Introduction
Swine influenza A viruses (SIV) belong to the genus influenza A viruses (IAV) of the family Orthomyxoviridae and are dangerous pathogens for pigs compared to influenza B, C, and D viruses [
1,
2]. The hemagglutinin (HA) and neuraminidase (NA) proteins of the IAV are applied to further classify them into subtypes [
3]. Although there are currently 18 HA subtypes and 11 NA subtypes, most of which circulate in wild birds, antigenic variation of influenza arises rapidly from drift and shift resulting in the creation of novel IAV [
4].
Pigs are regarded as “mixing vessels” for the production of IAV with pandemic potential because they have both SA2,3Gal and SA2,6Gal receptor residues dispersed throughout the respiratory tracts, whereas avian, swine and human IAV can undergo genetic reassortment [
5,
6]. Once the novel IAV is established in pigs, it will be dangerous to the animals and pose a risk of spreading to people and causing outbreaks and pandemics with introduction or reintroduction into humans [
7]. The emergence of the pandemic 2009 H1N1 (pdm09) viruses, which contains genetic fragments from avian, swine, and human, further emphasizes the risk by highlighting the swine in the production of influenza reassortant variants that could infect humans [
8].
With avian/human, human/swine, and human/avian/swine triple-reassortant (TP) lineage, Eurasian avian-like (EA) lineage SIV, and classical swine (CS) lineage SIV cocirculating in pigs, China possesses the most complicated SIV ecosystem [
9]. Based on lineage classification, six genotypes of G1–G6 EA reassortant were found in EA H1N1 viruses from 2011 to 2018 [
2]. The predominant emergent EA reassortant viruses (EA-H1N1) in pigs have pdm09 and TPs-derived internal genes and have shown a sharp increase since 2016 [
2]. EA-H1N1 is the predominant genotype in circulation in pigs detected across at least 10 provinces and possesses all of the essential hallmarks of a candidate pandemic virus [
10‐
12]. Therefore, understanding the evolution and pathogenicity of EA-H1N1 in China may help to guide management and pandemic preparedness strategies [
2].
In this study, we phylogenetically characterized SIV isolates obtained from pregnant sows with miscarriage and respiratory disease on farms in Heilongjiang, China. Additionally, to find out the virulence of the virus in mammals, the mouse mode was used to study the pathogenicity of the isolate.
Materials and methods
Nucleic acid extraction and pathogen detection
3 tissue samples including heart, lung, placenta, submaxillary lymph nodes and tonsil from miscarriage and respiratory disease in pregnant sows were collected at a period (September 2020 and October 2020) on the same pig farm in Heilongjiang province. Tissue samples were homogenized and diluted with sterile PBS, collected supernatant followed by centrifugation at 8000 g for 10 min at 4 °C to remove residual tissue debris. Next-generation sequencing (NGS) was used to detect pathogens that miscarriage and respiratory disease in pregnant sows or other common clinical pathogens. RNA was extracted using TRIzol reagent accordance with the manufacturer’s protocol. For RNA NGS, cDNA library was constructed using TR503-01 reagents according to the manufacturer’s instructions, and an oligonucleotide of known sequence was added to the sample for quality control. Sequencing libraries were generated using the Nextera XT DNA Library Prep Kit (Illumina) with dual index pairs. Libraries underwent amplification with the following conditions as previously reported [
13]: (i) 72 °C for five minutes and 95 °C for 30 s, (ii) up to 25 cycles of 95 °C for 15 s, 55 °C 30 s, and 72 °C for 60 s, (iii) 72 °C for 5 min. Then, the libraries were quantified using the KAPA universal complete kit (Roche, Swiss Confederation), pooled to equal concentration, and sequenced on an Illumina platform using paired-end 100 or 150 bp reads.
