu H (2021) Genetic analysis and biological characteristics of novel clade 2.3.4.4 reassortment H5N6 avian influenza viruses from poultry in eastern China in 2016. Int J Infect Dis 110:436–448

    Article  CAS  PubMed  Google Scholar 

  29. Yang F, Zhu L, Liu F, Cheng L, Yao H, Wu N, Wu H, Li L (2022) Generation and characterization of monoclonal antibodies against the hemagglutinin of H3N2 influenza A viruses. Virus Res 317:198815

    Article  CAS  PubMed  Google Scholar 

  30. Rui-Hua Z, Hong-Yu C, Ming-Ju X, Kai L, Hua-Lan C, Cun-Lian W, Dong W, Cun-Xin L, Tong X (2011) Molecular characterization and pathogenicity of swine influenza H9N2 subtype virus A/swine/HeBei/012/2008/(H9N2). Acta Virol 55:219–226

    Article  PubMed  Google Scholar 

  31. Lu B, Zhou H, Chan W, Kemble G, Jin H (2006) Single amino acid substitutions in the hemagglutinin of influenza A/Singapore/21/04 (H3N2) increase virus growth in embryonated chicken eggs. Vaccine 24:6691–6693

    Article  CAS  PubMed  Google Scholar 

  32. Wu HB, Lu RF, Peng XM, Peng XR, Cheng LF, Liu FM, Wu NP (2017) Characterization of novel reassortant influenza A (H5N2) viruses isolated from poultry in Eastern China, 2015. Front Microbiol 8:741

    Article  PubMed  PubMed Central  Google Scholar 

  33. Lyoo KS, Kim JK, Jung K, Kang BK, Song D (2014) Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses. Virol J 11:170

    Article  PubMed  PubMed Central  Google Scholar 

  34. Graaf-Rau A, Hennig C, Lillie-Jaschniski K, Koechling M, Stadler J, Boehmer J, Ripp U, Pohlmann A, Schwarz BA, Beer M, Harder T (2023) Emergence of swine influenza A virus, porcine respirovirus 1 and swine orthopneumovirus in porcine respiratory disease in Germany. Emerg Microbes Infect 12:2239938

    Article  PubMed  PubMed Central  Google Scholar 

  35. Wu R, Liu Z, Liang W, Yang K, Deng J, Duan Z, Zhou D, Xu D (2011) Serological and virological surveillance of swine H1N1 and H3N2 influenza virus infection in two farms located in Hubei province, central China. Zoonoses Public Health 58:508–513

    Article  CAS  PubMed  Google Scholar 

  36. Borkenhagen LK, Wang GL, Simmons RA, Bi ZQ, Lu B, Wang XJ, Wang CX, Chen SH, Song SX, Li M, Zhao T, Wu MN, Park LP, Cao WC, Ma MJ, Gray GC (2020) High risk of influenza virus infection among swine workers: examining a dynamic cohort in China. Clin Infect Dis 71:622–629

    Article  PubMed  Google Scholar 

  37. Houser KV, Pearce MB, Katz JM, Tumpey TM (2013) Impact of prior seasonal H3N2 influenza vaccination or infection on protection and transmission of emerging variants of influenza A(H3N2)v virus in ferrets. J Virol 87:13480–13489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR, Donatelli I, Kawaoka Y (2000) Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol 74:8502–8512

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Pascua PN, Song MS, Lee JH, Baek YH, Kwon HI, Park SJ, Choi EH, Lim GJ, Lee OJ, Kim SW, Kim CJ, Sung MH, Kim MH, Yoon SW, Govorkova EA, Webby RJ, Webster RG, Choi YK (2012) Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses. Proc Natl Acad Sci U S A 109:15900–15905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. He P, Wang G, Mo Y, Yu Q, Xiao X, Yang W, Zhao W, Guo X, Chen Q, He J, Liang M, Zhu J, Ding Y, Wei Z, Ouyang K, Liu F, Jian H, Huang W, Garcia-Sastre A, Chen Y (2018) Novel triple-reassortant influenza viruses in pigs, Guangxi, China. Emerg Microbes Infect 7:85

    Article  PubMed  PubMed Central  Google Scholar 

  41. Chen J, Fu X, Chen Y, He S, Zheng Y, Cao Z, Yu W, Zhou H, Su S, Zhang G (2014) Identification of four genotypes of H3N2 swine influenza virus in pigs from southern China. Arch Virol 159:2705–2709