Isolation and identification of SIV
SIV was isolated and expanded in embryonated chicken eggs. SIV-positive samples were filtered using 0.22 μm filters and then inoculated into 10-day-old embryonated chicken eggs at 37 °C for 72 h as previously described [
2,
14]. At 4 days post-infection (dpi), the virus was harvested and then inoculated into fresh embryonated chicken eggs. After successive generations, viral RNA was extracted. Reverse transcription was carried out using the Uni12 (AGCAAAAGCAGG) primer to detect SIV by RT-PCR [
15]. The virus was identified by RT-PCR using specific primers M1 gene of SIV (Additional file
1: Table S1). The subtype of the virus was identified by RT-PCR using subtype-specific primers [
16,
17]. After 3 generations, the virus was harvested and then infected MDCK (Madin-Darby canine kidney) cells containing 2 µg/mL TPCK-trypsin. MDCK cells were cultured in high-glucose DMEM (Dulbecco’s modified Eagle medium, HyClone, Marlborough, MA, USA) supplemented with 10%(v/v) FBS (heat-inactivated fetal bovine serum, Inner Mongolia opcel Biotechnology Co., Ltd., Inner Mongolia, China) for 50% tissue culture infective dose (TCID
50) and Indirect immunofluorescence assay (IFA). 1% chicken erythrocytes were prepared from EDTA anticoagulant and Alsever’s Solution to determine hemagglutination activity.
IFA was performed to detect viral activity of isolates. Briefly, Confluent with MDCK cells in 24-well plates were washed three times with PBS and infected with the isolates at 0.1 MOI in serum-free DMEM containing 2 µg/mL TPCK-trypsin for 16 h. The infected MDCK cells were washed 3 times with phosphate buffer containing Tween 20 (PBST) and then fixed with 4% paraformaldehyde solution for 30 min at 4 °C. The blocked cells were incubated with NP monoclonal antibody (Abcam, UK) for 2 h at 37 °C. Subsequently, the cells were washed with PBST 3 times and incubated with Alexa Fluor 488 goat anti-mouse antibody (ThermoFisher, Waltham, MA, USA) for 1 h at 37 °C. After intensive washing, the cells were analyzed under a fluorescence microscope (Ti-U-Nikon, Tokyo, Japan) with a video documentation system.
Western blot assay (WB) was performed to determine IAV. Briefly, the isolates were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked in blocking buffer containing 5% skimmed milk in Tris-buffered saline and Tween 20 (TBS-T) at 4 °C for 1 h, and incubated with the NP monoclonal antibody for 2 h at room temperature (RT). The HRP-labelled goat anti-mouse antibody (ABclonal, China) was used as the secondary antibody to incubate the membrane for 1 h at RT. After washing thrice with TBS-T, signals were visualized using the ECL chemiluminescence system. Protein bands were analyzed via Image Lab software 4.0.1.
Transmission electron microscopic (TEM) analysis was performed to observe the morphology of the SIV as reported [
18]. The isolates purified by ultra-high-speed centrifugation were negatively stained with phosphotungstic acid and ammonium molybdate on 400 mesh copper mesh (Beijingzhongjingkeyi Technology Co., Ltd, China), respectively, and images were recorded on a Hitachi microscope (HT7700, Japan) with a camera.
Whole-genome analysis of SIV
PCR was performed using segment-specific primers for eight genes (PB2, PB1, PA, HA, NP, NA, M, and NS) as reported previously [
19]. The PCR products were purified and sequenced as reported previously [
20]. DNA sequences were compiled and edited using Lasergene 7.1 (DNASTAR, Madison, WI, USA). Phylogenetic trees were generated by the distance-based neighbor-joining method using software MEGA 7.0 (Sinauer Associates, Inc., Sunderland, MA, USA). The reliability of the tree was assessed by bootstrap analysis with 1000 replicates.
Infection studies in mice
To evaluate the pathogenicity of isolates in mammalian hosts, the six-week-old female BALB/c mice were purchased from the Laboratory Animal Research Center of Huazhong Agricultural University (Wuhan, China) approved by the Ethical and Welfare Committee (HZAUMO-2022-0051) and were infected intranasally with 50 µL 106 50% egg infectious dose virus (EID50) after anesthesia with sodium pentobarbital Additionally, 5 mice infected with 50 µL of PBS were used as negative controls. All mice were monitored for mortality and weight change over 14 days daily and euthanized immediately with 30% or greater weight loss (considered as death). The lung, trachea, spleen, ovary, and kidney collected from the infected before the agonal stage death and control mice after 14 days (humanely euthanized by injecting an overdose of intraperitoneal sodium pentobarbital) were then taken for viral titer analysis in embryonated chicken eggs by Reed and Muench.
Histology and immunohistochemistry
The infected group before the agonal stage death and the PBS group following 14 dpi of virus isolates were humanely euthanized by injecting an overdose of intraperitoneal sodium pentobarbital and necropsied. The mice were aseptically necropsied, and the lung and trachea were collected and fixed in 10% neutral buffered formalin for HE staining [
21]. Immunohistochemistry was performed to identify the position of the virus in the lungs and trachea as reported previously [
22‐
24].
Statistical analysis
All the statistical analyses were performed using GraphPad Prism software. Comparisons among different groups were evaluated by one-way ANOVA. Data were expressed as the mean ± the standard deviation (SD). In all cases, p < 0.05 was considered as statistically significant difference.
Discussion
Swine influenza was first recognized as a disease of pigs during the human pandemic in 1918 [
31]. Pigs are an important host in IAV ecology, since they are susceptible to infection with avian, porcine, and human IAV. Avian/human, human/swine, and TP reassortant IAV have been isolated from pigs worldwide [
32]. The sequences of EA H1N1 SIVs from China between 2001 and 2020, including the 2009 pandemic H1N1 (A(H1N1) pdm09), and triple reassortant H1N2 (TP H1N2) influenza viruses, with SIVs generating 11 genotypes [
1,
2]. Presently, there are various subtypes of swine influenza virus in China. H1N1, H3N2 and H1N2 were the predominant genetic subtypes of the virus in pigs, and H4N8, H5N1, H6N6, H7N9, H9N2 and H10N8 SIV also were directly transmitted from to the pigs since 2009 to 2021 [
33‐
37]. In our study, a H1N1 virus (A/swine/Heilongjiang/GN/2020(H1N1)) was isolated from pigs with miscarriage and respiratory disease. Homology and phylogenetic analyses of whole-genome results demonstrated that the PB2, PB1, PA, NP, and M genes of the virus are confirmed to have high homology with pdm09-H1N1 viruses nucleotide sequence, and the HA and NA genes of the virus are closely related to the nucleotide sequence homology of EA-H1N1 viruses, while the NS gene of the virus is closely related to TP viruses from China (Additional file
1: Fig. S3). These data indicated that the origin of eight gene fragments of the virus is consistent with EA-H1N1 SIV as reported previously [
2], so A/swine/Heilongjiang/GN/2020 belongs to the EA-H1N1 genotype.
The deduced amino acid sequences of A/swine/Heilongjiang/GN/2020 and the reference viruses of H1N1 from different lineages were analyzed to determine whether the signature amino acids of HA1 genes associated with virulence and host adaptation had changed [
38]. The HA gene of A/swine/Heilongjiang/GN/2020 contained the motif PSIQSR/GLF at the cleavage site between HA1 and HA2 consistent with the characteristics of low pathogenic IAV as reported previously (Fig.
4) [
7,
25]. The glycosylation positions 195 (NNT) and 274 (NCT) of A/swine/Heilongjiang/GN/2020 are inconsistent with that of the CS lineage (Fig.
4), while their changes might enable the influenza virus to escape the host immune system as reported previously [
39,
40].
The binding preference of HA to the host SAα2,6Gal receptor is a critical determinant for the cross-species transmission of IAVs to humans [
26]. The HAs of human IAVs preferentially bind to the SAα2,6Gal receptor, while avian IAVs prefer the SAα2,3Gal receptor [
41]. EA-H1N1 replicate similarly to pdm/09-H1N1, both preferentially bind human-like SAα2,6Gal receptor and replicate efficiently in human airway epithelial cells, as reported [
2]. Amino acid residues at the receptor-binding position of HA1 (positions Q223 and G225) retained SAα2,6Gal receptor and SAα2,3Gal (G131-A135) in the virus, predicting that it had an affinity for mammalian cell-surface receptors that could adapt host to infect avian species, swine and humans by receptor-binding. The antigenic determinant domains (Sa, Sb, Ca1, Ca2, and Cb) were found to be involved in antibody binding [
27,
28], and the major antigenic determinant domains of A/swine/Heilongjiang/GN/2020 were similar to the reference influenza. However, in the antigenic site of Ca1, the mutation T204S was found in the virus (Fig.