    Article  CAS  PubMed  Google Scholar 

  42. Fan X, Zhu H, Zhou B, Smith DK, Chen X, Lam TT, Poon LL, Peiris M, Guan Y (2012) Emergence and dissemination of a swine H3N2 reassortant influenza virus with 2009 pandemic H1N1 genes in pigs in China. J Virol 86:2375–2378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yu H, Zhang PC, Zhou YJ, Li GX, Pan J, Yan LP, Shi XX, Liu HL, Tong GZ (2009) Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Biochem Biophys Res Commun 386:278–283

    Article  CAS  PubMed  Google Scholar 

  44. Kong WL, Huang LZ, Qi HT, Cao N, Zhang LQ, Wang H, Guan SS, Qi WB, Jiao PR, Liao M, Zhang GH (2011) Genetic characterization of H1N2 influenza a virus isolated from sick pigs in Southern China in 2010. Virol J 8:469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sun L, Zhang G, Shu Y, Chen X, Zhu Y, Yang L, Ma G, Kitamura Y, Liu W (2009) Genetic correlation between H3N2 human and swine influenza viruses. J Clin Virol 44:141–144

    Article  CAS  PubMed  Google Scholar 

  46. Peng X, Wu H, Xu L, Peng X, Cheng L, Jin C, Xie T, Lu X, Wu N (2016) Molecular characterization of a novel reassortant H1N2 influenza virus containing genes from the 2009 pandemic human H1N1 virus in swine from eastern China. Virus Genes 52:405–410

    Article  CAS  PubMed  Google Scholar 

  47. Karasin AI, Brown IH, Carman S, Olsen CW (2000) Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J Virol 74:9322–9327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang J, Wu M, Hong W, Fan X, Chen R, Zheng Z, Zeng Y, Huang R, Zhang Y, Lam TT, Smith DK, Zhu H, Guan Y (2016) Infectivity and transmissibility of avian H9N2 influenza viruses in pigs. J Virol 90:3506–3514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wu H, Yang F, Lu R, Xu L, Liu F, Peng X, Wu N (2018) Isolation and molecular characterization of an H5N1 swine influenza virus in China in 2015. Arch Virol 163:701–705

    Article  CAS  PubMed  Google Scholar 

  50. Li X, Fu Y, Yang J, Guo J, He J, Guo J, Weng S, Jia Y, Liu B, Li X, Zhu Q, Chen H (2015) Genetic and biological characterization of two novel reassortant H5N6 swine influenza viruses in mice and chickens. Infect Genet Evol 36:462–466

    Article  CAS  PubMed  Google Scholar 

  51. Wang X, Ilyushina NA, Lugovtsev VY, Bovin NV, Couzens LK, Gao J, Donnelly RP, Eichelberger MC, Wan H (2017) Amino acids in hemagglutinin antigenic site B determine antigenic and receptor binding differences between A(H3N2)v and ancestral seasonal H3N2 influenza viruses. J Virol 91:e01512-16

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.

Funding

This work was supported by grants from the National Science Foundation of the People’s Republic of China (Grant Number 32273092); the Zhejiang Provincial Natural Science Foundation of China (Grant Number LY24H190001); and the Fundamental Research Funds for the Central Universities (Grant Number 2022ZFJH003).

Author information

Authors and Affiliations

  1. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, and National Clinical Research Center for Infectious Diseases, School of Medicine, the First Affiliated Hospital, Zhejiang University, Hangzhou, 310003, China

    Fan Yang, Linfang Cheng, Fumin Liu, Hangping Yao, Nanping Wu & Haibo Wu

  2. Animal Husbandry and Veterinary Institute, Zhejiang Academy of Agricultural Science, Hangzhou, 310021, China

    Lihua Xu

Authors
  1. Fan Yang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Linfang Cheng
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Fumin Liu
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Hangping Yao
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Nanping Wu
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Lihua Xu
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Haibo Wu
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

FY and LC conducted the experiments, analyzed the data, and wrote the manuscript. FL, HY and NW contributed to the data analysis and manuscript writing. LX and HW reviewed and revised the manuscript. All authors have read and approved the final version of manuscript.

Corresponding author

Correspondence to Haibo Wu.

Ethics declarations

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (Approval No. 2019-39).

Competing interests

The authors declare that they have no competing interests.

Additional information

Handling editor: Marie Galloux.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, F., Cheng, L., Liu, F. et al. Genetic and molecular characterization of a novel reassortant H3N2 influenza virus from a sick pig in Eastern China in 2019. Vet Res 56, 38 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-025-01462-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-025-01462-7

Keywords

Veterinary Research

ISSN: 1297-9716