4), and the antibody produced by seasonal IAV H1N1 elicited limited cross-reactivity against the virus because of mutation of the antigenic determinant domains [
7].
Previous studies showed that EA-H1N1 viruses and their cases displayed increased pathogenicity [
11]. A recent study found that EA H1N1 viruses formed eight different genotypes through reassortment with viruses of other lineages circulating in humans and pigs, two of these genotypes (G4 and G5) were widely distributed in pigs as reported in 22 provinces in China between October 2013 and December 2019 [
14]. In our study, the respiratory disease in mice infected with the EA-H1N1(A/swine/Heilongjiang/GN/2020) resulted in greater weight loss and was lethal, which is similar to the outcome of mice infected with TP A/Ohio/2/07 (OH/2) virus [
42]. More interestingly, the replication of A/swine/Heilongjiang/GN/2020 was not restricted to respiratory tract tissues but could be systemically spread in mice. The A/swine/Heilongjiang/GN/2020 showed effective replication in lung, trachea, spleen and kidney of mice (Fig.
5C), which is consistent with the lethal and systemic transmission of EA-H1N1 viruses in mice [
43], and was inconsistent with pdm09 virus not causing lethal or exhibit extrapulmonary virus spread [
44]. Yang et al. reported that the EAH1N1 SIVs are preferentially bound to human-type receptors, which suggests that is needed to prevent the efficient transmission of EAH1N1 SIVs to humans as reported previously [
45]. Meng et al. found that EA H1N1 viruses obtained pathogenic in mice characteristics by accumulating mutations in its acidic polymerase (PA) gene that is predominant in the pdm09-H1N viruses [
46]. Wang et al. found that a single-amino-acid substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) in the HA1 protein completely abolished the respiratory droplet transmission of GX/18, whereas the substitution in the same position (G225E) in HA1 enabled HLJ/27 to transmit in guinea pigs [
47]. In our study, the PA gene of A/swine/Heilongjiang/GN/2020 belongs to the pdm09 lineage (Additional file
1: Figure S3C) and the amino acid residues at the receptor-binding pocket of HA1 (position Q223 and G225) retained configurations (SAα2,6Gal) (Fig.
4), which suggests that that the virus could be effectively transmitted to humans through mice. The characterization of reproductive failure in late-gestation sows is premature farrowing of stillborn and mummified fetuses, and the pathogen of which only was SIV and no other pathogens by NGS and PCR analysis. So, is miscarriage associated with significant proliferation of influenza virus in the ovary or uterus? We attempted to determine whether the virus could be isolated from the ovary of mice infected with A/swine/Heilongjiang/2020, but it was unfortunately unsuccessful (Fig.
5C). Pregnant sows have an increased risk of respiratory complications and reproductive failure related to influenza as reported previously [
48], which may also be responsible for uterine contractions constituting a threat of miscarriage or premature labor. Nonetheless, the mechanism of reproductive failure that EA-H1N1caused reproductive failure in late-gestation sows remains to be further explored in pigs. Histopathological and immunohistochemistry examinations showed that the virus could cause severe damage of respiratory tracts and proliferated effectively lungs and trachea (Fig.
5D, E). These results found that the EA H1N1 virus (A/swine/Heilongjiang/GN/2020) was highly pathogenic to mice and could be systemic spread in mice.
EA-H1N1 viruses have been a growing problem in pig farms since 2014, inevitably increasing their exposure to humans [
49]. Two human cases of EA-lH1N1 infection have been reported in 2016 and 2019 in China [
49,
50], and genetic analysis indicated that the two cases were caused by the EA-H1N1 virus [
2]. The two patients’ neighbors who reared pigs, suggesting that the EA-H1N1 virus could transmit from swine to humans [
49‐
51]. Thus, virological surveillance of EA-H1N1 is necessary for swine and human populations, which has fundamental implications for human public health.
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